A p53 transzkripciós faktor család szerepe teratogén vírusok sejtkárosító hatásának molekuláris mechanizmusában = The role of the p53 transcription factor family in the molecular mechanism of cytopatic effect elicited by teratogenic viruses

Kutatásaink célja a rubeolavírus (RV), a vesicularis stomatitis vírus (VSV) és a herpes simplex vírus (HSV) sejtkárosító és teratogén hatásának jobb megértése volt, ezért azt tanulmányoztuk, hogy ezen vírusok befolyásolják-e a p63 és a Bcl-2 családba tarozó fehérjék expresszióját. RV fertőzés hatására, mindegyik TAp63 izoforma szintje emelkedett, míg a deltaNp63alfa szintje csökkent Vero sejtekben. VSV fertőzés hatására, a Bcl-2 és a Bax-alfa szintje csökkent, a p53 és a p63 szintje nem változott, míg a p18 Bax expressziója jelentős mértékben emelkedett WK sejtekben. VSV-vel fertőzött HaCaT sejtekben a deltaNp63alfa és a mutáns p53 szintje csökkent, a Bcl-2 nem változott, míg a Bax-alfa és a p18 Bax expressziója fokozódott. HSV fertőzés hatására, a deltaNp63alfa szintje csökkent, míg néhány más p63 izoforma expressziója fokozódott. Továbbá, a HSV fertőzés a Bax-béta szintjének emelkedését váltotta ki. Eredményeink azt mutatják, hogy mindhárom vírus befolyásolja a p63 izoformák és a Bcl-2 családtagok arányát, de egymástól eltérő módon. A különböző p63 izoformák szintjének változása fontos szerepet játszhat ezen vírusok sejtkárosító és teratogén hatásában. | We have investigated the effects of rubella virus (RV), vesicular stomatitis virus and herpes simplex virus (HSV) on the expressions of p63 isoforms and Bcl-2 family members, in an effort to gain some insight into the cytopathic and teratogenic effects of these viruses. Our studies have shown that RV-infected Vero cells displayed highly elevated levels of each TAp63 isoform and a decreased level of deltaNp63alpha. The levels of Bcl-2 and Bax-alpha were decreased, p53 and p63 remained unaffected, while the expression of p18 Bax was increased in VSV-infected WK cells. The levels of deltaNp63alpha and mutated p53 were decreased, Bcl-2 remained unaffected, while the expressions of Bax-alpha and p18 Bax were increased in VSV-infected HaCaT cells. HSV-infected HaCaT cells exhibited decreased level of deltaNp63alpha and highly elevated levels of some other p63 isoforms. Moreover, HSV infection caused a significant increase in the expression level of Bax-beta. Taken together, our data demonstrate that RV, VSV and HSV infections alter the stoichiometric ratios of the p63 isoforms and Bcl-2 family members, but in different ways. The dysregulated patterns of p63 expression observed in the infected cells may represent mechanisms whereby these viruses exert their cytopathic and teratogenic effects

Involvement of p63 in the herpes simplex virus-1-induced demise of corneal cells

<p>Abstract</p> <p>Background</p> <p>The transcription factor p63 plays a pivotal role in the development and maintenance of epithelial tissues, including the ocular surface. In an effort to gain insight into the pathogenesis of keratitis caused by HSV-1, we determined the expression patterns of the p63 and Bax proteins in the Staatens Seruminstitute Rabbit Cornea cell line (SIRC).</p> <p>Methods</p> <p>SIRC cells were infected with HSV-1 at various multiplicities and maintained for different periods of time. Virus replication was measured by indirect immunofluorescence assay and Western blot analysis. Cell viability was determined by MTT assay. The apoptotic response of the infected cells was quantified by ELISA detecting the enrichment of nucleosomes in the cytoplasm. Western blot analysis was used to determine the levels of p63 and Bax proteins.</p> <p>Results</p> <p>Indirect immunofluorescence assays and Western blot analyses demonstrated the presence of HSV-1 glycoprotein D (gD) in the infected SIRC cell line, and the pattern of gD expression was consistent with efficient viral replication. The results of MTT and ELISA assays showed that HSV-1 elicited a strong cytopathic effect, and apoptosis played an important role in the demise of the infected cells. Mock-infected SIRC cells displayed the constitutive expression of ΔNp63α. The expressions of the Bax-β and TAp63γ isoforms were considerably increased, whereas the level of ΔNp63α was decreased in the HSV-1-infected SIRC cells. Experiments involving the use of acyclovir showed that viral DNA replication was necessary for the accumulation of TAp63γ.</p> <p>Conclusion</p> <p>These data suggest that a direct, virus-mediated cytopathic effect may play an important role in the pathogenic mechanism of herpetic keratitis. By disturbing the delicate balance between the pro-survival ΔN and the pro-apoptotic TA isoforms, HSV-1 may cause profound alterations in the viability of the ocular cells and in the tissue homeostasis of the ocular surface.</p

Propionibacterium acnes induces autophagy in keratinocytes: involvement of multiple mechanisms

Propionibacterium acnes is a dominant member of the cutaneous microbiota. Herein, we evaluate the effects of different P. acnes strains and propionic acid on autophagy in keratinocytes. Our results showed that P. acnes strain 889 altered the architecture of the mitochondrial network, elevated the levels of LC3B-II, Beclin-1 and phospho-AMPKalpha, stimulated autophagic flux, facilitated intracellular redistribution of LC3B, increased average number of autophagosomes per cell, and enhanced development of acidic vesicular organelles in the HPV-KER cell line. Propionic acid increased the level of phospho-AMPKalpha, enhanced lipidation of LC3B, stimulated autophagic flux, as well as facilitated translocation of LC3B into autophagosomes in HPV-KER cells. P. acnes strains 889, 6609 and heat-killed strain 889 also stimulated autophagosome formation in primary keratinocytes to varying degrees. These results indicate that cell wall components and secreted propionic acid metabolite of P. acnes evoke mitochondrial damage successively, thereby trigger AMPK-associated activation of autophagy, which in turn facilitates the removal of dysfunctional mitochondria and promotes survival of keratinocytes. Thus, we suggest that low-level colonization of hair follicles with non-invasive P. acnes strains, by triggering a local increase in autophagic activity, might exert a profound effect on several physiological processes responsible for the maintenance of skin tissue homeostasis

Long-read sequencing uncovers a complex transcriptome topology in varicella zoster virus

The procedure of finding novel transcriptional isoforms. The flowchart describes the pipeline used to detect novel TSSs and TES-s. (PDF 505 kb

IL-36α and Lipopolysaccharide Cooperatively Induce Autophagy by Triggering Pro-Autophagic Biased Signaling

Autophagy is an intracellular catabolic process that controls infections both directly and indirectly via its multifaceted effects on the innate and adaptive immune responses. It has been reported that LPS stimulates this cellular process, whereas the effect of IL-36α on autophagy remains largely unknown. We therefore investigated how IL-36α modulates the endogenous and LPS-induced autophagy in THP-1 cells. The levels of LC3B-II and autophagic flux were determined by Western blotting. The intracellular localization of LC3B was measured by immunofluorescence assay. The activation levels of signaling pathways implicated in autophagy regulation were evaluated by using a phosphokinase array. Our results showed that combined IL-36α and LPS treatment cooperatively increased the levels of LC3B-II and Beclin-1, stimulated the autophagic flux, facilitated intracellular redistribution of LC3B, and increased the average number of autophagosomes per cell. The IL36α/LPS combined treatment increased phosphorylation of STAT5a/b, had minimal effect on the Akt/PRAS40/mTOR pathway, and reduced the levels of phospho-Yes, phospho-FAK, and phospho-WNK1. Thus, this cytokine/PAMP combination triggers pro-autophagic biased signaling by several mechanisms and thus cooperatively stimulates the autophagic cascade. An increased autophagic activity of innate immune cells simultaneously exposed to IL-36α and LPS may play an important role in the pathogenesis of Gram-negative bacterial infections

Herpes Simplex Virus Infection Alters the Immunological Properties of Adipose-Tissue-Derived Mesenchymal-Stem Cells

The proper functioning of mesenchymal stem cells (MSCs) is of paramount importance for the homeostasis of the body. Inflammation and infection can alter the function of MSCs, which can also affect the regenerative potential and immunological status of tissues. It is not known whether human herpes simplex viruses 1 and 2 (HSV1 and HSV2), well-known human pathogens that can cause lifelong infections, can induce changes in MSCs. In non-healing ulcers, HSV infection is known to affect deeper tissue layers. In addition, HSV infection can recur after initially successful cell therapies. Our aim was to study the response of adipose-derived MSCs (ADMSCs) to HSV infection in vitro. After confirming the phenotype and differentiation capacity of the isolated cells, we infected the cells in vitro with HSV1-KOS, HSV1-532 and HSV2 virus strains. Twenty-four hours after infection, we examined the gene expression of the cells via RNA-seq and RT-PCR; detected secreted cytokines via protein array; and determined autophagy via Western blot, transmission electron microscopy (TEM) and fluorescence microscopy. Infection with different HSV strains resulted in different gene-expression patterns. In addition to the activation of pathways characteristic of viral infections, distinct non-immunological pathways (autophagy, tissue regeneration and differentiation) were also activated according to analyses with QIAGEN Ingenuity Pathway Analysis, Kyoto Encyclopedia of Genes and Genome and Genome Ontology Enrichment. Viral infections increased autophagy, as confirmed via TEM image analysis, and also increased levels of the microtubule-associated protein light chain 3 (LC3B) II protein. We identified significantly altered accumulation for 16 cytokines involved in tissue regeneration and inflammation. Our studies demonstrated that HSV infection can alter the viability and immunological status of ADMSCs, which may have implications for ADMSC-based cell therapies. Alterations in autophagy can affect numerous processes in MSCs, including the inhibition of tissue regeneration as well as pathological differentiation