Genetics, molecular and cell biology of apoptotic cell death

Apoptotic cell death is an integral part of development and cell turnover in multicellular organisms. Since early 1970’s, when apoptosis was defined on morphological basis, plethora of genes has been identified participating in initiation, execution and regulation of cell death. This article reviews these latest advances and describes our present understanding of the sequential events of apoptotic cell death, from the early steps of death receptor initiated and mitochondrial pathways to activation of caspases, and finally, the proper corpse clearance. It also discusses dysregulation of apoptosis, leading to various pathologies, such as cancer, autoimmune disease and neurodegenerative disorders

Csontrendszeri és genetikai elváltozások az idiopathiás scoliosis etiológiájában. = Bone density and genetic changes in etiology of idiopathic scoliosis.

Az idiopathiás scoliosis (IS) etiológiájának genetikai komponensét két megközelítéssel vizsgáltuk. Az első egy genomikus polimorfizmus vizsgálat volt amely a 17 kr. q21.32 régiójában lévő kromoszómapolimorfizmusra utalt. Nagyszámú IS-es betegből készült DNS adatbank további vizsgálata során azonban a kromoszómapolimorfizmust nem sikerült igazolni. A második megközelítés a kontroll és az IS-es betegek jobb és a bal oldali paravertebrális izmainak génexpressziós analízise volt. Közel 200 gén mutatott expresszióbeli eltérést a jobb és a bal oldali izombiopsziák vonatkozásásban. A génexpressziós eltérés igazolására 12 gént vizsgáltuk tovább quantitatív real-time PCR (QPCR) technikával. Három génre szűkült vuzsgálatunk (Hox7, CDK5, LEP), melyek közül a leptint (LEP) egyedi mintákon is vizsgálva igazolni tudtuk az expresszióbeli különbséget a két oldal között két beteg mintáin. Sajnos a megvizsgált két kontroll mintában ellenkező irányú eltérést detektáltunk, aminek nem tudjuk még a magyarázatát. A filamin gének mutációja kimutatható sceletális problémákban, így filamin B mutációk a csigolyafejlődési rendellenességek hátterében állhatnak. A filamin B gén exonjaira tervezett primerekkel direkt szekvenálás segítségével próbáltunk mutációkat keresni. Számos már ismert és eddig nem ismert SNP-t találtunk; egy esetben találtunk olyan eltérést, ami egyik szülőben sem volt megtalálható, és a betegeben egy ala-val cserével társul. | The genetic component of idiopathic scoliosis (IS) has been investigated in two different approaches. The first method was a genetic polymorphism investigation suggested a chromosome polymorphism in 17th chromosome q21.32 region. However, we did not succeed in proving the role of this chromosome polymorphism in a further analysis of a DNA data bank consisted of a huge number of IS patients. The second approach we used was the comparison of gene expression of left and right paravertebral muscles between IS patients and control individuals. Approximately 200 genes showed differences of gene expression related to right and left sided muscle biopsies. We did choose 12 genes to prove the discrepancy in gene expressivity by quantitative real-time PCR (QPCR). The analysis has been focused on three genes (Hox7, CDK5, LEP), of which the leptin (LEP) was assayed in individual samples and the difference of gene expressivity was showed between the two sides in two patients? samples. The mutation of filamin genes can be shown in human skeletal problems, e.g. filamin B gene mutation can be demonstrated in the background of vertebral malformations. We template to find mutation by direct sequenation with primers created on filamin B gene's exons. Several known and up to now unknowns SNPs were detected. Such a case was found as well, where the discrepancy was not showed in neither of the parents and this was associated with an ala change

Altered Cell Surface N-Glycosylation of Resting and Activated T Cells in Systemic Lupus Erythematosus

Altered cell surface glycosylation in congenital and acquired diseases has been shown to affect cell differentiation and cellular responses to external signals. Hence, it may have an important role in immune regulation; however, T cell surface glycosylation has not been studied in systemic lupus erythematosus (SLE), a prototype of autoimmune diseases. Analysis of the glycosylation of T cells from patients suffering from SLE was performed by lectin-binding assay, flow cytometry, and quantitative real-time PCR. The results showed that resting SLE T cells presented an activated-like phenotype in terms of their glycosylation pattern. Additionally, activated SLE T cells bound significantly less galectin-1 (Gal-1), an important immunoregulatory lectin, while other lectins bound similarly to the controls. Differential lectin binding, specifically Gal-1, to SLE T cells was explained by the increased gene expression ratio of sialyltransferases and neuraminidase 1 (NEU1), particularly by elevated ST6 beta-galactosamide alpha-2,6-sialyltranferase 1 (ST6GAL1)/NEU1 and ST3 beta-galactoside alpha-2,3-sialyltransferase 6 (ST3GAL6)/NEU1 ratios. These findings indicated an increased terminal sialylation. Indeed, neuraminidase treatment of cells resulted in the increase of Gal-1 binding. Altered T cell surface glycosylation may predispose the cells to resistance to the immunoregulatory effects of Gal-1, and may thus contribute to the pathomechanism of SLE

Licensing by Inflammatory Cytokines Abolishes Heterogeneity of Immunosuppressive Function of Mesenchymal Stem Cell Population

When mesenchymal stem cells (MSCs) are used for therapy of immunological pathologies, they get into an inflammatory environment, altering the effectiveness of the treatment. To establish the impact of environmental inflammatory factors on MSCs' immunofunction in the mirror of intrinsic heterogeneity of mouse MSC population, individual MSC clones were generated and characterized. Adipogenic but not osteogenic differentiation and pro-angiogenic activity of five independent MSC cell lines were similar. Regarding osteogenic differentiation, clones MSC3 and MSC6 exhibited poorer capacity than MSC2, MSC4, and MSC5. To study the immunosuppressive heterogeneity, in vitro and in vivo experiments have been carried out using T-cell proliferation assay and delayed-type hypersensitivity (DTH) response, respectively. A remarkable difference was found between the clones in their ability to inhibit T-cell proliferation in the following order: MSC2MSC5>MSC4>MSC3>>MSC6. Nevertheless, the differences between the immunosuppressive activities of the individual clones disappeared on pretreatment of the cells with pro-inflammatory cytokines, a procedure called licensing. Stimulation of all clones with IFN- and TNF- resulted in elevation of their inhibitory capability to a similar level. Nitric oxide (NO) and prostaglandin E2 (PGE2) were identified as major mediators of immunofunction of the MSC clones. The earlier findings were also supported by in vivo results. Without licensing, MSC2 inhibited DTH response, while MSC6 did not affect DTH response. In contrast, prestimulation of MSC6 with inflammatory cytokines resulted in strong suppression by this clone as well. Here, we have showed that MSC population is functionally heterogeneous in terms of immunosuppressive function; however, this variability is largely reduced under pro-inflammatory conditions

Novel role for galectin-1 in T-cells under physiological and pathological conditions

Secreted, extracellular galectin-1 (exGal-1) but not intracellular Gal-1 (inGal-1) has been described as a strong immunosuppressive protein due to its major activity of inducing apoptosis of activated T-cells. It has previously been reported that T-cells express Gal-1 upon activation, however its participation in T-cell functions has remained largely elusive. To determine function of Gal-1 expressed by activated T-cells we have carried out a series of experiments. We have shown that Gal-1, expressed in Gal-1-transgenic Jurkat cells or in activated T-cells, remained intracellularly indicating that Gal-1-induced T-cell death was not a result of an autocrine effect of the de novo expressed Gal-1. Rather, a particular consequence of the inGal-1 expression was that T-cells became more sensitive to exGal-1 added either as a soluble protein or bound to the surface of a Gal-1-secreting effector cell. This was also verified when the susceptibility of activated T-cells from wild type or Gal-1 knockout mice to Gal-1-induced apoptosis were compared. Murine T-cells expressing Gal-1 were more sensitive to the cytotoxicity of the exGal-1 than their Gal-1 knockout counterparts. We also conducted a study with activated T-cells from patients with systemic lupus erythematosus (SLE), a disease in which dysregulated T-cell apoptosis has been well described. SLE T-cells expressed lower amounts of Gal-1 than healthy T-cells and were less sensitive to exGal-1. These results suggested a novel role of inGal-1 in T-cells as a regulator of T-cell response to exGal-1, and its likely contribution to the mechanism in T-cell apoptosis deficiency in lupus

Characterization and therapeutic application of canine adipose mesenchymal stem cells to treat elbow osteoarthritis

Visceral adipose tissue (AT) obtained from surgical waste during routine ovariectomies was used as a source for isolating canine mesenchymal stem cells (MSCs). As determined by cytofluorimetry, passage 2 cells expressed MSC markers CD44 and CD90 and were negative for lineage-specific markers CD34 and CD45. The cells differentiated toward osteogenic, adipogenic, and chondrogenic directions. With therapeutic aims, 30 dogs (39 joints) suffering from elbow dysplasia (ED) and osteoarthritis (OA) were intra-articularly transplanted with allogeneic MSCs suspended in 0.5% hyaluronic acid (HA). A highly significant improvement was achieved without any medication as demonstrated by the degree of lameness during the follow-up period of 1 y. Control arthroscopy of 1 transplanted dog indicated that the cartilage had regenerated. Histological analysis of the cartilage biopsy confirmed that the regenerated cartilage was of hyaline type. These results demonstrate that transplantation of allogeneic adipose tissue-derived mesenchymal stem cells (AT-MSCs) is a novel, noninvasive, and highly effective therapeutic tool in treating canine elbow dysplasia. © 2017, Canadian Veterinary Medical Association. All rights reserved

Characterization and therapeutic application of canine adipose mesenchymal stem cells to treat elbow osteoarthritis

Visceral adipose tissue (AT) obtained from surgical waste during routine ovariectomies was used as a source for isolating canine mesenchymal stem cells (MSCs). As determined by cytofluorimetry, passage 2 cells expressed MSC markers CD44 and CD90 and were negative for lineage-specific markers CD34 and CD45. The cells differentiated toward osteogenic, adipogenic, and chondrogenic directions. With therapeutic aims, 30 dogs (39 joints) suffering from elbow dysplasia (ED) and osteoarthritis (OA) were intra-articularly transplanted with allogeneic MSCs suspended in 0.5% hyaluronic acid (HA). A highly significant improvement was achieved without any medication as demonstrated by the degree of lameness during the follow-up period of 1 y. Control arthroscopy of 1 transplanted dog indicated that the cartilage had regenerated. Histological analysis of the cartilage biopsy confirmed that the regenerated cartilage was of hyaline type. These results demonstrate that transplantation of allogeneic adipose tissue-derived mesenchymal stem cells (AT-MSCs) is a novel, noninvasive, and highly effective therapeutic tool in treating canine elbow dysplasia. © 2017, Canadian Veterinary Medical Association. All rights reserved

Routing Nanomolar Protein Cargoes to Lipid Raft-Mediated/Caveolar Endocytosis through a Ganglioside GM1‐Specific Recognition Tag

There is a pressing need to develop ways to deliver therapeutic macromolecules to their intracellular targets. Certain viral and bacterial proteins are readily internalized in functional form through lipid raft-mediated/caveolar endocytosis, but mimicking this process with protein cargoes at therapeutically relevant concentrations is a great challenge. Targeting ganglioside GM1 in the caveolar pits triggers endocytosis. A pentapeptide sequence WYKYW is presented, which specifically captures the glycan moiety of GM1 (K-D = 24 nm). The WYKYW-tag facilitates the GM1-dependent endocytosis of proteins in which the cargo-loaded caveosomes do not fuse with lysosomes. A structurally intact immunoglobulin G complex (580 kDa) is successfully delivered into live HeLa cells at extracellular concentrations ranging from 20 to 160 nm, and escape of the cargo proteins to the cytosol is observed. The short peptidic WYKYW-tag is an advantageous endocytosis routing sequence for lipid raft-mediated/caveolar cell delivery of therapeutic macromolecules, especially for cancer cells that overexpress GM1.Peer reviewe

Cell Delivery: Routing Nanomolar Protein Cargoes to Lipid Raft-Mediated/Caveolar Endocytosis through a Ganglioside GM1-Specific Recognition Tag (Adv. Sci. 4/2020)

In article number 1902621, Tamás A. Martinek and co-workers develop a pentapeptidic tag, which reads the glycan code of ganglioside GM1 and triggers lipid raft-mediated endocytosis, avoiding lysosomal entrapment. This carrier molecule can deliver macromolecular cargoes (e.g., IgG complexes) into live cells with the possibility to escape to the cytosol.Peer reviewe