An ex vivo gene therapy approach to treat muscular dystrophy using inducible pluripotent stem cells.

Duchenne muscular dystrophy is a progressive and incurable neuromuscular disease caused by genetic and biochemical defects of the dystrophin-glycoprotein complex. Here we show the regenerative potential of myogenic progenitors derived from corrected dystrophic induced pluripotent stem cells generated from fibroblasts of mice lacking both dystrophin and utrophin. We correct the phenotype of dystrophic induced pluripotent stem cells using a Sleeping Beauty transposon system carrying the micro-utrophin gene, differentiate these cells into skeletal muscle progenitors and transplant them back into dystrophic mice. Engrafted muscles displayed large numbers of micro-utrophin-positive myofibers, with biochemically restored dystrophin-glycoprotein complex and improved contractile strength. The transplanted cells seed the satellite cell compartment, responded properly to injury and exhibit neuromuscular synapses. We also detect muscle engraftment after systemic delivery of these corrected progenitors. These results represent an important advance towards the future treatment of muscular dystrophies using genetically corrected autologous induced pluripotent stem cells

SELECTIVE MEASUREMENT OF α SMOOTH MUSCLE ACTIN: WHY β-ACTIN CAN NOT BE USED AS A HOUSEKEEPING GENE WHEN TISSUE FIBROSIS OCCURS

Abstract Background Prevalence of fibroproliferative diseases, including chronic kidney disease is rapidly increasing and has become a major public health problem worldwide. Fibroproliferative diseases are characterized by increased expression of α smooth muscle actin (α-SMA) that belongs to the family of the six conserved actin isoforms showing high degree homology. The aim of the present study was to develop real-time PCRs that clearly discriminate α-SMA and ß-actin from other actin isoforms. Results Real-time PCRs using self-designed mouse, human and rat specific α-SMA or ß-actin primer pairs resulted in the specific amplification of the artificial DNA templates corresponding to mouse, human or rat α-SMA or ß-actin, however ß-actin showed cross-reaction with the housekeeping γ-cyto-actin. We have shown that the use of improperly designed literary primer pairs significantly affects the results of PCRs measuring mRNA expression of α-SMA or ß-actin in the kidney of mice underwent UUO. Conclusion We developed a set of carefully designed primer pairs and PCR conditions to selectively determine the expression of mouse, human or rat α-SMA and ß-actin isoforms. We demonstrated the importance of primer specificity in experiments where the results are normalized to the expression of ß-actin especially when fibrosis and thus increased expression of α-SMA is occur

A gyermekkori koronavírus-fertőzést követő sokszervi gyulladás diagnosztikája és kezelése

A SARS-CoV-2-fertőzés ritka gyermekkori szövődménye a sokszervi gyulladás, angol terminológiával paediatric inflammatory multisystem syndrome (PIMS). Két vagy több szerv érintettségével járó, súlyos tünetekkel induló betegségről van szó, amelynek tünetei átfedést mutatnak a Kawasaki-betegséggel, a toxikus sokk szindrómával és a makrofágaktivációs szindrómával. A PIMS-betegek intenzív terápiás osztályon vagy intenzív terápiás háttérrel rendelkező intézményben kezelendők, ahol biztosítottak a kardiológiai ellátás feltételei is. A szükséges immunterápia a klinikai prezentációtól függ. A jelen közleményben a szerzők a releváns nemzetközi irodalom áttekintését követően ajánlást tesznek a PIMS diagnosztikai és terápiás algoritmusára. Orv Hetil. 2021; 162(17): 652-667. Summary. Pediatric inflammatory multisystem syndrome (PIMS) is a rare complication of SARS-CoV-2 infection in children. PIMS is a severe condition, involving two or more organ systems. The symptoms overlap with Kawasaki disease, toxic shock syndrome and macrophage activation syndrome. PIMS patients should be treated in an intensive care unit or in an institution with an intensive care background, where cardiological care is also provided. The required specific immunotherapy depends on the clinical presentation. In this paper, after reviewing the relevant international literature, the authors make a recommendation for the diagnostic and therapeutic algorithm for PIMS. Orv Hetil. 2021; 162(17): 652-667

Discovery and Development of Toll-Like Receptor 4 (TLR4) Antagonists: A New Paradigm for Treating Sepsis and Other Diseases

Abstract. Sepsis remains the most common cause of death in intensive care units in the USA, with a current estimate of at least 750,000 cases per year, and 215,000 deaths annually. Despite extensive research still we do not quite understand the cellular and molecular mechanisms that are involved in triggering and propagation of septic injury. Endotoxin (lipopolysaccharide from Gram-negative bacteria, or LPS) has been implicated as a major cause of this syndrome. Inflammatory shock as a consequence of LPS release remains a serious clinical concern. In humans, inflammatory responses to LPS result in the release of cytokines and other cell mediators from monocytes and macrophages, which can cause fever, shock, organ failure and death. A number of different approaches have been investigated to try to treat and/or prevent the septic shock associated with infections caused by Gram-negative bacteria, including blockage of one or more of the cytokines induced by LPS. Recently several novel amphipathic compounds have been developed as direct LPS antagonists at the LPS receptor, TLR4. This review article will outline the current knowledge on the TLR4-LPS synthesis and discuss the signaling, in vitro pre-clinical and in vivo clinical evaluation of TLR4 antagonists and their potential use in sepsis and a variety of diseases such as atherosclerosis as well as hepatic and renal malfunction. KEY WORDS: drug discovery; LPS; sepsis; toll-like receptor antagonists

The study of the effect of immunosuppressive drugs on lipid metabolism

Introduction: Lipid abnormalities including increased total cholesterol, triglycerides, and lowdensity lipoprotein-cholesterol have been frequently reported in renal transplantation and could be involved in the high frequency of cardiovascular disease in this population. Immunosuppressive therapy appears to be a main factor that influences the post-transplant lipid profile. Cyclosporine A (CsA), rapamycin (RAPA), tacrolimus (TAC) and mycophenolate mofetil (MMF) are commonly used immunosuppressant in solid organ transplant patients. Several of these immunosuppressive agents including CsA, RAPA and TAC appear to have a significant effect on patient lipid level. Although RAPA does not seem to cause nephrotoxicity as commonly seen in patients treated with CsA or TAC, it seems to be associated with an incremental increase in triglyceride level. However, the immunosuppressive-induced hyperlipidemia has not been sufficiently described. Purpose: Our aim was to determine the effects of these drugs in vitro on key regulatory enzymes of lipid metabolism; Cholesteryl Ester Transfer Protein (CETP), hepatic lipase (HL) and lipoprotein lipase (LPL) activity within human plasma, as well as the in vivo effects of TAC on these enzymes in renal transplant patients. In addition, we also investigated the effects of RAPA and TAC on cholesterol efflux from human THP-l macrophages. Methods: The effects of CsA, TAC, RAPA and MMF on CETP, HL and LPL activity were first determined in vitro in human normolipidemic plasma and post-heparin normal human plasma, respectively. We further investigated the in vivo effects of TAC on these enzymes activities in renal transplant patients for one month following transplantation. The cholesterol efflux study was conducted independently to assess the effects of RAPA and TAC on ApoA-I- and HDL-mediated cholesterol efflux from human THP-l macrophages, as well as adenosine-triphosphate binding cassette (ABC)Al and ABCG1 protein expressions in these cells. Results: Our in vitro CETP study showed that CsA and RAPA induced CETP activity in human normolipidemic plasma in a dose-dependant manner. Although, none of these drugs, CsA, TAC, RAPA and MMF affected in vitro HL activity, these drugs suppressed the LPL activity in the post-heparin plasma. Unlike TAC, RAPA was shown to decrease apoAl-mediated cholesterol efflux and ABCA1 protein expression in human THP-l macrophages. In agreement with our in vitro result, our clinical study demonstrated that TAC significantly increased triglyceride levels and reduced the LPL activity in the renal transplant patients, regardless of the patients were on statin or not. Conclusions: These findings suggest that the increase in CETP activity, suppression in LPL activity and inhibition in the cholesterol efflux following either CsA, RAPA or TAC treatments observed in the present study may be associated with hypercholesterolemia and hypertriglyceridemia seen in patients administered these drugs.Medicine, Faculty ofPathology and Laboratory Medicine, Department ofGraduat

An ex vivo gene therapy approach to treat muscular dystrophy using inducible pluripotent stem cells.

Duchenne muscular dystrophy is a progressive and incurable neuromuscular disease caused by genetic and biochemical defects of the dystrophin-glycoprotein complex. Here we show the regenerative potential of myogenic progenitors derived from corrected dystrophic induced pluripotent stem cells generated from fibroblasts of mice lacking both dystrophin and utrophin. We correct the phenotype of dystrophic induced pluripotent stem cells using a Sleeping Beauty transposon system carrying the micro-utrophin gene, differentiate these cells into skeletal muscle progenitors and transplant them back into dystrophic mice. Engrafted muscles displayed large numbers of micro-utrophin-positive myofibers, with biochemically restored dystrophin-glycoprotein complex and improved contractile strength. The transplanted cells seed the satellite cell compartment, responded properly to injury and exhibit neuromuscular synapses. We also detect muscle engraftment after systemic delivery of these corrected progenitors. These results represent an important advance towards the future treatment of muscular dystrophies using genetically corrected autologous induced pluripotent stem cells