Impact of Circulating Lymphoma Cells on HLA Typing Outcomes in Patients with Diffuse Large B-Cell Lymphoma: A Case Report.

BACKGROUND: Recipient's high resolution HLA typing is required in allogeneic hematopoietic cell transplantation from unrelated donors, as well as for haploidentical family donors. For these purposes, Next-Generation Sequencing (NGS) methods are the gold standard. METHODS: We present a case of a patient with an incorrect HLA typing result caused by the population of circulating lymphoma cells. The first HLA examination was performed from peripheral blood (PB) using NGS in the active phase of diffuse large B-cell lymphoma with bone marrow involvement. RESULTS: Because of rare and inconclusive results, confirmed twice for the A* locus (A*02:32N), real-time polymerase chain reaction (RT-PCR)was performed. With RT-PCR method, we obtained more expected results according to the population allele frequency: in HLA-A locus (A*02:01) but also in DQB1 (DQB1*03:01, not as in NGS - DQB1*03:10). For the final verification, we used swab material and we obtained unambiguous NGS result with expected, frequent HLA-A*02:01 and DQB1*03:01 alleles corresponding to the RT-PCR result from PB. CONCLUSIONS: To conclude, we suspect that the discrepancies between NGS and RT-PCR results were caused by the presence of a significant amount of circulating lymphoma cells in the peripheral blood sample. Lymphomagenic mutations may involve the histocompatibility antigen coding region and affect HLA expressed on malignant cells. This finding may be relevant for the selection of test material in primary and confirmatory HLA testing in patients with active hematological malignancies because of the strong impact of incorrect HLA typing on the procedure of a donor selection.

Clinical Application of In Vitro Tests for COVID-19 Vaccine Delayed Hypersensitivity Diagnostics.

Drug hypersensitivity reactions can be classified as immediate or delayed. While diagnostic options for immediate reactions are well developed and standardized, delayed reactions (in many cases type IV according to Gell and Coombs) are a challenge for allergy work-up. In recent years, some in vitro markers have been proposed and used for delayed reactions, such as contact dermatitis. Primary strategy: Avoidance is difficult to achieve, especially for COVID-19 vaccinations, when immunity against infection is extremely important. The aim of our study was to evaluate the application of in vitro delayed hypersensitivity tests in COVID-19 vaccines. Seven patients with a positive history of severe delayed drug allergy were enrolled. Vein blood was collected to stimulate cells with the tested vaccines (Comirnaty, Janssen, Spikevax) and excipients with the assessment of CD40L, CD69, IL-2, IL-4, IL-6, IL-10, IFNgamma, TNFalfa, and intracellular markers: granulysin and INFgamma. In addition, basophile activation tests, patch tests, skin prick tests, and intradermal tests were performed with the tested vaccine. Finally, the decision was made to either administer a vaccine or resign. Two out of seven patients were considered positive for drug hypersensitivity in the in vitro test according to the high vaccine stimulation index measured with CD69 (6.91 and 12.18) and CD40L (5.38 and 15.91). All patch tests, BATs, and skin tests were negative. Serum interleukin measurements were inconclusive as the impact of the vaccine itself on the immunity system was high. Intracellular markers gave uncertain results due to the lack of stimulation on the positive control. CD69 and CD40L could be reliable in vitro markers for delayed hypersensitivity to COVID-19 vaccines. Patch tests, skin tests, BATs, and serum interleukins did not confirm their usefulness in our study.

Comparative Analysis of Fcγ and Complement Receptors Presence on Monocytes in Pulmonary Sarcoidosis and Tuberculosis.

Sarcoidosis (SA) is a granulomatous disorder, which mostly affects the lungs. Its clinical characteristics resemble tuberculosis (TB), but its treatment is different. The etiology of SA is unknown; however, mycobacterial antigens were proposed as environmental factors in its development. Due to previously revealed immunocomplexemia with mycobacterial antigens in the blood of our SA but not TB patients, and in the search for biomarkers for differential diagnosis of the two disorders, we studied the phagocytic activity of monocytes from both patients' groups with flow cytometry. With the use of this method, we also analyzed the occurrence of receptors for IgG (FcγR) and complement components (CR) at the surface of these monocytes, responsible for phagocytosis of immunocomplexes. We revealed a higher phagocytic activity of monocytes in both disorders, but an increased frequency of monocytes with FcγRIII (CD16) and decreased with CR1 (CD35) receptor in the blood of SA vs. TB patients. With regard to our other genetic study on FcγRIII variants in SA and TB, this may account for the decreased clearance of immunocomplexes and different immune responses in the two diseases. Thus, the presented analysis not only sheds light on the pathomechanisms of SA and TB but may also support their differential diagnosis.

T regulatory cells metabolism: The influence on functional properties and treatment potential.

CD4+CD25highFoxP3+ regulatory T cells (Tregs) constitute a small but substantial fraction of lymphocytes in the immune system. Tregs control inflammation associated with infections but also when it is improperly directed against its tissues or cells. The ability of Tregs to suppress (inhibit) the immune system is possible due to direct interactions with other cells but also in a paracrine fashion via the secretion of suppressive compounds. Today, attempts are made to use Tregs to treat autoimmune diseases, allergies, and rejection after bone marrow or organ transplantation. There is strong evidence that the metabolic program of Tregs is connected with the phenotype and function of these cells. A modulation towards a particular metabolic stage of Tregs may improve or weaken cells' stability and function. This may be an essential tool to drive the immune system keeping it activated during infections or suppressed when autoimmunity occurs.

Ageing-resembling phenotype of long-term allogeneic hematopoietic cells recipients compared to their donors.

BACKGROUND: Ageing is a complex phenomenon that leads to decreased proliferative activity, loss of function of the cells, and cellular senescence. Senescence of the immune system exacerbates individual's immune response, both humoral and cellular but increases the frequency of infections. We hypothesized that physiological ageing of adaptive immune system occurs in recipients of allogeneic hematopoietic cells transplant (allo-HCT) at faster rate when compared to their respective donors since the small number of donor cells undergo immense proliferative stress restoring recipients hematopoiesis. We compared molecular characterizations of ageing between recipients and donors of allo-HCT: telomeric length and immunophenotypic changes in main lymphocyte subsets - CD4+, CD8+, CD19+, CD56+. RESULTS: Median telomeric length (TL) of CD8+ lymphocytes was significantly longer in donors compared to recipients (on average 2,1 kb and 1,7 kb respectively, p = 0,02). Similar trends were observed for CD4+ and CD19+ although the results did not reach statistical significance. We have also found trends in the immunophenotype between recipients and donors in the subpopulations of CD4+ (naïve and effector memory), CD8+ Eomes+ and B-lymphocytes (B1 and B2). Lower infection risk recipients had also a significantly greater percentage of NK cells (22,3%) than high-risk patients (9,3%) p = 0,04. CONCLUSION: Our data do not support the initial hypothesis of accelerated aging in the long term all-HCT recipients with the exception of the recipients lymphocytes (mainly CD8+) which present some molecular features, characteristic for physiological ageing (telomeric shortening, immunophenotype) when compared to their respective donors. However, a history of lower infection numbers in HCT recipients seems to be associated with increased percentage of NK cells. The history of GVHD seems not to affect the rate of ageing. Therefore, it is safe to conclude that the observed subtle differences between recipients' and donors' cells result mainly from the proliferative stress in the early period after allo-HCT and the difference between hosts' and recipients' microenvironments.

Autoimmunity and Cancer-Two Sides of the Same Coin.

Autoimmune disease results from the immune response against self-antigens, while cancer develops when the immune system does not respond to malignant cells. Thus, for years, autoimmunity and cancer have been considered as two separate fields of research that do not have a lot in common. However, the discovery of immune checkpoints and the development of anti-cancer drugs targeting PD-1 (programmed cell death receptor 1) and CTLA-4 (cytotoxic T lymphocyte antigen 4) pathways proved that studying autoimmune diseases can be extremely helpful in the development of novel anti-cancer drugs. Therefore, autoimmunity and cancer seem to be just two sides of the same coin. In the current review, we broadly discuss how various regulatory cell populations, effector molecules, genetic predisposition, and environmental factors contribute to the loss of self-tolerance in autoimmunity or tolerance induction to cancer. With the current paper, we also aim to convince the readers that the pathways involved in cancer and autoimmune disease development consist of similar molecular players working in opposite directions. Therefore, a deep understanding of the two sides of immune tolerance is crucial for the proper designing of novel and selective immunotherapies.

Cytokines and chemokines multiplex analysis in patients with low disease activity rheumatoid arthritis.

Rheumatoid arthritis is a severe chronic autoimmune disorder that results from pathological activation of immune cells and altered cytokine/chemokine network. The aim of our study was to evaluate concentrations of chosen cytokines and chemokines in blood sera and synovial fluid samples isolated from low disease activity rheumatoid arthritis (RA) patients and osteoarthritis (OA) sufferers. Blood sera and synovial fluid samples have been obtained from 24 OA and 14 RA patients. Cytokines/chemokines levels have been determined using a Milliplex® Map 38-plex human cytokine/chemokine magnetic bead-based panel (Merck Millipore, Germany) and Luminex® MAGPIX® platform (Luminex USA). Low disease activity RA patients showed altered concentration of numerous cytokine/chemokine when compared to OA controls-they were characterized by, inter alia, increased: eotaxin/CCL11 (p = 0.037), GRO/CXCL1 (p = 0.037), IL-2 (p = 0.013), IL-4 (p = 0.017), IL-7 (p = 0.003), IL-8 (p = 0.0007) and GM-CSF (p = 0.037) serum levels, whilst MDC/CCL22 concentration was decreased in this group (p = 0.034). Eotaxin/CCL11 (p = 0.001), GRO/CXCL1 (p = 0.041), IL-10 (p = 0.003), GM-CSF (p = 0.01), IL-1RA (p = 0.0005) and VEGF (p = 0.01) concentrations in synovial fluid of RA females were also increased. Even with low disease activity score, RA patients exhibited increased concentrations of cytokines with pro- and anti-inflammatory activities, as well as numerous chemokines, growth factors and regulators of angiogenesis. Surprisingly, RA subjects also shown decreased concentration of CCL22 chemokine. The attempt to restore cytokine balance and tolerogenic environment is ineffective in RA sufferers even with good disease management. Distinguished factors could serve as possible indicators of disease progression even in low disease activity patients.

Mesenchymal stem cells transfer mitochondria to allogeneic Tregs in an HLA-dependent manner improving their immunosuppressive activity.

Cell-based immunotherapies can provide safe and effective treatments for various disorders including autoimmunity, cancer, and excessive proinflammatory events in sepsis or viral infections. However, to achieve this goal there is a need for deeper understanding of mechanisms of the intercellular interactions. Regulatory T cells (Tregs) are a lymphocyte subset that maintain peripheral tolerance, whilst mesenchymal stem cells (MSCs) are multipotent nonhematopoietic progenitor cells. Despite coming from different origins, Tregs and MSCs share immunoregulatory properties that have been tested in clinical trials. Here we demonstrate how direct and indirect contact with allogenic MSCs improves Tregs' potential for accumulation of immunosuppressive adenosine and suppression of conventional T cell proliferation, making them more potent therapeutic tools. Our results also demonstrate that direct communication between Tregs and MSCs is based on transfer of active mitochondria and fragments of plasma membrane from MSCs to Tregs, an event that is HLA-dependent and associates with HLA-C and HLA-DRB1 eplet mismatch load between Treg and MSC donors.

KIR Receptors as Key Regulators of NK Cells Activity in Health and Disease.

Natural killer (NK) cells are part of the cellular immune response. They target mainly cancer and virally infected cells. To a high extent cytotoxic activity of NK cells is regulated inter alia by signals from killer immunoglobulin-like receptors (KIR). The major histocompatibility complex (MHC) class I molecules are important ligands for KIR receptors. Binding of ligands (such as MHC I) to the KIR receptors has the important role in solid organ or hematopoietic cell transplantation. Of note, the understanding of the relationship between KIR and MHC receptors may contribute to the improvement of transplant results. Donor-recipient matching, which also includes the KIR typing, may improve monitoring, individualize the treatment and allow for predicting possible effects after transplantation, such as the graft-versus-leukemia effect (GvL) or viral re-infection. There are also less evident implications of KIR/MHC matching, such as with pregnancy and cancer. In this review, we present the most relevant literature reports on the importance of the KIR/MHC relationship on NK cell activity and hematopoietic stem cell transplantation (HSCT)/solid organ transplantation (SOT) effects, the risk of allograft rejection, protection against post-transplant cytomegalovirus (CMV) infection, pregnancy complications, cancer and adoptive therapy with NK cells.

Regulatory T Cells-Related Genes Are under DNA Methylation Influence.

Regulatory T cells (Tregs) exert a highly suppressive function in the immune system. Disturbances in their function predispose an individual to autoimmune dysregulation, with a predominance of the pro-inflammatory environment. Besides Foxp3, which is a master regulator of these cells, other genes (e.g., Il2ra, Ctla4, Tnfrsf18, Ikzf2, and Ikzf4) are also involved in Tregs development and function. Multidimensional Tregs suppression is determined by factors that are believed to be crucial in the action of Tregs-related genes. Among them, epigenetic changes, such as DNA methylation, tend to be widely studied over the past few years. DNA methylation acts as a repressive mark, leading to diminished gene expression. Given the role of increased CpG methylation upon Tregs imprinting and functional stability, alterations in the methylation pattern can cause an imbalance in the immune response. Due to the fact that epigenetic changes can be reversible, so-called epigenetic modifiers are broadly used in order to improve Tregs performance. In this review, we place emphasis on the role of DNA methylation of the genes that are key regulators of Tregs function. We also discuss disease settings that have an impact on the methylation status of Tregs and systematize the usefulness of epigenetic drugs as factors able to influence Tregs functions.

Cytokine Imprint in Preeclampsia.

The hallmark of preeclampsia (PE) is a shift toward persistent inflammatory response, accompanied by endothelial dysfunction. The driving forces in PE are proinflammatory cytokine and growth factors, in parallel with reduced functionality of anti-inflammatory effectors, like regulatory T cells are observed. Unfortunately, no conclusive mechanism underlying preeclampsia has been identified. For this reason, research on preeclampsia is needed to provide a state of the art understanding of the pathophysiology, identification of new diagnostics tools and the development of targeted therapies. The 68 patients were divided into three groups: gestational hypertension (GH) group (n = 19) and PE group (n = 28) and a control group (n = 21). We have tested a set of 53 cytokines, chemokines and growth factors in preeclampsia and gestational hypertension, and then compared them with normal pregnancies. Using a diagnostic test assessment characteristic parameters (IL-22, MDC/CCL22, IL-2/IL-4 ratio) have been identified and cut-off values have been proposed to diagnose preeclampsia. All parameters had high negative or positive predictive values, above 80%. In conclusion, we have proposed a potential set of immune parameters to diagnose preeclampsia.

Intestinal Microbiota in Common Chronic Inflammatory Disorders Affecting Children.

The incidence and prevalence rate of chronic inflammatory disorders is on the rise in the pediatric population. Recent research indicates the crucial role of interactions between the altered intestinal microbiome and the immune system in the pathogenesis of several chronic inflammatory disorders in children, such as inflammatory bowel disease (IBD) and autoimmune diseases, such as type 1 diabetes mellitus (T1DM) and celiac disease (CeD). Here, we review recent knowledge concerning the pathogenic mechanisms underlying these disorders, and summarize the facts suggesting that the initiation and progression of IBD, T1DM, and CeD can be partially attributed to disturbances in the patterns of composition and abundance of the gut microbiota. The standard available therapies for chronic inflammatory disorders in children largely aim to treat symptoms. Although constant efforts are being made to maximize the quality of life for children in the long-term, sustained improvements are still difficult to achieve. Additional challenges are the changing physiology associated with growth and development of children, a population that is particularly susceptible to medication-related adverse effects. In this review, we explore new promising therapeutic approaches aimed at modulation of either gut microbiota or the activity of the immune system to induce a long-lasting remission of chronic inflammatory disorders. Recent preclinical studies and clinical trials have evaluated new approaches, for instance the adoptive transfer of immune cells, with genetically engineered regulatory T cells expressing antigen-specific chimeric antigen receptors. These approaches have revolutionized cancer treatments and have the potential for the protection of high-risk children from developing autoimmune diseases and effective management of inflammatory disorders. The review also focuses on the findings of studies that indicate that the responses to a variety of immunotherapies can be enhanced by strategic manipulation of gut microbiota, thus emphasizing on the importance of proper interaction between the gut microbiota and immune system for sustained health benefits and improvement of the quality of life of pediatric patients.

Associations of TP53 codon 72 polymorphism with complications and comorbidities in patients with type 1 diabetes.

Wild-type TP53 plays an important role in the regulation of immune response and systemic inflammation. In type 1 diabetes (T1D), TP53 pathways are upregulated and an increased susceptibility to apoptosis is observed. We hypothesize that TP53 codon 72 polymorphism could be associated with complications and comorbidities in patients with T1D. We have investigated the associations of the TP53 codon 72 polymorphism with the T1D complications and comorbidities (retinopathy, nephropathy, hypertension, dyslipidemia, autoimmune thyroiditis, and celiac disease) in 350 patients. The key results of our approach are as follows: (1) In diabetic subjects, the Pro/Pro genotype is associated with an increased risk of microvascular complications, dyslipidemia, and celiac disease; (2) the Arg/Arg variant is associated with a decreased risk of autoimmune thyroiditis and celiac disease; (3) the Pro allele is associated with an increased risk of dyslipidemia, autoimmune thyroiditis, and celiac disease. Although further studies are required, our results for the first time indicate that the TP53 codon 72 polymorphism could be considered a genetic marker to predict the increased susceptibility to some T1D complications and comorbidities. KEY MESSAGES: We analyzed the TP53 codon 72 polymorphism in patients with T1D. Pro/Pro genotype is associated with an increased risk of microvascular complications, dyslipidemia, and celiac disease. The Arg/Arg variant is associated with a decreased risk of autoimmune thyroiditis and celiac disease. The Pro allele is associated with an increased risk of dyslipidemia, autoimmune thyroiditis, and celiac disease.

Immune Status Against Hepatitis B in Patients After Allogeneic Hematopoietic Cell Transplantation-Factors Affecting Early and Long-Lasting Maintenance of Protective Anti-HBs Titers.

The immunization of allogeneic hematopoietic cell transplantation (HCT) recipients against vaccine-preventable diseases is a part of posttransplantation guidelines. We conducted a prospective study to assess clinical and immunological parameters that would determine the response and long-term maintenance of protective antibody titers upon the hepatitis B virus (HBV) vaccination after HCT. The investigated variables included: vaccination of the HCT recipients and their donors prior to HCT, chronic graft versus host disease (cGVHD) and the timing of post-HCT vaccination, and B- and T-cell subtype status. Forty-two patients were immunized with three or more doses of recombinant hepatitis B surface antigen (rHBsAg) administered according to the individualized schedule of 0-1-2-6-(12) months. After vaccination, seroconversion was achieved in the whole group. The vaccines were categorized according to the antibody (Ab) titers as weak (WRs; 28.7%), good (GRs; 38%) or very good responders (VGRs; 3.3%). In multivariate logistic regression, severe cGVHD (OR= 15.5), and preceding donor immunization (OR= 0.13) were independent predictors of a weak response to vaccination. A prior belonging to the WR group impaired the durability of protection (OR= 0.17) at a median follow-up of 11.5 years. Patients with severe cGVHD showed a trend toward lower median Ab titers, although they required a higher rate of booster vaccine doses. All VGRs had CD4+ cells > 0.2 x 106/L. There was a lower mean rate of CD4+IL2+ lymphocytes in WRs. Vaccination demonstrated the immunomodulatory effect on B-cell and T-cell subsets and a Th1/Th2 cytokine profile, while shifts depended on a history of severe cGVHD and the type of vaccine responder. To conclude, vaccination of HCT donors against HBV allows a better response to vaccination in the respective HCT recipients. Double doses of rHBsAg should be considered in patients with cGVHD and in those not immunized before HCT. A dedicated intensified vaccination schedule should be administered to WRs.

Antigen-reactive regulatory T cells can be expanded in vitro with monocytes and anti-CD28 and anti-CD154 antibodies.

BACKGROUND: In recent years, therapies with CD4+CD25highFoxP3+ regulatory T cells (Tregs) have been successfully tested in many clinical trials. The important issue regarding the use of this treatment in autoimmune conditions remains the specificity toward particular antigen, as because of epitope spread, there are usually multiple causative autoantigens to be regulated in such conditions. METHODS: Here we show a method of generation of Tregs enriched with antigen-reactive clones that potentially covers the majority of such autoantigens. In our research, Tregs were expanded with anti-CD28 and anti-CD154 antibodies and autologous monocytes and loaded with a model peptide, such as whole insulin or insulin ß chain peptide 9-23. The cells were then sorted into cells recognizing the presented antigen. The reactivity was verified with functional assays in which Tregs suppressed proliferation or interferon gamma production of autologous effector T cells (polyclonal and antigen-specific) used as responders challenged with the model peptide. Finally, we analyzed clonotype distribution and TRAV gene usage in the specific Tregs. RESULTS: Altogether, the applied technique had a good yield and allowed us to obtain a Treg product enriched with a specific subset, as confirmed in the functional tests. The product consisted of many clones; nevertheless, the content of these clones was different from that found in polyclonal or unspecific Tregs. CONCLUSIONS: The presented technique might be used to generate populations of Tregs enriched with cells reactive to any given peptide, which can be used as a cellular therapy medicinal product in antigen-targeted therapies.

Podocytes-The Most Vulnerable Renal Cells in Preeclampsia.

Preeclampsia (PE) is a disorder that affects 3-5% of normal pregnancies. It was believed for a long time that the kidney, similarly to all vessels in the whole system, only sustained endothelial damage. The current knowledge gives rise to a presumption that the main role in the development of proteinuria is played by damage to the podocytes and their slit diaphragm. The podocyte damage mechanism in preeclampsia is connected to free VEGF and nitric oxide (NO) deficiency, and an increased concentration of endothelin-1 and oxidative stress. From national cohort studies, we know that women who had preeclampsia in at least one pregnancy carried five times the risk of developing end-stage renal disease (ESRD) when compared to women with physiological pregnancies. The focal segmental glomerulosclerosis (FSGS) is the dominant histopathological lesion in women with a history of PE. The kidney's podocytes are not subject to replacement or proliferation. Podocyte depletion exceeding 20% resulted in FSGS, which is a reason for the later development of ESRD. In this review, we present the mechanism of kidney (especially podocytes) injury in preeclampsia. We try to explain how this damage affects further changes in the morphology and function of the kidneys after pregnancy.

Therapy with CD4+CD25+ T regulatory cells - should we be afraid of cancer?

This review focuses on the role of regulatory T cells (Tregs) in the process of carcinogenesis. The controversy of this issue arose due to the increasing therapeutic use of Tregs in humans (inter alia, in the treatment of autoimmune diseases). It is mainly due to potential dangers related to immunosuppressive activity of these cells, especially regarding cancer. The natural function of regulatory T cells (which is the suppression of excessive activity of the immune system) is purportedly linked to an increased risk of cancer initiation. This work brings together and summarizes the most important reports of researchers dealing with this problem and attempts to explain doubts and fears related to Tregs and their uncertain connection with cancer initiation and progression. It is clearly shown that regulatory T cells are associated with acceleration of existing tumors (they are attracted by microenvironments created by cancer cells) but cannot initiate them on their own.

Synthesis and antiproliferative activity of new mycophenolic acid conjugates with adenosine derivatives.

New conjugates of mycophenolic acid (MPA) and adenosine derivatives were synthesized and assessed as potential immunosuppressants on Jurkat cell line and peripheral blood mononuclear cells (PBMC) from healthy donors. As compared to MPA, all compounds were found to be more active against Jurkat cell line. The antiproliferative activities were compared with MPA and adenosine, in both 2',3'-O-isopropylidene protected and free hydroxyl groups possessing forms. The obtained results were also discussed in terms of selectivity index, defined as SI = IC50/EC50.

Transcriptional profile of in vitro expanded human epidermal progenitor cells for the treatment of non-healing wounds.

BACKGROUND: Epidermal progenitor cells (EPCs) have been under extensive investigation due to their increasing potential of application in medicine and biotechnology. Cultured human EPCs are used in the treatment of chronic wounds and have recently became a target for gene therapy and toxicological studies. One of the challenges in EPCs culture is to provide a high number of undifferentiated, progenitor cells displaying high viability and significant biological activity. OBJECTIVES: The goal of this study was to characterize the in vitro cultured progenitor cells and to assess whether the cells with the progenitor phenotype are able to enhance wound healing. Additionally, we aimed to establish the complete procedure of the culture, analysis and clinical application of epidermal progenitor cells. METHODS: In this study we present a method of cell isolation and culture followed by a technique of transplantation of the cultured cells onto the wound bed. The applied isolation technique involves two enzymatic steps (dispase, trypsin) and it is characterized by a high yield of cells. The obtained cells were cultured in vitro up to the second passage in serum-free and xeno-free keratinocytes-dedicated medium. Key stem cell markers were determined with means of flow cytometry and quantitative real-time PCR. RESULTS: The in vitro expanded cells displayed high proliferative activity without features of neither apoptosis nor necrosis. The flow cytometry and transcriptomic analyses showed enhanced expression of stem cell markers (i.e. proteins: ΔNp63, CD29, CD49f and BNC1, CDKN1A transcripts) in the expanded cells. In the presented compassionate use study, cultured autologous cells from an oncological patient were suspended in fibrin sealant and transplanted directly to a non-healing wound, resulting in wound closure within 2 months. CONCLUSION: The cells cultured in serum-free media display epidermal stem cells features and a potential to stimulate wound healing. This promising procedure of isolation, culture and application warrants further clinical trials in the treatment of chronic wounds.