Prevalence of the M1UK sublineage among emm1 Streptococcus pyogenes invasive strains isolated in the Czech Republic from December 2022 to May 2023. / Výskyt sub-linie M1 UK mezi invazivními kmeny Streptococcus pyogenes typ emm1 izolovanými od prosince 2022 do kvetna 2023 v Ceské republice.

AIMS: Since December 2022, an increase in invasive disease caused by Streptococcus pyogenes has been observed in the Czech Republic, with a shift in the clinical presentation and age of patients. Unlike in previous years, invasive disease is more common in children and adolescents under 18 years of age and in previously healthy middle-aged adults. An increase has been noticed in the number of S. pyogenes isolates from primarily sterile sites such as haemoculture, cerebrospinal fluid, pleural effusion fluid, joint fluid, and postmortem specimens. Routine emm gene typing revealed emm1 to be the predominant emm type of S. pyogenes. Between January 2023 and July 2023, 46% of all S. pyogenes isolates from invasive cases were assigned to the emm1 type. The globally spread M1UK sublineage is characterized by differences in the expression of seven genes, including the streptococcal pyrogenic toxin A (speA) gene, compared to historical emm1 iGAS strains. The aim of this study is to determine whether the more toxigenic M1UK sublineage is associated with the increase in invasive disease in the Czech Republic. METHODS: Whole genome sequencing of 41 S. pyogenes isolates from patients with invasive disease recovered in the Czech Republic in 2018 and 2019 and from December 2022 to May 2023 was performed using the MiSeq instrument (Illumina). Bioinformatics analysis was performed using freely available online tools the Bacterial and Viral Bioinformatics Resource Center. RESULTS: Based on whole genome sequencing data of 41 emm1 isolates of S. pyogenes from patients with invasive infectious disease recovered in 2018 and 2019 and from December 2022 to May 2023, the M1UK sublineage was found to be predominant from December 2022 to May 2023. CONCLUSION: The reason for the spread of the M1UK sublineage in the Czech Republic late in 2022 and in the first half of 2023 is not entirely clear, but it may be related to reduced immunity due to limited GAS transmission during lockdowns, especially in children. Another factor that may have contributed to the high incidence of invasive infectious diseases is the seasonal circulation of respiratory viruses.

Detection of superantigens in Streptococcus pyogenes isolates based on whole genome sequencing data.

Streptococcus pyogenes causes a variety of human diseases ranging from uncomplicated respiratory tract and skin infections to severe invasive diseases possibly involving toxic shock syndrome. Besides the emm gene-encoded M protein, important virulence factors are pyrogenic exotoxins, referred to as superantigens. The National Reference Laboratory for Streptococcal Infections has newly introduced bioinformatics tools for processing S. pyogenes whole genome sequencing data. Using the SRST2 software and BV-BRC platform, WGS data of 10 S. pyogenes isolates from patients with invasive disease were analysed, and emm type, sequence type, and superantigen encoding gene profiles were determined. The Unicycler assembly pipeline with the SPAdes de novo assembler was used to assemble genome sequences from short reads.

Xenogeneic protein-free cultivation of mesenchymal stromal cells - towards clinical applications.

We have studied a rapid cultivation method for human mesenchymal stromal cells based on CellGroTM medium and human serum, supplemented with insulin, ascorbic acid, dexamethasone, epidermal growth factor, platelet-derived growth factor BB, macrophage colony-stimulating factor and fibroblast growth factor 2. This study has shown that rapid expansion of human multipotent mesenchymal stromal cells using human serum could not be achieved without addition of growth factors. Furthermore, we have found that insulin and, quite probably, epidermal growth factor may be omitted from our formula without loss of colony-forming capacity or total cell yield. On the other hand, dexamethasone, ascorbic acid and fibroblast growth factor 2 were necessary for the growth and colony-forming capacity of multipotent mesenchymal stromal cells, while platelet-derived growth factor BB prevented their differentiation into adipogenic lineage. Moreover, multipotent mesenchymal stromal cells cultivated in our system expressed higher levels of bone morphogenetic protein 2, but not bone morphogenetic protein 7, than cells cultivated in α-MEM with foetal bovine serum. This shows that our system promotes differentiation of mesenchymal cells towards osteogenic and chondrogenic lineages, making them more suitable for bone and cartilage engineering than cells grown in conventional media. Furthermore, we have proved that these cells may be conveniently cultivated in a closed system, in vessels certified for clinical use (RoboFlaskTM), making the transfer of our cultivation technology to good clinical practice easier and more convenient.

The potential of non-adrenergic, non-cholinergic targets in the treatment of interstitial cystitis/painful bladder syndrome.

Regulation of bladder function involves both divisions of the autonomic nervous system. However, in addition to the classical autonomic transmitters, noradrenaline and acetylcholine, other autonomic transmitters and other signalling components play important roles in physiology and pathophysiology of the lower urinary tract. Several substances of neuronal non-adrenergic, non-cholinergic (NANC) systems have already proven to considerably influence functional responses in the inflamed urinary bladder. Interstitial cystitis (IC) or painful bladder syndrome (PBS) is a chronic inflammatory bladder disease, characterized by urinary frequency, urgency and pelvic pain. IC/PBS is difficult to diagnose, especially because the etiology of the condition is largely unknown. Despite the unclear nature of the cause and manifestation of IC/PBS, it has been shown that the disease involves a significant NANC component. Here, we review the possible roles of ATP, adenosine, nitric oxide, vasoactive intestinal polypeptide, substance P, and pituitary adenylate cyclase-activating peptide in the contribution to IC/PBS development and manifestation of IC/PBS symptoms.

Coupled nitric oxide and autonomic receptor functional responses in the normal and inflamed urinary bladder of the rat.

Both divisions of the autonomic nervous system are involved in regulation of urinary bladder function. Several substances, other than noradrenaline and acetylcholine, seem to play important roles in physiology and pathophysiology of lower urinary tract. In the current study, we aimed to examine if there exist interplays between nitric oxide (NO) and autonomic transmitters and if such interactions vary in different parts of the urinary bladder in healthy and cyclophosphamide (CYP)-induced cystitic rats; when administered to the animals (100 mg/kg; i.p.), the cytotoxic CYP metabolite acrolein induces bladder inflammation. In the current study a series of in vitro functional studies were performed on detrusor muscle strip preparations. Stimulation with electrical field stimulation (EFS), methacholine, adenosine 5´-triphosphate (ATP), and adrenaline evoked contractile responses in isolated bladder preparations that were significantly reduced in cyclophosphamide (CYP)-treated rats. While the nitric oxide synthase inhibitor N(omega)-nitro-L-arginine (L-NNA; 10(-4) M) did not affect contractile responses in normal, healthy strip preparations, it significantly increased the contractile responses to EFS, methacholine and adrenaline, but not to ATP, in the bladders from the CYP-treated rats. In the CYP-treated rats, the ATP-evoked relaxatory part of its dual response (an initial contraction followed by a relaxation) was 6-fold increased in comparison with that of normal preparations, whereas the isoprenaline relaxation was halved in the CYP-treated. While L-NNA (10(-4) M) had no effect on the isoprenaline-evoked relaxations, it reduced the ATP-evoked relaxations in strip preparations from the bladder body of CYP-treated rats. Stimulation of beta(2)- and beta(3)-adrenoceptors evoked relaxations and both responses were reduced in cystitis, the latter to a larger extent. In the trigone, the reduced ATP-evoked contractile response in the inflamed strips was increased by L-NNA, while L-NNA had no effect on the ATP-evoked relaxations, neither on the relaxations in healthy nor on the larger relaxations in the inflamed trigone. The study shows that both contractile and relaxatory functions are altered in the state of inflammation. The parasympathetic nerve-mediated contractions of the body of the bladder, evoked by the release of ATP and acetylcholine, were substantially reduced in cystitis. The relaxations to beta-adrenoceptor and purinoceptor stimulation were also reduced but only the ATP-evoked relaxation involved NO.

[Xenogeneic protein free cultivation of mesenchymal multipotent stromal cells]. / Kultivace mezenchymových kmenových bunek bez xenogenních bílkovin.

PURPOSE OF THE STUDY: The aim of this study was to compare the standard laboratory method of cultivation of mesenchymal multipotent stromal cells (MSC) and a novel technique of rapid MSC expansion focused on simple clinical use. MATERIAL AND METHODS: Bone marrow mononuclear cells of donors were cultured for 14 days by the standard and the new cultivation method. The standard method (STD) was based on an alpha MEM medium supplemented with foetal calf serum (FCS). The new animal protein-free method (CLI) was based on the clinical grade medium CellgroTM, pooled human serum and human recombinant growth factors (EGF, PDGF-BB, M-CSF, FGF-2) supplemented with dexamethasone, insulin and ascorbic acid. The cell product was analyzed by flow cytometry. Furthermore, the cell products of STD and CLI methods were differentiated in vitro, and histochemical and immunohistochemical analyses, electron microscopy and elemental analysis were performed. Some cells were seeded on biodegradable scaffolds, in vivo implanted into immunodeficient mice for 6 weeks and evaluated by histological methods. RESULTS: Yields of the CLI method after 14 days of cultivation were 40-fold higher than those obtained by the STD technique (p<0.05). Cell products of both STD and CLI methods fulfilled the criteria of MSC in terms of antigen expression assessed by flow cytometry, as well as osteogenic, chondrogenic and adipogenic in vitro differentiation assays. Moreover, these cells seeded on three-dimensional scaffolds cultured in osteogenic medium produced mineral deposits and a fibrillar extracellular matrix seen with the electron microscope. Deposits examined by element analysis contained calcium and phosphorus at a ratio of 5 to 3, which corresponded to hydroxyapatite. The cell product seeded on biodegradable scaffolds and implanted into immunodeficient mice was able to form a bone-like calcified tissue with blood supply of mouse origin. DISCUSSION: The currently used methods of cultivation have certain disadvantages compared to the CLI technique, such as a longer cultivation period, need of primary expansion and reseeding and use of FCS with all its potential risks. High yields of cells obtained by the CLI method in a very short time make the use of cultured cells potentially suitable for an acute trauma management. Other therapeutic non-orthotopic applications of CLI-cultured cells have to be further investigated. CONCLUSIONS: The CLI method is unique, rapid, simple and lacking the addition of animal proteins. CLI-cultured cells fulfil the criteria of MSC. The CLI method potentially allows for closed system cultivation in good manufacturing practice (GMP) conditions. It seems to be easily transferable to good clinical practice compared to other protocols and should extend the possibilities of cell therapy and tissue engineering of cartilage and bone. The new method is protected by Czech patent 301 148 and by Europian patent EP 1999250 according to Czech and international laws.

Circulating endothelial precursor cells (EPC) in patients undergoing allogeneic haematopoietic progenitor cell transplantation.

We have studied the number of endothelial precursor cells in eighteen patients undergoing allogeneic haematopoietic stem cell transplantation. Endothelial precursor cells were evaluated by colony-forming assay and compared to healthy controls. Patients undergoing allogeneic haematopoietic stem cell transplantation had significantly lower numbers of endothelial precursor cells before the procedure than healthy controls. The numbers of endothelial precursor cells were even lower in the first year after the treatment and seemed to recover partially after twelve months, but even then, they were lower than in healthy volunteers. On the other hand, the number of circulating CD146+CD31+ mature endothelial cells were higher than in healthy controls after more than a one-year follow-up. We hypothesize that lower numbers of endothelial precursor cells and higher numbers of endothelial cells in patients undergoing allogeneic haematopoietic stem cell transplantation reflect ongoing endothelial damage, probably caused by immunological mechanisms, and that this longterm damage may explain the higher risk of cardiovascular events in allogeneic haematopoietic stem cell transplant survivors.

Can haloperidol disguise fever?

Haloperidol when applied intraperitoneally to cold-exposed febrile rabbits induces a strong hypothermic effect. This effect is due to the downward shift of the threshold central temperature for induction of cold thermogenesis and vasomotion. The shift occurs during the early phase of the fever and is less prominent during the late phase of the fever. The hypothermic effect of high doses of haloperidol can eliminate the increase of body temperature in febrile individuals.