Autophagy and Endoplasmic Reticulum Stress during Onset and Progression of Arrhythmogenic Cardiomyopathy

Arrhythmogenic cardiomyopathy (AC) is a heritable, potentially lethal disease without a causal therapy. AC is characterized by focal cardiomyocyte death followed by inflammation and progressive formation of connective tissue. The pathomechanisms leading to structural disease onset and progression, however, are not fully elucidated. Recent studies revealed that dysregulation of autophagy and endoplasmic/sarcoplasmic reticulum (ER/SR) stress plays an important role in cardiac pathophysiology. We therefore examined the temporal and spatial expression patterns of autophagy and ER/SR stress indicators in murine AC models by qRT-PCR, immunohistochemistry, in situ hybridization and electron microscopy. Cardiomyocytes overexpressing the autophagy markers LC3 and SQSTM1/p62 and containing prominent autophagic vacuoles were detected next to regions of inflammation and fibrosis during onset and chronic disease progression. mRNAs of the ER stress markers Chop and sXbp1 were elevated in both ventricles at disease onset. During chronic disease progression Chop mRNA was upregulated in right ventricles. In addition, reduced Ryr2 mRNA expression together with often drastically enlarged ER/SR cisternae further indicated SR dysfunction during this disease phase. Our observations support the hypothesis that locally altered autophagy and enhanced ER/SR stress play a role in AC pathogenesis both at the onset and during chronic progression.

Rare Neurological Diseases: an Overreview of Pathophysiology, Epidemiology, Clinical Features and Pharmacoeconomic Considerations in the Treating

Rare diseases (RD) are serious chronic diseases affecting small number of people compared to the general population. There are between 6000 and 8000 RDs, which affect about 400 million people worldwide. Drugs used for causal treatment of RDs are called orphan drugs. RDs bear great clinical and economic burden for patients, their families, healthcare systems and society overall. There are at least two reasons for the high cost of treatment of RDs. First, there is no causal therapy for majority of RDs, so exacerbations, complications, and hospitalizations in those patients are common. The second reason is high price of available orphan drugs, which are not cost-effective when traditional pharmacoeconomic evaluation is employed. The pharmacoeconomic aspect of the treatment of RDs is especially important in the field of neurology, since at least one fifth of all RDs is composed of neurological conditions. The aim of this paper was to provide a concise overview of the pathophysiological, epidemiological and clinical characteristics of some of the most important and common rare neurological diseases, with special reference to their impact on society and economy.

Epigenome-wide association study of human frontal cortex identifies differential methylation in Lewy body pathology

Parkinson’s disease (PD) and dementia with Lewy bodies (DLB) are closely related progressive disorders with no available causal therapy, neuropathologically characterized by intraneuronal aggregates of misfolded α-synuclein. To explore the role of DNA methylation changes in PD and DLB pathogenesis, we performed an epigenome-wide association study (EWAS) of 322 postmortem frontal cortex samples and replicated results in an independent set of 219 donors. We report novel differentially methylated replicating loci associated with Braak Lewy body stage near SFMBT2, PHYHIP, BRF1/PACS2 and DGKG. The DGKG locus also showed evidence of DNA methylation changes in the earliest, preclinical stage of disease. Differentially methylated probes were independent of known PD genetic risk alleles. Meta-analysis provided suggestive evidence for a differentially methylated locus within the chromosomal region affected by the PD-associated 22q11.2 deletion. Our findings elucidate novel disease pathways in PD and DLB and generate hypotheses for future molecular studies of Lewy body pathology.

Oxygen Toxicity to the Immature Lung—Part II: The Unmet Clinical Need for Causal Therapy

Oxygen toxicity continues to be one of the inevitable injuries to the immature lung. Reactive oxygen species (ROS) production is the initial step leading to lung injury and, subsequently, the development of bronchopulmonary dysplasia (BPD). Today, BPD remains the most important disease burden following preterm delivery and results in life-long restrictions in lung function and further important health sequelae. Despite the tremendous progress in the pathomechanistic understanding derived from preclinical models, the clinical needs for preventive or curative therapies remain unmet. This review summarizes the clinical progress on guiding oxygen delivery to the preterm infant and elaborates future directions of research that need to take into account both hyperoxia and hypoxia as ROS sources and BPD drivers. Many strategies have been tested within clinical trials based on the mechanistic understanding of ROS actions, but most have failed to prove efficacy. The majority of these studies were tested in an era before the latest modes of non-invasive respiratory support and surfactant application were introduced or were not appropriately powered. A comprehensive re-evaluation of enzymatic, antioxidant, and anti-inflammatory therapies to prevent ROS injury is therefore indispensable. Strategies will only succeed if they are applied in a timely and vigorous manner and with the appropriate outcome measures.

Use of Rifaximin in Irritable Bowel Syndrome Treatment

Introduction and purpose of the work: Irritable Bowel Syndrome (IBS) affects up to 10% of the population. The deterioration in health-related quality of life in IBS patients has been shown to be comparable to, and perhaps even more severe than, other serious chronic organic diseases such as inflammatory bowel disease, diabetes, hypertension, and end-stage renal disease. The aim of the study is to present a new method of IBS treatment.State of knowledge: There are four main types of IBS: Constipation-predominant IBS (IBS-C), Diarrhea-predominant IBS (IBS-D), Fluctuating IBS (IBS-M), and the unclassified form of IBS (IBS-U). Disturbances in entero-brain interactions play a key role in its multifactorial etiopathogenesis. In patients with IBS, quantitative and qualitative disturbances in the composition of the intestinal microbiota can be found. So far, it has not been possible to clearly establish the cause of the disease, so there is no possibility of causal therapy and effective cure. Both non-pharmacological methods and drugs, including rifaximin α, have been used in IBS therapy.Summary: Rifaximin has a positive effect on the symptoms of IBS. In clinical trials conducted in patients with IBS, the use of rifaximin for 2 weeks was associated with a reduction in the severity of joint symptoms, bloating, abdominal pain and discomfort, and an improvement in stool consistency within 4 and 12 weeks, and did not increase the risk of side effects. In the forms: with predominant diarrhea, mixed and unclassified, in order to reduce the overall symptoms as well as reduce the severity of flatulence and / or diarrhea, a 14-day therapy with rifaximin α is recommended.

Subkutane Allergen-Immuntherapie bei Hausstaubmilbenallergie – die Anwendung kann auch bei Kindern unter 5 Jahren sicher erfolgen

<b>Background:</b> Allergen immunotherapy (AIT) is the only causal therapy for IgE-mediated allergy. There is less evidence about the safety and efficacy of AIT especially subcutaneous immunotherapy (SCIT) in children under 5 years old. We aimed to investigate the side effects and associated risk factors of house dust mite (HDM) SCIT in preschool children with respiratory allergic diseases. <b>Methods:</b> The preschool children who had HDM-related allergic rhinitis with/without asthma were enrolled and undergone standardized HDM SCIT in our department from June 2013 to December 2019. Local reactions (LRs) and systemic reactions (SRs) were recorded and categorized according to World Allergy Organization recommendations. Demographic data and other therapeutic-related parameters were also recorded to investigate potential risk factors for these side effects. <b>Results:</b> A total of 91 children (60 boys, 65.93%; 31 girls, 34.07%; mean age 4.13 years old) were included in the study. Among the 91 patients, 3109 SCIT injections were recorded, 62/91 (68.13%) experienced 186 immediate LRs, 4 /91(4.40%) experienced 6 delayed LRs, 11/91 (12.09%) children experienced 44 immediate SRs, 21/44 (47.73%) were grade 1 SRs, 21/44 (47.73%) were grade 2, 2/44 (4.55%) were grade 3, no grade 4 or 5 SRs occurred. Furthermore, 1/91 (1.10%) experienced 1 delayed SRs, manifested by urticaria 2 days later after allergen injection. 9/91 (9.89%) experienced 2 or more times SRs. Multivariable logistic regression analysis showed BMI (OR 1.506; 95%CI 1.091 to 2.079; <i>p</i> &#x3c; 0.05) and sIgE against HDM (OR 1.497; 95%CI 1.082 to 2.071; <i>p</i> &#x3c; 0.05) were risk factors for LRs. No variable was found to correlate with SRs (all <i>p</i> &#x3e; 0.05). <b>Conclusions:</b> HDM subcutaneous immunotherapy is considered to be safe in preschool children with respiratory allergic diseases. Higher BMI and HDM sIgE level in children are risk factors for developing LRs. The incidence of SRs and the rate of severe SRs are low in preschool children.

MO036DAPAGLIFLOZIN RESCUES THE RENAL PHENOTYPE OF GLYCOGEN STORAGE DISEASE TYPE IB

Background and Aims Glycogenosis I type b (GsdI-b) is a rare metabolic disease and immune disorder characterized by hepato-renal glycogen accumulation caused by a deficiency in the Glucose-6-phosphate transporter (G6PT). G6PT transports glucose-6-phosphate (G6P) from cytoplasm to endoplasmic reticulum (ER) where a G6Pase catalyses the hydrolysis of G6P in glucose and phosphate. G6PT deficiency lead to impaired glucose homeostasis, myeloid disfunction and long-term risk of hepatocellular adenomas. No causal therapy is so far available for GSDI-b patients besides a dietary approach to control glycemia and the use of Granulocyte Colony-Stimulating Factor (GCSF) to improve neutropenia. Over time, these supports increase the chronicity of GSDI-b with some complications. A mouse model recapitulating the GDSI-b has been recently generated by inducing G6PT suppression after tamoxifen injection. Here, we characterized the renal phenotype of TM-G6PT-/- mice model focusing on the molecular mechanisms that lead to renal dysfunction. Finally, we evaluated the efficiency of Dapagliflozin, a selective inhibitor of SGLT2, on kidney functions in terms of therapeutic effect. Method Machine learning approach to computer based evaluation of renal morphology was used to analyze the renal sections from TM-G6PT-/- treated with or without dapagliflozin. Results: G6PT is expressed in all renal zones and a severe downregulation of G6PT mRNA expression in whole kidney of TM-G6PT-/- mice can be observed. TM-G6PT-/- mice show tubular vacuolization and overall cellular dysfunction of PT due to a high glycogen accumulation. TM-G6PT-/- mice manifest glycosuria, phosphaturia and polyuria associated with a down regulation of main transporters of PT cells. The urine concentrating defect is due to a primarily role of G6PT in CNT/CD cells confirmed by a downregulation of AQP2, main water channel along CD segments. This mouse model recapitulates the human GSD-Ib renal phenotype characterized by a disfunction of PT but also CNT/CD cells. In order to evaluate whether targeting the glucose metabolism would improve the renal phenotype of these mice we limited glucose flux across the apical membrane of PT cells, applying the SGLT2-inhibitor dapagliflozin to reduce new glycogen formation. After one month of treatment, Dapagliflozin prevents glycogen accumulation in TM-G6PT-/- mice and ameliorates the main dysregulated markers of PT function. This finding was paralleled by an improvement of the histological features of kidney morphology in dapagliflozin treated TM-G6PT-/- mice. Conclusion Our data provide evidence that treatment with dapagliflozin ameliorates intracellular glycogen storage and improves the renal functions in TM-G6PT-/- mice.

MO081THE INFLUENCE OF VITAMIN D3 ON PODOCYTE DIFFERENTIATION IN SITU AND IN VITRO

Background and Aims Dedifferentiation of podocytes affects their complex 3 D morphology and is the main initiator for the development of chronic kidney disease (CKD). Unfortunately, there is no causal therapy for CKD until today. Thus, inadequate and late treatment lead to end-stage renal disease which subsequently makes renal replacement therapy inevitable. To address this, new treatment options are of high significance for CKD patients. Recently, vitamin D3 (VitD) became a promising candidate, but it is controversially discussed. In the present study, we investigated the influence of VitD on podocyte differentiation and the related pathways in situ and in vitro. Method We combined a podocyte dedifferentiation model (GlomAssay) with an automated imaging procedure (Aquifer Imaging Machine). We analyzed cultured glomeruli from transgenic mice expressing cyan-fluorescent protein (CFP) under the control of the nephrin promoter which were treated with VitD and its` analogue (calcipotriol). In this model, the decreasing CFP fluorescence is as a read out for podocyte (de)differentiation. Additionally, VitD-, calcipotriol- and VitD receptor (VDR) inhibitor (PS121912)-treated glomeruli were investigated by RNA-Seq and LC-MS/MS to reveal the molecular effects of VitD on podocyte differentiation. Furthermore, we treated cultured murine podocytes with VitD, calcipotriol and PS121912 to elucidate the morphological and molecular changes by immunofluorescence staining, RT-qPCR and Western blot. Results VitD- and calcipotriol-treated glomeruli showed a significantly higher intensity of CFP fluorescence after 9 days, indicating higher level of nephrin compared to the control. This was verified by RT-qPCR and Western blot for nephrin and CFP. Additionally, we found an upregulation of VDR in VitD- and calcipotriol-treated glomeruli compared to controls. By transcriptomic and proteomic analysis, we identified molecular patterns that are specific for the different treated groups. Thus, we observed differential gene expression in VitD- and Wnt-signaling pathway as well as regulated genes that are essential for the actin cytoskeleton, focal adhesion formation and the slit membrane. Beside this, cultured podocytes showed a significant upregulation of the slit membrane protein nephrin, VDR and CYP24A1 by VitD. This is accompanied by an altered morphology of the podocytes due to a reorganization of the actin cytoskeleton. Conclusion Our results show that VitD influences podocyte differentiation in situ and in vitro by the regulation of specific signaling pathways.

Safety of house dust mite subcutaneous immunotherapy in preschool children with respiratory allergic diseases

Background Allergen immunotherapy (AIT) is the only causal therapy for IgE-mediated allergy. There is less evidence about the safety and efficacy of AIT especially subcutaneous immunotherapy (SCIT) in children under 5 years old. We aimed to investigate the side effects and associated risk factors of house dust mite (HDM) SCIT in preschool children with respiratory allergic diseases. Methods The preschool children who had HDM-related allergic rhinitis with/without asthma were enrolled and undergone standardized HDM SCIT in our department from June 2013 to December 2019. Local reactions (LRs) and systemic reactions (SRs) were recorded and categorized according to World Allergy Organization recommendations. Demographic data and other therapeutic-related parameters were also recorded to investigate potential risk factors for these side effects. Results A total of 91 children (60 boys, 65.93%; 31 girls, 34.07%; mean age 4.13 years old) were included in the study. Among the 91 patients, 3109 SCIT injections were recorded, 62/91 (68.13%) experienced 186 immediate LRs, 4 /91(4.40%) experienced 6 delayed LRs, 11/91 (12.09%) children experienced 44 immediate SRs, 21/44 (47.73%) were grade 1 SRs, 21/44 (47.73%) were grade 2, 2/44 (4.55%) were grade 3, no grade 4 or 5 SRs occurred. Furthermore, 1/91 (1.10%) experienced 1 delayed SRs, manifested by urticaria 2 days later after allergen injection. 9/91 (9.89%) experienced 2 or more times SRs. Multivariable logistic regression analysis showed BMI (OR 1.506; 95%CI 1.091 to 2.079; p < 0.05) and sIgE against HDM (OR 1.497; 95%CI 1.082 to 2.071; p < 0.05) were risk factors for LRs. No variable was found to correlate with SRs (all p > 0.05). Conclusions HDM subcutaneous immunotherapy is considered to be safe in preschool children with respiratory allergic diseases. Higher BMI and HDM sIgE level in children are risk factors for developing LRs. The incidence of SRs and the rate of severe SRs are low in preschool children.