Changes in the Concentrations of Proangiogenic Cytokines in Human Brain Glioma and Acute Lymphoblastic Leukemia.

Acute lymphoblastic leukemia (ALL) and glioma are some of the most common malignancies, with ALL most often affecting children and glioma affecting adult men. Proangiogenic cytokines and growth factors play an important role in the development of both of these tumors. Glioma is characterized by an extremely extensive network of blood vessels, which continues to expand mainly in the process of neoangiogenesis, the direct inducers of which are cytokines from the family of vascular endothelial growth factors, i.e., vascular endothelial growth factor (VEGF-A) and its receptor vascular endothelial growth factor receptor 2 (VEGF-R2), as well as a cytokine from the fibroblast growth factor family, fibroblast growth factor 2 (FGF-2 or bFGF). Growth factors are known primarily for their involvement in the progression and development of solid tumors, but there is evidence that local bone marrow angiogenesis and increased blood vessel density are also present in hematological malignancies, including leukemias. The aim of this study was to examine changes in the concentrations of VEGF-A, VEGF-R2, and FGF-2 (with a molecular weight of 17 kDa) in a group of patients divided into specific grades of malignancy (glioma) and a control group; changes of VEGF-A and FGF-2 concentrations in childhood acute lymphoblastic leukemia and a control group; and to determine correlations between the individual proteins as well as the influence of the patient's age, diet, and other conditions that may place the patient in the risk group. During the statistical analysis, significant differences in concentrations were found between the patient and control groups in samples from people with diagnosed glioma and from children with acute lymphoblastic leukemia, but in general, there are no significant differences in the concentrations of VEGF-A, VEGF-R2, and FGF-2 between different grades of glioma malignancy. Among individuals treated for glioma, there was no significant impact from the patient's gender and age, consumption of food from plastic packaging, frequency of eating vegetables and fruit, smoking of tobacco products, the intensity of physical exercise, or the general condition of the body (Karnofsky score) on the concentrations of the determined cytokines and receptor. The listed factors do not bring about an actual increase in the risk of developing brain glioma.

Effects of Combinatory In Vitro Treatment with Immune Checkpoint Inhibitors and Cytarabine on the Anti-Cancer Immune Microenvironment in De Novo AML Patients.

Despite substantial progress in the diagnostic and therapeutic procedures, acute myeloid leukaemia (AML) still constitutes a significant problem for patients suffering from its relapses. A comprehensive knowledge of the disease's molecular background has led to the development of targeted therapies, including immune checkpoint inhibitors, and demonstrated beneficial effects on several types of cancer. Here, we aimed to assess in vitro the potential of the immune checkpoint blockage for supporting anti-cancer responses to the AML backbone therapy with cytarabine. PBMCs of AML patients were collected at admission and, following the therapy, eight complete remission (CR) and eight non-responders (NR) subjects were selected. We assessed the effects of the in vitro treatment of the cells with cytarabine and the immune checkpoint inhibitors: anti-CTLA-4, anti-PD-1, anti-PD-L1. The study protocol allowed us to evaluate the viability of the cancer and the immune cells, proliferation status, phenotype, and cytokine release. Anti-PD-L1 antibodies were found to exert the most beneficial effect on the activation of T cells, with a concomitant regulation of the immune balance through Treg induction. There was no direct influence on the blast cells; however, the modulation of the PD-1/PD-L1 axis supported the expansion of lymphocytes. Changes in the response between CR and NR patients might result from the differential expression of PD-1 and PD-L1, with lower levels in the latter group. The tested blockers appear to support the anti-cancer immune responses rather than directly improve the effects of cytarabine. In conclusion, checkpoint proteins' modulators might improve the anti-cancer responses in the tumour environment.

The influence of calcitriol and methylprednisolone on podocytes function in minimal change disease in vitro model.

Minimal change disease (MCD), considered one of the major causes of nephrotic syndrome, is a complex pathological condition with disturbances in podocytes' foot processes. Numerous studies suggested the essential role of vitamin D3 in maintaining proper glomerulus function. However, the data on direct potential of that compound in reference to podocytes are scarce. Thus, here we assessed the influence of calcitriol (active vitamin D3) on podocyte function, apart from commonly used steroids (methylprednisolone). CIHP-1 podocyte cell line was used to implement the LPS-PAN-induced MCD in vitro model. Viability, podocyte-related slit diaphragm proteins, morphology, function as a barrier was evaluated using flow cytometry, RT-PCR, confocal microscopy, and TEER analysis. Calcitriol or methylprednisolone did not affect cell viability. Podocyte-related proteins demonstrated different responses to in vitro treatment compared to previously reported changes in total glomeruli. Podocyte morphology was partially restored in the presence of the tested compounds. In addition, TEER analysis revealed improvement of LPS-PAN-induced cells' function as a barrier when vitamin D3 or steroid was used. In conclusion, a significant potential for modulation of MCD in vitro model podocytes with calcitriol or selected steroids was reported. Further studies on vitamin D3 in context of podocyte-related phenomenon accompanying MCD are of great importance.

Effect of antiviral and immunomodulatory treatment on a cytokine profile in patients with COVID-19.

Background: The severity of COVID-19 is associated with an elevated level of a variety of inflammatory mediators. Increasing evidence suggests that the Th17 response contributes to the severity of COVID-19 pneumonia, whereas Th22 response plays a regulatory role in SARS-CoV-2 infection. Two main types of available COVID-19 treatments are antivirals and immunomodulatory drugs; however, their effect on a cytokine profile is yet to be determined. Methods: This study aim to analyse a cytokine profile in peripheral blood from patients with COVID-19 (n=44) undergoing antiviral or/and immunomodulatory treatment and healthy controls (n=20). Circulating CD4+ and CD8+ T cells and their intracellular expression of IL-17A and IL-22 were assessed by flow cytometry. Results: Initial results showed an overexpression of IL-17F, IL-17A, CCL5/RANTES, GM-CSF, IL-4, IL-10, CXCL-10/IP-10 and IL-6 in COVID-19 patients compared to healthy controls. Treatment with remdesivir resulted in a significant decline in concentrations of IL-6, IL-10, IFN-alpha and CXCL10/IP-10. Immunomodulatory treatment contributed to a significant downregulation of IL-10, IFN-alpha, CXCL10/IP-10 and B7-H3 as well as upregulation of IL-22 and IL-1 beta. A combination of an antiviral and immunomodulatory treatment resulted in a significant decrease in IL-17F, IL-10, IFN-alpha, CXCL10/IP-10 and B7-H3 levels as well as an increase in IL-17A and IL-1 beta. We found significantly higher percentage of both CD4+ and CD8+ T cells producing IL-17A and CD4+ T cells producing IL-22 in patients with COVID-19. Conclusion: Administration of antiviral or/and immunomodulatory treatment resulted in a significant downregulation of pro-inflammatory cytokine expression and an upregulation of T cell absolute counts in most cases, thus showing effectiveness of treatment in COVID-19. SARS-CoV-2 infection induced cytokine overexpression in hospitalized patients with COVID-19 as well as lymphopenia, particularly a decrease in CD4+ and CD8+ T cell counts. Moreover, despite the reduced counts of CD4+ and CD8+ T cells, both subsets showed overactivation and increased expression of IL-17A and IL-22, thus targeting Th17 response might alleviate inflammatory response in severe disease.

Excessive BMI is associated with higher C-peptide level at recognition but also with its greater loss in two years clinical observation in children with new onset type 1 diabetes.

Introduction: The prevalence of obesity in general pediatric population increases without sparing children with T1D. We intended to find factors associated with the possibility of preserving endogenous insulin secretion in individuals with long-standing T1D. At onset, higher BMI is associated with higher C-peptide level, which may indicate to be one of the favorable factors involved in preserving residual ß-cell function. The study determines the influence of BMI on C-peptide secretion in children newly diagnosed with T1D in two years observation. Methods: We assessed the possible relationship between selected pro- and anti-inflammatory cytokines, body mass at recognition and ß-cell function status. 153 pediatric patients with newly diagnosed T1D were divided into quartiles according to BMI-SDS index. We separated a group consisted of patients with BMI-SDS >1. Participants were followed up for two years and examined for changes in body weight, HbA1c, and insulin requirement. C-peptide was assessed at baseline and after two years. We evaluated the patients' levels of selected inflammatory cytokines at baseline. Results: Subjects with higher BMI-SDS presented higher serum C-peptide levels and lower insulin requirements at diagnosis than children with lower body weight. The two-year follow-up showed that C-peptide levels of obese patients dropped more rapidly than in children with BMI-SDS within normal limits. The group with BMI-SDS >1 showed the greatest decrease in C-peptide level. Despite statistically insignificant differences in HbA1c at diagnosis between the study groups, in the fourth quartile and BMI-SDS >1 groups, HbA1c as well as insulin requirements increased after two years. The levels of cytokines varied the most between BMI-SDS <1 and BMI-SDS >1 groups and were significantly higher within BMI-SDS >1 group. Discussion: Higher BMI, associated with enhanced levels of inflammatory cytokines, relates to preservation of C-peptide at T1D recognition in children but is not beneficial in the long term. A decrease in C-peptide levels combined with an increase in insulin requirements and in HbA1c among patients with high BMI occur, which may indicate a negative effect of excessive body weight on the long term preservation of residual ß-cell function. The process seems to be mediated by inflammatory cytokines.

The short-term and long-term effects of intranasal mesenchymal stem cell administration to noninflamed mice lung.

Mesenchymal stem cells (mesenchymal stromal cells; MSC)-based therapies remain a promising approach to treat degenerative and inflammatory diseases. Their beneficial effects were confirmed in numerous experimental models and clinical trials. However, safety issues concerning MSCs' stability and their long-term effects limit their implementation in clinical practice, including treatment of respiratory diseases such as asthma, chronic obstructive pulmonary disease, and COVID-19. Here, we aimed to investigate the safety of intranasal application of human adipose tissue-derived MSCs in a preclinical experimental mice model and elucidate their effects on the lungs. We assessed short-term (two days) and long-term (nine days) effects of MSCs administration on lung morphology, immune responses, epithelial barrier function, and transcriptomic profiles. We observed an increased frequency of IFNγ- producing T cells and a decrease in occludin and claudin 3 as a long-term effect of MSCs administration. We also found changes in the lung transcriptomic profiles, reflecting redox imbalance and hypoxia signaling pathway. Additionally, we found dysregulation in genes clustered in pattern recognition receptors, macrophage activation, oxidative stress, and phagocytosis. Our results suggest that i.n. MSCs administration to noninflamed healthy lungs induces, in the late stages, low-grade inflammatory responses aiming at the clearance of MSCs graft.

Immunological balance between Treg and Th17 lymphocytes as a key element of type 1 diabetes progression in children.

Type 1 diabetes (T1D) is autoimmune destruction of the beta cells of pancreatic islets. Due to complexity of that disease, the mechanisms leading to the tolerance breakdown are still not fully understood. Previous hypothesis of imbalance in the Th1 and Th2 cells as the main contributing factor has been recently changed towards role of other lymphocytes - regulatory (Treg) and IL-17A-producing (Th17). Our study aims to assess changes within Treg and Th17 cells in newly diagnosed T1D pediatric patients and their association with disease remission. Flow cytometry implementation allowed for Treg and Th17 analysis in studied groups and further combination with clinical and laboratory data. In addition, expression of diabetes-related genes was tested and evaluated in context of their association with studied lymphocytes. Initial results revealed that Treg and ratio Treg/Th17 are significantly higher in T1D than in healthy controls. Moreover, patients with lower HbA1c and daily insulin requirements demonstrated higher levels of Tregs. Similar tendency for insulin intake was also observed in reference to Th17 cells, together with high levels of these cells in patients demonstrating higher values for c-peptide after 2 years. In low-level Treg patients, that subset correlates with the c-peptide in the admission stage. In addition, higher levels of IL-10 were associated with its correlation with HbA1c and insulin dosage. In the context of gene expression, moderate associations were demonstrated in T1D subjects inter alia between CTLA4 and Treg or ratio Treg/Th17. Cumulatively, our data indicate a possible novel role of Treg and Th17 in mechanism of type 1 diabetes. Moreover, potential prognostic value of these populations has been shown in reference to diabetes remission.

A Proliferation-Inducing Ligand and B-Cell Activating Factor Are Upregulated in Patients with Essential Thrombocythemia.

A proliferation-inducing ligand (APRIL) and B-cell activating factor (BAFF) are cytokines belonging to the tumor necrosis factor family which play an essential role in B-cell maturation, differentiation, and survival. Recent evidence indicates their importance in hematological disorders; however, their function in essential thrombocytosis (ET) pathogenesis remains elusive. Therefore, we aimed to analyze the role of APRIL and BAFF in megakaryocytopoiesis in ET patients. We observed elevated levels of APRIL and BAFF in the plasma of ET patients compared with healthy controls, while no differences were found among patients with different JAK2(V617F) statuses. In addition, APRIL levels were positively associated with the number of platelets and WBC count. In the bone marrow, APRIL but not BAFF levels were higher in ET patients with the JAK2(V617F) mutation; however, JAK2(V617F)-negative patients showed slightly reduced levels of BAFF. In ET patients, we showed that the differentiation of CD34+ progenitor cells towards megakaryocytes induces the expression of both APRIL and BAFF. More importantly, APRIL neutralization significantly reduced platelet production. In conclusion, our data provide evidence that blocking APRIL signaling, which acts as an autocrine growth factor for terminal megakaryocytopoiesis, inhibits platelet production in ET patients, regardless of the status of JAK2(V617F) mutation.

The Phenotype of the Adipocytes Derived from Subcutaneous and Visceral ADMSCs Is Altered When They Originate from Morbidly Obese Women: Is There a Memory Effect?

Adipose tissue is an abundant source of mesenchymal stem cells (ADMSCs). Evidence has suggested that depot-specific ADMSCs (obtained from subcutaneous or visceral adipose tissue-subADMSCs or visADMSCs, respectively) account for differential responses of each depot to metabolic challenges. However, little is known about the phenotype and changes in metabolism of the adipocytes derived from ADMSCs of obese individuals. Therefore, we investigated the phenotypic and metabolic characteristics, particularly the lipid profile, of fully differentiated adipocytes derived from ADMSCs of lean and obese (with/without metabolic syndrome) postmenopausal women. We observed a depot-specific pattern, with more pronounced changes present in the adipocytes obtained from subADMSCs. Namely, chronic oversupply of fatty acids (present in morbid obesity) triggered an increase in CD36/SR-B2 and FATP4 protein content (total and cell surface), which translated to an increased LCFA influx (3H-palmitate uptake). This was associated with the accumulation of TAG and DAG in these cells. Furthermore, we observed that the adipocytes of visADMSCs origin were larger and showed smaller granularity than their counterparts of subADMSCs descent. Although ADMSCs were cultured in vitro, in a fatty acids-deprived environment, obesity significantly influenced the functionality of the progenitor adipocytes, suggesting the existence of a memory effect.

Circulating Hematopoietic (HSC) and Very-Small Embryonic like (VSEL) Stem Cells in Newly Diagnosed Childhood Diabetes type 1 - Novel Parameters of Beta Cell Destruction/Regeneration Balance and Possible Prognostic Factors of Future Disease Course.

AIMS/HYPOTHESIS: We aimed to evaluate hematopoietic stem cells (HSC) and very small embryonic-like stem cells (VSEL) mobilization to establish their role in residual beta cell function maintenance and partial remission occurrence in children newly diagnosed with type 1 diabetes. METHODS: We recruited 59 type 1 diabetic patients (aged 6-18 years) monitored for 2 years, and 31 healthy children as a control group. HSC and VSEL levels were assessed at disease onset in PBMC isolated from whole peripheral blood with the use of flow cytometry. An assessment of beta cell function was based on C-peptide secretion. Studied groups were stratified on the basis of VSEL, HSC and/or C-peptide median levels in regard to beta cell function and partial remission. RESULTS: Patients with higher stimulated C-peptide secretion at disease onset demonstrated lower levels of HSC (p < 0.05), while for VSEL and VSEL/HSC ratio higher values were observed (p < 0.05). Accordingly, after 2 years follow-up, patients with higher C-peptide secretion presented lower initial levels of HSC and higher VSEL/HSC ratio (p < 0.05). Patients with lower values of HSC levels demonstrated a tendency for better partial remission prevalence in the first 3 to 6 months after diagnosis. CONCLUSIONS: These clinical observations indicate a possible significant role of HSC and VSEL in maintaining residual beta cell function in type 1 diabetic patients.

Abdominoplasty Skin-Based Dressing for Deep Wound Treatment-Evaluation of Different Methods of Preparation on Therapeutic Potential.

The management of hard-to-heal wounds is a significant clinical challenge. Acellular dermal matrices (ADMs) have been successfully introduced to enhance the healing process. Here, we aimed to develop protocol for the preparation of novel ADMs from abdominoplasty skin. We used three different decellularization protocols for skin processing, namely, 1M NaCl and sodium dodecyl sulfate (SDS, in ADM1); 2M NaCl and sodium dodecyl sulfate (SDS, in ADM1); and a combination of recombinant trypsin and Triton X-100 (in hADM 3). We assessed the effectiveness of decellularization and ADM's structure by using histochemical and immunochemical staining. In addition, we evaluated the therapeutic potential of novel ADMs in a murine model of wound healing. Furthermore, targeted transcriptomic profiling of genes associated with wound healing was performed. First, we found that all three proposed methods of decellularization effectively removed cellular components from abdominoplasty skin. We showed, however, significant differences in the presence of class I human leukocyte antigen (HLA class I ABC), Talin 1/2, and chondroitin sulfate proteoglycan (NG2). In addition, we found that protocols, when utilized differentially, influenced the preservation of types I, III, IV, and VII collagens. Finally, we showed that abdominoplasty skin-derived ADMs might serve as an effective and safe option for deep wound treatment. More importantly, our novel dressing (ADM1) improves the kinetics of wound closure and scar maturation in the proliferative and remodeling phases of wound healing. In conclusion, we developed a protocol for abdominoplasty skin decellularization suitable for the preparation of biological dressings. We showed that different decellularization methods affect the purity, structure, and therapeutic properties of ADMs.

Regulatory B Cells Involvement in Autoimmune Phenomena Occurring in Pediatric Graves' Disease Patients.

Graves's disease is the most common type of autoimmune hyperthyroidism. Numerous studies indicate different factors contributing to the onset of the disease. Despite years of research, the exact pathomechanism of Graves' disease still remains unresolved, especially in the context of immune response. B cells can play a dual role in autoimmune reactions, on the one hand, as a source of autoantibody mainly targeted in the thyroid hormone receptor (TSHR) and, on the other, by suppressing the activity of proinflammatory cells (as regulatory B cells). To date, data on the contribution of Bregs in Graves' pathomechanism, especially in children, are scarce. Here, we investigated the frequencies of Bregs before and during a methimazole therapy approach. We reported higher Foxp3+ and IL-10+ Breg levels with CD38- phenotype and reduced numbers of CD38 + Foxp3 + IL-10+ in pediatric Graves' patients. In addition, selected Breg subsets were found to correlate with TSH and TRAb levels significantly. Noteworthy, certain subpopulations of Bregs were demonstrated as prognostic factors for methimazole therapy outcome. Our data demonstrate the crucial role of Bregs and their potential use as a biomarker in Graves' disease management.

Differential Response of MDA-MB-231 and MCF-7 Breast Cancer Cells to In Vitro Inhibition with CTLA-4 and PD-1 through Cancer-Immune Cells Modified Interactions.

Drugs targeting immune checkpoint molecules have been found effective in melanoma, lung cancer, and other malignancies treatment. Recent studies on breast cancer demonstrated the significance of inhibitory anti-CTLA-4 and anti-PD-1 in the regulation of disease progression. However, seemingly the same types of breast cancer do not always respond unambiguously to immunotherapy. Thus, here we set out to analyze the in vitro effects of inhibiting CTLA-4 and PD-1 on interactions between co-cultured lymphocytes and two selected breast adenocarcinoma cell lines. Breast cancer cells were co-cultured with lymphocytes to evaluate the effects of CTLA-4 and PD-1 inhibition. Proliferation, cell cycle, and viability assessment were measured in cancer cells. IFN-gamma, IL-10, perforin, granzyme B production, and CTLA-4 and PD-1 expression were analyzed in lymphocytes. We found that administration of anti-CTLA-4 improved the anti-cancer activity of T cells with reduced proliferation and viability of MDA-MB-231. Lack of response was observed in the context of MCF-7. In addition, differential expression of checkpoint proteins was found between studied cancer cells lines. Inhibition of molecules was followed by IL-10 and IFN-gamma decrease in lymphocytes co-cultured with MDA-MB-231, not demonstrated in reference to MCF-7. Furthermore, CTLA-4 blockage was associated with reduction of CTLA-4+ and PD-1+ lymphocytes in MDA-MB-231, with a significant increase in MCF-7, reduced by anti-PD-1. Altogether, our study revealed that anti-CTLA-4 and anti-PD-1 treatment can improve lymphocytes effects on breast cancer cells. Favorable effects seemed to be related to breast cancer cells features as differential responses were reported. Novel blocking antibodies strategies should be tested for more effective cancer inhibition.

Evaluating the Role of Circulating Dendritic Cells in Methimazole-Treated Pediatric Graves' Disease Patients.

Graves' disease (GD) is hyperthyroidism associated with organ-specific autoimmune inflammation. GD occurs more frequently in adults than in children; however, pediatric patients are a therapeutic challenge due to cycles of remissions and relapses requiring constant monitoring at every stage of treatment administered. Dendritic cells (DCs) are considered to be a link between innate and adaptive immunity. DCs, as antigen-presenting cells (APCs), are involved in antigen presentation to T lymphocytes, thereby initiating a shift towards effector cells. In accordance, DCs also participate in the modulation of tolerance to specific antigens. To date, the data on DCs' role in Graves' pathological processes are scarce. Therefore, here, we evaluated the frequencies and role of circulating DCs in GD pediatric patients treated with methimazole. Flow cytometric analysis was implemented to evaluate three subsets of dendritic cells and their correlation with clinical GD-related parameters. We found significantly higher levels of DC subsets in patients at diagnosis. Furthermore, methimazole treatment seemed to effectively reduce subsets of DCs, which, in addition, were found to differentially correlate with thyroid function. Our study shed new light on DCs' role in the pediatric GD pathomechanism. Further studies are required for the mechanistic assessment of DCs' exact role in disease progression and influence on thyroid function.