Ines Mrakovčić-Šutić

Early Changes in Frequency of Peripheral Blood Lymphocyte Subpopulations in Severe Traumatic Brain‐Injured Patients

Infections are leading causes of increased morbidity and mortality of severe traumatic brain‐injured (STBI) patients. The mechanism underlying the susceptibility to the infections is still unexplained. The purpose of the study was to investigate changes in frequency of leucocytes subpopulations in peripheral blood of patients with STBI during the course of intensive care treatment. Twenty patients with STBI were included in the study. Healthy age‐ and sex‐ volunteers served as control. Peripheral blood samples were taken from these patients at day 1, 4 and 7, and peripheral blood mononuclear cells (PBMC) were isolated. The percentage of T, B lymphocyte, NK and NKT cells as well as monocytes was analysed by simultaneous detection of surface antigens using fluorochrome‐conjugated monoclonal antibodies. The two major subsets of T lymphocytes (CD3+CD56−CD4+ and CD3+CD56−CD8+) and NK cells (CD3−CD56+dim and CD3−CD56+bright) were also analysed by flow cytometry. Extracranial infections were presented in 55% patients with STBI. At day 4, the percentage of T lymphocytes with cytotoxic phenotype significantly diminished and their numbers restored at day 7. The frequency of NKT cells showed the identical time‐dependent pattern, whereas the percentage of NK cells diminished on day 4 but did not restore after 7 days. The frequency of B lymphocytes did not change significantly during the time investigated, whereas the percentage of monocytes increased immediately after the injury and gradually diminished. The decrease in cells with cytotoxic phenotype might explain high incidence of susceptibility to infection of patients with STBI.

Expression of cytolytic protein–perforin in peripheral blood lymphocytes in severe traumatic brain injured patients

PURPOSE The purpose of this study was to investigate the changes of cytotoxic protein-perforin in peripheral blood lymphocytes in severe TBI patients and possible correlation between severity of TBI and perforin expression. METHODS Flow cytometry was used for simultaneous detection of intracellular perforin and cell surface antigens of peripheral blood lymphocytes of 20 severe TBI patients on day 1, 4 and 7 after the onset of injury. Peripheral blood mononuclear cells from 20 healthy volunteers were used as control. Clinical and laboratory parameters were also recorded. RESULTS There was a statistically significant decrease of perforin-positive lymphocytes including T, natural killer (NK) and NKT cells on day 4 as compared with day 1 after the brain injury or healthy controls. On day 7, perforin expression was restored in lymphocyte of cytotoxic phenotype (CD8(+) T lymphocytes, NK cells, and NKT cells) compared with day 1. High positive correlation was found between the severity of TBI and frequency of perforin-positive cells on day 4 when the occurrence of the intra-hospital infections was the highest. CONCLUSION Severe TBI significantly decreases perforin expression in T lymphocytes, NK and NKT cells, which indicate a possible mechanism underlying the high susceptibility to infections.

Metallothionein expression and tissue metal kinetics after partial hepatectomy in mice

To better elucidate previous results showing that partial hepatectomy noticeably changes the tissue content of zinc, calcium, magnesium, and iron(II) ions in regenerating the liver, thymus, and spleen, we report on the correlation of these metal tissue kinetics in these organs with the expression of metallothionein-I+II (MT-I+II) proteins and MT-I mRNA in early postoperative period (1, 2, 6, 12, and 24 h) after one-third hepatectomy (pHx). The results showed that 2 h after pHx the regenerating liver accumulated Zn2+, Ca2+, Mg2+, and Fe2+ ions while decreasing the concentration of all these metals in the spleen and of Zn2+ in the thymus. On the 24th h, a new high accumulation of Zn2+ and Ca2+ was seen in the regenerating liver and of Zn2+, Ca2+, and Fe2+ in the spleen. Simultaneously, MT-I mRNA increased in the liver and spleen. In hepatocytes and on several spleen and thymus mononuclear lymphatic cells, the increased expression of MT proteins was found mainly in the cytoplasm and nuclei. The areas expressing MTs in regenerating liver inversely correlated with those containing apoptotic cells, suggesting that these proteins participate in tissue restoration through reduction or increase of metal ions after injury to the liver.

Metal tissue kinetics in regenerating liver, thymus, spleen and submandibular gland after partial hepatectomy in mice

Liver regeneration after partial hepatectomy (pHx) is a well-defined process, which involves the concerted action of extra- and intracellular factors resulting in induction of cell replication and its inhibition at the time when the entire liver mass is restored. Concomitantly, the breakdown of previously maintained tolerance and the exposure of self-antigens lead to the activation of preimmune and immune repertoires, which participate in surveillance against aberrant cells and the re-establishment of previous morphostasis. Because, in these events, important biological function might have tissue minerals that are affecting the structural integrity and enzyme activities, transduction signals, transcription and replication factors during cell proliferation and apoptosis, as well as the development and maintenance of immune functions and cytokine production, in this study we analyzed tissue dynamics of zinc, ioron magnesium, and calcium in the liver, thymus, spleen, and submandibular gland in intact and pHx mice on the 1st, 2nd, 7th, and 15th d after one-third pHx, using microwave digestion and inductivity coupled plasma spectrometry. The data showed that pHx induces significant and interconnected changes in all of the estimated metals not only in the regenerating liver but also in the lymphatic tissues and submandibular gland, indicating their importance for the control of growth processes.

Syngeneic pregnancy induces overexpression of natural killer T cells in maternal liver.

Conditions such as stress, infection, autoimmune disease, etc. elevate the number and function of extrathymic T cells that are generated mainly in the liver. As primitive, self‐reactive clones of T cells that coexpress receptors of the natural killer (NK) lineage, they mediate cytotoxicity against altered self, malignant and infected cells and have the unique potential to rapidly secrete large amount of T helper 1 (Th1) or Th2 cytokines.

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.

Immunological consequences of lesions of nucleus basalis in rats

In this study we followed up the effects of acute (ten days) and chronic (two months) stereotaxic lesions of NB in rats on: 1.) cellularity of lymphatic organs, 2.) humoral and 3.) cellular immunity and 4.) in vitro functional reactivity of splenocytes to polyclonal mitogens. Experiments were done on (AoxDA) F1 male rats, aged 4 months and maintained in standard laboratory conditions. The coordinates for bilateral stereotaxic lesions were taken from atlas of Pellegrino. Electrolysis was performed unipolarly at the anode with 10 mA for 10s. Histological verification of the lesions was performed by the method of Wolf. For the evaluation of humoral or cell mediated immunity standard plaque forming cell assay to sheep red blood cells (SRBC) or transplantation of allogeneic skin were used. The data have shown that bilateral electrolytic lesions of NB cause the prolongation of rejection time to allogeneic skin, transplanted on tenth p.o. (15.8+-0.4 days versus 12.8+-0.5 in intact control, p<.001). Some prolongation of allograft survival was, however, seen also in group of rats in which electrodes were lowered in NB without turning off the electric current, indicating the consequences of mechanical lesions of NB on cell mediated immunity. Humoral immunity tested after sensitization of rats with SRBC, on tenth p.o. was found also to be slightly depressed and followed by depletion of thymus, but the values were on the border of statistical significance. Chronic electrolytic lesions of NB in rats which were not otherwise sensitized, revealed that in comparison to appropriate controls, the cellularity of thymus of NB lesioned rats is higher, while the cellularity of spleen is decreased. Although the tendency of hypoplasia of bone marrow seems to exist, the results are not statistically significant. The data imply that NB is not important only for several behavioural functions but also for mechanisms involved in the maintenance of normal immunological response and composition of lymphoid organs. It is obvious that NB might play an important role in the reestablishment of homeostasis after the initiation of an immune response.

Augmentation of NKT and NK cell-mediated cytotoxicity by peptidoglycan monomer linked with zinc

BACKGROUND: Peptidoglycan monomer (PGM), which was originally prepared by biosynthesis from culture fluids of penicillin-treated Brevibacterium divaricatum, is an immunostimulator, the activities of which might be improved by addition of zinc (Zn) to the basic molecule. METHODS: To test the possible cytotoxic effects of this new analogue, we analyzed the ability of PGM-Zn and PGM to change the phenotypic profile of hepatic and splenic mononuclear lymphatic cells and to affect the growth of malignant T-cell line YAC-1 and syngeneic thymocytes. RESULTS: Pretreatment of C57BL/6 mice primarily with PGM-Zn over 6 days (10/mg/kg intraperitoneally) significantly enhanced the proportions of NK1.1high+, CD4-CD8-, CD69+, and CD3intermediate/NK1.1+/IL2R-beta+ (NKT) cells in the liver, and major histocompatibility complex class II+, CD69+, and CD8+ cells in the spleen. Both types of cells were highly cytotoxic against YAC-1 and syngeneic thymocytes, increasing the destruction of YAC-1 by 70% on addition of hepatic cells and by 30% on addition of splenic cells. Destruction of thymocytes increased by 10 and 50%, respectively. CONCLUSION: The results point to PGM-Zn as a potent cytotoxicity-inducing agent, which also generates autoreactive NKT cells.

Crosstalk Between Enzyme Matrix Metalloproteinases 2 and 9 and Regulatory T Cell Immunity in the Global Burden of Atherosclerosis.

Changes in immune and inflammatory responses may play a crucial role in the development and progression of atherosclerosis, as an autoimmune, chronic and progressive inflammatory disease. Immunological activity and vascular inflammation during atherosclerosis can be modulated by autoimmune responses against self‐antigens, according to changeable risk factors (cholesterol, oxidized low‐density lipoprotein (ox‐LDL) in the vascular wall, fatty acids, etc.), and accompanied by accumulation of leucocytes and proinflammatory cytokines, which stimulate the transcription of matrix metalloproteinases (MMPs), whose concentration are increased in foam cell‐rich regions. Regulatory T cells (Tregs) represent a unique subpopulation of T cells specialized in the regulation of immune response and in the suppression of proatherogenic T cells. The aim of our study was to examine the interactions between the concentration of enzyme matrix metalloproteinases 2 and 9 (MMP‐2 and 9) in urine and the percentage of Tregs in peripheral blood of two groups of patients: with carotid artery stenosis (CAS), undergoing surgery and with mild atherosclerosis (A) from general practice. The method of enzyme immunoassay (ELISA) was used to determine enzyme MMP expression, and Tregs was examined by flow cytometric analysis. Our data have showed a large increase in the enzyme MMP‐2 and 9 in the urine of CAS and A patients in comparison with healthy controls and indicated this method as an easy marker for the monitoring of the development of atherosclerosis. Simultaneously, the diminished number of Tregs in the same patients pointed the importance of these regulatory mechanisms in the etiopathogenesis of atherosclerosis and possible Tregs‐mediated therapy.