Production of cytokines during interaction of peripheral blood mononuclear cells with autologous ovarian cancer cells or benign ovarian tumour cells.

Cytokines produced by tumour and immune cells may play a significant role in a modulation of immune cells response against tumour. We investigated an ability of peripheral blood mononuclear cells (PBMC) of patients with early and advanced stages of ovarian cancer and from non-cancer patients to produce various cytokines in the presence or absence of autologous ovarian cancer (OC) cells or benign ovarian tumour (BOT) cells. Activated PBMC of patients with advanced stage of cancer produced slight amount of interferon gamma (IFN-gamma) and what's more, the production of IFN-gamma was decreased in the presence of OC cells. PBMC of patients with ovarian cancer or benign ovarian tumour generated comparable amounts of interleukin 6 and 10 (IL-6, IL-10), and transforming growth factor beta1 (TGF-beta1). PBMC of the patients with cancer produced higher amount of tumour necrosis factor alpha (TNF-alpha) than PBMC of non-cancer patients. We demonstrated here that the reciprocal contact of OC cells from advanced cancer with autologous PBMC altered the direction of produced cytokines and leads to the down-regulation of IFN-gamma and TNF-alpha as well as to up-regulation of immunosuppressive (IL-10, TGF-beta1) and pro-inflammatory (IL-6) cytokines production.

Ovarian cancer cells modulate human blood neutrophils response to activation in vitro.

In cancer, numerous cells of both innate and adaptive immune systems are activated. Polymorphonuclear neutrophils are potent effector cells of inflammation that are an important component of tumour development and progression. The important signalling proteins that are involved in neutrophil functions are extracellular signal-regulated kinases 1/2 (ERK1/2). We investigated the reactive oxygen species (ROS) production, adhesive ability and CD11b/CD18 adhesion molecule expression on neutrophils isolated from peripheral blood of ovarian cancer patients and the in vitro response of these cells to stimuli and direct contact with ovarian cancer cells isolated from tumour. We found that functional activities of neutrophils isolated from patients with advanced stages of ovarian cancer (FIGO III/IV) were intensified in comparison to neutrophils isolated from healthy female volunteers. Neutrophils of cancer patients produce higher amounts of ROS in response to stimuli than those of control group. Unstimulated neutrophils of patients possess higher expression of CD11b/CD18 molecule that is accompanied by increased adhesive ability of these cells. Our results reveal that augmented functional activities of neutrophils may result from the intensification of ERK1/2 kinases phosphorylation. We found that interactions with ovarian cancer cells modulate neutrophil functions as a result of cell-to-cell direct contact. We conclude that ovarian cancer cells affect pro-inflammatory activities in neutrophils via influence of signalling pathways in response to stimuli. Our results suggest the possibility that neutrophils responding to contact with cancer cells contribute to the progression and metastatic potential of tumour cells.

Signal transduction pathways affected by nitric oxide donors during neutrophil functional response in vitro.

OBJECTIVE AND DESIGN: We investigated the intracellular signalling pathways by which nitric oxide (NO) donors: diethylamine/NO (DEA/NO) and 3-morpholinosydnonimine (SIN-1) regulate the functional response of human neutrophils to activating stimuli. METHODS: The phosphorylation and nitration of signalling proteins, cyclic GMP level, neutrophil respiratory burst and adhesive activities and CD11b/CD18 molecule expression on neutrophils in the presence and absence of soluble guanylate cyclase inhibitors were determined. RESULTS: NO donors showed strong inhibitory effect on activated neutrophils. NO donors nitrated the tyrosine residues in signalling proteins causing a decrease in tyrosine phosphorylation and neutrophils response to activation. Diethylamine/NO employed cyclic GMP as a signalling molecule in its action on neutrophils, whereas peroxynitrite anion donor affected neutrophil functions in a cGMP-independent manner. Moreover, we observed that peroxynitrite anion can overcome the nitric oxide molecule action. CONCLUSIONS: We conclude that each NO donor depending on its concentration and chemical nature may act on different elements of neutrophil signalling pathways capable of inducing distinct neutrophil functions.

Mannan-binding lectin (MBL) in women with tumours of the reproductive system.

Mannan-binding lectin (MBL) is an important factor of innate immunity contributing to the clearance of microorganisms. Recently, an antitumourigenic role of MBL has been suggested. We investigated mbl2 genotypes, MBL concentrations, and MBL-MASP-2 complex activity in patients with ovarian cancer. The expression of both mbl2 and masp-2 genes were investigated in ovarian tissue sections. Additionally, samples from patients with other malignant and benign tumours of the reproductive tract were tested. A significantly higher incidence of MBL deficiency/insufficiency-associated genotypes was found among patients with malignant disease compared to age-matched controls. Unexpectedly, no differences in median MBL level or MBL-MASP-2 complex activity were found between the groups. This was partly a reflection of higher MBL concentrations and MBL-MASP-2 activity in cancer patients compared with healthy women carrying corresponding genotypes. MBL-specific mRNA expression was detected in several normal and malignant ovarian tissues, as well as in ovarian epithelial cell lines. Intracellular staining with MBL-specific antibodies demonstrated the presence of MBL in ovarian cell lines, and in normal as well as malignant ovarian tissue sections. In contrast, MASP-2-specific mRNA expression was detected only in the ovary tissues of patients with malignant disease. No significant changes in MBL concentration during 3 months of chemotherapy were noticed. MBL was detected in ascites and in the fluid of benign ovarian cysts. Our findings may reflect anti-tumourigenic activity of MBL protein which might suggest potential therapeutic application. However, it cannot be excluded that mbl-2 mutant alleles may be in linkage disequilibrium with an unidentified tumour susceptibility gene(s).

The effect of interleukin-6 on hepatitis B virus replication in peripheral blood mononuclear cells in vitro.

Although the major target organ for hepatitis B virus (HBV) is the liver, the possibility of infection of peripheral blood mononuclear cells (PBMCs) with HBV has also been reported. This study was performed to analyze the course of HBV infection of PBMCs and to investigate the influence of interleukin-6 (IL-6) on the efficiency of infection of PBMCs with HBV in vitro. PBMCs isolated from a healthy donor were infected by exposing to a HBsAg-, HBeAg-positive serum in the presence or absence of exogenous IL-6. The efficiency of infection was estimated by HBV DNA determination in the cells and medium in the course of infection. The results of this study show that the presence of IL-6 during the PBMCs infection with HBV increased the efficiency of this infection.

[The use of flow cytometry for the determination of the hepatitis B virus pre-S1 envelop protein binding by human peripheral blood lymphocytes in vitro in the course of recovery]. / Zastosowanie cytometrii przeplywowej do oceny wiazania fragmentu Pre-S1 antygenu powierzchniowego wirusa zapalenia watroby typu B przez limfocyty ludzkiej krwi obwodowej in vitro u osób po przebyciu infekcji.

The course of hepatitis B virus (HBV) infection depends on the host immune response to various antigens of the virus, including hepatitis B surface antigen (HbsAg), which is present on the envelope of HBV and which contains Pre-S1, Pre-S2 and S proteins. The peripheral blood lymphocytes (PBL) from patients with chronic hepatitis B infection (CHB) and patients acutely infected with HBV and recovered completely (convalescents) were studied for detecting of Pre-S1 antigen binding. We used a cytometric method for measurement of the binding of fluorescein labeled Pre-S1 antigen (FITC/Pre-S1) to the PBL. The binding of FITC/Pre-S1 was determined on resting lymphocytes and on lymphocytes cultured in vitro for 5 days in the presence of Pre-S1 and phytohaemagglutinin-P (PHA-P). The expression of CD3 and CD19 molecules on the surface of PBLs simultaneously with the expression of FITC/Pre-S1 was also analysed using flow cytometry. We found that the Pre-S1 binding significantly depends on the state of the activation of lymphocytes. Specific stimulation of the convalescent lymphocytes with Pre-S1 resulted in an increase in the percentage of the FITC/Pre-S1 binding cells in relation to the unstimulated cultured and resting cells as well as to the cells from CHB patients. We suggest that flow cytometric measurement of Pre-S1 binding by lymphocytes may be useful indicator of the disease activity.

Nitric oxide generation from hydroxylamine in the presence of neutrophils and in the cell-free system.

Conversion of hydroxylamine (HA) to nitric oxide (NO) has been studied in the presence or absence of human neutrophils with or without myristate acetate phorbol (PMA), catalase (CAT), hydrogen peroxide (H2O2), and superoxide dismutase (SOD) and nitric oxide synthase (NOS) inhibitors. The generation of NO from HA in the presence of neutrophils was higher than in the cell-free system. We found that catalase did not influence the nitrite generation from HA in the cell-free system and in the presence of neutrophils. The H2O2 enhanced the NO generation from HA in the presence of neutrophils only. When catalase and H2O2 were added together, a high increase of NO generation from HA in both systems was observed. The addition of SOD decreased whereas addition of PMA enhanced the NO generation from HA in the presence of neutrophils. The presented data show the possible role of oxygen radicals in the decomposition of HA to NO. The addition of NOS inhibitors to the culture of neutrophils decreased the generation of nitrite from HA. Our results suggest that NO generation from HA, which is an intermediate in NO production from L-arginine, may be supported by an enzymatic pathway in which cellular NO synthase is involved.

Spontaneous apoptosis of neutrophils in whole blood and its relation to apoptosis gene proteins.

Apoptosis of neutrophils limits their pro-inflammatory potential. We tested the ability of fresh and cultured whole blood neutrophils to undergo spontaneous apoptosis and expression of p53, Fas/Apo-1, bcl-2 protein in the cells using flow cytometry. Neutrophil apoptosis was estimated using Annexin V and propidium iodide binding and verified under light microscopy. The percentage of early and late apoptotic neutrophils in the blood samples increased significantly after 20 h culture from 12.3 +/- 14.2% and 4.3 +/- 4.2% to 39.5 +/- 14% and 15.3 +/- 9.6%, respectively. The majority of late apoptotic neutrophils had altered morphology in FSC/SSC dot plot compared to alive or early apoptotic neutrophils. Cultured neutrophils presented markedly lower expression of bcl-2 protein compared to fresh blood cells: 211 +/- 321 median of fluorescence intensity (MFI) and 787 +/- 1152 MFI, respectively. The increased percentage of late apoptotic cells after culture paralleled the increase in the Fas/Apo-1 expression and negatively correlated with bcl-2 expression. We noted intracellular expression of p53 protein in neutrophils, although the expression did not correlate neither to the percentage of the apoptotic neutrophils, nor to the Fas/Apo-1 or bcl-2 expression. Our results suggested that neutrophil apoptosis is gene regulated, moreover, we present a possibility to assess the neutrophil apoptosis and cellular expression of the proteins of apoptosis related genes in whole blood samples.

The comparison of some biological activities of lipopolysaccharides obtained from smooth and rough Proteus mirabilis strains.

The biological activity of lipopolysaccharide (LPS) obtained from Proteus mirabilis smooth and rough strains was investigated. The tested endotoxins (differing in polysaccharide chain lenght) were isolated from wild S1959 strain as well as from its rough mutants Ra and Re. Induction of tumor necrosis factor-alpha (TNF-alpha), and nitric oxide production as well as activation of complement system by lipopolysaccharide are the pathophysiological reaction in a host response to gram-negative bacteria. In this study, it was found that S (S1959), Ra (R110) and Re (R45) chemotypes of LPS similarly induced the human neutrophils to release TNF-alpha. In contrast none of the LPS stimulated the neutrophils to synthesis of nitric oxide regardless of doses used and culture time. Te Re form of LPS showed the strongest anticomplementary activity.

CD3-/HLA-DR+, CD3+/HLA-DR+ cell levels and PHA stimulation of lymphocytes obtained from women with recurrent spontaneous abortions and subjected to partner's lymphocyte immunization.

It is assumed that 80% of recurrent spontaneous abortions (RSA) of unknown etiology are of immunological origin. The proposed experimental treatment of those patients could be immunization with paternal lymphocytes, though the exact mechanism through which this therapy exerts its effect is still unknown. Some authors suggest that lymphocyte alloimmunization alters the number and activity of particular subpopulations of peripheral blood lymphocytes. The aim of this study was to evaluate the proliferative O activity of lymphocytes stimulated with phytohemagglutinin (PHA) and the percentage of peripheral lymphocytes carrying CD3+/HLA-DR+ and CD3-/HLA-DR+ markers, present in 32 selected for this study RSA women subjected to paternal lymphocytes' alloimmunization performed prior to conception and in early pregnancy. We conclude that, the post-immunization changes seem to have different quantitative and qualitative character comparing to those seen in normal pregnancy. They seem to avert unfavourable immunological phenomena observed in RSA women, allowing for successful continuation of pregnancy.

Oxidative burst response of neutrophils primed with PreS1 antigen of hepatitis B virus in patients with chronic hepatitis B and convalescents.

The pathogenesis of liver damage in the course of hepatitis B virus (HBV) infection depends on the host's specific and non-specific immune response to various viral antigens. The role of polymorphonuclear neutrophils (PMN) in the natural immune reaction and during secondary microbial infections is well documented. Increased free radical production is associated with many pathological conditions such as shock, ischaemia or chronic inflammatory diseases. We studied the oxidative metabolism of neutrophils in patients with chronic HBV and after recovery (convalescents). The effect of the PreS1 fragment of HBV antigen on some neutrophil functions in vitro was also examined. There were significant differences in the values of spontaneous and stimulated oxidative burst of neutrophils, measured using luminol-chemiluminescence, in patients with HBV when compared with the convalescents. PreS1 antigen did not by itself induce the respiratory burst in human neutrophils but it potentiated their response to a second stimulus. Hence we observed a priming of neutrophils, for an enhanced respiratory burst, by PreS1 antigen.

Effect of pre-S1 antigen on human lymphocyte proliferative responses.

The peripheral blood mononuclear cells (PBMCs) from patients acutely infected with HBV and recovered completely (n = 20), patients with chronic hepatitis B infection (CHB)- (n = 10) and HBsAg-positive carriers (n = 9) and healthy individuals (n = 8) were studied for their in vitro proliferative response to a synthetic pre-S1(20-49)x4 antigen. PBMCs from convalescents showed significant proliferative response in the presence of synthetic pre-S1 antigen. PBMCs from CHB- and HBsAg-positive exhibited reduced proliferative response not only to Pre-S1 antigen but also to nonspecific mitogens. This study suggests that the immune recognition of pre-S1 antigen and response of PBMCs to the pre-S1 antigen may be an important part of the normal human response to HBV infection. Failure to clear the HBV infection with development of the chronic carrier state may be caused by the lack of an efficient pre-S1 antigen-specific response.

[Adhesion and aggregation of neutrophils in patients with unstable angina]. / Adherencja i agregacja neutrofilów u chorych z niestabilna dusznica bolesna.

The adherence and aggregation abilities of neutrophils (PMNs) were evaluated in thirty three patients with unstable angina, who were qualified for PTCA procedure. The control group consisted of forty one clinically healthy persons. The blood for investigations was obtained from coronary sinus and basilic vein just before the procedure, while in the control group from basilic vein only. The adherence of PMNs to plastic surface (rest and stimulated by PMA) was estimated in vitro according to Oez's et al. method by measuring optical density of generated formazan, whereas the aggregation of PMNs was evaluated using the leukergy test according to the method of Fleck in Berliner's and Aronson's modification. In patients with unstable angina statistically significant higher (p < 0.001) adherence of peripheral blood PMNs, compared with control groups was found (patients: rest-0.525 +/- 0.245, stimulated-0.839 +/- 0.419, control group: rest-0.260 +/- 0.129, stimulated-0.522 +/- 0.377). The aggregation of peripheral blood PMNs was significantly higher (p < 0.05) in the sick than in the control group (the sick-10.98 +/- 4.29%, controls-4.65 +/- 3.01%). No differences in investigated parameters of PMNs obtained from peripheral or coronary sinus blood were found.

[Generation of active oxygen species by neutrophils in patients with unstable angina pectoris]. / Generowanie aktywnych zwiazków tlenowych przez neutrofile u chorych z niestabilna dusznica bolesna.

The generation of O2-. and H2O2 by neutrophils (PMNs) obtained from peripheral and coronary sinus blood was investigated in twenty four patients with unstable angina, who were qualified for PTCA procedure. The control group consisted of twenty one clinically healthy persons. The blood for the investigations was obtained from coronary sinus and basilic vein just before the procedure, while in the control group from--basilic vein only. In patients with unstable angina statistically significant higher (p < 0.05) O2-. generation by peripheral blood PMNs (at rest and stimulated), compared with the control group, was found respectively (the sick: rest--11,09 +/- 1.92; stimulated--25.48 +/- 5.76 nmol/cell/min.; healthy: rest--7.98 +/- 1.06; stimulated--13.58 +/- 1.19 nmol/cell/min). No significant differences in O2-. generation between PMNs obtained from coronary sinus or peripheral blood were found. No differences in H2O2 generation by rest PMNs obtained from coronary sinus or peripheral blood were found in patients compared with the control group. The generation of H2O2 by PMA stimulated PMNs was higher in the sick without statistical significance.

Immunological status of competitive cyclists before and after the training season.

The total number of leucocytes, T lymphocyte subsets, mitogen induced proliferation of lymphocytes, Il-2 generation, adherence capacity and chemiluminescence of granulocytes were measured and a leukergy test performed in fifteen young cyclists. The investigations were carried out at rest at the beginning of a training season and after six months of intensive training and a racing season, cycling approximately 500 km a week. Baseline values of the tested immune parameters were within the range observed in 16 non-trained healthy controls except significantly increased non stimulated neutrophil chemiluminescence. The second cyclo-ergometer test in August showed a marked improvement in the performance capacity of the cyclists. Significant decrease in absolute numbers of CD3+ and CD4+ cells, diminished IL-2 generation and fMLP and PMA stimulated chemiluminescence of neutrophils were noted. Surprisingly, a marked increase in lymphocyte proliferation induced by PHA and anti-CD3 MoAb and normalisation in non stimulated neutrophil chemiluminescence were also observed at rest after the training season. We conclude that long-lasting intensive training may result in significant alterations in lymphocyte number and composition and in neutrophil oxidative burst capacity, but their actual significance for immunity is seen controversially.

[Influence of PTCA on neutrophil aggregation in patients with unstable angina pectoris (part II)]. / Wplyw zabiegu PTCA na agregacje granulocytów obojetnochlonnych u chorych z niestabilna dusznica bolesna (czesc II).

In twenty patients with unstable angina, the effects of percutaneous transluminal coronary angioplasty (PTCA) on aggregation of neutrophils (PMNs) obtained from coronary sinus (CS) and basilic vein (BV) were investigated. Blood samples for the investigations were taken from CS just before the procedure, within the first minute after the last balloon inflation and 20 minutes after PTCA; from BV--just before and 20 minutes after the procedure. The results were compared with those obtained from 12 patients with unstable angina (in whom coronary angiography was performed, but for various reasons angioplasty was desisted from) and with those obtained from 20 clinically healthy persons. In patients with unstable angina the significantly higher percent of aggregated PMNs from CS as well as from BV was found, compared with healthy persons. Within the first minute after the procedure, the significantly increased percentage of aggregated PMNs from CS compared with the results before the procedure, was observed (10.50 +/- 4.06% and 18.36 +/- 7.69%, p < 0.05), but 20 minutes after, the value of the investigated parameter decreased to 5.88 +/- 2.86% and was similar to that in healthy persons. One minute after the procedure, in patients in whom PTCA was performed, the significantly decreased PMNs number in the blood obtained from CS was found, compared with the results before PTCA (4295 +/- 840 and 3850 +/- 825; p < 0.05). Twenty minutes after the procedure no further decrease of PMNs number was observed. In patients with or without PTCA no changes in the number and aggregation of PMNs obtained from BV were observed.

Histamine releasing activity and its inhibition by supernatants from cultured lymphoid cells of Nippostrongylus brasiliensis-infected rats.

The influence of Nippostrongylus brasiliensis infection on the capability of HRF (Histamine Releasing Factor) generation by rat lymphoid cells in vitro has been studied. Spleen cells and thymocytes of normal and Nippostrongylus brasiliensis-infected rats were cultured in the presence of nonspecific mitogen (PHA) or specific N. brasiliensis antigen (NbAg), and cell-free supernatants fractionated by Sephadex G-75 chromatography were tested on homologous mast cells for histamine releasing activity. The results show that PHA-stimulated lymphoid cells from both normal and infected rats produced a factor releasing histamine from mast cells. Histamine releasing activity was not detected when lymphoid cells of N. brasiliensis-infected rats were cultured in the presence of NbAg. Moreover, supernatants of these cultures diminished HRF-induced histamine release from mast cells, suggesting the production of factor(s) inhibiting this release. This histamine release inhibiting activity was detected in fractions in Sephadex G-75 chromatography of supernatants from the cultures of NbAg-stimulated thymocytes of infected rats.

Effect of Tolpa Peat Preparation on some immune parameters in healthy volunteers. Preliminary data.

Investigations of some immune parameters were carried out in healthy humans receiving TPP in doses of 50, 100, 300 and 600 mg daily for 14 days. The percent and composition of T lymphocyte subsets were studied along with the ability to generate interleukin-1 (Il-1) and interleukin-2 (Il-2) as well as spontaneous and PHA stimulated expression of receptors for Il-2 (TAC) and transferrin receptor (Tr) on lymphocytes. Lymphocytes proliferation induced with mitogens and alloantigens, generation of both Tumour Necrosis Factor alpha(TNF alpha) by monocytes and superoxide radicals by granulocytes were estimated. A 50 mg dose of TPP changed neither the T-cell percent among mononuclear cells nor the composition of T-cell subsets. A higher dosage of 100 mg of TPP caused a significant increase of the percent of T-cells on the 14th day but did not influence their subset composition. On the 14th and 28th day of the observation a statistically insignificant decrease of IL-2 generation was observed but with no change of both TAC receptor expression on lymphocytes and the ability to generate IL-1 by monocytes. TPP in the doses of 50 and 100 mg exerted a slight immunomodulatory potential in healthy humans, however the immunological parameters assessed were within the standard values. In other immune parameters examined during the administration of higher TPP doses in human groups consisted of four volunteers considerable differences have been found but individual differences in a limited experimental group and the lack of a placebo group did not allow for definite conclusions but for preliminary data only.

Rat lymphoid cell--derived histamine and 5-hydroxytryptamine releasing factor.

The in vitro production of histamine releasing factor (HRF) by lymphoid cells of rats, both normal and infected with Nippostrongylus brasiliensis, has been studied. Spleen cells and thymocytes were cultured either alone or in the presence of mitogen (PHA, 10 and 50 micrograms/ml) and the dialysed cell-free supernatants were tested for histamine releasing activity on rat peritoneal and pleural mast cell in vitro. We found that spleen cells and thymocytes of normal rats stimulated with PHA in 24 h cultures generated a factor which released histamine and 5-hydroxytryptamine from mast cells, and this ability was potentiated following N. brasiliensis infection of rats - lymphoid cells donors. Pleural mast cells were more sensitive to the action of HRF than peritoneal cells. Rat HRF had an apparent m.w. of 50,000 to 70,000 daltons as determined by gel chromatography and was a heat stable protein inducing histamine release from homologous mast cells in a very rapid (complete in 1-2 min at 37 degrees C), dose and temperature dependent secretory process.

Effect of neutrophils on the growth of human pre-S1-reactive T-cells in vitro.

Granulocyte factor (GF) derives from the specific granules of polymorphonuclear neutrophils. GF possesses an immunoregulatory activity and augments the immune response to antigens in vitro and in vivo. The Pre-S1 sensitive T-cells reactive to Pre-S1 protein in vitro were developed from peripheral blood mononuclear cells of hepatitis B convalescent by in vitro Pre-S1 protein stimulation. The GF involvement in the activation and development of the Pre-S1 specific regulatory T-cells in vitro was studied.