Methodology of assessment of population satisfaction with the quality of medical service based on the adaptive polling technologyn

This article covers the problem of assessment of patient satisfaction as a parameter of the quality of medical service. The existing methods of assessment of patients' attitude to a medical institution, their benefits and disadvantages are considered. The methodology for the calculation of the satisfaction coefficient is proposed, which can be the basis for the application of the technology (depending on the type and amount of medical care received by the patient) of adaptive polling and analysis of the information received

Depletion of Foxp3(+) regulatory T cells is accompanied by an increase in the relative abundance of Firmicutes in the murine gut microbiome

A reciprocal interaction exists between the gut microbiota and the immune system. Regulatory T (Treg) cells are important for controlling immune responses and for maintaining the intestinal homeostasis but their precise influence on the gut microbiota is unclear. We studied the effects of Treg cell depletion on inflammation of the intestinal mucosa and analysed the gut microbiota before and after depletion of Treg cells using the DEpletion of REGulatory T cells (DEREG) mouse model. DNA was extracted from stool samples of DEREG mice and wild‐type littermates at different time‐points before and after diphtheria toxin application to deplete Treg cells in DEREG mice. The V3/V4 region of the 16S rRNA gene was used for studying the gut microbiota with Illumina MiSeq paired ends sequencing. Multidimensional scaling separated the majority of gut microbiota samples from late time‐points after Treg cell depletion in DEREG mice from samples of early time‐points before Treg cell depletion in these mice and from gut microbiota samples of wild‐type mice. Treg cell depletion in DEREG mice was accompanied by an increase in the relative abundance of the phylum Firmicutes and by intestinal inflammation in DEREG mice 20 days after Treg cell depletion, indicating that Treg cells influence the gut microbiota composition. In addition, the variables cage, breeding and experiment number were associated with differences in the gut microbiota composition and these variables should be respected in murine studies

GenoType CM Direct® and VisionArray Myco® for the Rapid Identification of Mycobacteria from Clinical Specimens

M. tuberculosis is the single infectious agent responsible for most deaths worldwide outside of pandemics. Diseases due to non-tuberculous mycobacteria (NTM) are increasing in many regions of the world. The two molecular assays GenoType CM direct® (GTCMd) (Bruker, Billerica, MA, USA) and VisionArray Myco® (VAM) (ZytoVision, Bremerhaven, Germany) are based on the DNA/DNA hybridization technique, and allow for the identification of tuberculous and the most clinically relevant non-tuberculous mycobacterial species from clinical specimens. We evaluated the performance of both assays for the identification of mycobacteria from 65 clinical specimens of 65 patients and compared it with the results of conventional culture. Based on conventional culture that recovered 37 mycobacterial isolates including 11 tuberculous and 26 NTM isolates, sensitivity, specificity, positive predictive value and negative predictive value were 89.2%, 81.5%, 86.8% and 84.6% for GTCMd and 73.0%, 96.3%, 96.4% and 72.2% for VAM. Additionally, GTCMd identified mycobacteria from five and VAM from one culture-negative sample. Both assays identified a mycobacterium in one sample overgrown by other microorganisms. Two M. abscessus subsp. abscessus isolates grown from culture were identified as M. chelonae by GTCMd assay. In conclusion, both assays improve the rapid identification of mycobacteria directly from clinical specimens

Impact of 5-aza-2'-deoxycytidine and epigallocatechin-3-gallate for induction of human regulatory T cells.

The epigenetic regulation of transcription factor genes is critical for T-cell lineage specification. A specific methylation pattern within a conserved region of the lineage specifying transcription factor gene FOXP3, the Treg-specific demethylated region (TSDR), is restricted to regulatory T (Treg) cells and is required for stable expression of FOXP3 and suppressive function. We analysed the impact of hypomethylating agents 5-aza-2'-deoxycytidine and epigallocatechin-3-gallate on human CD4(+)  CD25(-) T cells for generating demethylation within FOXP3-TSDR and inducing functional Treg cells. Gene expression, including lineage-specifying transcription factors of the major T-cell lineages and their leading cytokines, functional properties and global transcriptome changes were analysed. The FOXP3-TSDR methylation pattern was determined by using deep amplicon bisulphite sequencing. 5-aza-2'-deoxycytidine induced FOXP3-TSDR hypomethylation and expression of the Treg-cell-specific genes FOXP3 and LRRC32. Proliferation of 5-aza-2'-deoxycytidine-treated cells was reduced, but the cells did not show suppressive function. Hypomethylation was not restricted to FOXP3-TSDR and expression of master transcription factors and leading cytokines of T helper type 1 and type 17 cells were induced. Epigallocatechin-3-gallate induced global DNA hypomethylation to a lesser extent than 5-aza-2'-deoxycitidine, but no relevant hypomethylation within FOXP3-TSDR or expression of Treg-cell-specific genes. Neither of the DNA methyltransferase inhibitors induced fully functional human Treg cells. 5-aza-2'-deoxycitidine-treated cells resembled Treg cells, but they did not suppress proliferation of responder cells, which is an essential capability to be used for Treg cell transfer therapy. Using a recently developed targeted demethylation technology might be a more promising approach for the generation of functional Treg cells

Quantification of Regulatory T Cells in Septic Patients by Real-Time PCR–Based Methylation Assay and Flow Cytometry

<div><p>During sepsis, a relative increase of regulatory T (Treg) cells has been reported. Its persistence is associated with lymphocyte anergy, immunoparalysis and a poor prognosis. Currently, an exact quantification of human Treg cells based on protein expression of marker molecules is ambiguous, as these molecules are expressed also by activated non-regulatory T cells. Furthermore, no firm criteria for flow cytometer gate settings exist so far. Recently, a specific DNA methylation pattern within <em>FOXP3-TSDR</em> has been reported that allows distinguishing Treg and non-regulatory T cells, independent of their activation status. Using this epigenetic marker, we established a single-tube real-time PCR based methylation assay (QAMA) for relative quantification of Treg cells. Validation was performed on defined ratios of methylated and unmethylated target sequence and on mixtures of Treg and non-regulatory T cells. DNA-methylation was measured in CD4⁺ T cells isolated from blood samples of 30 septic patients and 30 healthy subjects and compared with results of Treg cell quantification by flow cytometry based on CD4⁺ CD25^hiCD127^low measurement. In septic patients both methods showed an increased ratio of Treg cells to all CD4⁺ T cells. In healthy individuals, the results obtained by both methods were clearly positively correlated. However, the correlation between both methods in septic patients was only weak. We showed that quantification of Treg cells by QAMA detects CD4⁺ T cells with unmethylated <em>FOXP3-TSDR</em>, hidden in the CD25^med/low fraction of flow cytometry. Given that unmethylated <em>FOXP3-TSDR</em> is the most specific feature of Treg cells to date, our assay precisely quantifies Treg cells, as it additionally detects those committed Treg cells, hidden in the CD25^med/low fraction of CD4⁺ cells. Furthermore, QAMA is a reliable method, which is easier to standardize among laboratories and can thus improve reproducibility of Treg cell quantification.</p> </div

Characteristics of the septic patients enrolled in the study.

<p>SAPS = Simplified Acute Physiology Score.</p

Illustration of the CD25 and CD4 gate settings used to sort various CD4⁺ populations.

<p>CD4⁺ CD25^med cells showed an unmethylated ratio of 4.4% (SD ±0.17%); CD4⁺ CD25⁻ cells, of 0.8% (SD ±0.60%); and CD4⁺ CD25^hiCD127^low cells of 99.8% (SD ±0.24%)with <i>FOXP3-TSDR</i> QAMA. Results were calculated of four experiments from four healthy men.</p

Characteristics of healthy subjects enrolled in the study.

<p>Characteristics of healthy subjects enrolled in the study.</p