Targeted expression of heme oxygenase-1 in satellite cells improves skeletal muscle pathology in dystrophic mice.

BACKGROUND: Adult muscle-resident myogenic stem cells, satellite cells (SCs), that play non-redundant role in muscle regeneration, are intrinsically impaired in Duchenne muscular dystrophy (DMD). Previously we revealed that dystrophic SCs express low level of anti-inflammatory and anti-oxidative heme oxygenase-1 (HO-1, HMOX1). Here we assess whether targeted induction of HMOX1 affect SC function and alleviates hallmark symptoms of DMD. METHODS: We generated double-transgenic mouse model (mdx;HMOX1Pax7Ind) that allows tamoxifen (TX)-inducible HMOX1 expression in Pax7 positive cells of dystrophic muscles. Mdx;HMOX1Pax7Ind and control mdx mice were subjected to 5-day TX injections (75 mg/kg b.w.) followed by acute exercise protocol with high-speed treadmill (12 m/min, 45 min) and downhill running to worsen skeletal muscle phenotype and reveal immediate effects of HO-1 on muscle pathology and SC function. RESULTS: HMOX1 induction caused a drop in SC pool in mdx;HMOX1Pax7Ind mice (vs. mdx counterparts), while not exaggerating the effect of physical exercise. Upon physical exercise, the proliferation of SCs and activated CD34- SC subpopulation, was impaired in mdx mice, an effect that was reversed in mdx;HMOX1Pax7Ind mice, however, both in vehicle- and TX-treated animals. This corresponded to the pattern of HO-1 expression in skeletal muscles. At the tissue level, necrotic events of selective skeletal muscles of mdx mice and associated increase in circulating levels of muscle damage markers were blunted in HO-1 transgenic animals which showed also anti-inflammatory cytokine profile (vs. mdx). CONCLUSIONS: Targeted expression of HMOX1 plays protective role in DMD and alleviates dystrophic muscle pathology.

Compromised diabetic heart function is not affected by miR-378a upregulation upon hyperglycemia.

BACKGROUND: Cardiac-abundant microRNA-378a (miR-378a) is associated with postnatal repression of insulin-like growth factor 1 receptor (IGF-1R) controlling physiological hypertrophy and survival pathways. IGF-1/IGF-1R axis has been proposed as a therapeutic candidate against the pathophysiological progress of diabetic cardiomyopathy (DCM). We ask whether hyperglycemia-driven changes in miR-378a expression could mediate DCM progression. METHODS: Diabetes mellitus was induced by streptozotocin (STZ) (55 mg/kg i.p. for 5 days) in male C57BL/6 wild type (miR-378a+/+) and miR-378a knockout (miR-378a-/-) mice. As a parallel human model, we harnessed human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM miR378a+/+ vs. hiPSC-CM miR378a-/-) subjected to high glucose (HG) treatment. RESULTS: We reported miR-378a upregulation in cardiac diabetic milieu arising upon STZ administration to wild-type mice and in HG-treated hiPSC-CMs. Pro-hypertrophic IGF-1R/ERK1/2 pathway and hypertrophic marker expression were activated in miR-378a deficiency and upon STZ/HG treatment of miR-378a+/+ specimens in vivo and in vitro suggesting miR-378a-independent hyperglycemia-promoted hypertrophy. A synergistic upregulation of IGF-1R signaling in diabetic conditions was detected in miR-378a-/- hiPSC-CMs, but not in miR-378a-/- hearts that showed attenuation of this pathway, pointing to the involvement of compensatory mechanisms in the absence of miR-378a. Although STZ administration did not cause pro-inflammatory or pro-fibrotic effects that were detected in miR-378a-/- mice, the compromised diabetic heart function observed in vivo by high-resolution ultrasound imaging upon STZ treatment was not affected by miR-378a presence. CONCLUSIONS: Overall, data underline the role of miR-378a in maintaining basal cardiac structural integrity while pointing to miR-378a-independent hyperglycemia-driven cardiac hypertrophy and associated dysfunction.

CD4+ regulatory T cells lacking Helios and Eos.

The transcriptional regulators that drive regulatory T (Treg) cell development and function remain partially understood. Helios (Ikzf2) and Eos (Ikzf4) are closely-related members of the Ikaros family of transcription factors. They are highly expressed in CD4+ Treg cells and functionally important for Treg cell biology, as mice deficient for either Helios or Eos are susceptible to autoimmune diseases. However, it remains unknown if these factors exhibit specific or partially redundant functions in Treg cells. Here we show that mice with germline deletions of both Ikzf2 and Ikzf4 are not very different from animals with single Ikzf2 or Ikzf4 deletions. Double knockout Treg cells differentiate normally, and efficiently suppress effector T cell proliferation in vitro. Both Helios and Eos are required for optimal Foxp3 protein expression. Surprisingly, Helios and Eos regulate different, largely non-overlapping, sets of genes. Only Helios is required for proper Treg cell aging, as Helios deficiency results in reduced Treg cell frequencies in the spleen of older animals. These results indicate that Helios and Eos are required for distinct aspects of Treg cell function.

Generation of human induced pluripotent stem cell lines with HMOX1 promoter polymorphism and CRISPR/Cas9-mediated deletion of exon 50 of DMD gene.

Duchenne muscular dystrophy (DMD), originating from the lack of functional dystrophin, clinically manifests as devastating disease of skeletal muscles with progressive cardiac involvement. HMOX1 promoter polymorphism may reflect different activity of heme oxygenase-1 (HO-1) that may be critical for DMD progression. Here we generated human induced pluripotent stem cell (hiPSC) lines from healthy donors-derived peripheral blood mononuclear cells with different variants of HMOX1 promoter (GT repeats), and engineered by CRISPR/Cas9-mediated deletion of exon 50 of DMD gene. Such in vitro model could add to molecular understanding of DMD and verify the prognostic value of HMOX1 promoter polymorphism.

DNA damage repair kinase DNA-PK and cGAS synergize to induce cancer-related inflammation in glioblastoma.

Cytosolic DNA promotes inflammatory responses upon detection by the cyclic GMP-AMP (cGAMP) synthase (cGAS). It has been suggested that cGAS downregulation is an immune escape strategy harnessed by tumor cells. Here, we used glioblastoma cells that show undetectable cGAS levels to address if alternative DNA detection pathways can promote pro-inflammatory signaling. We show that the DNA-PK DNA repair complex (i) drives cGAS-independent IRF3-mediated type I Interferon responses and (ii) that its catalytic activity is required for cGAS-dependent cGAMP production and optimal downstream signaling. We further show that the cooperation between DNA-PK and cGAS favors the expression of chemokines that promote macrophage recruitment in the tumor microenvironment in a glioblastoma model, a process that impairs early tumorigenesis but correlates with poor outcome in glioblastoma patients. Thus, our study supports that cGAS-dependent signaling is acquired during tumorigenesis and that cGAS and DNA-PK activities should be analyzed concertedly to predict the impact of strategies aiming to boost tumor immunogenicity.

Tablet-assisted objective structured spotter practical examination (TOSSPE): Advantages of an innovative anatomy spotter examination method for medical student assessment.

The affordances of technology-based assessments, like the objectively structured practical examination, have become an integral part of gross anatomy courses. The Department of Anatomy Faculty of Medicine at the University of Warmia and Mazury developed and introduced an application for tablet devices which has been implemented in student examinations and assessments, called the tablet-assisted objective structured spotter practical examination. It was created to simplify the educational process and to build a rich learning environment, facilitating deep learning for students through examination and feedback data. The method consists of cadaver stations with traditional corresponding pin spotters in an expanded tablet application. It not only provides instant feedback on various observations of teaching-learning skills but has also positively affected the entire process of education. The method provides an unbiased evaluation of knowledge and understanding of the anatomy course, ensuring objectivity and standardization. The current study was performed on a total of 608 first-year medical students in Polish and English divisions and focused on the observed advantages since the new method was introduced. Outcomes indicate that after the implementation of the method for both the Polish and English divisions' first-year medical students, the mean score of examinations significantly increased compared to other teaching-learning methods. The study highlights that students were excited about the implementation of the new method and identified its many benefits. It is recognized that technological development and the digital environment offer a range of opportunities and added value versus traditional assessment activities, methods, and processes.

The multifaceted view of heart problem in Duchenne muscular dystrophy.

Dystrophin is a large protein serving as local scaffolding repetitively bridging cytoskeleton and the outside of striated muscle cell. As such dystrophin is a critical brick primarily in dystrophin-associated protein complex (DAGC) and in a larger submembranous unit, costamere. Accordingly, the lack of functional dystrophin laying at the root of Duchenne muscular dystrophy (DMD) drives sarcolemma instability. From this point on, the cascade inevitably leading to the death of myocyte begins. In cardiomyocytes, intracellular calcium overload and related mitochondrial-mediated cell death mainly contribute to myocardial dysfunction and dilation while other protein dysregulation and/or mislocalization may affect electrical conduction system and favor arrhythmogenesis. Although clinically DMD manifests as progressive muscle weakness and skeletal muscle symptoms define characteristic of DMD, it is the heart problem the biggest challenge that most often develop in the form of dilated cardiomyopathy (DCM). Current standards of treatment and recent progress in respiratory care, introduced in most settings in the 1990s, have improved quality of life and median life expectancy to 4th decade of patient's age. At the same time, cardiac causes of death related to DMD increases. Despite preventive and palliative cardiac treatments available, the prognoses remain poor. Direct therapeutic targeting of dystrophin deficiency is critical, however, hindered by the large size of the dystrophin cDNA and/or stochastic, often extensive genetic changes in DMD gene. The correlation between cardiac involvement and mutations affecting specific dystrophin isoforms, may provide a mutation-specific cardiac management and novel therapeutic approaches for patients with CM. Nonetheless, the successful cardiac treatment poses a big challenge and may require combined therapy to combat dystrophin deficiency and its after-effects (critical in DMD pathogenesis). This review locates the multifaceted heart problem in the course of DMD, balancing the insights into basic science, translational efforts and clinical manifestation of dystrophic heart disease.

Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes, in Contrast to Adipose Tissue-Derived Stromal Cells, Efficiently Improve Heart Function in Murine Model of Myocardial Infarction.

Cell therapies are extensively tested to restore heart function after myocardial infarction (MI). Survival of any cell type after intracardiac administration, however, may be limited due to unfavorable conditions of damaged tissue. Therefore, the aim of this study was to evaluate the therapeutic effect of adipose-derived stromal cells (ADSCs) and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) overexpressing either the proangiogenic SDF-1α or anti-inflammatory heme oxygenase-1 (HO-1) in a murine model of MI. ADSCs and hiPSCs were transduced with lentiviral vectors encoding luciferase (Luc), GFP and either HO-1 or SDF-1α. hiPSCs were then differentiated to hiPSC-CMs using small molecules modulating the WNT pathway. Genetically modified ADSCs were firstly administered via intracardiac injection after MI induction in Nude mice. Next, ADSCs-Luc-GFP and genetically modified hiPSC-CMs were injected into the hearts of the more receptive NOD/SCID strain to compare the therapeutic effect of both cell types. Ultrasonography, performed on days 7, 14, 28 and 42, revealed a significant decrease of left ventricular ejection fraction (LVEF) in all MI-induced groups. No improvement of LVEF was observed in ADSC-treated Nude and NOD/SCID mice. In contrast, administration of hiPSC-CMs resulted in a substantial increase of LVEF, occurring between 28 and 42 days after MI, and decreased fibrosis, regardless of genetic modification. Importantly, bioluminescence analysis, as well as immunofluorescent staining, confirmed the presence of hiPSC-CMs in murine tissue. Interestingly, the luminescence signal was strongest in hearts treated with hiPSC-CMs overexpressing HO-1. Performed experiments demonstrate that hiPSC-CMs, unlike ADSCs, are effective in improving heart function after MI. Additionally, long-term evaluation of heart function seems to be crucial for proper assessment of the effect of cell administration.

The Contribution of IgG Glycosylation to Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) and Complement-Dependent Cytotoxicity (CDC) in Hashimoto's Thyroiditis: An in Vitro Model of Thyroid Autoimmunity.

Antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) are involved in destruction of thyroid tissue in Hashimoto's thyroiditis (HT). N-glycosylation of the Fc fragment affects the effector functions of IgG by enhancing or suppressing the cytotoxicity effect. The aim of the present study was to assess the impact of HT-specific IgG glycosylation in ADCC and CDC, using in vitro models. The normal thyroid Nthy-ori 3-1 cell line and thyroid carcinoma FTC-133 cells were used as the target cells. Peripheral blood mononuclear cells (PBMCs) from healthy donors and the HL-60 human promyelotic leukemia cell line served as the effector cells. IgG was isolated from sera of HT and healthy donors and then treated with α2-3,6,8-neuraminidase to cut off sialic acids (SA) from N-glycans. We observed more intensive cytotoxicity in the presence of IgG from HT patients than in the presence of IgG from healthy donors. Removal of SA from IgG N-glycans increased ADCC intensity and reduced CDC. We conclude that the enhanced thyrocyte lysis resulted from the higher anti-TPO content in the whole IgG pool of HT donors and from altered IgG glycosylation in HT autoimmunity.

Neuroimaging results, short-term assessment of psychomotor development and the risk of autism spectrum disorder in extremely premature infants (≤28 GA) - a prospective cohort study (preliminary report).

OBJECTIVE: Infants ≤28 GA are at particular risk of psychomotor and neurological developmental disorder. They also remain at a higher risk of developing autism spectrum disorder (ASD), characterized by persistent deficits in communication/social interactions and restricted, repetitive behaviors, activities and interests. Monitoring their development by a team of specialists (a neurologist, psychologist, psychiatrist) allows us to make an early diagnosis and to implement appropriate therapy. Neuroimaging studies during the neonatal period may be helpful in clarifying diagnosis and prognosis. Objective: The aim of the study was to search for the interrelation between the results of neuroimaging and the neurological, psychological and psychiatric evaluation at the age of 2. PATIENTS AND METHODS: Material and methods: Neonates born at ≤28 weeks between 01.06.2013 and 31.12.2015 and hospitalized at NICU were enrolled. We present the results of the first 12 children who have attained 2 years of corrected age and have undergone both neuroimaging, and neurological, psychological and psychiatric assessments. Transfontanel ultrasound was performed according to general standards, MRI between 38 and 42 weeks of corrected age. Neurological examination based on the Denver scale, ASD screening with use of the STAT test and psychological DSR assessment were performed at 2 years of corrected age. RESULTS: Results: Median GA was 26 weeks and median weight 795 g. The ultrasound examination was normal in 9 cases (75%) and MRI in 4 (33%). Abnormalities in the cerebellum were the main additional information found in MRI as compared to US. Neurological examination was normal in 8 infants (67#37;), in 4 of whom neuroimaging was normal. In 4 (33%) infants the neurological examination was abnormal. Psychomotor development at an average level or above was found in seven (58#37;) children. In 4 of them neuroimaging was normal, whereas 3 had ventricular dilatation and haemorrhagic infarct. There were no abnormalities within the cerebellum in this group. In the remaining 5 children (42#37;) psychomotor development was rated as delayed. All of them had cerebellar haemorrhage. An increased risk of ASD was observed in 4 children who developed cerebellar hemorrhage. CONCLUSION: Conclusions: 1. The use of MRI at a term-equivalent age may contribute to the prognosis of neurodevelopmental outcomes in extremely premature infants, allowing risk stratification and thus enhancing early monitoring of a child's development and functional status 2. There is a clear tendency towards abnormal psychomotor development and positive screening for ASD to co-occur with abnormal MRI findings in the cerebellum.

Ikaros mediates gene silencing in T cells through Polycomb repressive complex 2.

T-cell development is accompanied by epigenetic changes that ensure the silencing of stem cell-related genes and the activation of lymphocyte-specific programmes. How transcription factors influence these changes remains unclear. We show that the Ikaros transcription factor forms a complex with Polycomb repressive complex 2 (PRC2) in CD4(-)CD8(-) thymocytes and allows its binding to more than 500 developmentally regulated loci, including those normally activated in haematopoietic stem cells and others induced by the Notch pathway. Loss of Ikaros in CD4(-)CD8(-) cells leads to reduced histone H3 lysine 27 trimethylation and ectopic gene expression. Furthermore, Ikaros binding triggers PRC2 recruitment and Ikaros interacts with PRC2 independently of the nucleosome remodelling and deacetylation complex. Our results identify Ikaros as a fundamental regulator of PRC2 function in developing T cells.

Biomechanical characteristics of the porcine denticulate ligament in different vertebral levels of the cervical spine-preliminary results of an experimental study.

BACKGROUND: Few studies exist on the mechanical properties of denticulate ligaments and none report the variation in these properties at different levels of the spine. The aim of this study was to perform an experimental determination of load-extension and stress-strain characteristics of the denticulate ligament and to establish if their properties change at different vertebral levels of the cervical spine. METHOD: The study was carried out on a total of 98 porcine denticulate ligament samples dissected from seven fresh porcine cervical spinal cord specimens. All of the samples were subjected to an uniaxial tensile test at a speed of 2mm/min, during which the load-extension characteristics were registered. RESULTS: The analysis revealed a decrease of the failure force in the caudal orientation indicated by significant differences between the C1 (1.04±0.41N) and C7 (0.55±0.12N) vertebral levels (P=0.037). The average ultimate force that broke the denticulate ligaments was 0.88N. The mean value of Young׳s modulus was 2.06MPa with a minimum of 1.31MPa for C7 and maximum of 2.46MPa for C5. CONCLUSIONS: The values of the denticulate ligament failure force in samples from different cervical vertebrae levels differ significantly. The presented data should be taken into consideration during numerical modelling of the human cervical spinal cord.

The relationship between structural aspects of self-concept and psychosocial adjustment in adolescents from alcoholic families.

Sixty adolescents from alcoholic families living in two large cities in Poland were examined in 2008 and 2009. Richness, stability, and certainty of their self-concepts, as well as levels of school adjustment, anxiety, and depression, were evaluated using a set of questionnaires. In a series of bivariate analyses, the strongest associations found were between richness of the self-concept and the social withdrawal syndrome, and between stability of the self-concept and depression. Both relationships remained significant, using multiple regression models, after controlling for possible confounding factors. Possible explanations and implications for the findings, as well as the study's limitations, are noted and discussed.

RNAimmuno: a database of the nonspecific immunological effects of RNA interference and microRNA reagents.

The RNAimmuno database was created to provide easy access to information regarding the nonspecific effects generated in cells by RNA interference triggers and microRNA regulators. Various RNAi and microRNA reagents, which differ in length and structure, often cause non-sequence-specific immune responses, in addition to triggering the intended sequence-specific effects. The activation of the cellular sensors of foreign RNA or DNA may lead to the induction of type I interferon and proinflammatory cytokine release. Subsequent changes in the cellular transcriptome and proteome may result in adverse effects, including cell death during therapeutic treatments or the misinterpretation of experimental results in research applications. The manually curated RNAimmuno database gathers the majority of the published data regarding the immunological side effects that are caused in investigated cell lines, tissues, and model organisms by different reagents. The database is accessible at http://rnaimmuno.ibch.poznan.pl and may be helpful in the further application and development of RNAi- and microRNA-based technologies.

Recent advances in understanding of the immunological off-target effects of siRNA.

Short interfering RNAs (siRNAs) are the most commonly used RNA interference (RNAi) triggers. They hold promise as potent therapeutic tools, as demonstrated by recent successful in vivo experiments. However, in addition to triggering intended sequence-specific silencing effects, the reagents of RNAi technology can often cause side effects, including immunological off-target effects. The cellular sensors of foreign RNA, such as RIG-I or Toll-like receptors, involved in innate immune antiviral responses, are activated by RNAi reagents. Stimulation of these pathways results in changes in the cellular transcriptome and proteome that can lead to the inhibition of cell division and growth and eventually apoptosis. An additional undesired effect in the context of research applications may be the misinterpretation of experimental results. To date, a number of the specific features of siRNA structure, sequence and delivery mode that are responsible for these effects have been identified. This knowledge may be helpful in designing safer gene-silencing reagents. In this article we discuss the recent developments in the field of non-specific toxic effects caused by RNAi triggers and their delivery vehicles. These data are critically discussed and evaluated, taking advantage of relevant information compiled in the recently launched RNAimmuno database (http://rnaimmuno.ibch.poznan.pl).