Antidermatophytic effect of Bacillus mojavensis SZMC 22228 and its secreted chymotrypsin-like protease

The aim of the present study was to investigate the antifungal effect of Bacillus mojavensis SZMC 22228 against different dermatophytes and to isolate the antidermatophytic compound from the bacterial ferment broth. B. mojavensis SZMC 22228 and its cell-free ferment broth effectively inhibited the growth of clinical reference strain of Microsporum canis, Microsporum gypseum, Trichophyton mentagrophytes, Trichophyton rubrum and Trichophyton tonsurans in agar diffusion test in vitro. An antidermatophytic, ~25 kDa protein (B. mojavensis SZMC 22228 antidermatophyitc protein, BMAP) was purified from the antifungally active, cell-free ferment broth using size exclusion and ion-exchange chromatography. BMAP showed antifungal effect against all of the investigated dermatophytes both in agar diffusion and broth microdilution susceptibility tests. M. gypseum proved to be the most susceptible dermatophyte to BMAP (MIC=40 μg/ml), all the other investigated fungi were less susceptible (MIC=80 μg/ml). The enzymatic activity of this protein was investigated in microtiter plate assay using hydrolase specific chromogenic substrates. BMAP showed high proteolytic activity towards N-Succ-Ala-Ala-Pro-Phe-pNA, and proved to be a chymotrypsin-like protease. These results suggest that the antidermatohytic activity of B. mojavensis SZMC 22228 correlates with its chymotrypsin-like protease production. After further investigations, the purified BMAP could be a promising base of a novel antidermatophytic strategy

Mesterséges bőrszövetek a kutatásban és a gyógyításban [Tissue engineered skin products in research and therapeutic applications]

Összefoglaló. A bőrpótlóknak mind a klinikumban, mind a gyógyszerkutatásokban kiemelt szerepük van. Ezek a kezdetleges mesterséges bőrszövetek segíthetik a bőr regenerálódását, modellezhetik a főbb funkciókat, de megvannak a korlátaik is, mechanikailag sérülékenyek, és nem tartósak. A legtöbb bőrpótló vagy acelluláris, vagy csak egy-két sejttípust tartalmaz. Az eredeti bőrrel megegyező szerkezetű, teljesen funkcionális mesterséges bőrszövet a mai napig nem létezik. A háromdimenziós szövetnyomtatás megoldást kínálhat erre a problémára is, hiszen a bőrszövet minden sejtes eleme felhasználható, megfelelő hidrogélek és biotinták segítségével pedig olyan komplex struktúrák hozhatók létre, amelyek képesek a bőr teljes funkcionális repertoárját biztosítani. Ez nemcsak klinikai szempontból kiemelt jelentőségű, hanem a preklinikai kísérletek esetében kiválthatja az állatmodelleket és számos toxikológiai vizsgálatot is. Orv Hetil. 2022; 163(10): 375-385. Summary. Skin substitutes have a prominent role in therapeutic applications and drug research. These simple artificial skin tissues can support skin regeneration, in vitro they can model the main functions of the skin but they also have limitations such as being mechanically vulnerable and not durable enough. Most skin substitutes are either acellular or contain only one or two cell types. Fully functional artificial skin substitute with the same structure as the original skin has not been produced to this day. Three-dimensional tissue bioprinting can also offer a solution to this problem, as all cellular elements of skin tissue can be used, and with the help of appropriate hydrogels and bioinks, complex structures can be created that can provide a complete functional repertoire of the skin. It is important not just in the clinical therapeutic use, but it can also trigger the replacement of animal models and a number of toxicological studies in preclinical trials. Orv Hetil. 2022; 163(10): 375-385

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

Mesterséges bőrszövetek a kutatásban és a gyógyításban

A bőrpótlóknak mind a klinikumban, mind a gyógyszerkutatásokban kiemelt szerepük van. Ezek a kezdetleges mesterséges bőrszövetek segíthetik a bőr regenerálódását, modellezhetik a főbb funkciókat, de megvannak a korlátaik is, mechanikailag sérülékenyek, és nem tartósak. A legtöbb bőrpótló vagy acelluláris, vagy csak egy-két sejttípust tartalmaz. Az eredeti bőrrel megegyező szerkezetű, teljesen funkcionális mesterséges bőrszövet a mai napig nem létezik. A háromdimenziós szövetnyomtatás megoldást kínálhat erre a problémára is, hiszen a bőrszövet minden sejtes eleme felhasználható, megfelelő hidrogélek és biotinták segítségével pedig olyan komplex struktúrák hozhatók létre, amelyek képesek a bőr teljes funkcionális repertoárját biztosítani. Ez nemcsak klinikai szempontból kiemelt jelentőségű, hanem a preklinikai kísérletek esetében kiválthatja az állatmodelleket és számos toxikológiai vizsgálatot is

Antidermatophytic effect of Bacillus mojavensis SZMC 22228 and its secreted chymotrypsin-like protease

The aim of the present study was to investigate the antifungal effect of Bacillus mojavensis SZMC 22228 against different dermatophytes and to isolate the antidermatophytic compound from the bacterial ferment broth. B. mojavensis SZMC 22228 and its cell-free ferment broth effectively inhibited the growth of clinical reference strain of Microsporum canis, Microsporum gypseum, Trichophyton mentagrophytes, Trichophyton rubrum and Trichophyton tonsurans in agar diffusion test in vitro. An antidermatophytic, ~25 kDa protein (B. mojavensis SZMC 22228 antidermatophyitc protein, BMAP) was purified from the antifungally active, cell-free ferment broth using size exclusion and ion-exchange chromatography. BMAP showed antifungal effect against all of the investigated dermatophytes both in agar diffusion and broth microdilution susceptibility tests. M. gypseum proved to be the most susceptible dermatophyte to BMAP (MIC=40 μg/ml), all the other investigated fungi were less susceptible (MIC=80 μg/ml). The enzymatic activity of this protein was investigated in microtiter plate assay using hydrolase specific chromogenic substrates. BMAP showed high proteolytic activity towards N-Succ-Ala-Ala-Pro-Phe-pNA, and proved to be a chymotrypsin-like protease. These results suggest that the antidermatohytic activity of B. mojavensis SZMC 22228 correlates with its chymotrypsin-like protease production. After further investigations, the purified BMAP could be a promising base of a novel antidermatophytic strategy

Toward better drug development: Three-dimensional bioprinting in toxicological research

The importance of three-dimensional (3D) models in pharmacological tests and personalized therapies is significant. These models allow us to gain insight into the cell response during drug absorption, distribution, metabolism, and elimination in an organ-like system and are suitable for toxicological testing. In personalized and regenerative medicine, the precise characterization of artificial tissues or drug metabolism processes is more than crucial to gain the safest and the most effective treatment for the patients. Using these 3D cell cultures derived directly from patient, such as spheroids, organoids, and bioprinted structures, allows for testing drugs before administration to the patient. These methods allow us to select the most appropriate drug for the patient. Moreover, they provide chance for better recovery of patients, since time is not wasted during therapy switching. These models could be used in applied and basic research as well, because their response to treatments is quite similar to that of the native tissue. Furthermore, they may replace animal models in the future because these methods are cheaper and can avoid interspecies differences. This review puts a spotlight on this dynamically evolving area and its application in toxicological testing