Differential expansion of circulating human MDSC subsets in patients with cancer, infection and inflammation

Background Myeloid-derived suppressor cells (MDSC) are a functional myeloid cell subset that includes myeloid cells with immune suppressive properties. The presence of MDSC has been reported in the peripheral blood of patients with several malignant and non-malignant diseases. So far, direct comparison of MDSC across different diseases and Centers is hindered by technical pitfalls and a lack of standardized methodology. To overcome this issue, we formed a network through the COST Action Mye-EUNITER (www.mye-euniter.eu) with the goal to standardize and facilitate the comparative analysis of human circulating MDSC in cancer, inflammation and infection. In this manuscript, we present the results of the multicenter study Mye-EUNITER MDSC Monitoring Initiative, that involved 13 laboratories and compared circulating MDSC subsets across multiple diseases, using a common protocol for the isolation, identification and characterization of these cells. Methods We developed, tested, executed and optimized a standard operating procedure for the isolation and immunophenotyping of MDSC using blood from healthy donors. We applied this procedure to the blood of almost 400 patients and controls with different solid tumors and non-malignant diseases. The latter included viral infections such as HIV and hepatitis B virus, but also psoriasis and cardiovascular disorders. Results We observed that the frequency of MDSC in healthy donors varied substantially between centers and was influenced by technical aspects such as the anticoagulant and separation method used. Expansion of polymorphonuclear (PMN)-MDSC exceeded the expansion of monocytic MDSC (M-MDSC) in five out of six solid tumors. PMN-MDSC expansion was more pronounced in cancer compared with infection and inflammation. Programmed death-ligand 1 was primarily expressed in M-MDSC and e-MDSC and was not upregulated as a consequence of disease. LOX-1 expression was confined to PMN-MDSC. Conclusions This study provides improved technical protocols and workflows for the multi-center analysis of circulating human MDSC subsets. Application of these workflows revealed a predominant expansion of PMN-MDSC in solid tumors that exceeds expansion in chronic infection and inflammation

Effects of catechin and cyclophosphamide derivatives on leukemic cells

Galusan (-)-epigalokatechiny (EGCG) jest pochodną katechiny najliczniej występującą w zielonej herbacie. Mafosfamid (MAF) jest pochodną cyklofosfamidu i należy do grupy związków alkilujących nowej generacji. Celem pracy było określenie działania EGCG i MAF na patologiczne komórki hematopoetyczne. Badania wykonano na komórkach ludzkiej promielocytarnej linii komórkowej HL-60. Komórki HL-60 eksponowano na działanie EGCG w dawkach 5 μg, 10 μg, 25 μg, 50 μg, 75 μg, oraz 100 μg w przeliczeniu na ml medium hodowlanego, natomiast MAF w dawkach 1 μg, 2,5 μg, 5 μg, 10 μg, 15 μg, 20 μg, 25 μg i 50 μg w przeliczeniu na ml medium hodowlanego. Analizowano także efekty kombinowanego działania EGCG i MAF na komórki leukemiczne. Funkcjonalne i morfologiczne zmiany zachodzące w komórkach HL-60 określono przy użyciu metody spektrofotometrii i cytometrii przepływowej po 24 i 48 godzinach po ich potraktowaniu. Aktywność metaboliczną komórek określono stosując test MTT (bromku 3[4,5-dimetylo-2-ilo]-2,5-difenylotetrazolu) i test FDA/PI (dioctan fluoresceiny / jodek propidyny), a wielkość komórek i gęstość analizując wartości FSC (ang. forward scatter channel) i SSC (ang. side scatter channel). Pochodne katechiny i cyklofosfamidu spowodowały czasowe zmiany w aktywności metabolicznej komórek określonej ilością utworzonego formazanu przy zastosowaniu testu MTT oraz procentowymi ilościami komórek w populacjach FDA+/PI-, FDA-/PI-, FDA-/PI+ przy użyciu testu FDA/PI, jak również czasowe zmiany w wielkości i ziarnistości komórek określane procentową ilością komórek w populacjach FSC high/SSC low, FSC low/ SSC high, FSC high/SSC high. Przeciwleukemiczny potencjał EGCG i MAF był zależny od zastosowanego związku, jego dawki oraz od punktu czasowego, w którym przeprowadzano analizy. W przypadku kombinowanego działania EGCG i MAF na komórki HL-60 zaobserwowano nasilenie aktywności przeciwnowotworowej. Wyniki przeprowadzonych badań są pierwszymi danymi ukazującymi efekty kombinowanego działania mafosfamidu i galusanu (-)-epigalokatechiny na ludzkie komórki leukemiczne.(-)-Epigallocatechin gallate (EGCG) is the major catechin found in green tea. Mafosfamide (MAF) is a cyclophosphamide derivative and belongs to the new generation alkylating agents. The aim of the study was to determine the alone and combined effects of EGCG and MAF on pathological hematopoitic cells.The investigations were conducted on human promyeolocytic HL-60 cells. The leukemic cells were exposed to the action of EGCG at doses 5 μg, 10 μg, 25 μg, 50 μg, 75 μg, 100 μg per 1 ml of medium and MAF at doses 1 μg, 2,5 μg, 5 μg, 10 μg, 15 μg, 20 μg, 25 μg, 50 μg per 1 ml of medium. The effects of EGCG and MAF on HL-60 cells were analyzed at 24 and 48 h after their exposure. The research was conducted using spectrophotometric and flow cytometric methods. The MTT assay, FDA/PI method and FSC?SSC measurement were used. After exposure of HL-60 cells to the EGCG and MAF the functional and morphological changes were observed. The various patterns of temporary alteration in the cell viability, cell membrane damage, the cell size and granularity were found. These are the first data that show the combined action of EGCG and MAF on human leukemic cells

The Legal Aspects of In Vitro Fertilisation (selected issues)

Przedmiotem niniejszej pracy są prawne aspekty zapłodnienia pozaustrojowego. Pracę rozpoczyna omówienie procedury in vitro oraz jej charakteru prawnego, czyli czy jest to lecznicza czynność medyczna, czy nieterapeutyczna czynność lekarska. Następnie zarysowane zostały problemy prawne, które niesie ze sobą stosowanie tej metody. Stanowiło to przyczynek do poszukiwania rozwiązań prawnych w międzynarodowych regulacjach i orzecznictwie, ze szczególnym uwzględnieniem systemów Rady Europy i Unii Europejskiej. Zaprezentowano także rozstrzygnięcia przyjęte w wybranych obcych porządkach prawnych. W dalszej części pracy dokonano analizy początku ochrony życia i zdrowia ludzkiego na gruncie Konstytucji i prawa karnego w odniesieniu do embrionu in vitro. Opisane zostały próby uregulowania medycznie wspomaganej prokreacji, które były podejmowane na przestrzeni ostatnich lat. Na końcu omówione zostały rozwiązania przyjęte w ustawie o leczeniu niepłodności.The subject of this thesis are the legal aspects of in vitro fertilisation. It disscusses the in vitro procedure and its legal nature, that is whether it is a therapeutical intervention or a non-therapeutical medical act. The legal problems of this method are then described. It was to look for some legal solutions in international law and judicature, especially in the Council of Europe and the European Union legal systems. The regulations in selected foreign countries are also presented. The rest of the dissertation focuses on the beginning of the protection of human life and health on the grounds of the Constitution and criminal law, with special emphasis on its implications for the fertilised embryo. The projects of legal regulation of the medically assisted procreation that have been practised in the past few years are also described. The thesis ends with an analysis of the solutions presented in the Infertility Treatment Act

Secretory leukocyte protease inhibitor is present in circulating and tissue-recruited human eosinophils and regulates their migratory function

Eosinophils and secretory leukocyte protease inhibitor (SLPI) are both associated with Th2 immune responses and allergic diseases, but whether the fact that they are both implicated in these conditions is pathophysiologically related remains unknown. Here we demonstrate that human eosinophils derived from normal individuals are one of the major sources of SLPI among circulating leukocytes. SLPI was found to be stored in the crystalline core of eosinophil granules, and its dislocation/rearrangement in the crystalline core likely resulted in changes in immunostaining for SLPI in these cells. High levels of SLPI were also detected in blood eosinophils from patients with allergy-associated diseases marked by eosinophilia. These include individuals with eosinophilic granulomatosis with polyangiitis (EGPA) and atopic dermatitis (AD), who were also found to have elevated SLPI levels in their plasma. In addition to the circulating eosinophils, diseased skin of AD patients also contained SLPI-positive eosinophils. Exogenous, recombinant SLPI increased numbers of migratory eosinophils and supported their chemotactic response to CCL11, one of the key chemokines that regulate eosinophil migratory cues. Together, these findings suggest a role for SLPI in controlling Th2 pathophysiologic processes via its impact on and/or from eosinophils

Differential expansion of circulating human MDSC subsets in patients with cancer, infection and inflammation

Background Myeloid-derived suppressor cells (MDSC) are a functional myeloid cell subset that includes myeloid cells with immune suppressive properties. The presence of MDSC has been reported in the peripheral blood of patients with several malignant and non-malignant diseases. So far, direct comparison of MDSC across different diseases and Centers is hindered by technical pitfalls and a lack of standardized methodology. To overcome this issue, we formed a network through the COST Action Mye-EUNITER (www.mye-euniter.eu) with the goal to standardize and facilitate the comparative analysis of human circulating MDSC in cancer, inflammation and infection. In this manuscript, we present the results of the multicenter study Mye-EUNITER MDSC Monitoring Initiative, that involved 13 laboratories and compared circulating MDSC subsets across multiple diseases, using a common protocol for the isolation, identification and characterization of these cells. Methods We developed, tested, executed and optimized a standard operating procedure for the isolation and immunophenotyping of MDSC using blood from healthy donors. We applied this procedure to the blood of almost 400 patients and controls with different solid tumors and non-malignant diseases. The latter included viral infections such as HIV and hepatitis B virus, but also psoriasis and cardiovascular disorders. Results We observed that the frequency of MDSC in healthy donors varied substantially between centers and was influenced by technical aspects such as the anticoagulant and separation method used. Expansion of polymorphonuclear (PMN)-MDSC exceeded the expansion of monocytic MDSC (M-MDSC) in five out of six solid tumors. PMN-MDSC expansion was more pronounced in cancer compared with infection and inflammation. Programmed death-ligand 1 was primarily expressed in M-MDSC and e-MDSC and was not upregulated as a consequence of disease. LOX-1 expression was confined to PMN-MDSC. Conclusions This study provides improved technical protocols and workflows for the multi-center analysis of circulating human MDSC subsets. Application of these workflows revealed a predominant expansion of PMN-MDSC in solid tumors that exceeds expansion in chronic infection and inflammation