The manual differential count of leukocytes is a common analysis in the hematological laboratory. It is used for morphological assessment of the blood cells and can get valuable information according to diagnosis of hematological diseases. The microscopy assessment is dependent of a good staining result of the blood smear in order to get the best conditions to differentiate the different cell types and detect morphological or pathological findings.The aim of this study was to determine if it was possible to change the staining method without any compromises to the quality of the staining results. For this study 25 whole blood samples were collected, and blood smears was made and stained with four different staining protocols, including the one currently used in the routine practice at laboratoriemedicin Sundsvall. The samples were examined by three biomedical scientists and the staining quality of the cells was graded on a four-point scale. The statistical results with Friedmans and Wilcoxons signed-rank test showed differences between the methods on the nuclear and cytoplasm of lymphocytes and the nuclear of monocytes and neutrophils. The recommended staining protocol from the manufacturer was the method that had highest frequency of statistically significant differences compared to the other methods for those cell types. The differences were in favour for the other methods, and the current method showed the best performance. In conclusion it’s not recommended to continue the study with the manufacturer´s staining protocol, but its valuable to continue compare the best performed staining protocols.

Psuedothrombocytopenia, the presence of platelet clumping on the peripheral blood film, is usually considered to rule out the diagnosis of immune thrombocytopenia (ITP) or other causes of increased platelet destruction and to be a laboratory artifact of no clinical significance. We report a case of concurrent psuedothrombocytopenia and ITP. While the examination of the peripheral blood film remains a critical element of the diagnostic approach to thrombocytopenia, this case emphasizes the importance of confirming the relevance of morphologic findings by additional testing.

Malignant melanoma is an invasive type of skin cancer with high mortality rates, if not detected promptly. The mortality trends are generally linked to multiple dysplastic nevi, positive family history, genetic susceptibility and phenotypic features including fair skin, freckles, numerous atypical nevi, light coloured hair and eyes, inability to tan and prolonged exposure to ultraviolet radiation B (UVB). To date, the major anti-cancer therapeutics for melanoma include surgery, chemotherapy, radiotherapy, and immunotherapy. Recently, extracellular vesicles, especially exosomes, have been highlighted for their therapeutic benefits in numerous chronic diseases such as cancer. Exosomes display multifunctional properties, including inhibition of cancer cell proliferation and initiation of apoptosis. Hence, this study aimed to evaluate the genotoxicity and cytotoxicity of cord blood stem cell-derived (CBSC) exosomes on 6 samples of peripheral blood lymphocytes taken from healthy individuals and melanoma patients and on 3 samples of melanoma (CHL-1) cells. The limited number of samples was due to the time limitations and restrictions that were in place due to the COVID-19 pandemic. In this in vitro study, the optimal concentration of CBSC-derived exosomes (0, 100, 200, 300, 400 μg/ml protein at 24, 48 and 72h treatments) was confirmed by the CCK-8 assay. CBSC exosomes (300 μg/ml) were used to treat lymphocytes and CHL-1 cells in the Comet assay and evaluated using the real-time polymerase chain reaction (qPCR) and Western blotting (WB). The data of the CCK-8 and Comet assays illustrated that exosomes exerted genotoxic effects on CHL-1 cells (CCK-8 assay, ****p < 0.0001), (Comet assay, *p <0.05, **p < 0.01). However, the data portraying a reduction in the viability of lymphocytes needs further investigation as the number of samples was limited, therefore, further clarification is required. Importantly, no significant adverse effect was observed in healthy lymphocytes when treated with the same exosomes (p = ns). When further challenged with UVA+B radiation, the exosomes did not induce any genoprotective effect on ROS-induced CHL-1 cells, compared to the positive control (p = ns). Our data insinuates that the damage might be caused by inducing apoptosis. The anti-tumourigenic potential of exosomes was observed by activating the p53-mediated apoptotic pathway in CHL-1 cells, up-regulating p53, p21 and caspase 3 and down-regulating BCL-2 at mRNA (**p < 0.01, ***p <0.001, ****p <0.0001) and protein levels (*p < 0.05, **p <0.01). The potency of CBSC exosomes in inhibiting cancer progression in CHL-1 cells whilst causing no harm to the healthy lymphocytes makes it an ideal potential candidate for anti-cancer therapy. More samples are required to evaluate the therapeutic effect of exosomes on lymphocytes from cancer patients to fully understand their mechanism of action.

Obesity is a health issue of both individual and global importance. Evidence from rodent literature suggests that dietary preferences for fat and sugar might influence dopaminergic signaling in the brain and thus executive cognition. These diet-related changes could provide a mechanistic basis potentially explaining obesity-promoting behaviour. However, valid evidence for this link in humans is still scarce. This thesis aimed to add to this gap by studying dopamine-related gene expression profiles in peripheral cells and executive cognition in a human sample (n = 75). The results provide indications for an association between dietary preference and alterations in dopamingeric sigaling on a peripheral gene expression level even though the group differences were not statistically significant. A link to cognition could not be established with the methods applied. Yet, several targets for future research are suggested to further explore this interplay.

<p>Several labs have previously demonstrated that humoral immune responses at one mucosal tissue can disseminate to other mucosal sites, giving rise to the theory of the common mucosal immune system (CMIS). Evidence that demonstrates a similar link between systemic immune responses and mucosal protection is lacking despite indications that both mucosal and systemic memory B cells share common circulatory pathways. The focus of this study is to determine the contribution of blood-borne B cells to mucosal immunity in humans, and how B cells trafficking through blood can be induced to traffic to mucosal tissues.</p> <p>To address these aims, I have performed several analyses of active and inactive peripheral blood memory B cell (MBC) populations in normal, healthy humans. Analysis of recently activated blood B cells confirmed the revealed that the majority of pre-plasma cells (PPC) in blood of healthy, normal humans secrete IgA, and that the majority of IgA- positive PPC secrete primarily polymeric IgA. A large fraction of blood PPC also express CCR10, and this population contains the highest fraction of IgA-expressing and pIgA-secreting PPC in blood. In contrast, most CCR10- PPC secrete IgG, although a small fraction secretes and stains positive for IgA, and analysis of α4β7expression by blood MBC revealed that CCR10 is more inclusive of IgA-switched and pIgA-secreting PPC in blood. The data presented in this study demonstrate that IgA+/CCR10+ PPC represent the mucosal subset of PPC in the blood of healthy humans, and can be investigated as representative of recent or ongoing mucosal immune responses.</p> <p><em>In vitro </em>polyclonal stimulation of blood MBC through CD40 was able to induce CCR10 expression by blood MBC treated with IL-21, IL-2, IL-10 with additions of other germinal center (GC) cytokines such as IL-5 and TGF-β enhancing CCR10 induction. Surprisingly, CCR10 expression by IgG MBC stimulated under these conditions was similar to that of IgA MBC, demonstrating that CCR10 expression is not limited to IgA- switched B cell clones. The results of this study demonstrate that CCR10 expression is inducible by many GC factors, and importantly, is not limited to IgA-switched B cells.</p> <p>Systemic immunization of healthy volunteers with tetanus toxoid/diphtheria vaccine induced a robust systemic IgG response, but also resulted in a post-immunization mobilization of IgA PPC in blood. These PPC were not specific for tetanus or diphtheria, and in several volunteers showed specificity for mucosal pathogens such as poliovirus (PV) and herpes simplex virus (HSV) glycoproteins. In addition, stimulation of anti-HSV IgG memory was also observed. Thus we have demonstrated that a systemic immune response is capable of inducing antibody relevant to mucosal immunity, possibly exposing a mechanism through which systemic and mucosal humoral immune responses are linked.</p> <p>The studies presented here demonstrate the presence of mucosal B cell memory in blood and thus provide new insight into ways to assess and manipulate mucosal immunity.</p> / Doctor of Philosophy (Medical Science)

no / Recent studies have gained positive results using nanoparticles (NPs) in treating atherosclerosis on animals. But their toxicity and application in treating other heart diseases such as heart failure and endocarditis still need proper investigation. Gold nanoparticles (Au-NPs) were chosen as model substances as they have been successfully used in treating cancer. In this study, we use both H9C2 myoblasts and rat peripheral blood mononuclear cells to determine the influence of Au-NP size on their cytotoxicity and cell apoptosis. H9C2 cells were treated with Au-NPs of a diameter of 5, 20, 40 and 100nmfor 24 hrs before their cell viabilities tested by MTT assay, cell apoptosis measured by flow cytometry, and the generation of reactive oxygen species (ROS) detected by Fluorometric Intracellular ROS Kit. Distribution of the Au-NPs and their effects on the structure of mitochondria and lysosome were detected by electron microscopy. In addition, we obtained rat peripheral blood mononuclear cells and treated them with Au-NPs same with H9C2 cell line. Our results showed NPs of 5, 40, and 100 nm reduced cell viabilities on H9C2 cells while20nm showed no change on cell viability (Ctrl: 100±8.2 vs 20nm: 95.39±9.13, P>0.05, n=6) and some protect effect on ISO induced H9C2 cells apoptosis (ISO: 100±13.5 vs 20nm: 80.19±17.36, P>0.05, n=6). All size of Au-NPs reduced cell viabilities on rat peripheral blood mononuclear cells while 40nm showed the least reduction on cell viability (Ctrl: 100.0±3.0 vs 40nm: 76.31±3.68, P<0.001, n=6) and significant protect effect on ISO-induced rat peripheral monocytes apoptosis (ISO: 100±1.86 vs 40nm: 45.34±10.32, P<0.05, n=6). In addition, 20nm Au-NP showed some protect effect on ROS generation on ISO-induced H9C2 cells (ISO: 100±3.79 vs 20nm: 94.84±4.98, P>0.05, n=6), while 40nm produced more ROS (ISO: 100±3.79 vs 40nm: 141.63±42.81, P>0.05, n=6). Electron microscopy detection showed correlated results in structure. These results on H9C2 cell line are basically in agreeable to our animal study. The protective effect of 20nm may due to its ability to protect ISO-induced ROS generation. The results on rat peripheral monocytes are slightly different to those on H9C2 cells. Further investigation need to focus on the role of NPs size on cell apoptosis by detecting autophagy specific protein through western blotting. / Abstract of conference paper.

Cellular stress is central to the understanding of pathological mechanisms and the development of new therapeutic strategies and serves as a biomarker for disease progression in neurodegeneration, diabetes, cancer, cardiovascular and other chronic diseases. The common cellular stress assay (CSA) based on Seahorse technology in peripheral blood mononuclear cells (PBMCs) shows inconsistent results, which prevents its use as a biomarker for the progression of chronic diseases. Therefore, the aim of this study was to investigate potential factors that affect the CSA in PBMCs. We measured the CSA parameters in PBMCs from study participants and compared the results according to the potential factors, namely, the PBMC isolation method, age, seasonal variation and the gender of the study participants. PBMCs were isolated by OptiPrep® and RobosepTM-S methods. PBMCs isolated with the OptiPrep method showed much higher extracellular acidification and higher respiration compared to Robosep-isolated cells. Moreover, OptiPrep-isolated cells showed a higher number of outliers for the proton production rate (PPR) and a high respiratory quotient, indicating impurities with other cells, such as platelets, and technical inconsistencies. PBMCs from older individuals showed higher maximal respiration, spare capacity and extracellular acidification than younger participants. Additionally, in winter, maximal respiration and spare capacity decreased. From spring until early autumn, spare capacity and maximal respiration continuously increased. Elderly males also showed higher basal respiration, spare capacity and extracellular acidification than females. In conclusion, the findings of this study clearly demonstrate that the results of CSA parameters measured in PBMCs are influenced by the PBMC isolation method, age, seasonal variation and gender. Therefore, we recommend that researchers and physicians properly interpret the results of CSA parameters in PBMCs by considering these factors. It is important to use separate CSA evaluation standards based on the isolation method, age, gender and season-dependent factors. To assess the cellular stress situation in PBMCs, both extracellular acidification and mitochondrial respiration should be taken into account. Further study of additional factors, such as mitochondrial mass, should be conducted to improve the measurement of CSA parameters for the assessment of the real mitochondrial fitness.

BACKGROUND: The course of disease for patients with idiopathic pulmonary fibrosis (IPF) is highly heterogeneous. Prognostic models rely on demographic and clinical characteristics and are not reproducible. Integrating data from genomic analyses may identify novel prognostic models and provide mechanistic insights into IPF. METHODS: Total RNA of peripheral blood mononuclear cells was subjected to microarray profiling in a training (45 IPF individuals) and two independent validation cohorts (21 IPF/10 controls, and 75 IPF individuals, respectively). To identify a gene set predictive of IPF prognosis, we incorporated genomic, clinical, and outcome data from the training cohort. Predictor genes were selected if all the following criteria were met: 1) Present in a gene co-expression module from Weighted Gene Co-expression Network Analysis (WGCNA) that correlated with pulmonary function (p < 0.05); 2) Differentially expressed between observed "good" vs. "poor" prognosis with fold change (FC) >1.5 and false discovery rate (FDR) < 2 %; and 3) Predictive of mortality (p < 0.05) in univariate Cox regression analysis. "Survival risk group prediction" was adopted to construct a functional genomic model that used the IPF prognostic predictor gene set to derive a prognostic index (PI) for each patient into either high or low risk for survival outcomes. Prediction accuracy was assessed with a repeated 10-fold cross-validation algorithm and independently assessed in two validation cohorts through multivariate Cox regression survival analysis. RESULTS: A set of 118 IPF prognostic predictor genes was used to derive the functional genomic model and PI. In the training cohort, high-risk IPF patients predicted by PI had significantly shorter survival compared to those labeled as low-risk patients (log rank p < 0.001). The prediction accuracy was further validated in two independent cohorts (log rank p < 0.001 and 0.002). Functional pathway analysis revealed that the canonical pathways enriched with the IPF prognostic predictor gene set were involved in T-cell biology, including iCOS, T-cell receptor, and CD28 signaling. CONCLUSIONS: Using supervised and unsupervised analyses, we identified a set of IPF prognostic predictor genes and derived a functional genomic model that predicted high and low-risk IPF patients with high accuracy. This genomic model may complement current prognostic tools to deliver more personalized care for IPF patients.

CHARLES UNIVERSITY IN PRAGUE Faculty of Pharmacy in Hradec Králové Department of Biological and Medical Sciences Study program: Health Care Bioanalytics Candidate: Bc. Nikola Fučíková Supervisor: doc. MUDr. Lukáš Smolej, Ph.D. Title of diploma thesis: Prognostic significance of atypical leukemic cell morphology in chronic lymphocytic leukemia The aim of this thesis is to evaluate the prognostic significance of atypical cell morphology and smudge cells in patients with untreated chronic lymphocytic leukemia. We performed differential leukocytes count and classified lymphocytes as typical and atypical in a cohort of 101 patients (median age, 66 years; males, 69%, Rai III/IV stages, 18%). For atypical CLL, we used the 15% threshold and 59% of patients were classified as atypical CLL (aCLL). For smudge cells, we chose the 30% threshold and 33% of patients were classified as smudge cells positive. Patients in early clinical Rai stage (0) had significantly higher number of smudge cells (p=0.04). We didn't find a significant association between aCLL / smudge cells with modern prognostic indicators. We didn't find a relationship between aCLL and the time to first-line therapy (p=0.394). However, patients with aCLL had a significantly shorter overall survival (p=0.0397). There was a trend toward shorter...

Inflammatory bowel diseases (IBD) are an autoimmune illnesses affecting gastrointestinal tract. The main types include ulcerative colitis and Crohn's disease. Recently, primary sclerosing cholangitis (PSC) has also been associated with IBD. PSC is a chronic liver disease associated with bile duct stenosis. The exact pathogenesis and etiology of these diseases is not clear, despite the great efforts of the scientific community. They are multifactorial diseases that are associated with dysbiosis of intestinal microbiota. Their diagnosis is based on for patients unpleasant endoscopic examinations and therefore the search for new serum biomarkers is needed and appreciated target of scientific interest. In the first part of diploma thesis, we focused on the reactivity of peripheral blood cells of IBD patients to 10 selected representatives of typical intestinal microbiota: Lactobacillus plantarum, Bifidobacterium adolescentis, Blautia coccoides, Roseburia intestinalis, Eubacterium rectale, Faecalibacterium prausnitzii, Ruminococcus flavefaciens, Bacteroides thetaiotaomicron, Prevotella ruminicola and Escherichia coli. Reactivity of CD, UC and PSC- IBD patients was increased after stimulation with Faecalibacterium, Lactobacillus and Prevotella. However, we got low percentage of cytokine-producing cells,...