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Intracellular protein phosphorylation is a critical step in cellular activation stimulated by the binding of various ligands to cell surface receptors. This process is initiated by activation of specific protein-tyrosine kinases associated with intracellular domains of the respective ligand receptor. JAK-STATs pathway is one of the main pathways in the cell activation process and given their important role in various PIDs, STATs proteins have been extensively studied in immune function in health and disease. Therefore, our work has focused on investigating and evaluating STATs activation and establishing flow cytometric methods to assess their phosphorylation to be a surrogate marker as a fast and sensitive diagnostic tool to current methods such as WB. At the first, we studied STAT1 and STAT3 activation and established a flow cytometric procedure to analyze variations of INF-α- and IL-6-induced STAT1 and STAT3 phosphorylation in T cells from whole blood, respectively (publication ΙΙ). To examine whether our results were specific, the samples were also analyzed by WB in parallel. After that, we validated the normal values of pSTAT1 and pSTAT3 based on 21 healthy adult controls according to an appropriate validation process. We showed that, in contrast to the conventional methods like WB, our assay offers a diagnostic benefit by avoiding labor and time consumption, with the advantage of achieving an earlier diagnosis, which potentially leads to improve treatment decisions; hence, patient’s outcome (publication ΙΙ). Furthermore, we verified FCM-based pSTAT1 and pSTAT3 profiling established here in patients group suffering from CMC and HIES. Our results demonstrated that pSTAT1 and pSTAT3 assay is an effective tool to identify and characterize well-known PIDs such as CMC and HIES, respectively (publication ΙΙ). Next, we introduced a fast and straightforward flow cytometric assay for the assessment of T cell proliferation, based on the staining of phosphorylated STAT5A (publication ΙΙΙ). We showed that pSTAT5A represents an appropriate approach to predict the behavior of T cells upon activation by CD3/CD28 and PHA. FCM-based pSTAT5A profiling is an intracellular flow cytometric method, enabling the early and reliable detection of T cell proliferation without long time incubation (within 24 h instead to 5 days). Importantly, measurement of pSTAT5A represents a new principle to assess T cell proliferation. It reveals important information on T cell biology by using series of kinetics and different kinds of T cell stimulation. For instance: [1] after stimulation via CD3/CD28 and negative pSTAT5A and T cell proliferation, the immune defect could be occurred in the whole signaling cascade (TCR-IL-2 transcription-JAK3-STAT5), [2] After stimulation via external IL-2 and negative pSTAT5A, the immune defect could be localized in the signaling cascade (IL-2R – JAK3 – STAT5), [3] After stimulation via external IL-2 and positive pSTAT5A, the immune defect could be localized in the signaling cascade (TCR - IL-2 transcription) (publication ΙΙΙ). We showed a strong correlation between the STAT5A phosphorylation and the percentage of dividing cells (publication ΙΙΙ). Later on, we used the measurement established here to investigate whether the phosphorylation of STAT5A is an appropriate candidate for predicting CMV specific T cell proliferation. It is well-known that CMV specific T cells expand with CMV reactivation and are probably prerequisite for control and protection. We demonstrated that CMV specific pSTAT5A detection represents a fast and straightforward diagnostic tool to assess CMV specific T cell proliferation without requisite several days’ culture (publication ΙV). Furthermore, we showed a positive correlation between the percentage of pSTAT5A+ T cells vs. (1) CMV-IgG concentrations vs. (2) the percentage of expanded T cells and vs. (3) the percentage of initial CMV specific T cells (publication ΙV). Finally, we evaluated the diagnostic value of pSTAT5 assay and determined the percentage of pSTAT5A+ T cells cut-off value at which pSTAT5 assay has the greatest diagnostic potency. Our data showed that a cut-off value of 9.1 % could be used to assess CMV specific T cell proliferation with a specificity and sensitivity of 100% and 73%, respectively. We verified measurement established here by CMV specific T cells stimulation in three selected patients diagnosed with CARMIL2-mutation and suffering from chronic CMV infection. Our results showed that the complete and the partial deficiency of CMV and CD3/CD28 stimulated pSTAT5A correlated with the complete and the partial deficiency of CMV and CD3/CD28 stimulated T cell proliferation, respectively (publication ΙV). In conclusion, disorders in JAKs-STATs signal pathways in T cells may result in insufficient response to stimulants. Therefore, FCM-based pSTATs profiling is an effective tool for clinical laboratory diagnostics [1] to understand the susceptibility to recurrent opportunistic infections [2] to rapidly identify T cell proliferation [3] to investigate tumor-specific responses of CD8 T effector and memory cells (56) and finally [4] to identify and distinguish well-known PIDs like CARMIL-2 mutations, CMC, AD-HIES or AR-HIES.

Acute lymphoblastic leukaemia (ALL) is the most frequent childhood malignancy. One of the recent improvements in ALL treatment was the introduction of minimal residual disease (MRD) monitoring that enables risk stratification based treatment adaptation. The same MRD monitoring helps to choose relapse treatment, to guide indication for stem cell transplantation (SCT) and allows for a more personalized management of patients undergoing SCT. One of the main routes of MRD levels detection is characterisation of leukemic blasts using flow cytometry. However, flow cytometry is limited by its mainly manual expertise-based analysis. Such analysis is subjective and clearly insufficient for current complex data. While new computational tools are available for multidimensional flow cytometry data, there is an urgent need to test and adapt them for the use in clinical environment. The goal of this thesis is to detect immunophenotypes associated with leukaemia and their development by leveraging machine-assisted analysis of a set of diagnostic files selected based on information about more than three hundred thousand of multiparameter flow cytometry datasets. Advanced bioinformatic tools will help to detect blast and healthy haematopoietic populations, to derive their immunophenotypes and to identify individual...

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Since late 2019 a new coronavirus, SARS-CoV2, has spread through the world and caused a pandemic. The virus causes the heterogenous disease Covid-19, causing flu- like symptoms for most people but can cause Acute Respiratory Distress Syndrome and fatal outcome in some patients. The mechanism is not clearly understood, but depletion of the immunological response has been seen in patients with severe Covid-19. This study will focus on T- lymphocyte, T- cell subtypes, B- lymphocyte, NK- cell and intracytoplasmic interleukin subsets in hospitalized Covid-19 patients to examine immune phenotypes that could be related to, or cause, more severe disease. The method used is multicolour flow cytometry on whole blood and frozen PBMC. Comparison of fraction of each cell type from total cell count, with regard of underlying diseases, age, sex and other factors were done using multivariate regression analysis to evaluate mechanisms correlated to severe disease. The results showed overall lower lymphocyte counts in Covid- patients versus healthy controls. Lower lymphocyte counts were also observed in men versus women. Lower levels of NK-cells and B-cells were found in people in ages over 60 years.  Higher glucose levels were associated with lower B-cell and T-regulatory cell counts, while diabetes patients showed higher T-regulatory cell counts. No differences in proinflammatory cytokines were found between patient groups but were slightly higher in patients versus healthy controls. The conclusion of this study is that dysfunction of the immune system caused by infection of SARS-CoV2 gives lymphocytopenia and more severe outcome.

No description available.

This thesis addresses multiple approaches to investigating mechanisms of immune linked disease. There are four projects outlined below which describe the work of these investigations. First, educating students about techniques to study disease and therapies is an important area of research. Flow cytometry is a common technique in immunology and its versatility and high throughput abilities can be applied to many fields. While it is very useful, flow cytometry is a complex technique that requires training to operate and understand, and there are very few reports about administering effective training. This thesis outlines the first report of a full semester university course about flow cytometry. Students who completed the course reported increased confidence in their skill levels in conceptual, technical and analytical areas. Second, in the fight against cancer, immunotherapies may provide the necessary adaptability to successfully combat many cancer types. By strengthening and educating the immune system, clinicians can help patients fight cancer without resorting to harmful chemotherapeutics, or immunotherapies can be used in tandem with current treatments. Chimeric antigen receptor (CAR) T cells and checkpoint blockade are two of the most successful immunotherapies. CAR T cells combine the extraordinary binding ability of an antibody with T cell signaling molecules via genetic engineering, for a faster and more efficient cancer killing version of the patient's own T cells. These have been remarkably successful, but results depend on the specific signaling co-receptors that are included in the design. Increased understanding of co-receptor function could help in making CAR T cell design more specific, and enable CAR T cells to be effective against more types of cancers. Metabolic function is crucial in understanding T cell therapeutics because T cells need to use energy efficiently enough to compete with ravenous cancer cells. This thesis outlines an ongoing investigation into a co-receptor's effect on CAR T cell metabolism, suggesting that co-receptors can alter CAR T cell metabolism by increasing maximal respiration. Third, CD5 is a negative regulatory co-receptor on T cells that can modulate T cell activation. Related inhibitory co-receptors (PD-1 and CTLA-4) are currently being effectively blocked as checkpoint therapies to reactivate T cells towards cancerous cells. This thesis outlines ongoing work investigating CD5's impact on cellular metabolism. We have found that T cells without CD5 are hypermetabolic as compared to normal naïve T cells. CD5 deficient T cells also have higher maximal respiration, higher basal respiration and higher glycolytic capacity. These differences are also present transiently after non-specific activation. Thus, CD5 significantly regulates the ability of a T cell to use energy, suggesting that CD5 may be a good target for creating more efficient T cell immunotherapies. Fourth, in a separate project, this thesis examines environmental causes of disease. Asthma and allergies are common and growing problems in children and adults. Evaporative cooling can be a less expensive alternative to central cooling, but its effects on allergens and other bioaerosols in the home remains unclear. This project examines the relationship between evaporative cooling and bioaerosols (dust mites, bacterial endotoxin, and fungal β-(1→3)-D-glucans) in low income homes in Utah. We report significantly higher levels of these bioaerosols, particularly fungi in homes with evaporative cooling after adjusting for home-specific factors.

Thrombocytes are functional equivalents of mammalian platelets and also possess megakaryocyte features. It has been shown earlier that hox genes play a role in megakaryocyte development. Our earlier microarray analysis showed five hox genes, hoxa10b, hoxb2a, hoxc5a, hoxc11b and hoxd3a, were upregulated in zebrafish thrombocytes. However, there is no comprehensive study of genome wide scan of all the hox genes playing a role in megakaryopoiesis. I first measured the expression levels of each of these hox genes in young and mature thrombocytes and observed that all the above hox genes except hoxc11b were expressed equally in both populations of thrombocytes. hoxc11b was expressed only in young thrombocytes and not in mature thrombocytes. The goals of my study were to comprehensively knockdown hox genes and identify the specific hox genes involved in the development of thrombocytes in zebrafish. However, the existing vivo-morpholino knockdown technology was not capable of performing such genome-wide knockdowns. Therefore, I developed a novel cost- effective knockdown method by designing an antisense oligonucleotides against the target mRNA and piggybacking with standard control morpholino to silence the gene of interest. Also, to perform knockdowns of the hox genes and test for the number of thrombocytes, the available techniques were both cumbersome or required breeding and production of fish where thrombocytes are GFP labeled. Therefore, I established a flow cytometry based method of counting the number of thrombocytes. I used mepacrine to fluorescently label the blood cells and used the white cell fraction. Standard antisense oligonucleotide designed to the central portion of each of the target hox mRNAs, was piggybacked by a control morpholino and intravenously injected into the adult zebrafish. The thrombocyte count was measured 48 hours post injection. In this study, I found that the knockdown of hoxc11b resulted in increased number of thrombocytes and knockdown of hoxa10b, hoxb2a, hoxc5a, and hoxd3a showed reduction in the thrombocyte counts. I then screened the other 47 hox genes in the zebrafish genome using flow sorting method and found that knockdown of hoxa9a and hoxb1a also resulted in decreased thrombocyte number. Further, I used the dye DiI, which labels only young thrombocytes at specific concentrations and observed that the knockdown of hoxa10b, hoxb2a, hoxc5a, hoxd3a, hoxa9a and hoxb1a, lead to a decrease in young thrombocytes; whereas hoxc11b knockdown lead to increase in number of young thrombocytes. Using bromodeoxyuridine, I also showed that there is increase in release of young thrombocytes into peripheral circulation in hoxc11b knockdown fish which suggests that hoxc11b significantly promotes cell proliferation rather effecting apoptosis. In conclusion, I found six hox genes that are positive regulators and one hox gene is a negative regulator for thrombocyte development.

Glypican-3 (GPC3), the cellular membrane proteoglycan, has been established as a tumor biomarker for early diagnosis of hepatocellular carcinoma (HCC). GPC3 is highly expressed in more than 70% HCC tissues detected by antibody-based histopathological systems. Recently, aptamers, a short single-strand DNA or RNA generated from systematic evolution of ligands by exponential enrichment (SELEX), were reported as potential alternatives in tumor-targeted imaging and diagnosis. In this study, a total of 19 GPC3-bound aptamers were successfully screened by capillary electrophoresis (CE)-SELEX technology. After truncated, AP613-1 was confirmed to specifically target GPC3 with a dissociation constant (KD) of 59.85 nM. When modified with a phosphorothioate linkage, APS613-1 targeted GPC3 with a KD of 15.48 nM and could be used as a specific probe in living Huh7 and PLC/PRF/5 imaging, GPC3-positive cell lines, but not in L02 or A549, two GPC3-negative cell lines. More importantly, Alexa Fluor 750-conjugated APS613-1 could be used as a fluorescent probe to subcutaneous HCC imaging in xenograft nude mice. Our results indicated that modified AP613-1, especially APS613-1, was a potential agent in GPC3-positive tumor imaging for HCC early diagnosis.

DNA damage is a form of cell stress and injury. Increased systemic DNA damage is related to the pathogenic development of neurodegenerative diseases. Depression occurs in a relatively high percentage of patients suffering from degenerative diseases, for whom antidepressants are often used to relieve depressive symptoms. However, few studies have attempted to elucidate why different groups of antidepressants have similar effects on relieving symptoms of depression. Previously, we demonstrated that neurotoxins N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP4)- and camptothecin (CPT) induced the DNA damage response in SH-SY5Y cells, and DSP4 caused cell cycle arrest which was predominately in the S-phase. The present study shows that CPT treatment also resulted in similar cell cycle arrest. Some classic antidepressants could reduce the DNA damage response induced by DSP4 or CPT in SH-SY5Y cells. Cell viability examination demonstrated that both DSP4 and CPT caused cell death, which was prevented by spontaneous administration of some tested antidepressants. Flow cytometric analysis demonstrated that a majority of the tested antidepressants protect cells from being arrested in S-phase. These results suggest that blocking the DNA damage response may be an important pharmacologic characteristic of antidepressants. Exploring the underlying mechanisms may allow for advances in the effort to improve therapeutic strategies for depression appearing in degenerative and psychiatric diseases.