[Cell senescence, a new target for respiratory viral infections: From influenza virus to SARS-CoV-2]. / La sénescence cellulaire, nouvelle cible des infections virales respiratoires : du virus influenza au SARS-CoV-2.

The accumulation of senescent cells in tissues is a key process of aging and age-related diseases, including lung diseases such as chronic obstructive pulmonary disease, lung fibrosis, or cancer. In recent years, the spectrum of respiratory diseases associated with cellular senescence has been broadened, in particular acute viral pulmonary infections, foremost among which is coronavirus disease 2019 (COVID19), which is particularly severe in the elderly or in subjects with comorbidities. Influenza virus infection, which strikes more severely at the extreme ages of life, is also associated with severe pulmonary senescence. Cellular senescence potentially represents an original target for attacking these diseases, although its specific mechanisms remain largely misunderstood. New anti-senescent therapeutic approaches are thus proposed during severe viral pulmonary infections, with the aim of preventing acute effects and/or, in the longer term, pulmonary sequelae.

A calcium-sensitive promoter construct for gene therapy.

Targeting diseased cells is a challenging issue in both pharmacological and biological therapeutics. Gene therapy is emerging as a novel approach for treating rare diseases and for illnesses for which there is no other alternative. An important limitation of gene therapy has been the off-target effects and therefore efforts have been focused on increasing the specificity of gene transfer to the targeted organ. Here, we describe a promoter containing six nuclear factor of activated T cells (NFAT) consensus sequences, which is as efficient as the cytomegalovirus (CMV) promoter to drive expression in vascular smooth muscle cells both in vitro and in vivo. In contrast to the CMV promoter it is activated in a Ca(2+)-dependent manner after endoplasmic reticulum depletion and allows the transgene expression only in proliferative/diseased cells. Overexpression of sarco/endoplasmic reticulum (SR/ER) Ca(2+) ATPase 2a under the control of this NFAT promoter inhibits restenosis after angioplasty in rats. In conclusion, this promoter may be useful for gene therapy in vascular proliferative diseases and other diseases involving upregulation of the NFAT pathway.

SERCA2a gene transfer prevents intimal proliferation in an organ culture of human internal mammary artery.

Coronary restenosis, a major complication of percutaneous balloon angioplasty, results from neointimal proliferation of vascular smooth muscle cells (VSMCs). The sarco/endoplasmic reticulum calcium ATPase 2a isoform (SERCA2a), specific to contractile VSMCs, has been reported previously to be involved in the control of the Ca(2+)-signaling pathways governing proliferation and migration. Moreover, SERCA2a gene transfer was reported to inhibit in vitro VSMC proliferation and to prevent neointimal thickening in a rat carotid injury model. The aim of this study was to evaluate the potential therapeutic interest of SERCA2a gene transfer for prevention of in-stent restenosis using a ex vivo model of human left internal mammary artery (hIMA) intimal thickening. Left hIMAs, obtained at the time of aorto-coronary bypass surgeries, were subjected to balloon dilatation followed by infection for 30 min with adenoviruses encoding either human SERCA2 and green fluorescence protein (GFP) or control gene (ß-galactosidase, ß-gal) and GFP. Proliferation of subendothelial VSMCs and neointimal thickening were observed in balloon-injured hIMA maintained 14 days in organ culture under constant pressure and perfusion. SERCA2a gene transfer prevented vascular remodeling and significantly (P<0.01, n=5) reduced neointimal thickening in injured arteries (intima/media ratio was 0.07±0.01 vs 0.40±0.03 in ß-gal-infected arteries). These findings could have potential implications for treatment of pathological in-stent restenosis.

Efficient transduction of vascular smooth muscle cells with a translational AAV2.5 vector: a new perspective for in-stent restenosis gene therapy.

Coronary artery disease represents the leading cause of mortality in the developed world. Percutaneous coronary intervention involving stent placement remains disadvantaged by restenosis or thrombosis. Vascular gene therapy-based methods may be approached, but lack a vascular gene delivery vector. We report a safe and efficient long-term transduction of rat carotid vessels after balloon injury intervention with a translational optimized AAV2.5 vector. Compared with other known adeno-associated virus (AAV) serotypes, AAV2.5 demonstrated the highest transduction efficiency of human coronary artery vascular smooth muscle cells (VSMCs) in vitro. Local delivery of AAV2.5-driven transgenes in injured carotid arteries resulted in transduction as soon as day 2 after surgery and persisted for at least 30 days. In contrast to adenovirus 5 vector, inflammation was not detected in AAV2.5-transduced vessels. The functional effects of AAV2.5-mediated gene transfer on neointimal thickening were assessed using the sarco/endoplasmic reticulum Ca(2+) ATPase isoform 2a (SERCA2a) human gene, known to inhibit VSMC proliferation. At 30 days, human SERCA2a messenger RNA was detected in transduced arteries. Morphometric analysis revealed a significant decrease in neointimal hyperplasia in AAV2.5-SERCA2a-transduced arteries: 28.36±11.30 (n=8) vs 77.96±24.60 (n=10) µm(2), in AAV2.5-green fluorescent protein-infected, P<0.05. In conclusion, AAV2.5 vector can be considered as a promising safe and effective vector for vascular gene therapy.

Enhanced cardiac function in transgenic mice expressing a Ca(2+)-stimulated adenylyl cyclase.

The predominant functional adenylyl cyclases normally expressed in cardiac tissue and coupled to beta-adrenergic receptors are inhibited by micromolar Ca(2+) concentration. To modify the overall balance of activities, we have generated transgenic mice expressing the Ca(2+)-stimulatable adenylyl cyclase type 8 (AC8) specifically in the heart. AC activity is increased by at least 7-fold in heart membranes from transgenic animals and is stimulated by Ca(2+) in the same range of concentration that inhibits the endogenous activity. Moreover, the in vivo basal protein kinase A activity was augmented 4-fold. Overexpression of AC8 in the heart has no detrimental consequences on global cardiac function. Basal heart rate and contractile function, measured by noninvasive echocardiography, were unchanged. In contrast, on release of parasympathetic tone, the intrinsic contractility is heightened and unresponsive to further beta-adrenergic receptor stimulation. AC8 transgenic mice thus represent an original model to investigate the relative influence of Ca(2+) and cAMP on cardiac function within a phenotype of enhanced cardiac contractility and relaxation.

Axial stretch-dependent cation entry in dystrophic cardiomyopathy: Involvement of several TRPs channels.

In Duchenne muscular dystrophy (DMD), deficiency of the cytoskeletal protein dystrophin leads to well-described defects in skeletal muscle but also to dilated cardiomyopathy (DCM). In cardiac cells, the subsarcolemmal localization of dystrophin is thought to protect the membrane from mechanical stress. The dystrophin deficiency leads to membrane instability and a high stress-induced Ca(2+) influx due to dysregulation of sarcolemmal channels such as stretch-activated channels (SACs). In this work divalent cation entry has been explored in isolated ventricular Wild Type (WT) and mdx cardiomyocytes in two different conditions: at rest and during the application of an axial stretch. At rest, our results suggest that activation of TRPV2 channels participates to a constitutive basal cation entry in mdx cardiomyocytes.Using microcarbon fibres technique, an axial stretchwas applied to mimic effects of physiological conditions of ventricular filling and study on cation influx bythe Mn(2+)-quenching techniquedemonstrated a high stretch-dependentcationic influx in dystrophic cells, partially due to SACs. Involvement of TRPs channels in this excessive Ca(2+) influx has been investigated using specific modulators and demonstratedboth sarcolemmal localization and an abnormal activity of TRPV2 channels. In conclusion, TRPV2 channels are demonstrated here to play a key role in cation influx and dysregulation in dystrophin deficient cardiomyocytes, enhanced in stretching conditions.

Different expression of adenylyl cyclase isoforms after retinoic acid induction of P19 teratocarcinoma cells.

We have investigated the adenylyl cyclase (AC) activity and gene expression in retinoic acid (RA)-primed murine P19 teratocarcinoma cells, which recapitulate in vitro the first stages of neuroectodermal formation. Here we show that the P19 stem cells possess a basal Ca2+/CaM-stimulated AC activity, which increases about 10-fold after RA induction. The rise of AC activity is associated with a stage-specific up-regulation of AC2, AC5 and AC8 mRNAs and a down-regulation of AC3 mRNA. P19 cells provide a powerful model to investigate the role and specific regulation of AC isoforms during neuronal differentiation.

[DNA from human lymphocytes contains a sequence, homologous to immunoglobulin gene kappa]. / DNK, vykhodiashchaia iz limfotsitov cheloveka, soderzhit posledovatel'nosti, gomologichnye immunoglobulinovomu genu kappa.

DNA released by human lymphocytes and hyman blood plasma DNA were examined by the electrophoresis, electron microscopy and Southern blot hybridization. The data obtained suggested that DNA released by lymphocytes contains covalently closed circular molecules. By the technique of the Southern blot analysis it was shown that DNA released by lymphocytes and human blood plasma DNA contain discretely sized molecules homologous to the C kappa fragment of the human Ig gene.

[Calcium and magnesium ion-dependent endonuclease 37 kDa is activated during colchicine-induced apoptosis in HL-60 cells]. / Zavisimaia ot ionov kal'tsiia i magniia éndonukleaza 37 kDa aktiviruetsia pri indutsirovannom kolkhitsinom apoptoze v kletkakh HL-60.

With the aim of revealing endonuclease(s), activating in apoptotic cells, a nuclease activity of nuclear protein extract from human leukemic HL-60 cells treated with colchicine was studied. After a 30 h incubation with colchicine, a specific DNA fragmentation--a multiple of 200 base pair subunits--was clearly observed that was an evidence for the endonuclease activation. After 48 h almost the whole DNA was fragmented. At the same time histones appeared the major proteins of nuclear extract. Using the method for detection of nuclease activity in a gel, protein 37 kDa was identified as a Ca2+, Mg(2+)-dependent endonuclease. The maximum increase in nuclease activity occurred after a 24 h incubation with colchicine. A low activity of this protein was also found in control cells. These data suggest that in apoptotic cells activation of constantly expressed nucleases may occur, rather than a new synthesis of apoptotic nucleases.

[The participation of endonuclease in the formation of extrachromosomal DNA and the possible mechanisms of the occurrence of gene amplification]. / Uchastie éndonukleazy v orbazovanii ékstrakhromosomnoi DNK i vozmozhnye mekhanizmy vozniknoveniia amplifikatsii genov.

We examined the extrachromosomal DNA (exDNA, Hirt fraction) in ethidium bromide sensitive and resistant cells of line L929. The exDNA amount is greater in the latter. The amount of exDNA in L929 cells makes 0.19% of the total cellular DNA; the exDNA amounts in cells, resistant to 5 and 50 micrograms/ml ethidium bromide are 0.22 and 0.33%, resp. Using labelling by BudR, it is shown that approximately 16% exDNA in L cells constituted amplified sequences to be excreting to the culture medium. The Zn-independent endogenous nuclease is activated in the resistant cells. The treatment with cycloheximide (50 micrograms/ml) resulted in the increase in the exDNA amount and in the activation of Zn-independent endonuclease. The data obtained suggested that the activation of Zn-independent endonuclease may lead to the increase in the exDNA amount and determine presumably a high rate of cell adaptability to environmental conditions.

[The amplification and overexpression of mdr-family genes in ethidium bromide-resistant Chinese hamster CHO-K1 cells and in the hybrids of sensitive and resistant cells]. / Amplifikatsiia i sverkhékspressiia genov semeistva mdr v ustoichivykh k bromistomu étidiiu kletkakh kitaiskogo khomiachka CHO-K1 i v gibridakh chuvstvitel'nykh i ustoichivykh kletok.

Stable mutant cells Cebr-1 and Cebr-2, resistant to ethidium bromide (EB) in concentration of 2 micrograms/ml, have been isolated by a multistep selection in Chinese hamster ovary cells. It was shown that Cebr-1 and Cebr-2 cells acquired a cross-resistance to unrelated drugs. Stable changes in the structure of chromosomes 1, 2, 5 and 8 were revealed by karyological analysis. Overexpression and amplification of mdr genes were detected in Cebr-2 cells using Northern RNA and Southern DNA blot hybridization. Two independent hybrids Hybr-1 and Hybr-2 were obtained by fusion of Cebr-2 cells with Chinese hamster lung V-79 RJK cells, sensitive to EB. Hybr-2 cells were characterized by the same level of EB-resistance as Cebr-2 cells. Hybr-1 cells have a lower level of EB-resistance than Cebr-2 cells. Hybr-2 cells have demonstrated amplification and overexpression of mdr gene, the same as in Cebr-2 cells, whereas in Hybr-1 cells no mdr gene amplification was observed, but the level of mdr gene expression was higher than in sensitive cells. The data suggest that resistance of Chinese hamster cells to EB is mediated by amplification and overexpression of mdr genes.

[The amplification and overexpression of the mdr genes in Chinese hamster CHLV-79 RJK cells resistant to ethidium bromide correlate with the presence of karyotypic markers of the amplification]. / Amplifikatsiia i sverkhékspressiia genov mdr v kletkakh kitaiskogo khomiachka CHLV-79 RJK, ustoichivykh k bromistomu étidiiu, sochetaetsia s nalichiem kariotipicheskikh markerov amplifikatsii.

Evidence is provided that selection of the Chinese hamster cells CHLV-79 RJK with ethidium bromide results in amplification and overexpression of mdr family genes, one of which is encoding a transmembrane P-glycoprotein reducing the intracellular drug concentration. It is likely that the amplified copies are located at or near the sites of resident mdr gene localization to look as an abnormal chromosomal banding pattern in chromosome 1q26. In the following selection steps to higher drug concentration, the cells are keeping the degree of amplification, but mdr gene expression increases by many times. The data suggest that the resistance of the Chinese hamster cells CHLV-79 RJK to higher concentrations of ethidium bromide may be achieved via a variety of mechanisms, including as well mdr gene amplification as transcriptional regulation of these genes.

[The isolation and genetic characteristics of the new cell line of mouse myeloma spEBR-5 selected for ethidium bromide resistance and characterized by multiple drug resistance]. / Poluchenie i geneticheskaia kharakteristika novoi linii kletok mielomy myshi spEBR-5, otselektirovannykh na ustoichivosti k bromistomu étidiiu i kharakterizuiushchikhsia mnozhestvennoi lekarstvennoi ustoichivost'iu.

Stable mutant cells spEBR-5, resistant to ethidium bromide in concentration of 5 micrograms/ml, have been isolated by multistep selection in mouse myeloma cells sp2/0-Ag14. The spEBR-5 cells, selected with ethidium bromide, appeared to be cross resistant to unrelated drugs in connection with mdr/lb gene amplification and overexpression. Five specific chromosomal markers were detected in the karyotype of spEBR-5 cells as a result of chromosome structural rearrangement. No cytological manifestation of gene amplification such as HCR of chromosomes or DMs was found. A diffuse location of amplified sequences in chromosome(s) is suggested. The new mutant cell lines spEBR-5 can be used as a model for investigation of multidrug resistance mechanisms.

Resistance to adriamycin in human chronic promyeloleukemia line K562 correlates with directed genome destabilization--amplification of MDR1 gene and nonrandom changes in karyotype structure. / Ustoichivost' kletok khronicheskogo promieloleikoza cheloveka linii K562 k adriamitsinu korreliruet s napravlennoi destabilizatsiei genoma--amplifikatsiei gena MDR1 i ne sluchainymi izmeneniiami v strukture genotipa.

Three independent subclones (B2, B3 and C9) of human myelogenous leukemia cell line K562 selected with adriamycin (ADM) were analysed. Cross-resistance of these ADM-resistant cells was examined for a resistance to the number of drugs including colchicine, actinomycin D and ethidium bromide. MDR 1 gene amplification in B3 and C9 subclones was detected using Southern-hybridization with specific probe. Additional genetical material was found in genomes of resistant cells by analysis of G-banded metaphase chromosomes. An extraordinarily long marker chromosome was observed in every C9 metaphase plate. The character of this chromosome G-banding suggests that it may be a derivative of chromosome 5 containing a large homogeneously staining region (HSR) in locus 5q15. Both B2 and B3 subclones expressed double minute chromosomes (DMs) in 5% of cells. In the course of a prolonged cultivation (about 2 years) of C9 cells in the presence of ADM a progressive karyotype destabilization was observed: the frequency of new markers formation in C9 cells increased, cells having additional copies of marker chromosome with HSR appeared, the length of HSR varied, coexistence of HSR and DMs being found in several C9 cells. These karyotypical changes may be regarded as patterns of genome destabilization due to the multidrug resistance of K562/ADM cells.

[The karyotypic variability of Chinese hamster CHLV-79 RJK cells characterized by multiple drug resistance resulting from the amplification of the mdr gene family]. / Izmenchivost' kariotipa kletok kitaiskogo khomiachka CHLV-79 RJK, kharakterizuiushchikhsia mnozhestvennoi lekarstvennoi ustoichivost'iu, obuslovlennaia amplifikatsiei genov semeistva mdr.

Variability in karyotype structure of Chinese hamster lung V-79 RJK cells and of their six cell sublines, selected for increasing concentrations of ethidium bromide (EB), was investigated in addition to the number of mdr gene copies in cells, both EB sensitive and resistant. It is shown that EB resistant cells exhibit cross-resistance to different drugs resulting from mdr genes amplification. Southern DNA blot hybridization has shown that in Vebr-2 cells (the 1st step of selection) the number of mdr gene copies increased by 10 times, whereas in Vebr-30 cells (the 6th step of selection) the number of mdr gene copies remained the same as in Vebr-2 cells. The level of mdr genes expression in Vebr-30 cells being higher than in Vebr-2 cells. In Vebr-2 cells, homogeneously and differentially stained regions (HSRs) were detected in loci 1p31 and 1q26 of chromosome 1 material (markers Z1 and Z6, respectively). On the following selection steps (prolonged cultivation or increased drug concentration) additional HSRs appeared in chromosome 2 (locus 2qter), in derivatives of chromosome 5 (marker Z7, locus Z7pter) and chromosome X (marker Z2, locus Z2qter). In the course of prolonged cultivation, chromosome 2 and derivatives of chromosomes 1, 2, 5 and X, in which HSRs were found, participated in the formation of new markers resulting from deletions, inversions, insertions and translocations of the chromosomal material. It is supposed that mdr genes amplification in V-79 RJK cells resistant to EB may be regarded as a factor inducing subsequent genome destabilization and eventual progressive changes in the karyotype structure.

[Use of interstitial antibiotic electrophoresis with chronic pneumonia patients]. / Primenenie vnutritkanevogo élektroforeza antibiotikov u bol'nykh khronicheskoi pnevmoniei.

Interstitial electrophoresis of antibiotics included in the complex of curative measures was shown to be highly effective for the treatment of chronic pneumonia. The conclusion was made on the basis of an analysis of results of treatment of 128 patients with chronic pneumonia.

Adenylyl cyclase activity and gene expression during mesodermal differentiation of the P19 embryonal carcinoma cells.

DMSO-primed P19 pluripotent cells, which recapitulate the first stages of mammalian cardiogenesis and endodermal formation, were used as an in vitro model to analyze the variations in activity and expression of the different adenylyl cyclase (AC) isoforms during the early events of embryonic cell differentiation. Here, we show that the total AC activity, which increases up to 10-fold after differentiation of P19 cells, is mainly associated with increases in AC2, AC5, and AC6 mRNA levels. Particularly, the marked increase in AC5 mRNA correlates with the appearance of beating cardiomyocytes and with the transcription of the atrial myosin light chain (MLC1A) gene which encodes a protein specifically involved in the cardiac muscle cell contractile phenotype. Together, the results strongly suggest that 1) a rise in cyclic AMP (cAMP) may be associated with cardiomyocyte and endodermal cell differentiation during mammalian embryogenesis; and 2) AC5 gene expression starts very early during normal mouse cardiogenesis and correlates with the differentiation of cardiomyocytes.