Emerging evidence suggests the immune system has a role in preventing cancer, and in advanced cancer evidence of immune dysfunction is widespread. This project focused on cytotoxic T lymphocyte antigen 4 (CTLA4), a key negative regulator of T cell activation found on dedicated regulatory T cells (Treg) and activated T lymphocytes, and asked whether modulation of immune control with anti-CTLA4 blockade led to significant anti-tumour activity. Clinical and laboratory investigation of anti-CTLA4 blockade using tremelimumab in a phase II trial of second-line therapy in advanced oesophageal and gastric adenocarcinomas was combined with an attempt to establish a suitable pre-clinical model based on therapeutic vaccination against the tumour associated antigen (TAA) 5T4.Eighteen patients received tremelimumab. Most drug-related toxicity was mild but there was a single death due to bowel perforation. Four patients had stable disease with clinical benefit; one achieved a partial response after eight cycles (25.4 months) and remains well on study after four years. Markers of regulatory phenotype, forkhead box protein 3 (FoxP3) and CTLA4, doubled transiently in CD4+CD25high lymphocytes in the first month after tremelimumab before returning to baseline. In contrast, CTLA4 increased in CD4+CD25low/negative lymphocytes throughout the cycle of treatment. Post-treatment expanded Treg expressed FoxP3 without interleukin-2 and their defining suppressive function was not abolished despite prolonged anti-CTLA4 blockade. De novo proliferative responses to TAA 5T4 (8 of 18 patients) and carcinoembryonic antigen (CEA; 5 of 15) were detected. Patients with a post-treatment CEA proliferative response had median survival of 17.1 months compared to 4.6 months for non-responders (p=0.002). Baseline interleukin-2 release after T lymphocyte activation was higher in patients with clinical benefit and toxicity. Heterologous mouse 5T4 (m5T4) vaccination showed some evidence of weak therapeutic benefit, but all tumour models investigated had rapidly lethal kinetics. Specific m5T4 immune responses could be detected by serum antibody ELISA and IFN-gamma ELISPOT assays in naive animals but were lower frequency than published responses to h5T4, and were further attenuated in tumour-bearing animals. The addition of anti-CTLA4 blockade did not result in significant augmentation of m5T4 specific immunity after vaccination in non tumour-bearing animals and combination treatment was ineffective as therapy in this autologous model. Results are discussed in the context of emerging immunotherapeutics in melanoma and prostate cancer. In the absence of supportive data from the model system it would not be appropriate to pursue combination heterologous 5T4 vaccine with anti-CTLA4 blockade, but in view of the unusual durability of the best response to tremelimumab, and in vitro evidence of enhanced proliferative responses to relevant TAA, further investigation of drug activity may be warranted in metastatic gastric and oesophageal second-line treatment.

Glioblastoma multiforme (GBM), the most common and malignant brain tumor, is characterized by high molecular and cellular heterogeneity within and among tumors. Parameters such as invasive growth, infiltration of immune cells and endothelial proliferation contribute in a systemic manner to maintain the malignancy. Studies in this thesis show that the expression of Sox2 is correlated with Sox21 in human gliomas. We demonstrate that an upregulation of Sox21 induces loss of proliferation, apoptosis and differentiation in glioma cells in vitro and in vivo and seems to correlate with decreased Sox2 expression. Induced expression of Sox21 in vivo significantly reduces the tumor size and increase the survival extensively, suggesting that Sox21 can act as a tumor suppressor Our studies indicate that the balance of Sox21-Sox2 in glioma cells is decisive of either a proliferative or a non-proliferative state. Several TGFß family members have an important role in glioma development. TGFß promotes proliferation and tumorigenicity whereas BMPs mostly inhibit proliferation. We demonstrate that BMP7 can induce the transcription factor Snail in glioma cells and that this reduces the tumorigenicity with a concomitant increase in invasiveness. Thus, we have identified a mechanism to the double-edged sword of proliferation versus invasiveness in GBM, the latter contributing to relapse in patients. Experimental gliomas were induced with the Sleeping Beauty (SB) model in mice with different immunological status of their T cells. The tumors that developed were either GBMs or highly diffuse in their growth, reminiscent of gliomatosis cerebri (GC). GC is a highly uncommon form of glioma characterized by extensive infiltrative growth in large parts of the brain. It is an orphan disease and today there is practically a total lack of relevant experimental models. The SB system would constitute a novel experimental model to study the mechanisms behind the development of diffusely growing tumors like GC. The presence or absence of T cells did not affect tumor development. The work in this thesis demonstrates that the proliferative and the invasive capacities of glioma cells can be dissociated and that the SB model constitutes an excellent model to study the highly proliferative cells in GBMs versus the highly invasive cells in diffuse tumors like .GC.

Influenza is a serious illness worldwide, causing high morbidity and mortality. 10-20% of world population fall ill with influenza each year and 250 000 - 500 000 people die annually. The most efficacious protection to date is vaccination. Current vaccines are efficient only one season because of fast mutation rate of influenza virus. The effort to create an effective vaccine faces lack of potent adjuvant, which can adequately stimulate and modulate immune system to protect organism from virus infection. Moreover, todays vaccines administered parenterally do not induce immune response on mucosal surfaces. Bacillus firmus, a Gram-positive non-pathogenic bacterium, has strong immmune-modulating properties and is able to induce cross-protection when administered with influenza virus antigens. Immunization with Bacillus firmus stimulates production of neutralizing antibodies, but other mechanisms of its action remain to be elucidated. To better understand the mechanisms how is antiviral immunity enhanced by Bacillus firmus (delipidated fraction, DBF), the effect of immunization with DBF only was studied on mouse model. In last decade it has become obvious that intranasal immunization can induce both systemic and mucosal immune response and in case of influenza it can induce cross-protection. Therefore...

Les lymphocytes Th17 forment une population de cellules T CD4+ pro-inflammatoires, impliqués non seulement dans l'élimination d'agents pathogènes, mais ayant aussi un rôle délétère dans l'induction de maladies inflammatoires chroniques. Ils expriment spécifiquement le récepteur de chimiokines CCR6, qui a pour ligand le CCL20 mais aussi les β-defensine-1, 2 et 3, peptides ayant une activité antimicrobienne. Les cellules souches mésenchymateuses (CSMs) représentent une population cellulaire hétérogène exerçant diverses propriétés immunomodulatrices.Les résultats obtenus dans ce travail de thèse montrent que l'environnement inflammatoire contribue à augmenter l'adhésion des lymphocytes Th17 aux CSMs, et qu'elle est régulée par l'interaction du CCR6 avec ses ligands ; que les CSMs exercent, en partie via la sécrétion de PGE2, des effets anti-inflammatoires en faisant acquérir un phénotype régulateur aux lymphocytes Th17 différenciés, soulignant ainsi la plasticité de ces derniers.De plus, nous avons montré que les lymphocytes Th17 activés par l'antigène produisent du CCL20 et induisent, via la production de l'IL-17 et de l'IL-22, la sécrétion d'hBD-2, mais pas celle des hBD-1 et 3, par des kératinocytes épidermiques humains et de la peau reconstituée; que le CCL20, ainsi que la hBD-2, induisent l'arrêt de ces cellules sur l'endothélium enflammé in vitro en conditions de cisaillement. Finalement, l'activation spécifique d'antigène des lymphocytes Th17 entraîne une perte de l'expression de CCR6, ce qui provoque ainsi un état transitoire de non réponse à une nouvelle stimulation de ces cellules avec les ligands de CCR6, permettant leur migration ultérieure hors du tissu enflammé. / Th17 cells form a population CD4+ T cells with strong pro-inflammatory properties that are not only involved in the clearance of pathogens, but also play a deleterious role of in the pathogenesis of inflammatory disease. Th17 cells specifically express CCR6, a chemokine receptor that binds to its unique chemokine ligand, CCL20, as well as to human β-defensin (hBD)-1, 2 and 3, peptides with anti-microbial activity. Mesenchymal stem cells (MSC) represent a heterogenous population that exert broad immunomodulatory effects.The results from the studies carried out during this thesis show that the inflammatory environment contributes to increased adhesion of Th17 cells to MSCs, which is mediated via the interaction of CCR6 with its ligands, and that MSCs exert, in part via the secretion of PGE2, anti-inflammatory effects through the induction of a T regulatory cell phenotype in fully differentiated tissue-infiltrating Th17 cells, thereby underscoring the plasticity of the latter cells.Furthermore, the results show that antigen-activated Th17 cells produce CCL20 and induce, via the production of both IL-17 and IL-22, the secretion of hBD-2, but not 1 and 3, by normal human epidermal keratinocytes and reconstituted skin, and that CCL20, as well as hBD-2, induce arrest of these cells onto inflamed endothelium in vitro under conditions of shear stress. Finally, antigen-specific activation of Th17 cells also causes a loss of CCR6 expression from their cell surface and thus results in a transitory state of non-responsiveness to further stimulation of these cells with CCR6 ligands, which is likely to permit their subsequent migration out of inflamed tissue.

The aim of the study To investigate relationship between inflammation of the airways (bronchi) and pulmonary function. Objectives 1. To examine the levels of peripheral blood cytokines of patients suffering from COPD during exacerbation and remission, in order to detect non-invasive marker/markers of COPD that will enable us to differentiate infectious origin of exacerbation from non-infectious. 2. To investigate relationship between radiology changes of the lungs and respiratory function in patients suffering from chronic obstructive pulmonary disease. 3. To find out whether it is possible to evaluate changes of airway inflammation using classical inflammation and bacterial markers in patients suffering from COPD. To evaluate dependence of these markers on COPD clinical phase. 4. To evaluate the count of regulatory T lymphocytes (CD4+CD25+), as cells, possibly decreasing inflammation, in blood of the patients suffering from COPD and compare the results with results obtained from the group of patients who are not suffering from COPD. 5. To examine the amount of cells with CD25+ marker in mucosa of bronchi of patients suffering from COPD and compare this result with the result obtained in the control groups of non-smokers and smokers who are not suffering from COPD. Scientific novelty In practical work, the problem of timely diagnosis of exacerbation of the disease remains still important, despite significant achievements in research and treatment of COPD. The search of... [to full text] / Tyrimo tikslas: ištirti kvėpavimo takų (bronchų) uždegimo sąsajas su plaučių funkcija. Tyrimo uždaviniai 1. Ištirti asmenų, sergančių LOPL, periferinio kraujo citokinų koncentraciją ligos paūmėjimo ir remisijos metu, tikintis rasti neinvazinį LOPL paūmėjimo žymenį (žymenis), leisiantį atskirti infekcinę paūmėjimo kilmę nuo neinfekcinės. 2. Ištirti radiologinių plaučių pokyčių ir kvėpavimo funkcijos rodiklių sąsajas sergant lėtine obstrukcine plaučių liga. 3. Ištirti, ar apie kvėpavimo takų uždegimo pokyčius sergant LOPL galima spręsti iš klasikinių uždegimo ir bakterijų žymenų pokyčių kraujyje. Įvertinti šių kraujo žymenų priklausomybę nuo LOPL klinikinės fazės. 4. Nustatyti T reguliacinių limfocitų (CD4+CD25+), kaip galimai uždegimą slopinančių ląstelių, kiekį LOPL sergančių ligonių kraujyje ir palyginti su LOPL nesergančių asmenų grupe. 5. Ištirti CD25+ žymenį turtinčių ląstelių kiekį LOPL sergančių ligonių bronchų gleivinėje ir palyginti jį su LOPL nesergančių rūkančių ir nerūkančių asmenų kontrolinėmis grupėmis. Mokslinė darbo reikšmė ir naujumas. Nežiūrint didelių pasiekimų tiriant ir gydant LOPL, praktiniame darbe iki šiol išlieka savalaikio ligos paūmėjimo nustatymo problema. Nuolat ieškoma potencialių neinvazinių žymenų, kurie galėtų padėti laiku diagnozuoti LOPL paūmėjimą ir galimai nustatyti jo kilmę (bakterinis ar imuninis). Daug tikimasi iš citokinų, kaip potencialių LOPL paūmėjimo žymenų. Nors pavieniuose darbuose tirta IL-10 ir TNF-α koncentracija LOPL... [toliau žr. visą tekstą]

Tyrimo tikslas: ištirti kvėpavimo takų (bronchų) uždegimo sąsajas su plaučių funkcija. Tyrimo uždaviniai 1. Ištirti asmenų, sergančių LOPL, periferinio kraujo citokinų koncentraciją ligos paūmėjimo ir remisijos metu, tikintis rasti neinvazinį LOPL paūmėjimo žymenį (žymenis), leisiantį atskirti infekcinę paūmėjimo kilmę nuo neinfekcinės. 2. Ištirti radiologinių plaučių pokyčių ir kvėpavimo funkcijos rodiklių sąsajas sergant lėtine obstrukcine plaučių liga. 3. Ištirti, ar apie kvėpavimo takų uždegimo pokyčius sergant LOPL galima spręsti iš klasikinių uždegimo ir bakterijų žymenų pokyčių kraujyje. Įvertinti šių kraujo žymenų priklausomybę nuo LOPL klinikinės fazės. 4. Nustatyti T reguliacinių limfocitų (CD4+CD25+), kaip galimai uždegimą slopinančių ląstelių, kiekį LOPL sergančių ligonių kraujyje ir palyginti su LOPL nesergančių asmenų grupe. 5. Ištirti CD25+ žymenį turtinčių ląstelių kiekį LOPL sergančių ligonių bronchų gleivinėje ir palyginti jį su LOPL nesergančių rūkančių ir nerūkančių asmenų kontrolinėmis grupėmis. Mokslinė darbo reikšmė ir naujumas. Nežiūrint didelių pasiekimų tiriant ir gydant LOPL, praktiniame darbe iki šiol išlieka savalaikio ligos paūmėjimo nustatymo problema. Nuolat ieškoma potencialių neinvazinių žymenų, kurie galėtų padėti laiku diagnozuoti LOPL paūmėjimą ir galimai nustatyti jo kilmę (bakterinis ar imuninis). Daug tikimasi iš citokinų, kaip potencialių LOPL paūmėjimo žymenų. Nors pavieniuose darbuose tirta IL-10 ir TNF-α koncentracija LOPL... [toliau žr. visą tekstą] / The aim of the study To investigate relationship between inflammation of the airways (bronchi) and pulmonary function. Objectives 1. To examine the levels of peripheral blood cytokines of patients suffering from COPD during exacerbation and remission, in order to detect non-invasive marker/markers of COPD that will enable us to differentiate infectious origin of exacerbation from non-infectious. 2. To investigate relationship between radiology changes of the lungs and respiratory function in patients suffering from chronic obstructive pulmonary disease. 3. To find out whether it is possible to evaluate changes of airway inflammation using classical inflammation and bacterial markers in patients suffering from COPD. To evaluate dependence of these markers on COPD clinical phase. 4. To evaluate the count of regulatory T lymphocytes (CD4+CD25+), as cells, possibly decreasing inflammation, in blood of the patients suffering from COPD and compare the results with results obtained from the group of patients who are not suffering from COPD. 5. To examine the amount of cells with CD25+ marker in mucosa of bronchi of patients suffering from COPD and compare this result with the result obtained in the control groups of non-smokers and smokers who are not suffering from COPD. Scientific novelty In practical work, the problem of timely diagnosis of exacerbation of the disease remains still important, despite significant achievements in research and treatment of COPD. The search of... [to full text]

Type 1 Diabetes (T1D) is organ-specific autoimmune disease which causes pancreatic beta cells to be irreversibly destroyed. The only possible treatment represents life-lasting insulin administration. The real trigger of destructive insulitis isn't known. T1D is a multi- factorial disease involving both external and internal factors in the disease pathogenesis. The presence of autoreactive T lymphocytes in pancreas is necessary for development of diabetes. T regulatory cells have protective function in the destructive insulitis. The aim of this diploma thesis was to study cord blood T regulatory cells and their connection to type 1 diabetes development. We tried to find the difference among T regulatory cells in mononuclear cord blood cells (CBMC) in different study groups. Samples were collected from mothers suffering from T1D, gestational diabetes. Healthy controls were tested as well. Sixty-eight samples of cord blood were included in the study among the years 2009 - 2011. Samples were divided into 3 groups (CBMC from children born to T1D mothers, mothers with gestational diabetes and healthy mothers without T1D). CBMC were ana- lysed by flow cytometry. T regulatory cells (defined as CD4+CD25+) were isolated by magnetic separation (MACS). The functional capacity of these cells was studied as well by...

Type 1 diabetes (T1D) is an autoimmune disease with multifactorial aetiology that involves an attack of self-reactive cytotoxic CD8 lymphocytes on insulin-producing beta cells in the pancreas. In the T1D pathophysiology, both innate and adaptive immunity mechanisms cooperate in the development of inflammation leading to autoimmune destruction. Autoreactive T lymphocytes are the canonical destructors of the beta cells, and B cells produce autoantibodies; the innate immunity cells are considered the initiators of the pathological autoimmune reaction by promoting T and B cell activation. Here, we provide evidence of both innate and adaptive immunity cell types dysregulation in patients with T1D, and that these changes occur before the onset of the disease. The changes in T regulatory lymphocytes (Tregs) and B cell subpopulations occur already in asymptomatic T1D first-degree relatives. During the first year after the onset of the disease, there is a gradual decrease in the neutrophil numbers in the periphery, which probably infiltrate the pancreas. We have focused more closely on the innate immunity dysregulation and its contribution to T1D pathogenesis. Initially, we describe that neutrophil products called neutrophil extracellular traps (NETs) are able to induce IFNγ-producing T cells through...