Neurological Diseases and Prevalence of Antineuronal Antibodies in Patients with Autoimmune Polyendocrine Syndrome Type 1 - A National Cohort Study.

Autoimmune polyendocrine syndrome type 1 (APS-1) is a rare monogenic disease caused by mutations in the autoimmune regulator gene. Although the disease-associated autoantibodies mostly target endocrine organs, autoantibodies from patients with APS-1 bind also to rat brain structures. The patients often have GAD65-antibodies, that can cause autoimmune encephalitis. However, neurological manifestations of APS-1 have not been systematically explored. We conducted a retrospective chart review on 44 Finnish patients with APS-1 (median age 38 years, 61% females) and collected all their neurological diagnoses. To assess the prevalence of serum antineuronal antibodies in APS-1, serum samples of 24 patients (median age 36 years, 63% females) were analyzed using a fixed cell-based assay. Of the 44 APS-1 patients, 10 (23%) had also received a diagnosis of a neurological disease. Of these neurological comorbidities, migraine (n = 7; 16%), central nervous system infections (n = 3; 7%), and epilepsy (n = 2; 5%) were the most prevalent. Other diagnoses recorded for single patients were axonal sensorimotor polyneuropathy, essential tremor, idiopathic intracranial hypertension, ischemic stroke, and trigeminal neuralgia. Serum antineuronal antibodies were detected in 42% of patients tested (10/24, 50% females, median age 42 years), GAD65 antibodies being the most common finding. Antibodies against glycine and aquaporin 4 were found in low titers. In four patients, relatively high titers of GAD65 antibodies without coexisting type 1 diabetes were found, but none presented with GAD65-encephalitis. Our study suggests an association between APS-1 and neurological disorders, the mechanisms of which are to be further investigated.

Affinity chromatography reveals direct binding of the GATA4-NKX2-5 interaction inhibitor (3i-1000) with GATA4.

Heart failure is a serious medical condition with a poor prognosis. Current treatments can only help manage the symptoms and slow the progression of heart failure. However, there is currently no cure to prevent and reverse cardiac remodeling. Transcription factors are in a central role in various cellular processes, and in the heart, GATA4 and NKX2-5 transcription factors mediate hypertrophic responses and remodeling. We have identified compounds that modulate the synergistic interaction of GATA4 and NKX2-5 and shown that the most promising compound (1, 3i-1000) is cardioprotective in vitro and in vivo. However, direct evidence of its binding site and mechanism of action has not been available. Due to the disordered nature of transcription factors, classical target engagement approaches cannot be utilized. Here, we synthesized a small-molecule ligand-binding pulldown probe of compound 1 to utilize affinity chromatography alongside CETSA, AlphaScreen, and molecular modeling to study ligand binding. These results provide the first evidence of direct physical binding of compound 1 selectively to GATA4. While developing drugs that target transcription factors presents challenges, advances in technologies and knowledge of intrinsically disordered proteins enable the identification of small molecules that can selectively target transcription factors.

Epstein-Barr Virus in the Cerebrospinal Fluid and Blood Compartments of Patients With Multiple Sclerosis and Controls.

BACKGROUND AND OBJECTIVES: Epstein-Barr virus (EBV) infection is a major risk factor of multiple sclerosis (MS). We examined the presence of EBV DNA in the CSF and blood of patients with MS and controls. We analyzed whether EBV DNA is more common in the CSF of patients with MS than in controls and estimated the proportions of EBV-positive B cells in the CSF and blood. METHODS: CSF supernatants and cells were collected at diagnostic lumbar punctures from 45 patients with MS and 45 HLA-DR15 matched controls with other conditions, all participants were EBV seropositive. Cellular DNA was amplified by Phi polymerase targeting both host and viral DNA, and representative samples were obtained in 28 cases and 28 controls. Nonamplified DNA from CSF cells (14 cases, 14 controls) and blood B cells (10 cases, 10 controls) were analyzed in a subset of participants. Multiple droplet digital PCR (ddPCR) runs were performed per sample to assess the cumulative EBV positivity rate. To detect viral RNA as a sign of activation, RNA sequencing was performed in blood CD4-positive, CD8-positive, and CD19-positive cells from 21 patients with MS and 3 controls. RESULTS: One of the 45 patients with MS and none of the 45 controls were positive for EBV DNA in CSF supernatants (1 mL). CSF cellular DNA was analyzed in 8 independent ddPCRs: EBV DNA was detected at least once in 18 (64%) of the 28 patients with MS and in 15 (54%) of the 28 controls (p = 0.59, Fisher test). The cumulative EBV positivity increased steadily up to 59% in the successive ddPCRs, suggesting that all individuals would have reached EBV positivity in the CSF cells, if more DNA would have been analyzed. The estimated proportion of EBV-positive B cells was >1/10,000 in both the CSF and blood. We did not detect viral RNA, except from endogenous retroviruses, in the blood lymphocyte subpopulations. DISCUSSION: EBV-DNA is equally detectable in the CSF cells of both patients with MS and controls with ddPCR, and the probabilistic approach indicates that the true positivity rate approaches 100% in EBV-positive individuals. The proportion of EBV-positive B cells seems higher than previously estimated.

Cervical lymph node diameter reflects disease progression in multiple sclerosis.

BACKGROUND: Multiple sclerosis (MS) is an autoimmune disease against the central nervous system (CNS), where B cells activate in the deep cervical lymph nodes (CLNs) before migrating to the CNS. CLN diameter in head magnetic resonance imaging (MRI) is an unexplored possible biomarker for disease activity. METHODS: We measured CLN axial diameter from head MRIs of patients with active stable relapsing-remitting MS (a-RRMS-stable, n = 26), highly active stable RRMS (ha-RRMS-stable, n = 23), RRMS patients directly after a relapse (RRMS-relapse, n = 64) and follow-up MRIs from the same patients (r-RRMS-follow-up, n = 26). MRIs of primary headache syndrome patients (n = 38) served as a control group. We evaluated the correlation between CLN diameter and clinical data. RESULTS: Increases in EDSS in approximately 2 year-follow up after imaging was connected to smaller CLN diameter at imaging (correlation coefficient -0.305, p = 0.009). In a regression model, age did not show a significant effect to CLN diameter in MS patients. Enlarged CLNs of over 10 mm diameter were more common in patients with shorter disease duration (p = 0.013). The largest CLN axial diameter in RRMS-relapse group was smaller than in the control group (p = 0.005), whereas MS subgroups of the study did not differ in CLN diameter. CONCLUSIONS: CLN diameter appears to reflect disease duration and disease progression in MS, in line with compartmentalization of immunological activity to the CNS in time. Decrease in CLN diameter was seen also during relapse. CLN axial diameter in MRI shows promise as a feasible biomarker for assessing MS disease activity.

Exploring JC polyomavirus sequences and human gene expression in brain tissue of patients with progressive multifocal leukoencephalopathy.

Progressive multifocal leukoencephalopathy (PML) is a rare neurological condition associated with reactivation of dormant JC polyomavirus (JCPyV). In this study, we characterized gene expression and JCPyV rearrangements in PML brain tissue. Infection of white matter astrocytes and oligodendrocytes as well as occasional brain cortex neurons was shown. PML brain harbored exclusively rearranged JCPyV variants. Viral transcripts covered the whole genome on both strands. Strong differential expression of human genes associated with neuroinflammation, blood-brain-barrier permeability and neurodegenerative diseases was shown. Pathway analysis revealed wide immune activation in PML brain. The study provides novel insights into the pathogenesis of PML.

Switching of hypertrophic signalling towards enhanced cardiomyocyte identity and maturity by a GATA4-targeted compound.

BACKGROUND: The prevalence of heart failure is constantly increasing, and the prognosis of patients remains poor. New treatment strategies to preserve cardiac function and limit cardiac hypertrophy are therefore urgently needed. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are increasingly used as an experimental platform for cardiac in vitro studies. However, in contrast to adult cardiomyocytes, hiPSC-CMs display immature morphology, contractility, gene expression and metabolism and hence express a naive phenotype that resembles more of a foetal cardiomyocyte. METHODS: A library of 14 novel compounds was synthesized in-house and screened for GATA4-NKX2-5 reporter activity and cellular toxicity. The most potent compound, 3i-1262, along with previously reported GATA4-acting compounds, were selected to investigate their effects on hypertrophy induced by endothelin-1 or mechanical stretch. Morphological changes and protein expression were characterized using immunofluorescence staining and high-content analysis. Changes in gene expression were studied using qPCR and RNA sequencing. RESULTS: The prototype compound 3i-1262 inhibited GATA4-NKX2-5 synergy in a luciferase reporter assay. Additionally, the isoxazole compound 3i-1262 inhibited the hypertrophy biomarker B-type natriuretic peptide (BNP) by reducing BNP promoter activity and proBNP expression in neonatal rat ventricular myocytes and hiPSC-CMs, respectively. Treatment with 3i-1262 increased metabolic activity and cardiac troponin T expression in hiPSC-CMs without affecting GATA4 protein levels. RNA sequencing analysis revealed that 3i-1262 induces gene expression related to metabolic activity and cell cycle exit, indicating a change in the identity and maturity status of hiPSC-CMs. The biological processes that were enriched in upregulated genes in response to 3i-1262 were downregulated in response to mechanical stretch, and conversely, the downregulated processes in response to 3i-1262 were upregulated in response to mechanical stretch. CONCLUSIONS: There is currently a lack of systematic understanding of the molecular modulation and control of hiPSC-CM maturation. In this study, we demonstrated that the GATA4-interfering compound 3i-1262 reorganizes the cardiac transcription factor network and converts hypertrophic signalling towards enhanced cardiomyocyte identity and maturity. This conceptually unique approach provides a novel structural scaffold for further development as a modality to promote cardiomyocyte specification and maturity.

Evaluating the long term effectiveness of a Mediterranean marine protected area to tackle the effects of invasive and range expanding herbivorous fish on rocky reefs.

Here we provide evidence, along an 8-year period time-series based on multifaceted data from a Mediterranean marine protected area (MPA), whether protection can tackle invasive and range expanding herbivore fishes, and their effects on the algal resource availability, taking into account the population trends of predatory fishes, fisheries catches of herbivore fishes and sea surface temperature (SST) through time. Our findings pointed out that an ineffective in restoring top-down control process MPA may facilitate, rather than alleviate, the sudden and enduring population burst of invasive and range-expanding herbivorous fishes at tipping points of abrupt change. This subsequently results in the deterioration of rocky reef habitats and the depletion of algal resources, with the tipping points of abrupt change for algal and herbivore fish species not overlapping chronologically. As sea temperature increases, ineffective or recently established MPAs may inadvertently facilitate the proliferation of invasive and range-expanding species, posing a significant challenge to management effectiveness and conservation objectives.

High Epstein-Barr virus capsid antigen IgG level associates with the carriership of CD8+ T cell somatic mutations in the STAT3 SH2 domain.

High carrier prevalence of STAT3 SH2 domain somatic mutations was recently discovered in CD8+ T cells. We found these low-allele-fraction clones in 26% of donors, without difference between multiple sclerosis (MS) patients and controls. Here we tested whether anti-viral antibodies associate with the carriership of these mutant clones. We compared antibody responses against common viruses in mutation carriers vs. non-carriers. Plasma samples of 152 donors (92 MS patients, 60 controls) were analyzed for antibodies against cytomegalovirus (CMV), Epstein-Barr virus (EBV), human herpesvirus-6A and parvovirus B19. The mutation carrier status associated with EBV VCA IgG level (p = 0.005) and remained significant after logistic regression (p = 0.036). This association was contributed similarly by MS patients and controls. These results suggest that EBV contributes to the generation or growth of these clones. The pathogenic role of the STAT3 mutant clones in MS is presently unclear, but their detailed characterization warrants further study.

Complete remission of central nervous system manifestations of IgG4-related disease with rituximab - a case report.

IgG4-related disease (IgG4-RD) is an emerging immune-mediated chronic fibrotic disease characterized by tumour-like mass formation. Reports of brain parenchymal involvement in IgG4-RD are rare and complete treatment-related remission of lesions has never been reported. Here, we present a woman in her mid-50s who developed headache and seizures. Brain magnetic resonance imaging revealed frontal bilateral pachymeningitis and a left frontal lobe parenchymal lesion, and pathologic findings were consistent with an IgG4-RD central nervous system manifestation. She had a history of tumour-like growth around the right optic nerve, orbital and maxillary cavities treated successfully with corticosteroids 28 years ago, and was receiving infliximab as a maintenance therapy for uveitis for the last 14 years. After initial high-dose corticosteroid treatment, the patient was treated with rituximab, and after 3 months, the patient presented with complete remission of IgG4-RD lesions and associated symptoms. This case illustrates the chronic, decades-spanning nature of IgG4-RD, and a complete response to rituximab even with intracerebral mass lesions that had emerged despite the use of infliximab, a therapy previously reported successful in IgG4-RD.

Association between resilience and frailty in older age: Findings from the Helsinki Birth Cohort Study.

OBJECTIVES: Resilience, a capacity to cope with adversity, has been linked to better functioning and health in older age. However, little is still known about resilience in relation to frailty. We explored whether resilience would be associated with frailty in older age and if we would observe differences in association between resilience and frailty according to the type of adversity. METHODS: The study included 681 participants from the Helsinki Birth Cohort Study, born in Helsinki between 1934 and 1944. Adversities in older age and resilience were assessed between 2015 and 2018 with the Hardy-Gill resilience scale, scores ranging from 0 (low) to 18 (high resilience). Frailty was assessed in 2017-18 by using a deficit accumulation-based Frailty Index with a scale from 0 to 1. Adversities were coded into categories by using a data-driven approach. A linear regression analysis was used to explore the association between resilience and frailty. RESULTS: Resilience was inversely associated with frailty in older age (ß -0.009, 95% CI -0.011 to -0.007, p<0.001). The association was observed for all other type of adversities except adversity in relationships and economical adversity. DISCUSSION: A higher resilience was related to lower levels of frailty in older age. Differences in association between resilience and frailty were observed according to the type of adversity. Focusing on the type of adverse events and the capacity to "bounce back" after an adversity in older age may reveal new perspectives on how to prevent and postpone frailty.

A skewed ratio of free light chains is more common in patients with late-onset than early-onset myasthenia gravis.

Myasthenia gravis (MG) is an autoantibody-mediated neuromuscular disease with an unpredictable clinical course. Serum free light chains (FLCs) have risen as a promising biomarker for MG, but their role in different subtypes of MG and in predicting disease progression is still uncharted. We investigated plasma from 58 generalized MG patients during post-thymectomy follow-up to determine κ and λ FLC and κ/λ ratio. In a subcohort of 30 patients, we examined the expression of 92 proteins associated with immuno-oncology using Olink. We further studied the ability of FLCs or proteomic markers to differentiate disease severity. Patients with late-onset MG (LOMG) displayed significantly higher mean κ/λ ratio than patients with early-onset MG (P = 0.004). Inducible T-cell co-stimulator ligand (ICOSLG), matrix metalloproteinase 7 (MMP7), hepatocyte growth factor (HGF), and arginase 1 (ARG1) were differentially expressed in MG patients compared to healthy controls. There were no significant associations between clinical outcomes and FLCs or the assayed proteins. In conclusion, an elevated κ/λ ratio suggests long-lasting aberrant clonal plasma cell function in LOMG. Immuno-oncology-related proteomic analysis showed alterations in immunoregulatory pathways. Our findings pinpoint the FLC ratio as a biomarker for LOMG and call for further investigation of the immunoregulatory pathways in MG.

Clinically relevant increases in serum neurofilament light chain and glial fibrillary acidic protein in patients with Susac syndrome.

BACKGROUND AND PURPOSE: Serum levels of neurofilament light chain (sNfL) and glial fibrillary acidic protein (sGFAP) are promising neuro-axonal damage and astrocytic activation biomarkers. Susac syndrome (SS) is an increasingly recognized neurological condition and biomarkers that can help assess and monitor disease evolution are highly needed for the adequate management of these patients. sNfL and sGFAP levels were evaluated in patients with SS and their clinical relevance in the relapse and remission phase of the disease was assessed. METHODS: As part of a multicentre study that enrolled patients diagnosed with SS from six international centres, sNfL and sGFAP levels were assessed in 22 SS patients (nine during a relapse and 13 in remission) and 59 age- and sex-matched healthy controls using SimoaTM assay Neurology 2-Plex B Kit. RESULTS: Serum NfL levels were higher than those of healthy controls (p < 0.001) in SS patients and in both subgroups of patients in relapse and in remission (p < 0.001 for both), with significantly higher levels in relapse than in remission (p = 0.008). sNfL levels showed a negative correlation with time from the last relapse (r = -0.663; p = 0.001). sGFAP levels were slightly higher in the whole group of patients than in healthy controls (p = 0.046) and were more pronounced in relapse than in remission (p = 0.013). CONCLUSION: In SS patients, both sNFL and sGFAP levels increased compared with healthy controls. Both biomarkers had higher levels during clinical relapse and much lower levels in remission. sNFL was shown to be time sensitive to clinical changes and can be useful to monitor neuro-axonal damage in SS.

Incidental demyelination in magnetic resonance imaging and 10-year risk of multiple sclerosis: A data lake cohort study.

BACKGROUND AND PURPOSE: There is an absence of data from large population-based cohort studies on the incidence of radiologically isolated syndrome (RIS). The incidence of RIS and the subsequent risk for multiple sclerosis (MS) were investigated. METHODS: A population-based, retrospective cohort study was conducted using a data-lake-based analysis of digitalized radiology reports. All brain and spinal cord magnetic resonance imaging (MRI) in people aged 16-70 during the years 2005-2010 (n = 102,224) were screened using optimized search terms to detect cases with RIS. The subjects with RIS were followed up until January 2022. RESULTS: The cumulative incidence of RIS was 0.03% when all MRI modalities were included and 0.06% when only brain MRI was included according to MAGNIMS 2018 recommendation criteria. With the Okuda 2009 criteria, the respective figures were 0.03% and 0.05% (86% concordance). The overall risk for MS after RIS was similar, 32% by using the MAGNIMS and 32% by using the Okuda definition of RIS. Individuals aged <35.5 years exhibited the most significant predisposition to MS (80%), whilst those >35.5 years had less than 10% risk of MS. MS diagnosed after RIS constituted 0.8% of the incident MS cases in the population during 2005-2010. CONCLUSIONS: A population-wide context was provided for the incidence of RIS and its relationship to MS. MAGNIMS recommendations were only slightly more sensitive to detect RIS compared to the Okuda criteria. RIS has a subtle effect on the overall incidence of MS, yet the risk for MS in individuals under the age of 35.5 years is substantial.

The role of Real-World Data and evidence in oncology medicines approved in EU in 2018-2019.

Use of Real-World Data (RWD) has gained the interest of different stakeholders in cancer care. The aim of this study was to identify and describe the use of RWD/RWE during the pre-authorization phase of products authorized by the EMA in 2018 and 2019 (n = 111), with the focus on oncology medicines (n = 24). Information was extracted from the European Public Assessment Report (EPAR) summaries and recorded for 5 stages (11 categories) of the drug development lifecycle (discovery, early development, clinical development, registration/market launch, lifecycle management). Specific chapters of full EPAR were reviewed to substantiate the findings on RWD/RWE use in clinical trial design, efficacy, safety, and effectiveness evaluation. RWD/RWE is present in all stages of the oncology drug development; 100.0 % in discovery, 37.5 % early development, 58.3 % in clinical development, 62.5 % in registration decision and 100.0 % in post-authorization lifecycle management. Examples showed that trial design supported by RWD/RWE included use of open label/single arm studies; efficacy was about using either comparison of results to historical controls, supplying survey data obtained outside the clinical trial or utilizing expert panel advice; safety about including literature findings in evidence; and effectiveness on comparison of trial results of the given product to historical data or existing standard of care. The findings of this study provide specific insights into how RWD/RWE is used in development of cancer therapeutics, how it contributes to regulatory decision making and can guide further policy developments in this field.

Early clinical features of new-onset refractory status epilepticus (NORSE) in adults.

BACKGROUND: The aim of this study was to identify early clinical features of patients with new-onset refractory status epilepticus (NORSE) that could direct the treatment in the first days of hospitalisation. METHODS: A retrospective cohort study of adult NORSE patients treated in the intensive care units of Helsinki University Hospital 2007-2018. RESULTS: We found 19 adult NORSE patients who divided into three subgroups on the basis of their clinical features: viral encephalitis (n = 5, 26%), febrile infection-related epilepsy syndrome (FIRES) (n = 6, 32%) and afebrile NORSE (n = 8, 42%). FIRES and afebrile NORSE patients remained without confirmed etiology, but retrospectively two paraneoplastic and two neurodegenerative causes were suspected in the afebrile NORSE group. Viral encephalitis patients were median 64 years old (IQR 55-64), and four (80%) had prodromal fever and abnormal findings in the first brain imaging. FIRES patients were median 21 years old (IQR 19-24), all febrile and had normal brain imaging at onset. In the afebrile NORSE group, median age was 67 (IQR 59-71) and 50% had prodromal cognitive or psychiatric symptoms. FIRES patients differed from other NORSE patients by younger age (p = 0.001), respiratory prodromal symptoms (p = 0.004), normal brain MRI (p = 0.044) and lack of comorbidities (p = 0.011). They needed more antiseizure medications (p = 0.001) and anesthetics (p = 0.002), had a longer hospital stay (p = 0.017) and more complications (p < 0.001). CONCLUSIONS: Among febrile NORSE patients, FIRES group was distinctive due to patients' young age, prodromal respiratory symptoms and normal first brain imaging. These features should be confirmed by subsequent studies as basis for selecting patients for early intensive immunotherapy.

Association of lifetime major depressive disorder with enhanced attentional sensitivity measured with P3 response in young adult twins.

Major depression is associated with alterations in the auditory P3 event-related potential (ERP). However, the persistence of these abnormalities after recovery from depressive episodes, especially in young adults, is not well known. Furthermore, the potential influence of substance use on this association is poorly understood. Young adult twin pairs (N = 177) from the longitudinal FinnTwin16 study were studied with a psychiatric interview, and P3a and P3b ERPs elicited by task-irrelevant novel sounds and targets, respectively. Dyadic linear mixed-effect models were used to distinguish the effects of lifetime major depressive disorder from familial factors and effects of alcohol problem drinking and tobacco smoking. P3a amplitude was significantly increased and P3b latency decreased, in individuals with a history of lifetime major depression, when controlling the fixed effects of alcohol abuse, tobacco, gender, twins' birth order, and zygosity. These results suggest that past lifetime major depressive disorder may be associated with enhanced attentional sensitivity.

Ectopic germinal centers in the thymus accurately predict prognosis of myasthenia gravis after thymectomy.

The ability of thymic histopathology to predict the long-term impact of thymectomy in non-thymomatous myasthenia gravis (NTMG) is mainly uncharted. We applied digital pathology to quantitatively characterize differences of thymic histology between early-onset (EOMG) and late-onset MG (LOMG) and to investigate the role of thymic changes for thymectomy outcomes in MG. We analyzed 83 thymic H&E slides from thymectomized NTMG patients, of which 69 had EOMG and 14 LOMG, using digital pathology open-access software QuPath. We compared the results to the retrospectively assessed clinical outcome at two years after thymectomy and at the last follow-up visit where complete stable remission and minimal use of medication were primary outcomes. The automated annotation pipeline was an effective and reliable way to analyze thymic H&E samples compared to manual annotation with mean intraclass correlation of 0.80. The ratio of thymic tissue to stroma and fat was increased in EOMG compared to LOMG (p = 8.7e-07), whereas no difference was observed in the ratio of medulla to cortex between these subtypes. AChRAb seropositivity correlated with the number of ectopic germinal centers (eGC; p = 0.00067) but not with other histological areas. Patients with an increased number of eGCs had better post-thymectomy outcomes at two years after thymectomy (p = 0.0035) and at the last follow-up (p = 0.0267). ROC analysis showed that eGC area predicts thymectomy outcome in EOMG with an AUC of 0.79. Digital pathology can thus help in providing a predictive tool to the clinician, the eGC number, to guide the post-thymectomy treatment decisions in EOMG patients.

GSK3ß Serine 389 Phosphorylation Modulates Cardiomyocyte Hypertrophy and Ischemic Injury.

Prior studies show that glycogen synthase kinase 3ß (GSK3ß) contributes to cardiac ischemic injury and cardiac hypertrophy. GSK3ß is constitutionally active and phosphorylation of GSK3ß at serine 9 (S9) inactivates the kinase and promotes cellular growth. GSK3ß is also phosphorylated at serine 389 (S389), but the significance of this phosphorylation in the heart is not known. We analyzed GSK3ß S389 phosphorylation in diseased hearts and utilized overexpression of GSK3ß carrying ser→ala mutations at S9 (S9A) and S389 (S389A) to study the biological function of constitutively active GSK3ß in primary cardiomyocytes. We found that phosphorylation of GSK3ß at S389 was increased in left ventricular samples from patients with dilated cardiomyopathy and ischemic cardiomyopathy, and in hearts of mice subjected to thoracic aortic constriction. Overexpression of either GSK3ß S9A or S389A reduced the viability of cardiomyocytes subjected to hypoxia-reoxygenation. Overexpression of double GSK3ß mutant (S9A/S389A) further reduced cardiomyocyte viability. Determination of protein synthesis showed that overexpression of GSK3ß S389A or GSK3ß S9A/S389A increased both basal and agonist-induced cardiomyocyte growth. Mechanistically, GSK3ß S389A mutation was associated with activation of mTOR complex 1 signaling. In conclusion, our data suggest that phosphorylation of GSK3ß at S389 enhances cardiomyocyte survival and protects from cardiomyocyte hypertrophy.

Comorbidities worsen the prognosis of generalized myasthenia gravis post-thymectomy.

BACKGROUND: The effect of comorbidities on the prognosis of myasthenia gravis (MG) remains unclear. In particular, the role of other autoimmune diseases (AD) is controversial. METHODS: In this retrospective single-center cohort study, we investigated 154 consecutive generalized thymectomized MG patients, with a mean follow-up time of 8.6 (±5.0) years post-thymectomy. Comorbidities diagnosed at any timepoint were retrieved from medical records and Charlson comorbidity index (CCI) scores were calculated. Patients were categorized into subgroups MG alone (n = 45) and MG with any comorbidity (n = 109); the latter was further categorized into MG with other ADs (n = 33) and MG with non-AD comorbidities (n = 76). The endpoints analyzed were complete stable remission (CSR), minimal need for medications, and need for in-hospital treatments. RESULTS: CSR was more frequent in MG alone than in MG with any comorbidity group (26.7% vs 8.3%, p = 0.004). Minimal need for medication was reached more often in the MG alone than in the MG with non-AD comorbidities group (p = 0.047). Need for in-hospital treatments was lower in the MG alone group than in MG patients with any comorbidity (p = 0.046). Logistic regression analysis revealed that lower CCI scores increased the likelihood of CSR (p = 0.033). Lower CCI scores were more prevalent both in patients with minimal need for medication and in patients who did not need in-hospital treatments (p < 0.001). CONCLUSIONS: Patients with generalized MG and comorbidities have a poorer prognosis than patients with MG alone during almost 9 years follow-up after thymectomy. AD comorbidities appeared not to translate into a higher risk compared to other comorbidities.

GATA-targeted compounds modulate cardiac subtype cell differentiation in dual reporter stem cell line.

BACKGROUND: Pharmacological modulation of cell fate decisions and developmental gene regulatory networks holds promise for the treatment of heart failure. Compounds that target tissue-specific transcription factors could overcome non-specific effects of small molecules and lead to the regeneration of heart muscle following myocardial infarction. Due to cellular heterogeneity in the heart, the activation of gene programs representing specific atrial and ventricular cardiomyocyte subtypes would be highly desirable. Chemical compounds that modulate atrial and ventricular cell fate could be used to improve subtype-specific differentiation of endogenous or exogenously delivered progenitor cells in order to promote cardiac regeneration. METHODS: Transcription factor GATA4-targeted compounds that have previously shown in vivo efficacy in cardiac injury models were tested for stage-specific activation of atrial and ventricular reporter genes in differentiating pluripotent stem cells using a dual reporter assay. Chemically induced gene expression changes were characterized by qRT-PCR, global run-on sequencing (GRO-seq) and immunoblotting, and the network of cooperative proteins of GATA4 and NKX2-5 were further explored by the examination of the GATA4 and NKX2-5 interactome by BioID. Reporter gene assays were conducted to examine combinatorial effects of GATA-targeted compounds and bromodomain and extraterminal domain (BET) inhibition on chamber-specific gene expression. RESULTS: GATA4-targeted compounds 3i-1000 and 3i-1103 were identified as differential modulators of atrial and ventricular gene expression. More detailed structure-function analysis revealed a distinct subclass of GATA4/NKX2-5 inhibitory compounds with an acetyl lysine-like domain that contributed to ventricular cells (%Myl2-eGFP+). Additionally, BioID analysis indicated broad interaction between GATA4 and BET family of proteins, such as BRD4. This indicated the involvement of epigenetic modulators in the regulation of GATA-dependent transcription. In this line, reporter gene assays with combinatorial treatment of 3i-1000 and the BET bromodomain inhibitor (+)-JQ1 demonstrated the cooperative role of GATA4 and BRD4 in the modulation of chamber-specific cardiac gene expression. CONCLUSIONS: Collectively, these results indicate the potential for therapeutic alteration of cell fate decisions and pathological gene regulatory networks by GATA4-targeted compounds modulating chamber-specific transcriptional programs in multipotent cardiac progenitor cells and cardiomyocytes. The compound scaffolds described within this study could be used to develop regenerative strategies for myocardial regeneration.