Marin Dominovic

Extracellular vesicles in blood, milk and body fluids of the female and male urogenital tract and with special regard to reproduction

Abstract Extracellular vesicles (EVs) are released from almost all cells and tissues. They are able to transport substances (e.g. proteins, RNA or DNA) at higher concentrations than in their environment and may adhere in a receptor-controlled manner to specific cells or tissues in order to release their content into the respective target structure. Blood contains high concentrations of EVs mainly derived from platelets, and, at a smaller amount, from erythrocytes. The female and male reproductive tracts produce EVs which may be associated with fertility or infertility and are released into body fluids and mucosas of the urogenital organs. In this review, the currently relevant detection methods are presented and critically compared. During pregnancy, placenta-derived EVs are dynamically detectable in peripheral blood with changing profiles depending upon progress of pregnancy and different pregnancy-associated pathologies, such as preeclampsia. EVs offer novel non-invasive diagnostic tools which may reflect the situation of the placenta and the foetus. EVs in urine have the potential of reflecting urogenital diseases including cancers of the neighbouring organs. Several methods for detection, quantification and phenotyping of EVs have been established, which include electron microscopy, flow cytometry, ELISA-like methods, Western blotting and analyses based on Brownian motion. This review article summarises the current knowledge about EVs in blood and cord blood, in the different compartments of the male and female reproductive tracts, in trophoblast cells from normal and pre-eclamptic pregnancies, in placenta ex vivo perfusate, in the amniotic fluid, and in breast milk, as well as their potential effects on natural killer cells as possible targets.

Cell death mechanisms at the maternal-fetal interface: insights into the role of granulysin.

During mammal pregnancy, a sensitive balance between hormones, cytokines, humoral factors, and local cellular interactions must be established. Cytotoxic cells infiltrating the decidua are heavily equipped with cytolytic molecules, in particular perforin and granulysin. Granulysin is especially abundant in NK cells which are able to spontaneously secrete high quantities of granulysin. Besides being a potent bactericidal and tumoricidal molecule, granulysin is also found to be a chemoattractant and a proinflammatory molecule. The precise role(s) of granulysin at the maternal-fetal interface has not been elucidated yet. It is possible that it behaves as a double-edged sword simultaneously acting as an immunomodulatory and a host defense molecule protecting both the mother and the fetus from a wide spectrum of pathogens, and on the other hand, in case of an NK cell activation, acting as an effector molecule causing the apoptosis of semiallograft trophoblast cells and consequently leading to various pregnancy disorders or pregnancy loss.

Granulysin expression and the interplay of granulysin and perforin at the maternal-fetal interface.

Granulysin (GNLY) is a cytolytic/apoptotic molecule highly expressed in immune cells, particularly NK cells, at the maternal-fetal interface. The primary function of GNLY is to carry out lysis or apoptosis induction in target cells, tumor cells or cells infected by intracellular pathogens. To exert some of its functions GNLY needs to collaborate with perforin. The purpose of this study was to determine: (a) the expression of GNLY at the gene and protein levels at the maternal-fetal interface, (b) the relationship(s) between GNLY and perforin, and (c) GNLY secretion by NK cells stimulated by the NK-sensitive K562 cell line and its HLA-C and HLA-G transfectants. GNLY and perforin genes were found to be highly activated at the interface. GNLY mRNA was present at significantly higher levels compared with other cytolytic/apoptotic molecules. Confocal microscopy analysis showed that most first trimester pregnancy decidual lymphocytes simultaneously contained both GNLY and perforin protein in their cytoplasm, with a punctuate pattern consistent with granule localization. In contrast to peripheral blood, in unstimulated decidual lymphocytes GNLY and perforin rarely co-localized (10% of GNLY-positive cells and 20% of perforin-positive cells were positive for both proteins). Contact between decidual lymphocytes and K562 cells caused GNLY and perforin to be expressed in the same granules (approximately 50% co-localization), i.e., to attain the pattern seen in peripheral blood lymphocytes. The abundant GNLY secretion by decidual NK cells compared with peripheral blood NK cells after 2h of contact with the NK-sensitive K562 cells and K562 transfectants was striking.

Mucins help to avoid alloreactivity at the maternal fetal interface.

During gestation, many different mechanisms act to render the maternal immune system tolerant to semi-allogeneic trophoblast cells of foetal origin, including those mediated via mucins that are expressed during the peri-implantation period in the uterus. Tumour- associated glycoprotein-72 (TAG-72) enhances the already established tolerogenic features of decidual dendritic cells with the inability to progress towards Th1 immune orientation due to lowered interferon (IFN)-γ and interleukin (IL)-15 expression. Mucine 1 (Muc 1) supports alternative activation of decidual macrophages, restricts the proliferation of decidual regulatory CD56+ bright natural killer (NK) cells, and downregulates their cytotoxic potential, including cytotoxic mediator protein expression. Removing TAG-72 and Muc 1 from the eutopic implantation site likely contributes to better control of trophoblast invasion by T cells and NK cells and appears to have important immunologic advantages for successful implantation, in addition to mechanical advantages. However, these processes may lead to uncontrolled trophoblast growth after implantation, inefficient defence against infection or tumours, and elimination of unwanted immunocompetent cells at the maternal-foetal interface. The use of mucins by tumour cells to affect the local microenvironment in order to avoid the host immune response and to promote local tumour growth, invasion, and metastasis confirms this postulation.

Colocalization of Granulysin Protein Forms with Perforin and LAMP‐1 in Decidual Lymphocytes During Early Pregnancy

Granulysin (GNLY) occurs in two forms, which have molecular weights of 9 and 15 kDa. We analyzed the cytotoxic potential of decidual lymphocytes (DLs) and peripheral blood lymphocytes (PBLs) based on the forms of GNLY that colocalizes with perforin (PER) and LAMP‐1 following activation.

Comparative study of frequency of different lymphocytes subpopulation in peripheral blood of patients with prostate cancer and benign prostatic hyperplasia

ZusammenfassungZIEL: Benigne Prostatahyperplasie (BPH) und Prostatakrebs (PC) sind die häufigsten urologischen Erkrankungen bei Männern über fünfzig und sie wurden vor kurzem für das Ergebnis einer gestörten Immunantwort gehalten. Trotz vieler Studien zur auf T-Zellen basierten Anti-Tumor-Immunität, kann die Rolle der angeborenen Immunzellen bei BPH und PC noch immer schlecht verstanden werden. In dieser Studie wurde die Frequenz von verschiedenen Leukozytensubpopulationen im peripheren Blut von sowohl BPH- und PC-Patienten als auch von gesunden Probanden analysiert und gegenseitig verglichen. METHODEN: 60 Probanden wurden in eine Querschnittsstudie eingeschlossen (20 Patienten mit BPH, 20 Patienten mit PC und 20 gesunde Probanden). Mononukleäre Zellen des peripheren Blutes (PBMC) wurden isoliert und sowohl der Prozentsatz von T-Lymphozyten, natürlichen Killerzellen (NK) und natürlichen Killer-T-Zellen (NKT) als auch der Prozentsatz von Subsets der T-Lymphozyten [CD3+CD56–CD4+, TReg (CD4+CD25+FoxP3+) und CD3+CD56–CD8+] und der NK-Zellen (CD3–CD56+dim und CD3–CD56+bright) wurden mit Durchflusszytometrie analysiert. Auch der intrazelluläre Inhalt von Interleukin-4 (IL-4) und Gamma-Interferon (IFN-γ) wurde in T-Lymphozyten, NK- und NKT-Zellen gefunden. ERGEBNISSE: Das Prozent von T-Lymphozyten und deren Subsets in Lymphozyten des peripheren Blutes unterschied sich nicht unter den untersuchten Gruppen, während die Frequenz der regulatorischen T-Zellen (TReg) die höchste bei der PC-Patienten war. Höherer Anteil der B-Lymphozyten und NKT-Zellen wurde in Lymphozyten des peripheren Blutes von BPH-Patienten beobachtet. Der Anteil der NK-Zellen und deren Subsets unterschied sich nicht unter den untersuchten Gruppen. Eine negative Korrelation zwischen dem PSA-Wert und dem Prozentsatz der T-Lymphozyten und NK-Zellen wurde nur bei PC-Patienten bemerkt. Sehr positive Korrelation zwischen dem PSA-Wert und dem Prozentsatz der regulatorischen T-Zellen (TReg) stellte man bei PC-Patienten fest. SCHLUSSFOLGERUNG: Unterschiedliche Frequenz der verschiedenen Lymphozytensubpopulationen im peripheren Blut von gesunden Männern und BPH- und PC-Patienten könnte für das Auftreten und Fortschreiten der Prostatahyperplasie oder des Tumors verantwortlich sein. Aufgrund der Tumorfähigkeit, T-Zell-Immunantwort zu unterdrücken, könnten die Zellen der angeborenen Immunität (NKT-Zellen und regulatorische T-Zellen) die zentrale Rolle in der Immunpathogenese von PC und BPH spielen.SummaryPURPOSE: Benign prostatic hyperplasia (BPH) and prostate cancer (PC) are the most common urologic diseases among men over fifty and, until recently, they were considered to be caused by the impaired immune response. Despite many studies designed to investigate T-cell-based antitumor immunity, the role of innate immune cells in BPH and PC is still poorly understood. In this study the frequency of different leukocytes subpopulation in peripheral blood of BPH, PC patients and in healthy volunteers was analysed and compared. METHODS: In a cross-sectional study 60 subjects were enrolled (20 patients with BPH or with PC and 20 healthy volunteers). Peripheral blood mononuclear cells (PBMC) were isolated and the percentage of T lymphocytes, natural killer (NK) and NKT cells, as well as subsets of T lymphocytes [CD3+CD56–CD4+, Tregs (CD4+CD25+FoxP3+) and CD3+CD56–CD8+] and NK cells (CD3–CD56+dim and CD3–CD56+bright) were analysed by flow cytometry. Intracellular content of interleukin-4 (IL-4) and interferon gamma (IFNγ in T lymphocytes, NK and NKT cells were also detected. RESULTS: The percentage of T lymphocytes and their subsets in peripheral blood lymphocytes did not differ among investigated groups, while the frequency of Tregs was the highest in PC patients. The percentage of NK cell and their subsets did not differ among investigated groups. Negative correlation between PSA value, percentage of T lymphocytes and NK cells was observed only in PC patients. Highly positive correlation between the PSA value and the percentage of Tregs was found in PC patients. CONCLUSION: Different frequencies in distinctly lymphocyte subpopulation in peripheral blood of healthy men, BPH and PC patients could be responsible for occurrence and progression of prostatic hyperplasia or tumour. Due to the ability of tumours to suppress the cognate T cell immune response, the cells of innate immunity (NKT and Tregs) may be playing a key role in the immunopathogenesis of PC and BPH.

Different Perforin Expression in Peripheral Blood and Prostate Tissue in Patients with Benign Prostatic Hyperplasia and Prostate Cancer

Perforin (P) is a prototypical cytotoxic molecule involved in cell‐mediated immunity against various pathogens, alloantigens and particularly different tumours. The purpose of this study was to determine P expression in different lymphocyte subpopulations isolated from peripheral blood and prostate tissue of patients with benign prostatic hyperplasia (BPH) and prostate cancer (PCa) and compare it with the P expression found in the control group. Twenty subjects were recruited in each of the groups. Prostate mononuclear cells of the BPH and PCa tissues were isolated by enzymatic digestion and gradient density centrifugation, whereas peripheral blood mononuclear cells were isolated by gradient density centrifugation alone. Cells and tissue samples were labelled using monoclonal antibodies against P and different surface antigens (CD3, CD4, CD8 and CD56) and analysed by immunofluorescence and flow cytometry. Total P expression in peripheral blood lymphocytes did not differ significantly between BPH/PCa patients and control group, although the BPH and PCa tissue showed lower P expression level. A negative correlation between prostate‐specific antigen levels and the overall percentage of P+, CD3+CD56−P+, and CD3−CD56+P+ cells in the prostate tissue was observed only in patients with PCa. Our findings indicate that the low frequency of P+ lymphocytes, including T, NKT and NK cells, in the prostate tissue of patients with BPH and, particularly, PCa could be the consequence of local tissue microenvironment and one of the mechanisms involved in the pathogenesis of prostate hyperplasia following malignant alteration.

Potential role of heat-shock protein 70 and interleukin-15 in the pathogenesis of threatened spontaneous abortions.

The role of HSP70 and both its constitutive (Hsc) and inducible (Hsp) forms in the pathogenesis of threatened spontaneous abortions was investigated.

Possible role of granulysin in pathogenesis of osteoarthritis

Increased presence of immune mediator and cytotoxic/apoptotic molecule granulysin was noticed in different tissues during pathological processes with the domination of Th1 over Th2 mediated immunity. Beside granulysin expression in T and NKT cells, activated NK cells are thought to be the major source of chemotactic 15 kDa and cytotoxic 9 kDa granulysin in vivo. As NK cells are the principal joints tissue-infiltrating lymphocyte subset, we hypothesized that granulysin mediated human cell death (apoptosis) could be responsible for the relatively silent damage of the joints tissue without clinically notable signs of systemic inflammation in the patients with osteoarthritis (OA). The analyzes of the presence and frequency of granulysin expressing lymphocytes at protein and gene levels in peripheral blood and synovial samples and/or the samples of joints tissue after the joint replacement therapy in patients with OA could give the initial insight to evaluate our hypothesis. It would be of the particular interest to differentiate the expression of 9 kDa and 15 kDa granulysin forms in the effector cells, since only the shorter form exhibits cytotoxic properties. The measurement of granulysin mediated early apoptosis in human NK sensitive K562 cells could be suitable in vitro model for evaluating granulysin activity. Furthermore, disturbed balance of pro-inflammatory and anti-inflammatory cytokines in OA patients, could influence the level of the granulysin expression. Having in mind that the granulysin and its regulation is still unknown in the pathogenesis of OA, it could be worth to explore this important pro-inflammatory, cytotoxic/apoptotic mediator.

Immunoregulation by Cytolytic Pathways, Mucins and Progesterone at the Maternal-Fetal Interface

The maternal-fetal interface of pregnant mammals is characterized by a sensitive balance between hormones, cytokines, humoral factors and cellular interactions. Progesterone activity leads to the transformation of endometrial cells into the decidual phenotype and ensures the integrity of the maternal-fetal interface during trophoblast invasion and placenta maturation. Communication between the genomic and non-genomic progesterone-regulated signaling pathways could be of critical importance for the establishment of a correct endocrine-immune interaction in the human endometrium during the establishment and maintenance of pregnancy. Cytolytic cells (NK and T lymphocytes) infiltrating the decidua of pregnancy are heavily equipped with cytolytic molecules perofrin and granulysin. These molecules have very important role(s) in the maternal tolerance of fetoplacental unit, control of trophoblast invasion, defense against infective agents and malignancies, as well as immunomodulatory functions. Mucins MUC- 1 and TAG-72 are important features of secretory phase decidual reaction, while their dissapearance from the surface of the epithelial cells enables apposition, adhesion and infiltration of high quality blastocysts at the precise area at the time of the implantation. Muc-1 and TAG-72 induce antiinflammatory orientation of the decidual antigen presenting cells.