Celiac disease is a systemic autoimmune affliction triggered by dietary gluten in genetically predisposed subjects. Current estimation of the prevalence of celiac disease is approximately 1% for the global population. These data are consistent with current figures from the Czech Republic (prevalence of 1.06% in 2023). Celiac disease can be associated with several complications including malignancy. Exact epidemiology data on T-cell lymphoma and small intestinal adenocarcinoma in the Czech Republic over the period 2010–2023 are also provided. Unlike the no-biopsy approach in children, despite current non-conclusive discussion and besides serology, upper gastrointestinal endoscopy, biopsies, and histology are mandatory for proper diagnosis and initial assessment of the severity of celiac disease in adults. However, there are so far no unified and integrated recommendations for the follow-up of adult celiac disease. Although the risk of malignancy in celiac disease is relatively low, especially in a strict gluten-free diet, it might nevertheless be further decreased by an individualized surveillance. Endoscopy controls with biopsies for histology, immunohistochemistry, and flow cytometry are essential. The purpose of this article was to review the risk and diagnostics of malignancy in celiac disease. A proposal for the individualized surveillance of premalignant gastrointestinal lesions in adult celiac disease was drafted.

Abstract Introduction The impact of chemoimmunotherapy (CIT) on immunoglobulin (Ig) quantities in patients with chronic lymphocytic leukemia (CLL) has not been extensively studied. Methods We analyzed Ig levels in 45 stable patients with indolent CLL (without indication for treatment) and 87 patients with progressive disease before first‐line treatment. Fifty‐five patients were evaluated again after the treatment with CIT. Results We observed significantly lower levels of all Ig classes and subclasses in patients with progressive disease compared to patients with indolent disease. After treatment, median IgA increased from 0.59 g/L to 0.74 g/L (p = 0.0031). In stable patients, lower IgA2 was associated with shorter time to first treatment, although it did not reach statistical significance (p = 0.056). Shorter overall survival was observed in patients with progressive disease and lower IgG2 (p = 0.043). Surprisingly, among the patients with progressive CLL, unmutated IGHV genes were associated with higher levels of IgG, IgG1 and IgM, while TP53 mutation and/or 17p deletion were associated with higher levels of IgA and IgA1. Conclusions CIT may lead to increase in IgA levels. Hypogammaglobulinemia is more common in patients with progressive CLL and unmutated IGHV or TP53 dysfunction.

The authors review the essential role of modern cryobanks in the development of cryotechnology before the start of the clinical cell and tissue transplantation programme and in the routine support of these programmes within the legislative framework settled by the European Union directives. Two clinical programmes are presented as examples: the autologous and allogeneic haematopoietic progenitor cell (HPC) transplantation programme and the Czech national cryopreserved allogeneic vascular tissue (VT) transplantation programme supported by the cryobank of the University Hospital Hradec Králové, which is a part of the European Union Tissue Establishment registered under the codes CZ000425, CZ000426 and CZ000427. An overview of necessary approvals to be obtained before the start of clinical programmes is presented, the high quality of cryopreservation technology and high level of cryobank staff safety is demonstrated, and the crucial role of the cryobank in clinical HPC and VT transplantation programmes is highlighted.

: This study investigates the activation of human immune cells (T, B, and NK cells, and granulocytes) by the ethanolic and aqueous extracts of Scutellaria baicalensis root (SBR) and to identify the compounds responsible for such an effect. The cell activation was determined by expression of the cell surface glycoprotein CD69 (cluster of differentiation 69), as measured by flow cytometry. The content of the main flavonoids in SBR extracts was estimated by high performance liquid chromatography with a diode array detector. NK cells were substantially activated by the SBR ethanolic extract (25–200 µg/mL), whereas B cells were activated on a lower scale. NK cells were slightly activated by the SBR aqueous extract (100–200 µg/mL). Wogonoside and baicalin, two major flavonoids present in the SBR extracts, exhibited the highest activation effect on NK cells. The content of baicalin, wogonoside, and baicalein accounted for 201.2, 47.7, and 3.4 mg/g in the ethanolic extract; and for 150.4, 33.6, and 4.6 mg/g in the aqueous extract, respectively. This study shows the immune-stimulating properties of SBR extracts and identifies their active compounds. This data may support the potential utilization of SBR for strengthening the human immune system against bacterial and viral infections.

Introduction: To date, there is not generally accepted and universal indicator of activity, and functional integrity of the small intestine in patients with coeliac disease. The aim of our study was to investigate whether serum concentrations of the non-essential amino acids citrulline and ornithine might have this function. Methods: We examined serum citrulline and ornithine concentrations in a subgroup of patients with proven coeliac disease and healthy controls (blood donors). Results: A total of 94 patients with coeliac disease (29 men, mean age 53 ± 18 years; 65 women, mean age 44 ± 14 years) and 35 healthy controls (blood donors) in whom coeliac disease was serologically excluded (10 men, mean age 51 ± 14 years; 25 women, mean age 46 ± 12 years) were included in the study. Significantly lower concentrations of serum ornithine were found in patients with coeliac disease (mean 65 ± 3 μmol/L; median 63 μmol/L, IQR 34 μmol/L, p < 0.001). No statistically nor clinically significant differences were found in the citrulline concentrations between the study and control group. Conclusions: Serum ornithine (but not citrulline) may be useful for assessing the functional status of the small intestine in uncomplicated coeliac disease. Further studies involving more detailed analysis of dietary and metabolic changes in patients will be needed to reach definitive conclusions.

AbstractBackgroundIn chronic lymphocytic leukemia (CLL), changes in the peripheral blood lymphocyte subsets play an important role in disease progression and infectious complications. The impact of chemoimmunotherapy (CIT) on these changes has not been extensively studiedMethodsWe used multi‐color flow cytometry, to prospectively measure absolute and relative numbers of CD4+ and CD8+ T‐cells and their subsets in 45 patients with indolent untreated CLL, 86 patients indicated for first‐line treatment, and 34 healthy controls. In 55 patients, we analyzed the impact of CITResultsCLL patients had a significant increase in most cell populations in comparison to controls. Progression of CLL was characterized by significantly elevated counts with the exception of a lower percentage of naïve T‐cells. After treatment, the percentage of naïve T‐cells further decreased at the expense of effector memory T‐cells (TEM). In patients with indolent CLL, higher percentages of naïve CD4+ (p = 0.0026) and naïve CD8+ (p = 0.023) T‐cells were associated with a longer time to first treatment (TTFT). The elevation of CD4+ central memory T‐cells (TCM) (p = 0.27) and TEM (p = 0.003) counts and a higher percentage of CD4+ TEM (p = 0.0047), were linked with shorter TTFT. In treated patients, increased regulatory T‐cells count was associated with shorter time to next treatment (TTNT) (p = 0.042), while higher CD4+ TCM count with shorter TTNT (p = 0.035) and shorter overall survival (p = 0.041).ConclusionOur results indicate that naïve cell depletion and CD4+ TCM and TEM increases are detrimental to CLL patients' prognosis.

In this chapter we consider the particular preservation storage procedures applied to a range of cell types used to produce cell-based medicines. Specifically, it deals with the scientific considerations for preserving each cell type and the kinds of cryopreservation protocols used to successfully preserve these different cell types. The cell types addressed include both those commonly in current use for patient treatment, such as whole blood and hematopoietic stem cells and also examples of new cell-based medicines including tissue progenitor cells (MSCs), (The use of the term Mesenchymal Stem Cell (MSC) has been hotly debated in the literature as it actually applies to several different cell types. The term "tissue specific progenitor cells" has been proposed as a more accurate term (Robey 2017) and where the abbreviation "MSC" appears elsewhere in this chapter it can be assumed this is a reference to Mesenchymal Stromal cells or the more generic term for this group of cell types Tissue-Specific Progenitor Cells (TSPCs).) tissue engineered constructs, CAR-T cells and pluripotent stem cells. However, the chapter does not consider the preservation and storage of organs or cells and tissues used in reproductive medicine. A second part of the chapter addresses best practice in meeting regulatory requirements for preservation and storage of both unfrozen and cryopreserved materials, including core requirements for the design of storage facilities. Also considered is best practice for packaging and shipment of cell-based medicines and their reception at the clinic and control within the hospital environment under pharmacy rules. The authors have used examples of regulatory documents primarily from the European Union and the USA, but also include reference to key international standards and WHO guidance.

Background: Lymphocyte differentiation is regulated by coordinated actions of cytokines and signaling pathways. IL-21 activates STAT1, STAT3, and STAT5 and is fundamental for the differentiation of human B cells into memory cells and antibody-secreting cells. While STAT1 is largely nonessential and STAT3 is critical for this process, the role of STAT5 is unknown. Objectives: This study sought to delineate unique roles of STAT5 in activation and differentiation of human naive and memory B cells. Methods: STAT activation was assessed by phospho-flow cytometry cell sorting. Differential gene expression was determined by RNA-sequencing and quantitative PCR. The requirement for STAT5B in B-cell and CD4(+) T-cell differentiation was assessed using CRISPR-mediated STAT5B deletion from B-cell lines and investigating primary lymphocytes from individuals with germline STAT5B mutations. Results: IL-21 activated STATS and strongly induced SOCS3 in human naive, but not memory, B cells. Deletion of STAT5B in B-cell lines diminished IL-21-mediated SOCS3 induction. PBMCs from STAT5B-null individuals contained expanded populations of immunoglobulin class-switched B cells, CD21(lo)Tbet(+) B cells, and follicular T helper cells. IL-21 induced greater differentiation of STAT5B-deficient B cells into plasmablasts in vitro than B cells from healthy donors, correlating with higher expression levels of transcription factors promoting plasma cell formation. Conclusions: These findings reveal novel roles for STAT5B in regulating IL-21-induced human B-cell differentiation. This is achieved by inducing SOCS3 to attenuate IL-21 signaling, and BCL6 to repress class switching and plasma cell generation. Thus, STAT5B is critical for restraining IL-21-mediated B-cell differentiation. These findings provide insights into mechanisms underpinning B-cell responses during primary and subsequent antigen encounter and explain autoimmunity and dysfunctional humoral immunity in STAT5B deficiency.

56 Meeting of the Society for Low Temperature Biology 7-9 October 2020 (online), in association with UCLouvain, Belgium RECOVERY OF ERYTHROCYTE OXYGEN TRANSPORT FUNCTION AFTER HYPOTHERMIC STORAGE UNDER THE ACTION OF HUMAN CORD BLOOD LOW-MOLECULAR FRACTION AND THE ACTOVEGIN

Global demand for natural pigments has increased in the past few years. Myxoxanthophyll, a glycosylated monocyclic carotenoid, is a pigment that occurs naturally in cyanobacteria but no scalable isolation process has been developed to obtain it from its natural source to date. In this study, myxoxanthophyll was isolated from unicellular cyanobacterium Synechocystis salina (S. salina) using high-performance countercurrent chromatography (HPCCC), where the lower phase of the biphasic solvent system composed of n-heptane–ethanol–water (2:4:4, v/v/v) was used as a mobile phase, whereas its upper phase was the stationary phase. For the HPCCC isolation, a multi-injection method was developed, and four consecutive sample injections (70 mg each) were performed, obtaining, in total, 20 mg of myxoxanthophyll, which was finally purified with high-performance liquid chromatography (HPLC). Overall, a final myxoxanthophyll yield of 15 mg (98% purity) was obtained. The target pigment showed a weak antioxidant and tyrosinase inhibitory effect, and exhibited immune-stimulating properties by activating human granulocytes. The results presented here form a basis for the large-scale production of myxoxanthophyll, and show the potential benefits of this pigment for human health.

Background Human mesenchymal stem cells (hMSCs) have tremendous potential in regenerative medicine, making it desirable to cryopreserve and bank them to increase their access and availability. Objective This research is part of a clinical trial performed on six patients that aimed to use advanced therapy medicinal products (ATMPs) based on hMSCs in patients undergoing repeated total hip replacement. Materials and methods To compare the characteristics of fresh and frozen hMSCs, we used the trypan blue exclusion test (cell viability), flow cytometry (cell viability and phenotyping), sterility determinations and the clonogenic assay of cell proliferation. Results Cryopreserved hMSCs showed good quality parameters after thawing in comparison with fresh hMSCs in suspension. When using a medium containing dimethyl sulfoxide (DMSO), the viability was higher than 90% in all cases. The cell purity determined by flow cytometry was also acceptable. Conclusion These initial results show that the prepared cryopreserved ATMP exhibited good viability and phenotype characteristics.

Purpose Deficiency of marginal-zone B cells was observed in peripheral blood (PB) of SLE pts in clinical remission or low disease activity (LDA)1. Goal of the prospective, comparative, long-term study is follow-up of this phenomenon. Methods Forty five adult SLE (ACR/1982, updated 1997) pts in complete remission or LDA2 and 10 age- and sex-matched healthy controls (HC) were enrolled in "month 0', and SLE also after twelve-months ("month 12') and 36-months ("month 36') period; overlap syndromes, infection, renal failure and monoclonal gammopathy in SLE were excluded. The DuraClone IM panel (Beckman Coulter) was used to identify CD19+CD27+IgM+ B cell subpopulation in PB samples by flow cytometry navios (Beckman Coulter) with software analysis using Kaluza version 1.2.; data obtained were expressed in relative% of PB lymphocytes and absolute values x106/L, and processed using Medcalc-Statistical Software programme. Results Significant differences (p =0.002 - <0.001) were obtained between absolute values of CD19+CD27+IgM+ B cells in HC (median 31.36, 95% CI 21.49 – 63.35) and SLE "month 0' (median 13.17, 95% CI 7.87 – 17.09), SLE "month 12' (median 10.56, 95% CI 7.24 – 16.04), and SLE "month 36' (median 9.66, 95% CI 7.22 – 13.21), but not between values obtained in SLE "month 0', "month 12' and "month 36' (p>0.05); not significant differences were found using analysis according to relative% of B cells under study (p>0.05). Conclusion Data obtained demonstrated a long-term deficiency of marginal-zone B cells in PB of SLE pts in complete remission or LDA; susceptibility to infection should be supposed, but further studies are necessary. Acknowledgement Research project PROGRES Q40-15, Charles Univ. Faculty of Medicine, Hradec Králové, Czech Republic. References Hrncir Z, et al. Clin Exper Rheumatol2016;34(S99);S–63. Fanouriakis A, et al. Ann Rheum Dis 2019;78;736–745.

Functional hyposplenism is a condition accompanying many diseases including autoimmune disorders and lymphomas. Hyposplenism is also commonly found in adult coeliac disease (up to 20 % of non-complicated and up to 80 % of complicated disease). Hyposplenism is associated with an increased risk of severe infections ( Streptococcus pneumoniae , Neisseria meningitidis and Haemophilus influenzae ). The aim of this prospective study was to investigate memory B lymphocytes as an indirect biomarker of functional hyposplenism. A total of 42 patients with coeliac disease (11 men, 31 women; mean age 49±14 years) and 10 healthy controls, blood donors (2 men, 8 women; mean age 39±7 years) were enrolled into the study. Nobody underwent previous splenectomy and no individual suffered from immunodeficiency. The DuraClone IM panel was used to identify B lymphocytes subpopulations in peripheral blood samples by flow cytometry Navios (Beckman Coulter) with software analysis using Kaluza version 1.2. Patients with coeliac disease and controls did not differ in basic parameters of leukocyte and total lymphocyte blood count. Switched memory B lymphocytes (CD19+CD27+IgD-), non-switched memory / marginal-zone-like B lymphocytes (CD19+CD27+IgD+) and IgM memory B lymphocytes (CD19+CD27+IgM++) were significantly lower in coeliac disease compared to controls. Follicular (naive) B lymphocytes were not significantly different between coeliac disease and controls. In conclusion, dysfunction of memory B lymphocytes can be responsible for an increased risk of severe bacterial infections in coeliac disease. Patients with coeliac disease with dysfunction of memory B lymphocytes are clearly indicated for anti-pneumococcal vaccination.

Background: Glatiramer acetate (GA) is an effective treatment for the earliest stages of multiple sclerosis (MS)—clinically isolated syndrome (CIS) or clinically definite MS (CDMS). Objective: This study aims to determine the differences in the lymphocyte population (at baseline and the course of five years) between confirmed sustained progression (CSP) and non-CSP groups and to identify potential biomarkers among these parameters that can predict a positive response to the treatment. Methods: Twelve male and 60 female patients were included in the study. Peripheral blood samples were collected before and five years after treatment with GA. The authors compared lymphocyte parameters between the CSP and non-CSP groups by statistical analyses. Univariate and penalized logistic regression models were fitted to identify the best lymphocyte parameters at baseline and their combination for potential biomarkers. Subsequently, the ROC analysis was used to identify cut-offs for selected parameters. Results: The parameter CD4+/CD45RO+ was identified as the best single potential biomarker, demonstrating the ability to identify patients with CSP. Moreover, a combination of four lymphocyte parameters at baseline, relative lymphocyte counts, CD3+/CD69+, CD4+/CD45RO+, and CD4+/CD45RA+ab, was identified as a potential composite biomarker. This combination explains 23% of the variability in CSP, which is better than the best univariate parameter when compared to CD4+/CD45RO+ at baseline. Conclusions: The results suggest that other biomarkers can help monitor the conditions of patients and predict a favourable outcome.

Background:Chronic lymphocytic leukemia (CLL) is typically associated with a significant immune deficiency resulting in elevated risk of infections ‐ the leading cause of death in CLL patients (pts). However, many specific mechanisms contributing to suppression of immunity in CLL are not yet well understood.Aims:Comprehensive assessment of immunological parameters in patients with CLL.Methods:We prospectively enrolled 126 CLL pts: 42 with clinically stable disease not requiring treatment (median age, 65 years; males, 50%; low/medium/high modified Rai risk, 59/36/5%) and 84 with CLL progression indicated for therapy (median age, 68 years; males, 64%; low/medium/high modified Rai risk, 0/43/57%). In addition, parameters of immunity were compared before and after first‐line chemoimmunotherapy in 39 pts (FCR, n = 13; BR, n = 15; G‐CLB, n = 5; R‐CLB, n = 6). Control group consisted of 46 healthy blood donors (median age, 40 years; males, 59%). Cellular subpopulations (CLL cells, T cells, Th and Tc cells, NK cells and polyclonal B cells) were quantified by flow cytometry (cytometer Navios, Beckman Coulter). Humoral immunity was evaluated by quantification of serum immunoglobulin concentrations (total IgG, IgG subclasses 1–4, IgA 1–2 and IgM) using Immunochemistry Image System (Beckman Coulter). Activation of complement was assessed by Wieslab Complement System Screen (Euro Diagnostica). Serum concentrations of immunity‐regulating cytokines (IL‐4, IL‐5, IL‐6, IL‐10, IL‐17A, IL‐17F, IL‐33 and IFN‐γ) were determined by xMAP multiplex technology (Luminex).Results:Serum IL‐4, IL‐6 and IL‐10 were elevated in CLL compared to controls (all p <0.0001). Polyclonal B cells were decreased (p < 0.0001) whereas Tc cells were increased (p = 0.019 and p < 0.0001) in stable and progressive CLL in comparison to controls. Both CLL subgroups had significantly lower IgG, A and M when compared to controls (all p < 0.0001). Progressive CLL pts had significantly higher numbers of NK cells (p = 0.0003) and stable CLL pts significantly higher IFN‐γ (p = 0.01) vs controls. Pts with progressive CLL had significantly lower concentrations of IgG, A, and M (p = 0.0012, p < 0.0001 and p = 0.027) than stable pts. Chemoimmunotherapy resulted in significant decline of polyclonal B cells, NK cells, Th cells, Tc cells and T regs (all p < 0.0001) but serum IgA and complement activation (both classical and alternative pathway) increased after therapy (p = 0.048, p = 0.002 and p = 0.0017).Summary/Conclusion:Conclusions: The present study identified multiple defects in cellular as well as humoral immunity in CLL pts. Clinical progression of CLL was associated with further decline of immune parameters. Cytoreductive therapy resulted in significant decrease in all physiological lymphocyte subpopulations; however, several markers of humoral immunity improved after treatment. The enrollment into the study continues and further analyses are underway including assessment of association of immune defects with infections and changes in immunological parameters over longer follow‐up. Supported by project PROGRES Q40/08, by DRO Ministry of Health (UHHK, 00179906) and by League Against Cancer Prague.image

In Europe the issue of regulation of safety and quality of tissue grafts was first discussed at the EATB meeting in Leuven in 1995. In the years 2004 and 2006 the pertinent Directives of the European Parliament and Council and of the European Commission were issued. In the Czech Republic the harmonized national legal norms were issued in 2008 and the State Institute for Drug Control was authorized to grant licences for activities of Tissue (TE) and Procurement (PE) Establishments. The TE of the University Hospital Hradec Kralove was granted this licence in 2011. The harmonization process lasted 22 years and was completed in 2017, when the TE was put on the list of the EU Tissue Establishments (EUTE). The TE was given three EUTE identification codes: CZ 000425 for reproductive tissues, CZ 000426 for haematopoietic tissues and CZ 000427 for other tissues, such as vascular tissues, bones, ligaments, tendons or amnia.