Investigation of the possible functions of PACAP in human trophoblast cells.

Pituitary adenylate cyclase activating polypeptide (PACAP) is an endogenous neuropeptide having a widespread distribution both in the nervous system and peripheral organs including the female reproductive system. Both the peptide and its receptors have been shown in the placenta but its role in placental growth, especially its human aspects, remains unknown. The aim of the present study was to investigate the effects of PACAP on invasion, proliferation, cell survival, and angiogenesis of trophoblast cells. Furthermore, cytokine production was investigated in human decidual and peripheral blood mononuclear cells. For in vitro studies, human invasive proliferative extravillous cytotrophoblast (HIPEC) cells and HTR-8/SVneo human trophoblast cells were used. Both cell types were used for testing the effects of PACAP on invasion and cell survival in order to investigate whether the effects of PACAP in trophoblasts depend on the examined cell type. Invasion was studied by standardized invasion assay. PACAP increased proliferation in HIPEC cells, but not in HTR-8 cells. Cell viability was examined using MTT test, WST-1 assay, and annexin V/propidium iodide flow cytometry assay. Survival of HTR-8/SVneo cells was studied under oxidative stress conditions induced by hydrogen peroxide. PACAP as pretreatment, but not as co-treatment, significantly increased the number of surviving HTR-8 cells. Viability of HIPEC cells was investigated using methotrexate (MTX) toxicity, but PACAP1-38 could not counteract its toxic effect. Angiogenic molecules were determined both in the supernatant and the cell lysate by angiogenesis array. In the supernatant, we found that PACAP decreased the secretion of various angiogenic markers, such as angiopoietin, angiogenin, activin, endoglin, ADAMTS-1, and VEGF. For the cytokine assay, human decidual and peripheral blood lymphocytes were separated and treated with PACAP1-38. Th1 and Th2 cytokines were analyzed with CBA assay and the results showed that there were no significant differences in control and PACAP-treated cells. In summary, PACAP seems to play various roles in human trophoblast cells, depending on the cell type and microenvironmental influences.

Changes in the expression of pituitary adenylate cyclase-activating polypeptide in the human placenta during pregnancy and its effects on the survival of JAR choriocarcinoma cells.

Pituitary adenylate cyclase-activating polypeptide (PACAP), a neuropeptide with survival-promoting actions, has been observed in endocrine organs and is thought to play a role in reproductive functions, including pregnancy. PACAP occurs in two forms, 27 and 38 amino acid residues, with PACAP38 being the predominant form in human tissues. In the present study, we determined the concentrations of PACAP38 and PACAP27 in first-trimester and full-term human placentas using radioimmunoassay. We found high levels of PACAP38 and lower levels of PACAP27 in different parts of the full-term human placenta. PACAP38 content increased in the placenta during pregnancy, both on the maternal side and on the fetal side. The effects of PACAP on the survival of JAR human choriocarcinoma cells were investigated using flow cytometry and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) cell viability assay in cells exposed to the widely used chemotherapeutic agent methotrexate (MTX). It was found that PACAP neither influenced the survival of JAR cytotrophoblast cells nor affected cellular response to the death-inducing effect of the chemotherapeutic agent MTX. The present observations further support the significance of PACAP in the human placenta. The observation that PACAP did not influence the effects of MTX may have future clinical importance, showing that PACAP does not decrease the effects of certain chemotherapeutic agents.

Deficient leucocyte antisedimentation is related to post-stroke infections and outcome.

BACKGROUND: Patients with stroke are more susceptible to infections, suggesting possible deficiencies of early immune responses, particularly of leucocytes. AIMS: To serially examine leucocyte antisedimentation rate (LAR), a simple test to detect activation of leucocytes, and correlate it with S100beta, procalcitonin and outcome in patients with acute ischaemic events. METHODS: Venous blood samples were taken from 61 healthy volunteers and 49 patients with acute ischaemic events (acute ischaemic stroke (AIS), n = 38; transient ischaemic attack (TIA), n = 11) within 6 hours, at 24 and 72 hours after onset of symptoms. RESULTS: LAR was significantly higher in acute ischaemic events compared to controls within 6 hours after onset of stroke regardless of post-stroke infections. In addition, the increase of LAR was delayed and attenuated in TIA in contrast to AIS. A deficiency in early increase of LAR was associated with post-stroke infections and a poor outcome, measured by the Glasgow Outcome Scale in AIS. There was a positive correlation between LAR and S100beta at 72 hours after the onset of ischaemic stroke. Increased levels of S100beta at 24 and 72 hours after stroke were associated with poor outcome. CONCLUSIONS: An early activation of leucocytes indicated by an increase of LAR is characteristic of acute ischaemic cerebrovascular events. A delayed and ameliorated leucocyte activation represented by LAR is characteristic of TIA in contrast to stroke. Deficient early activation predisposes to post-stroke infections related to poor outcome. In addition, the extent of tissue injury correlates with the magnitude of innate immune responses.

The role of Vdelta2+T-cells in patients with active Mycobacterium tuberculosis infection and tuberculin anergy.

SETTING: Tuberculosis (TB) is one of the most common major infectious diseases. In humans, acquired protective immunity to Mycobacterium tuberculosis depends on T-cells and involves multiple T-cell subsets; however, the pathways used by T-cells to restrict the growth of M. tuberculosis are poorly understood. OBJECTIVE: To investigate the possible role of Vdelta2+T-cells and regulatory T-cells in the immune response to M. tuberculosis. As Vdelta2+T-cell function has been shown to be impaired in patients with M. tuberculosis infection, we investigated the percentage of perforin and Fas ligand (FasL) positive Vdelta2+T-cells and the possible role of activating and inhibitory natural killer (NK) cell receptors as well as that of regulatory T-cells in the control of tuberculin responsiveness. RESULTS: Tuberculin-negative patients demonstrated decreased perforin expression and increased FasL expression, which could not be explained by dysregulation of NK cell receptor expression or altered regulatory T-cell function. CONCLUSION: Altered cytotoxic capacity and apoptotic potential of Vdelta2+T-cells provide a plausible explanation for defective cellular immune functions in M. tuberculosis-infected anergic patients.

Dendritic cells therapy confers a protective microenvironment in murine pregnancy.

The fetal-placental unit is a semi-allograft and immunological recognition of pregnancy, together with the subsequent response of the maternal immune system, is necessary for a successful pregnancy. Dendritic cells (DC) show a biological plasticity that confers them special characteristics regulating both immunity and tolerance. Therapy employing DC proved to diminish the abortion in the DBA/2J-mated CBA/J females; however, the underlying mechanisms remain unknown. Here, we evaluated whether DC therapy influences the presence of immunoregulatory populations of cells at the fetal-maternal interface. To address this hypothesis, we analysed the pregnancy-protective CD8, gammadelta cell populations as well as transforming growth factor (TGF)-beta1 and progesterone-induced blocking factor (PIBF) expression at the fetal-maternal interface from abortion-prone female mice that had previously received adoptive transfer of syngeneic DC. Syngeneic DC therapy induced an increase in the number of CD8 and gammadelta cells. Additionally, an upregulation of TGF-beta1 and PIBF expression could be detected after DC transfer. We suggest that DC therapy differentially upregulates a regulatory/protective population of cells at the fetal-maternal interface. It is reasonable to assure that this mechanism would be responsible for the lower abortion rate.

Progesterone-dependent immunomodulation.

The biological effects of progesterone are mediated by a 34-kDa protein named the progesterone-induced blocking factor (PIBF). PIBF, synthesized by lymphocytes of healthy pregnant women in the presence of progesterone, inhibits arachidonic acid release as well as NK activity, and modifies the cytokine balance. Within the cell the full-length PIBF is associated with the centrosome, while secretion of shorter forms is induced by activation of the cell. PIBF induces nuclear translocation of STAT6 as well as PKC phosphorylation and exerts a negative effect on STAT4 phosphorylation. The concentration of PIBF in pregnancy urine is related to the positive or negative outcome of pregnancy; furthermore, premature pregnancy termination is predictable by lower than normal pregnancy PIBF values. In vivo data suggest the biological importance of the above findings. Treatment of pregnant Balb/c mice with the antiprogesterone RU 486 results in an increased resorption rate, which is associated with the inability of spleen cells to produce PIBF. High resorption rates induced by progesterone receptor block as well as those due to high NK activity are corrected by simultaneous PIBF treatment.

Gamma/delta T cell subsets in patients with active Mycobacterium tuberculosis infection and tuberculin anergy.

Earlier data suggest that gamma/delta T cells may play an important role in the immune response to Mycobacterium tuberculosis. The aim of this study was to determine the percentage of different gamma/delta subsets in peripheral blood of active tuberculosis patients with a positive or negative tuberculin reaction. Thirty-eight patients infected with M. tuberculosis and 22 healthy controls were included in the study. Venous blood was taken before starting antimycobacterial treatment. Lymphocytes were reacted with monoclonal antibodies specific for different gamma/delta V chains (Vdelta1, Vdelta2, Vgamma9 and Vgamma4). The results were analysed in the context of tuberculin reactivity and X-ray findings. Our results revealed a selective loss of Vgamma9/Vdelta2 T cells in the peripheral blood of tuberculin-negative patients with active tuberculosis compared to healthy controls, while the ratio of Vgamma9/Vdelta2 T cells in the peripheral blood of patients with a positive skin test did not differ from that of healthy controls. These findings demonstrate a relationship between the loss of the major M. tuberculosis-reactive subset of gammadelta T cells and the absence of tuberculin reactivity. The data are consistent with the hypothesis that gammadelta T cells play a role in the protective immune response to M. tuberculosis infection.

Progesterone as an immunomodulatory molecule.

Increased progesterone sensitivity of pregnancy lymphocytes is due to activation-induced appearance of progesterone binding sites in the lymphocytes. Following recognition of fetally derived antigens gamma/delta TCR+ cells develop progesterone receptors. Progesterone binding results in the synthesis of a mediator protein named the progesterone-induced blocking factor (PIBF). PIBF by acting on the phospholipase A2 enzyme interferes with arachidonic acid metabolism, induces a Th2 biased immune response, and by controlling NK activity exerts an anti-abortive effect.

Natural killer cell activity and cytokine production after in vitro immunoglobulin treatment of lymphocytes derived from pregnant women with or without risk for spontaneous abortion.

OBJECTIVE: To investigate the possible mechanism of action effective in immunoglobulin G (IgG) treatment of recurrent spontaneous abortion (RSA). The effect in vitro of a commercially available intravenous immunoglobulin (IvIg) on the rate of interleukin (IL)-10 and IL-12 positive cells (Th1/Th2 balance) and on natural killer (NK) cell activity in populations of peripheral lymphocytes of healthy pregnant women and women at risk for premature pregnancy termination was studied. Primary habitual aborters as well as women showing clinical symptoms (bleeding or regular uterine contractions) of threatened premature pregnancy termination were included. METHODS: Lymphocytes of 20 pregnant women were tested. Five different batches of an IvIg with reported immunomodulatory potential were used at a concentration of 10 mg/mL. Cytokine profiles of the lymphocytes were determined by immunocytochemistry. For testing of NK cell activity, the 4 hr single cell cytotoxicity assay was used. RESULTS: Incubation with IgG of lymphocytes from recurrent spontaneous aborters concomitantly and significantly decreased the rate of IL-12 positive cells (P < 0.01) and increased the rate of IL-10 positive cells (P < 0.01), whereas such treatment had no significant effect on lymphocytes of pregnant women not at risk of abortion. Dialysis or heat treatment (56 degrees C, 30 min) of the IgG preparations did not modify the effect. Elevated NK cell activity of women at risk for premature pregnancy termination significantly decreased after IgG incubation of cells in all cases, whereas NK cell activity of normal pregnancy lymphocytes was not altered. CONCLUSION: This study suggests that incubation of peripheral lymphocytes from RSA patients with polyclonal polyspecific IgG alters cytokine profiles and NK activity while the same treatment does not affect lymphocytes of healthy pregnant women. These data might add to the understanding of mechanisms of action of IvIg in prevention of recurrent pregnancy loss.

The role of gamma/delta T cells in progesterone-mediated immunomodulation during pregnancy: a review.

PROBLEM: To determine if pregnancy is recognized by the immune system and if inadequate recognition of fetal antigens might result in failed pregnancy. METHOD OF STUDY: Review of literature and current data. RESULTS: In the decidua gamma/delta TCR positive cells significantly increase in number. A subset of gamma/delta T cells reacts with nonpolymorphic Class I or Class I like molecules. Trophoblast recognition is mediated by the V gamma 1 subset which recognize a conserved mammalian sequence on the trophoblast. Almost all gamma/delta T cells in the decidua are activated and use the V delta 1 chain, whereas the majority of human peripheral gamma/delta lymphocytes expresses V gamma 9/V delta 2 TCR. Peripheral gamma/delta T cells of healthy pregnant women preferentially use V gamma V delta 1 chains, on the other hand, those of recurrent aborters use the V gamma 9V delta 2 combination. Signaling via the V gamma 1.4V delta 1 receptor induces a Th2 type response, whereas activation of the lymphocytes via the V gamma 9V delta 2 receptor results in increased IL-12 production and natural killer (NK) activity. In the presence of progesterone, activated lymphocytes synthesize the progesterone induced blocking factor (PIBF), which inhibits NK activity and exerts an anti abortive effect in vivo. Decidual CD56+ and gamma delta+ cells are to a high extent the same population. CONCLUSION: All decidual CD56+ cells express PIBF, thus it cannot be excluded that local production of this substance contributes to low decidual NK activity and thus to the success of the pregnancy.

Th2 biased immune response in cases with active Mycobacterium tuberculosis infection and tuberculin anergy.

This study was aimed at investigating the immunologic relationship between cytokine production pattern and tuberculin negativity in patients with active Mycobacterium tuberculosis infection. After classifying patients by the extent of pulmonary involvement and the size of the tuberculin reaction, we evaluated the rate of cytokine positivity in peripheral blood to determine whether there is a characteristic cellular immune reaction pattern which could partly explain the tuberculin negativity in some of these cases. The significance of tuberculin anergy occurring in some cases with M. tuberculosis infection is still not clear. We investigated the ratio of IL- 4, IL-10, IL-12, CD-4, CD-8 expressing lymphocytes in the peripheral blood of patients with active M. tuberculosis infection and correlated the percentage of the reactive cells with the positivity or negativity of tuberculin skin reactions. Twenty-eight patients were included in the study, with 11 healthy volunteers serving as controls. 10 ml of venous blood was drawn before starting anti-mycobacterial treatment. A tuberculin skin test was performed, introducing intracutaneously 5 TU PPD on the forearm with results evaluated after 72 h. Consistent with the reactivity or non-reactivity of the tuberculin skin test, we found a significantly higher ratio of IL-4 and IL-10 positive lymphocytes and a significantly lower ratio of IL-12 in the peripheral blood of patients with tuberculin anergy than in that of tuberculin positive patients or healthy donors. There was no difference in the ratio of the CD-4 CD-8 positive lymphocytes among the three groups. To evaluate whether the differences could be explained by the degree of pulmonary tubercular involvement, we classified the patients into three groups according to the extent and type of X-ray findings. Seven out of eight tuberculin negative patients were classified as grade III, whereas in the tuberculin positive group only seven out of 20 fell in this category. There was no significant correlation between the radiological grade of the patients and the examined in vitro parameters unless the tuberculin reactivity of each patients was also considered. Tuberculin anergy may reflect an inappropriate immune response to the intracellular pathogen. The high percentage of IL-4 and IL-10 positive lymphocytes together with a low percentage of IL-12 positive lymphocytes in the peripheral blood of anergic patients suggests a Th2 biased immune response during the early course of the disease.

Cytokine production by lymphocytes in pregnancy.

PROBLEM: In the presence of progesterone lymphocytes of pregnant women release a 34-kDa protein named the progesterone-induced blocking factor (PIBF). PIBF mediates the immunomodulatory and anti-abortive effects of progesterone and its presence is related to the outcome of pregnancy. PIBF induces production of Th2 type cytokines by activated lymphocytes. The in vivo relationship between PIBF- and cytokine production of pregnancy lymphocytes and the outcome of pregnancy was investigated. METHOD OF STUDY: Interleukin (IL)-12 and IL-10 production and PIBF expression in peripheral lymphocytes of 111 healthy pregnant women and 120 women at risk for premature pregnancy termination were detected by immunocytochemistry. RESULTS: We found increased IL-12 and low PIBF and IL-10 expression on lymphocytes of "risk" patients, and a high rate of IL-10 and PIBF positivity on lymphocytes from healthy pregnant women. The cytokine production pattern of the lymphocytes was related to the presence or absence of previous abortions as well as to the outcome of pregnancy. CONCLUSION: These data suggest the involvement of an altered cytokine production pattern in the immunologic effects of progesterone.

Progesterone and non-specific immunologic mechanisms in pregnancy.

PROBLEM: Progesterone-dependent immunomodulation is one of the mechanisms that enables pregnancy to proceed to term. Immunologic effects of progesterone are mediated by a protein named the progesterone-induced blocking factor (PIBF). Among other effects this protein inhibits natural killer (NK) activity and displays an anti-abortive effect in mice. Recently, we have shown that PIBF induces a Th2 shift in vitro. The present study was aimed at investigating the in vivo effect of PIBF on cytokine production, as well as the relationship between cytokine production, NK activity, and pregnancy loss. METHOD OF STUDY: Balb-c mice on day 8.5 of pregnancy were injected intraperitoneally with 0.5 mg of rabbit anti-PIBF immunoglobulin G (IgG). Another group of mice was simultaneously treated with anti-NK monoclonal antibodies. Mice treated with the same amount of normal rabbit serum or untreated mice of similar gestational age were used as controls. The animals were sacrificed and their uteri were inspected. The ratio of living and resorbed embryos was determined. NK activity as well as cytokine expression on the spleen cells were determined by immunocytochemistry and enzyme-linked immunoadsorbent assay (ELISA). RESULTS: Mitogen-activated spleen cells from anti-PIBF-treated mice produced significantly (P < 0.001) less IL-10 than those of pregnant control mice. A significantly higher percentage (P < 0.001) of spleen cells from anti-PIBF-treated mice expressed interferon-gamma (IFN gamma) as determined by immunocytochemistry, than those of untreated pregnant mice. There was a positive relationship between the percentage of IFN gamma-positive spleen cells and resorption rates, and an inverse relationship between the latter and interleukin-10 (IL-10) production. All these effects were corrected by treatment with anti-NK antibodies. CONCLUSION: Our data suggest that PIBF contributes to the success of gestation via cytokine-mediated inhibition of NK activity.

Gamma/delta T lymphocytes in Mycobacterium tuberculosis infection.

BACKGROUND: Data on the percentage of gamma/delta T lymphocytes in the peripheral blood of patients infected with Mycobacterium tuberculosis are few and contradictory. The percentage of gamma/delta T lymphocytes in the peripheral blood of tuberculin positive and tuberculin negative patients with Mycobacterium tuberculosis infection and healthy controls was compared. METHODS: Thirty six patients infected with Mycobacterium tuberculosis and 11 healthy controls were studied. Lymphocytes were separated, cytocentrifuged onto glass microscope slides, and reacted with anti-gamma/delta monoclonal antibody. The percentage of gamma/delta positive cells was determined by microscopic counting of 300 lymphocytes. RESULTS: No difference was found in the percentage of gamma/delta T lymphocytes between patients and controls. However, when the patients were divided into two groups according to reactivity or non-reactivity in the Mantoux skin reaction a higher percentage of gamma/delta T lymphocytes was found in the peripheral blood of patients with tuberculin anergy than in tuberculin positive patients or controls. CONCLUSIONS: Higher gamma/delta T cell counts are found in tuberculin negative patients with tuberculosis than in tuberculin positive patients or tuberculin positive controls. The high gamma/delta T cell counts in tuberculin anergic patients may reflect a shift in the immune response in a Th2 direction characterised by increased antibody production and decreased cell mediated responses.

The immunological pregnancy protective effect of progesterone is manifested via controlling cytokine production.

PROBLEM: This study was aimed at investigating the involvement of an altered cytokine pattern in the immunomodulatory and anti-abortive effects of a progesterone-induced immunomodulatory protein (PIBF). METHOD: PIBF expression on lymphocytes of healthy pregnant women and from women at risk for premature pregnancy termination was determined. In sera of the same women TNF alpha was quantified by a bioassay using L929 cells. NK activity was determined by a single cell cytotoxicity assay. Cytokine production of the lymphocytes or murine spleen cells was measured by ELISA or detected by immunocytochemistry. In pregnant mice endogenous PIBF activity was neutralized by anti-PIBF IgG. RESULTS: Sera of women at risk for premature pregnancy termination contained significantly higher concentrations of TNF alpha than those from healthy pregnant women and PIBF expression on the lymphocytes was inversely related to serum concentration of TNF alpha. Increased NK activity of lymphocytes after neutralization of endogenous PIBF activity is corrected by anti-IL 2 treatment and PIBF inhibits IL 12 expression on activated lymphocytes. PIBF increases IL-10 production by activated spleen cells. In pregnant mice, neutralization of endogenous PIBF activity by specific antibody results in increased resorption rate and reduced splenic IL-10 production. CONCLUSIONS: Our data allow the assumption that via blocking IL-12 production PIBF inhibits NK activation with a concomitant reduction of TNF alpha levels. Disturbances in this system might lead to the expression of the known synergistic effect of IL-12 and TNF alpha, resulting in a Th 1 type cytokine dominance and pregnancy termination.