Investigation of systemic inflammatory response in first trimester pregnancy failure.

BACKGROUND: The contribution of local and systemic inflammation to the pathophysiology of sporadic first trimester miscarriages remains unclear. The objective of this study was to investigate the inflammatory response in the circulation of women presenting with first trimester miscarriage. METHODS: Levels of tumour necrosis factor alpha (TNFα), TNF receptors 1 and 2, interferon gamma (IFNγ), interleukin (IL)-6 and IL-10 were assayed using cytometric bead arrays in plasma samples from 29 euploid and 21 aneuploid missed miscarriages, 35 normal pregnant controls and 31 non-pregnant women (NPW). Whole blood flow cytometry was carried out with samples from 17 euploid and 16 aneuploid miscarriages, 18 pregnant controls and 13 NPW. RESULTS: The plasma of women with euploid miscarriage contained significantly higher circulating levels of TNFα (P < 0.005), IFNγ (P < 0.005), IL-6 (P < 0.005) and IL-10 (P < 0.01) than that of pregnant controls, irrespective of gestational age. Significantly (P < 0.05) higher TNF-R1 levels at 6-9 weeks, and significantly higher TNFα/IL-6 (P < 0.001) and significantly lower TNFα/IL-10 (P < 0.001) and IFNγ/IL-10 (P < 0.001) ratios at 10-14 weeks, were also found in euploid miscarriage cases compared with pregnant controls. TNFα/IL-10 ratio in plasma was significantly (P < 0.05) lower in miscarriages with an abnormal karyotype than those with normal karyotype. Normal pregnant women had a significantly higher plasma level of IFNγ (P < 0.01) and IFNγ/IL-10 ratio (P < 0.005), a significantly (P < 0.005) lower TNF-R1 level, and a significant (P < 0.05) increase in stimulated TNFα in monocytes, compared with NPW. CONCLUSIONS: Our data confirm that there is an inflammatory reaction in normal pregnancy compared with the non-pregnant state, which may be disrupted during miscarriage.

Fall in implantation rates following ICSI with sperm with high DNA fragmentation.

BACKGROUND: There is considerable uncertainty as to the significance of a high sperm DNA fragmentation index (DFI) for achieving a successful pregnancy. METHODS: The sperm DFI of 124 patients undergoing 192 IVF cycles and of 96 patients undergoing 155 ICSI cycles was determined using the sperm chromatin structure assay on neat sperm. RESULTS: The rate of continuing pregnancies in ICSI cycles (but not in IVF cycles) showed significant negative correlation (r = -0.184, P = 0.022) with the DFI value. A threshold value of DFI which showed a significant difference (P = 0.005) in rate of continuing pregnancies between higher and lower DFI levels was found for ICSI cycles to be > or = 19%, but no such threshold was found for IVF cycles. However, if the threshold of > or = 30% was used for IVF cycles there was a non-significant lowering of the rates of continuing pregnancy and implantation at the higher DFI levels. DFI level had no effect on fertilization rate or on the percentage of embryos having more than 4 cells at Day 3 after fertilization. A high DFI level had a marked significant effect (P = 0.001) on implantation rate in ICSI cycles but not in IVF cycles. A significant positive correlation (r = 0.268, P = 0.001) between DFI and sperm midpiece defects was also noted in the ICSI patients. CONCLUSIONS: These observations may help to resolve the issues about how, and to what extent, sperm DNA damage impacts upon the success of IVF and ICSI procedures.

Relationship between male reproductive hormones, sperm DNA damage and markers of oxidative stress in infertility.

This study investigated the relationship between male reproductive hormones and sperm DNA damage and markers of oxidative stress in men undergoing infertility evaluation for male factor (n = 66) and non-male factor (n = 63) infertility. Semen samples were analysed for DNA fragmentation index (DFI). Serum samples were analysed for FSH, inhibin B, anti-Müllerian hormone (AMH), testosterone and total antioxidant capacity (TAC). Serum inhibin B was significantly lower in the male factor group compared with the non-male factor group. Inhibin B showed a positive correlation with sperm concentration and motility, and serum AMH showed a positive correlation with sperm concentration and semen volume. DFI was 3-fold higher in the male factor group and showed a negative correlation with sperm motility. Blood plasma TAC was negatively related to sperm concentration. The results confirm that AMH and inhibin B are markers of Sertoli cell function. Sperm DNA damage is moderately increased in male factor infertility, and is negatively associated with sperm motility. A negative association between antioxidant activity and sperm concentration suggests that even minimal oxidative stress may influence sperm concentration. However, there was no significant relationship between hormone concentrations, sperm DNA damage and total antioxidant capacity, suggesting other mechanisms for sperm dysfunction.

The PI3 kinase, p38 SAP kinase, and NF-kappaB signal transduction pathways are involved in the survival and maturation of lipopolysaccharide-stimulated human monocyte-derived dendritic cells.

As a dendritic cell (DC) matures, it becomes more potent as an antigen-presenting cell. This functional change is accompanied by a change in DC immunophenotype. The signal transduction events underlying this process are poorly characterized. In this study, we have investigated the signal transduction pathways involved in the lipopolysaccharide (LPS)-induced maturation of human monocyte-derived DCs (MoDCs) in vitro. We show that exposure of immature MoDCs to LPS activates the p38 stress-activated protein kinase (p38SAPK), extracellular signal-regulated protein kinase (ERK), phosphoinositide 3-OH kinase (PI3 kinase)/Akt, and nuclear factor (NF)-kappaB pathways. Studies using inhibitors demonstrate that PI3 kinase/Akt but not the other pathways are important in maintaining survival of LPS-stimulated MoDCs. Inhibiting p38SAPK prevented activation of the transcription factors ATF-2 and CREB and significantly reduced the LPS-induced up-regulation of CD80, CD83, and CD86, but did not have any significant effect on the LPS-induced changes in macropinocytosis or HLA-DR, CD40, and CD1a expression. Inhibiting the NF-kappaB pathway significantly reduced the LPS-induced up-regulation of HLA-DR as well as CD80, CD83, and CD86. Inhibiting the p38SAPK and NF-kappaB pathways simultaneously had variable effects depending on the cell surface marker studied. It thus appears that different aspects of LPS-induced MoDC maturation are regulated by different and sometimes overlapping pathways.

Monocyte-derived dendritic cells do not proliferate and are not susceptible to retroviral transduction.

Dendritic cells may be generated ex vivo from CD34+ progenitor cells or peripheral blood mononuclear cells. Initial reports suggested that monocyte-derived dendritic cells (MoDCs) arise from a proliferating precursor and several groups subsequently reported successful retroviral transduction of these cells, again implying that cell division occurs. As this is of importance in the development of immunotherapy protocols, we investigated whether monocytes proliferate as they differentiate into MoDCs and also their susceptibility to retroviral transduction. During MoDC differentiation, there was a 51 +/- 12% reduction in cell number, 98% of cells were in G0/G1, no DNA synthesis was detectable and the cell cycle regulatory proteins pRb and p130 were in the hypophosphorylated forms observed in non-cycling cells. As expected from these results, MoDCs were refractory to transduction with a GALV1 pseudotyped Moloney murine leukaemia virus (MoMLV)-based retroviral vector. In contrast, generation of DCs from purified CD34 progenitors was accompanied by rapid entry into the cell cycle and a 41.1-fold cell expansion at the end of 14 d culture.

Preferential induction of apoptosis of leukaemic cells by farnesol.

Farnesol preferentially inhibits proliferation and induces apoptosis of tumour-derived but not non-transformed cell lines. We investigated whether farnesol induces apoptosis of blasts from patients with acute myeloid leukaemia (AML) and leukaemic cell lines, as compared with normal, human primary haemopoietic cells. We show that 30 microM farnesol causes apoptosis of leukaemic cell lines of T- and B-lymphocyte, myeloid or erythroid lineages and primary blasts obtained from patients with AML. However, the same concentration did not kill primary monocytes, or quiescent or proliferating T-lymphocytes. We conclude that farnesol selectively kills AML blasts and leukaemic cell lines in preference to primary haemopoietic cells.

Modulation of E2F activity in primary mouse B cells following stimulation via surface IgM and CD40 receptors.

Since signals via CD40 and the B cell receptor are known to synergize to induce B cell activation, we have analyzed the pocket protein/E2F complexes in mouse B lymphocytes following stimulation by anti-IgM, anti-CD40, alone or together. We find that E2F4 and DP1 form the predominant E2F heterodimers in the G0 and G1 phases of the cell cycle, complexed with hypophosphorylated p130. During late G1 and S phase this complex is replaced by at least three different E2F complexes, one of which is an E2F complex containing p107 or pRB as well as two "free" E2F complexes consisting of E2F4/DP1 and E2F1-3/DP1. These effects were mirrored by the levels and phosphorylation status of the three pocket proteins. We also observed an increase in electrophoretic mobility of DP1 and E2F4 as B cells progressed from G0 into early G1, resulting from their dephosphorylation. This is known to correlate with a decrease in DNA binding capacity of these proteins and could also be important for derepression of genes negatively regulated through E2F sites in their promoters. These results therefore indicate that the pRB/E2F pathway integrates proliferative signals emanating from the sIgM and CD40 receptors.

Pure populations of transduced primary human cells can be produced using GFP expressing herpes virus vectors and flow cytometry.

Herpes simplex virus (HSV) has often been suggested as a vector for gene delivery to the nervous system although it is also capable of infecting many other cell types. HSV also has the ability to package large genetic insertions so the expression of multiple genes from a single virus is possible. Here we show that a green fluorescent protein (GFP) expressing HSV1 vector can transduce two primary human cell types--quiescent human CD34+ hematopoietic progenitor cells and dendritic cells--which are both hard to transduce by other means. We also show that GFP is an effective marker when expressed from an HSV vector in vivo in the mouse brain. When GFP is expressed together with a second gene (in this case lacZ) from a single virus, transduced GFP-positive CD34+ hematopoietic progenitor cells or dendritic cells can both be generated at an effective efficiency of 100% for the second gene. Here transduction with the vector is combined with flow cytometry allowing GFP-positive cells to be sorted from the untransduced population. Such completely transduced populations of quiescent CD34+ hematopoietic progenitor and dendritic cells cannot easily be achieved by other means, and might thus allow experimental or therapeutic protocols to be carried out requiring high-level transduction which would not otherwise be possible. Such an approach using HSV vectors might also be applicable to other cell types for which transduction is as yet unreliable or of low efficiency.

p130, p107, and pRb are differentially regulated in proliferating cells and during cell cycle arrest by alpha-interferon.

We have determined how the phosphorylation of the retinoblastoma family (pRb, p107, and p130) is governed in individual cell cycle phases of Daudi B-cells during cell cycle exit triggered by alpha-interferon (alpha-IFN). alpha-IFN causes dephosphorylation of pRb and loss of p130 phosphorylated Form 3. However, the change in p130 phosphorylation in response to alpha-IFN occurs before dephosphorylation of pRb is complete because loss of p130 Form 3 occurs throughout the cell cycle prior to complete arrest in G1, whereas pRb is dephosphorylated only in G1. In contrast, p107 is dephosphorylated and is then depleted from cells as they exit the cell cycle. p130, predominantly in Form 1, and hypophosphorylated pRb bind an E2F DNA binding site; p130 complexes E2F-4, whereas pRb binds both E2F-4 and E2F-1. The phosphorylated forms of E2F-4 that bind to the E2F DNA site are different from hyperphosphorylated E2F-4, which predominates in primary hemopoietic cells in G0. We conclude that although cell cycle arrest induced by alpha-IFN may be mediated in part by formation of a complex containing p130 and E2F-4, alpha-IFN does not induce hyperphosphorylation of E2F-4, which characterizes primary hemopoietic cells in G0.

The effects of interleukin-8 on neutrophil fMetLeuPhe receptors, CD11b expression and metabolic activity, in comparison and combination with other cytokines.

The effect of the chemotactic cytokine, IL-8, on neutrophil function was compared with that of of other cytokines, GM-CSF, G-CSF TNF alpha and IFN-gamma. IL-8 rapidly stimulated a three-fold enhancement of the fMLP-stimulated respiratory burst, but this priming effect was transient compared with the slower and sustained effects of GM-CSF and IFN gamma. Apart from G-CSF, IL-8 was the weakest priming agent and was weaker than GM-CSF in priming arachidonic acid metabolism stimulated by calcium ionophore. When incubated in combination, IL-8 and TNF alpha were highly synergistic in their effects on respiratory burst priming, whereas IL-8 and GM-CSF showed little synergy. In contrast, IL-8 was as potent as GM-CSF at increasing the expression of neutrophil chemotactic peptide receptors and the beta 2 integrin, CD11b. The latter was maximally upregulated within 5 min of stimulation with IL-8, whereas the effect of GM-CSF was much slower. The kinetics of neutrophil respiratory burst priming by IL-8 were the same when measured in whole blood samples and in purified cell suspensions, and IL-8 dose-response curves were similar, showing that the low affinity IL-8 receptors on erythrocytes do not rapidly sequester circulating IL-8. The data suggest that IL-8 plays a minor role in priming neutrophil function and that a more major activity is the regulation of neutrophil adhesion and migration.

Pentoxifylline at clinically achievable levels inhibits FMLP-induced neutrophil responses, but not priming, upregulation of cell-adhesion molecules, or migration induced by GM-CSF.

Pentoxifylline (PTX) administered after bone-marrow transplantation reduces procedure-related organ damage mediated by TNF alpha. GM-CSF is also given post-transplant to stimulate earlier neutrophil recovery. Because PTX has been shown to inhibit neutrophil function, we sought to determine whether it also inhibited the effects of GM-CSF on neutrophil activity. The study confirmed that PTX at clinically achievable concentration (5-10 mumol/l) attenuated the responses of human neutrophils to chemotactic peptide, whereas it did not inhibit the effect of GM-CSF on neutrophil function even at high concentrations. In experiments with human neutrophils, neither the direct effects of GM-CSF such as stimulation of migration and increased expression of CD11b, nor the priming effects of GM-CSF on the respiratory burst, were inhibited by PTX. In experiments with monkeys, intravenous administration of PTX did not block subsequent GM-CSF-induced neutrophil CD11b upregulation or phagocyte margination, even when near millimolar plasma levels of pentoxifylline were obtained. The retention of cytokine-stimulated activities suggests that PTX will not compromise the response of neutrophils to stimuli from infectious foci.

The effect of inhibition of leukotriene synthesis on the activity of interleukin-8 and granulocyte-macrophage colony-stimulating factor.

The cytokines interleukin-8 (IL-8) and granulocyte-macrophage colony-stimulating factor (GM-CSF) enhanced the extracellular release of arachidonate metabolites from ionophore-stimulated neutrophils by 145 +/- 10% (mean +/- S.E.M., n = 13) and 182 +/- 11% (n = 16), respectively. To determine whether enhanced leukotriene production mediates the effects of these cytokines on neutrophil activity, two different specific arachidonate 5-lipoxygenase (5-LO) inhibitors, piriprost and MK-886, were used to inhibit leukotriene synthesis. Neither inhibitor affected the upregulation of CD11b beta(2)-integrin expression or priming of superoxide generation stimulated by IL-8 and GM-CSF. It is concluded that leukotrienes do not mediate either the direct or priming effects of these cytokines and that these classes of anti-inflammatory drugs are therefore unlikely to inhibit the effects of IL-8 and GM-CSF on neutrophil activation.