Developability considerations for bispecific and multispecific antibodies.

Bispecific antibodies (bsAb) and multispecific antibodies (msAb) encompass a diverse variety of formats that can concurrently bind multiple epitopes, unlocking mechanisms to address previously difficult-to-treat or incurable diseases. Early assessment of candidate developability enables demotion of antibodies with low potential and promotion of the most promising candidates for further development. Protein-based therapies have a stringent set of developability requirements in order to be competitive (e.g. high-concentration formulation, and long half-life) and their assessment requires a robust toolkit of methods, few of which are validated for interrogating bsAbs/msAbs. Important considerations when assessing the developability of bsAbs/msAbs include their molecular format, likelihood for immunogenicity, specificity, stability, and potential for high-volume production. Here, we summarize the critical aspects of developability assessment, and provide guidance on how to develop a comprehensive plan tailored to a given bsAb/msAb.

Treating murine inflammatory diseases with an anti-erythrocyte antibody.

Treatment of autoimmune and inflammatory diseases typically involves immune suppression. In an opposite strategy, we show that administration of the highly inflammatory erythrocyte-specific antibody Ter119 into mice remodels the monocyte cellular landscape, leading to resolution of inflammatory disease. Ter119 with intact Fc function was unexpectedly therapeutic in the K/BxN serum transfer model of arthritis. Similarly, it rapidly reversed clinical disease progression in collagen antibody-induced arthritis (CAIA) and collagen-induced arthritis and completely corrected CAIA-induced increase in monocyte Fcγ receptor II/III expression. Ter119 dose-dependently induced plasma chemokines CCL2, CCL5, CXCL9, CXCL10, and CCL11 with corresponding alterations in monocyte percentages in the blood and liver within 24 hours. Ter119 attenuated chemokine production from the synovial fluid and prevented the accumulation of inflammatory cells and complement components in the synovium. Ter119 could also accelerate the resolution of hypothermia and pulmonary edema in an acute lung injury model. We conclude that this inflammatory anti-erythrocyte antibody simultaneously triggers a highly efficient anti-inflammatory effect with broad therapeutic potential.

Antibody-mediated immune suppression is improved when blends of anti-RBC monoclonal antibodies are used in mice.

Although the prevention of hemolytic disease of the fetus and newborn is highly effective using polyclonal anti-D, a recombinant alternative is long overdue. Unfortunately, anti-D monoclonal antibodies have been, at best, disappointing. To determine the primary attribute defining an optimal antibody, we assessed suppression of murine red blood cell (RBC) immunization by single-monoclonal antibodies vs defined blends of subtype-matched antibodies. Allogeneic RBCs expressing the HOD antigen (hen egg lysozyme [HEL]-ovalbumin-human transmembrane Duffy(b)) were transfused into naïve mice alone or together with selected combinations of HEL-specific antibodies, and the resulting suppressive effect was assessed by evaluating the antibody response. Polyclonal HEL antibodies dramatically inhibited the antibody response to the HOD antigen, whereas single-monoclonal HEL antibodies were less effective despite the use of saturating doses. A blend of monoclonal HEL-specific antibodies reactive with different HEL epitopes significantly increased the suppressive effect, whereas a blend of monoclonal antibodies that block each other's binding to the HEL protein did not increase suppression. In conclusion, these data show that polyclonal antibodies are superior to monoclonal antibodies at suppressing the immune response to the HOD cells, a feature that can be completely recapitulated using monoclonal antibodies to different epitopes.

CD44 Antibody Inhibition of Macrophage Phagocytosis Targets Fcγ Receptor- and Complement Receptor 3-Dependent Mechanisms.

Targeting CD44, a major leukocyte adhesion molecule, using specific Abs has been shown beneficial in several models of autoimmune and inflammatory diseases. The mechanisms contributing to the anti-inflammatory effects of CD44 Abs, however, remain poorly understood. Phagocytosis is a key component of immune system function and can play a pivotal role in autoimmune states where CD44 Abs have shown to be effective. In this study, we show that the well-known anti-inflammatory CD44 Ab IM7 can inhibit murine macrophage phagocytosis of RBCs. We assessed three selected macrophage phagocytic receptor systems: Fcγ receptors (FcγRs), complement receptor 3 (CR3), and dectin-1. Treatment of macrophages with IM7 resulted in significant inhibition of FcγR-mediated phagocytosis of IgG-opsonized RBCs. The inhibition of FcγR-mediated phagocytosis was at an early stage in the phagocytic process involving both inhibition of the binding of the target RBC to the macrophages and postbinding events. This CD44 Ab also inhibited CR3-mediated phagocytosis of C3bi-opsonized RBCs, but it did not affect the phagocytosis of zymosan particles, known to be mediated by the C-type lectin dectin-1. Other CD44 Abs known to have less broad anti-inflammatory activity, including KM114, KM81, and KM201, did not inhibit FcγR-mediated phagocytosis of RBCs. Taken together, these findings demonstrate selective inhibition of FcγR and CR3-mediated phagocytosis by IM7 and suggest that this broadly anti-inflammatory CD44 Ab inhibits these selected macrophage phagocytic pathways. The understanding of the immune-regulatory effects of CD44 Abs is important in the development and optimization of therapeutic strategies for the potential treatment of autoimmune conditions.

IgG-Mediated Immune Suppression to Erythrocytes by Polyclonal Antibodies Can Occur in the Absence of Activating or Inhibitory Fcγ Receptors in a Full Mouse Model.

Polyclonal anti-D has been used to prevent RhD-negative mothers from becoming immunized against RhD positive fetal erythrocytes, and this mechanism has been referred as Ab or IgG-mediated immune suppression (AMIS). Although anti-D has been highly successful, the inhibitory mechanisms remain poorly understood. Two major theories behind AMIS involve the binding of IgG to activating or inhibitory FcγR, which can induce either erythrocyte clearance or immune inhibition, respectively. In this work, we explored the absolute role of activating and inhibitory FcγR in the AMIS mechanism using the HOD mouse model of RBC immunization. HOD mice contain a RBC-specific recombinant protein composed of hen egg lysozyme (HEL), OVA and human transmembrane Duffy Ag, and erythrocytes from HOD mice can stimulate an immune response to HEL. To assess the contribution of activating and inhibitory FcγR to AMIS, C57BL/6 versus FcRγ-chain(-/-) or FcγRIIB(-/-) mice were used as recipients of HOD-RBC alone or together with anti-HEL Abs (i.e., AMIS) and the resulting immune response to HEL evaluated. We show that anti-HEL polyclonal Abs induce the same degree of AMIS effect in mice lacking these IgG binding receptors as compared with wild-type mice. In agreement with this, F(ab')2 fragments of the AMIS Ab also significantly reduced the Ab response to the HOD cells. In conclusion, successful inhibition of in vivo Ab responses to HOD-RBC by polyclonal IgG can occur independently of activating or inhibitory FcγR involvement. These results may have implications for the understanding of RhD prophylaxis.

CD44 antibody-mediated amelioration of murine immune thrombocytopenia (ITP): mouse background determines the effect of FcγRIIb genetic disruption.

BACKGROUND: Several monoclonal antibodies to CD44 can successfully ameliorate murine immune thrombocytopenia (ITP). As these antibodies may be a potential replacement for intravenous immune globulin (IVIG) in the treatment of ITP and other autoimmune diseases, an understanding of their mechanisms of action is important. The role of the inhibitory Fc receptor (FcγRIIb) in the mechanism of action of IVIG and therapeutic CD44 antibodies remains uncertain. To assess if FcγRIIb in splenic macrophages plays a critical role in the action of these two therapeutics, splenectomized mice and mice genetically deficient in FcγRIIb on different backgrounds were evaluated. STUDY DESIGN AND METHODS: Thrombocytopenia was induced in FcγRIIb-deficient mice on B6;129S, C57BL/6, and BALB/C backgrounds, as well as splenectomized mice and control mice by platelet (PLT) antibody. PLT counts were enumerated before and after treatment with anti-CD44, red blood cell antibodies, or IVIG. RESULTS: Anti-CD44 is ineffective at inhibiting thrombocytopenia in B6;129S FcγRIIb-deficient mice but, like IVIG, is effective in splenectomized mice and FcγRIIb-deficient mice on the BALB/C and C57BL/6 background. CONCLUSION: These data suggest that 1) the B6;129S background itself is unlikely to be the sole reason for anti-CD44's inability to function in B6;129S FcγRIIb-deficient mice, 2) the simple loss of macrophage FcγRIIb expression alone is insufficient to explain anti-CD44 ameliorative function, and 3) a combination of mouse background genes in addition to FcγRIIb genetic disruption may affect the ability of anti-CD44 to function therapeutically. Similarities between IVIG and anti-CD44 mechanisms suggest that patients responsive to IVIG may also potentially respond to anti-CD44 treatment.

Antibody-mediated immune suppression of erythrocyte alloimmunization can occur independently from red cell clearance or epitope masking in a murine model.

Anti-D can prevent immunization to the RhD Ag on RBCs, a phenomenon commonly termed Ab-mediated immune suppression (AMIS). The most accepted theory to explain this effect has been the rapid clearance of RBCs. In mouse models using SRBC, these xenogeneic cells are always rapidly cleared even without Ab, and involvement of epitope masking of the SRBC Ags by the AMIS-inducing Ab (anti-SRBC) has been suggested. To address these hypotheses, we immunized mice with murine transgenic RBCs expressing the HOD Ag (hen egg lysozyme [HEL], in sequence with ovalbumin, and the human Duffy transmembrane protein) in the presence of polyclonal Abs or mAbs to the HOD molecule. The isotype, specificity, and ability to induce AMIS of these Abs were compared with accelerated clearance as well as steric hindrance of the HOD Ag. Mice made IgM and IgG reactive with the HEL portion of the molecule only. All six of the mAbs could inhibit the response. The HEL-specific Abs (4B7, IgG1; GD7, IgG2b; 2F4, IgG1) did not accelerate clearance of the HOD-RBCs and displayed partial epitope masking. The Duffy-specific Abs (MIMA 29, IgG2a; CBC-512, IgG1; K6, IgG1) all caused rapid clearance of HOD RBCs without steric hindrance. To our knowledge, this is the first demonstration of AMIS to erythrocytes in an all-murine model and shows that AMIS can occur in the absence of RBC clearance or epitope masking. The AMIS effect was also independent of IgG isotype and epitope specificity of the AMIS-inducing Ab.

The effects of magnesium sulfate on placental vascular endothelial growth factor expression in preeclampsia.

OBJECTIVE: To evaluate the effect of magnesium sulfate (MgSO(4)) on placental expression levels of vascular endothelial growth factor (VEGF). MATERIALS AND METHODS: Cotyledons of term normotensive and preeclamptic placentas were dually perfused for 6 h, with MgSO(4) (6-7 mg%) in the maternal reservoir [normotensive (n = 3); preeclamptic (n = 4)] and with the control medium (without MgSO(4)) [normotensive (n = 3); preeclamptic (n = 6)]. After perfusion, placental tissue samples were collected from four different placental compartments (amnion, chorion, placental villous and decidua). The collected placental tissues were homogenized and examined for VEGF by ELISA. Statistical significance was determined using a two-way analysis of variance. RESULTS: After perfusion with control medium, significantly lower levels of VEGF were detected in the chorion and placental villous compartments of preeclamptic placentas (70 ± 24 pg/g protein and 29 ± 11 pg/g protein; respectively), as compared with normotensive placentas (172 ± 80 pg/g protein and 51 ± 17 pg/g protein; respectively; p < 0.05). Exposure of preeclamptic placentas to MgSO(4) resulted in decreased VEGF levels by the amnion (57 ± 26 pg/g protein), as compared with the control group (153 ± 62 pg/g protein) (p < 0.05). On the other hand, MgSO(4) significantly increased VEGF levels by the placental villous and the decidua (58 ± 15 pg/g protein, 70 ± 29 pg/g protein; respectively), as compared with the control group (29 ± 11 pg/g protein, 33 ± 14 pg/g protein; respectively) (p < 0.01, p < 0.05; respectively). Exposure to MgSO(4) did not affect VEGF levels in normotensive placentas. CONCLUSION: Reduced levels of VEGF are expressed by some placental compartments in preeclampsia compared with normotensive pregnancy. Perfusion with MgSO(4) affects VEGF expression differently by preeclamptic and normotensive placentas. Increased production of placental VEGF in preeclampsia may play a role in the therapeutic action of MgSO(4).

Perfusion with magnesium sulfate increases sFlt-1 secretion only in the fetal side of placenta of women with preeclampsia.

OBJECTIVE: To examine the effect of magnesium sulfate (MgSO(4)) on sFlt (soluble fms-like tyrosine kinase)-1 in the fetal and maternal compartments of normotensive and preeclamptic placentas. METHODS: Cotyledons of term normotensive and preeclamptic placentas were dually perfused for six hours, with control medium and MgSO(4) (6-7 mg %) in the maternal reservoir. Perfusate sFlt-1 concentrations were measured. RESULTS: Median sFlt-1 concentration was higher in the maternal than in the fetal side in both groups and perfusion media (p < 0.0001). When perfused with control medium, the maternal side median sFlt-1 concentration was higher in the preeclampsia than in the control group (p < 0.0001). After perfusion with MgSO(4), the median maternal and fetal sides perfusate sFlt-1 concentration were higher in the preeclampsia than in the control group (p < 0.0001). In comparison to perfusion with control medium, the median sFlt-1 concentration of normal pregnant women decreased in the fetal and increased in the maternal side. In the preeclampsia group, only median fetal side sFlt-1 concentration increased. CONCLUSION: In contrast to normal pregnant women, perfusion with MgSO(4) of preeclamptic placentas did not increase their sFlt-1 concentration. This may indicate that MgSO(4) role may be limited to its anti-eclamptic and does not affect the anti-angiogenic state associated with preeclampsia.

Placental secretion of interleukin-1 and interleukin-1 receptor antagonist in preeclampsia: effect of magnesium sulfate.

Preeclampsia is a pregnancy-specific disorder characterized by hypertension and systemic endothelial dysfunction. Interleukin (IL)-1ß is a possible mediator of maternal endothelial dysfunction in preeclampsia. Serum IL-1ß as well as its natural inhibitor IL-1 receptor antagonist (IL-1Ra) were reported to be increased in women with preeclampsia. In the current study, we addressed the role of the placenta in controlling the circulatory levels of IL-1ß and its natural inhibitor IL-1Ra in preeclampsia, and the possible effect of magnesium sulfate (MgSO(4)) on these levels. Using an ex vivo placental perfusion system, placentas from preeclamptic (n = 9) and normotensive (n = 6) pregnancies were perfused in presence or absence of MgSO(4). Perfusate samples were collected from the maternal and the fetal circulations of the perfusion system, and IL-1ß and IL-1Ra were examined by enzyme-linked immunoassay (ELISA). Preeclamptic placentas secreted higher levels of IL-1ß (P < 0.001), and a tendentious higher levels of IL-1Ra, mainly into the maternal circulation, as compared with normotensive placentas, although no differences in IL-1ß:IL-1Ra ratio were detected. However, there was only tendentious increase in the secretion levels of IL-1ß or IL-1Ra into the fetal circulation of preeclamptic placentas, when compared with normotensive placentas. Administration of MgSO(4) to preeclamptic placentas resulted in an attenuation of the increased secretion of IL-1ß into the maternal circulation (P < 0.001), and in a tendentious reduction in IL-1Ra. However, IL-1ß:IL-1Ra ratio in preeclamptic placentas was not affected by MgSO(4). Interestingly, exposure of normotensive placenta to MgSO(4) resulted only in increased levels of IL-1Ra in the maternal circulation, without affecting IL-1ß levels or IL-1ß:IL-1Ra ratio. These findings suggest that the placenta may contribute to the elevation in serum IL-1ß and IL-1Ra in preeclampsia by increased secretion of these cytokines into the maternal circulation, and that MgSO(4) is able to attenuate this increased secretion of IL-1ß, and possibly IL-1Ra, in preeclampsia.

Magnesium sulfate normalizes placental interleukin-6 secretion in preeclampsia.

Interleukin-6 (IL-6) is one of the main proinflammatory mediators of hypertension and endothelial dysfunction in preeclampsia. In this study, we investigated the capacity of the preeclamptic placenta to secrete IL-6 and the effect of magnesium sulfate (MgSO(4)) on it. Placentas from normotensive (37-40 weeks) and preeclamptic (36-40 weeks) pregnancies were dually perfused for 6 h in the absence [normotensive (n = 3); preeclamptic (n = 4)] and presence [normotensive (n = 3); preeclamptic (n = 4)] of MgSO(4). Perfusate samples from the maternal and the fetal circulations were collected at each 30 min throughout the perfusion period and examined for IL-6 by enzyme-linked immunoassay. Statistical analysis was performed using the 2-way analysis of variance. In the absence of MgSO(4), IL-6 levels in the maternal and the fetal circulations of preeclamptic placentas (4.2 ± 1.3 and 0.9 ± 0.5 pg/mL/g cotyledon; respectively) were significantly higher, when compared with normotensive placentas (1.9 ± 0.5 and 0.2 ± 0.2 pg/mL/g cotyledon; respectively) (P < 0.05). Addition of MgSO(4) to the perfusate of normotensive placentas did not affect IL-6 secretion. However, exposure of preeclamptic placentas to MgSO(4) resulted in decreased IL-6 levels in the maternal circulations (1.7 ± 0.3 pg/mL/g cotyledon), when compared with the control group (P < 0.05). In the fetal circulation, the addition of MgSO(4) resulted only in a nonstatistical significant tendency toward decreased IL-6 levels, when compared with the control group. Our findings indicate that the perfused preeclamptic placenta secretes increased levels of IL-6 into the fetal and the maternal circulations and that MgSO(4) may normalize these increased secreted IL-6 levels.

Possible therapeutic effect of magnesium sulfate in pre-eclampsia by the down-regulation of placental tumor necrosis factor-alpha secretion.

OBJECTIVE: To examine the effect of magnesium sulfate (MgSO(4)) on tumor necrosis factor-alpha (TNF-alpha) secretion by preeclamptic placentas. STUDY DESIGN: Cotyledons of six, term, normotensive and ten, pre-eclamptic placentas were dually perfused for six hours (6h), with MgSO(4) (6-7 mg %) in the maternal reservoir [normotensive (n = 3); pre-eclamptic (n = 5)], and with control medium (without MgSO(4)) [normotensive (n = 3); pre-eclamptic (n = 5)]. Perfusate samples from the maternal and the fetal circulations were collected every 30 min throughout the 6h of perfusion, and examined for TNF- alpha levels using ELISA. Statistical significance was determined using a 2-way analysis of variance. RESULTS: Pre-eclamptic placentas perfused with control medium (without MgSO(4)) secreted higher levels of TNF-alpha into the fetal and the maternal circulations (1.60 +/- 0.59 pg/mL/g of cotyledon and 14.28 +/- 2.69 pg/mL/g of cotyledon, respectively), as compared to the fetal and maternal circulations of normotensive placentas (0.25 +/- 0.09 pg/mL/g of cotyledon and 6.73 +/- 1.11 pg/mL/g of cotyledon, respectively) (p < 0.01). Addition of MgSO(4) to normotensive placentas did not affect TNF-alpha levels in the fetal or maternal circulations. However, exposure of pre-eclamptic placentas to MgSO(4) significantly decreased TNF-alpha levels in both the fetal (0.89 +/- 0.09 pg/mL/g of cotyledon versus 1.6 +/- 0.59 pg/mL/g of cotyledon; p < 0.05) and the maternal circulations (4.74 +/- 2.78 pg/mL/g of cotyledon versus 14.28 +/- 2.69 pg/mL/g of cotyledon; p < 0.01). CONCLUSION: Down-regulation of placental TNF-alpha secretion by MgSO(4) in pre-eclampsia might indicate a possible therapeutic effect for this agent in reducing maternal, endothelial dysfunction and in improving neonatal outcome in pre-eclampsia, by reducing TNF-alpha levels in maternal and fetal circulations.

Nitrofurantoin transport by placental choriocarcinoma JAr cells: involvement of BCRP, OATP2B1 and other MDR transporters.

OBJECTIVE: To determine the role of BCRP in nitrofurantoin (NF) transport in JAr cells and the possible contribution of OATP2B1, P-gp and MRPs to this transport. METHODS: Cells were incubated with various BCRP, P-gp, MRPs, organic anion transporting polypeptide (OAT) and OATP2B1 inhibitors for 15 min, followed by incubation for 30 min with NF, with or without the inhibitors mentioned earlier. NF cytotoxicity was examined using neutral red (NR) assay. Intracellular NF levels were analyzed by HPLC. RESULTS: NR assay showed that incubation conditions with NF (as carried out in our experiments) were not cytotoxic. Incubation with specific inhibitors of BCRP (FTC, Chrysin and Novobiocin), showed a significant increase in NF accumulation in the cells. Inhibitors of OATP2B1 (EGCG and BSP) had no influence on NF accumulation. Specific inhibitors of P-gp and MRPs (Verapamil and Indomethacin, respectively) also had no influence on NF accumulation in JAr cells. CONCLUSIONS: NF is probably a specific substrate of BCRP, and BCRP has a major active role in NF transport in JAr cells. For the first time, we showed, that P-gp, MRPs, and the OATP2B1, probably have a negligible contribution to NF transport in JAr cells.

Progesterone levels in cesarean and normal delivered term placentas.

BACKGROUND: One of the most important hormones synthesized by the placenta during pregnancy is progesterone. The regulating mechanisms of progesterone synthesis and the mechanism responsible for the spontaneous onset of labor in women are still not fully understood. Progesterone is thought to have been involved in human parturition. The objective of this study was to compare the levels of progesterone in the human placentas, at the end of the gestation (37-41 weeks) in vaginal versus cesarean deliveries, and to evaluate the pattern of progesterone accumulation, instantly following its synthesis by the human placenta at the end of the pregnancy. METHODS: Progesterone levels in human placental tissue were determined by immunochemiluminescent analysis, following tissue homogenization. Progesterone secretion and accumulation pattern in the placental tissue was demonstrated using the ex vivo, closed, dual perfusion system of isolated human placental cotyledon. RESULTS: Immunochemiluminescent analysis of progesterone levels in human normal and cesarean-delivered placentas showed that placentas following normal vaginal delivery store higher concentrations of progesterone, and produce progesterone more intensively. Results obtained from 120-min perfusions (of vaginal and cesarean-delivered placentas) showed that progesterone tended to accumulate in the maternal rather than the fetal compartment. CONCLUSIONS: These data indicate that progesterone levels continuously rise till the end of pregnancy, with no apparent drop in progesterone levels during the labor process. In addition, progesterone is released from the syncytiotrophoblast preferably into the maternal component of the placental tissue.

Lipopolysaccharide differently affects prostaglandin E2 levels in fetal and maternal compartments of perfused human term placenta.

The aim of the present study was to examine the effect of lipopolysaccharide (LPS) on the levels of prostaglandin E(2) (PGE(2)) in the perfusates of the fetal and the maternal compartments of perfused human term placental tissue. Term placentas were perfused for 10h in the absence [control, (n=4)] and presence of LPS [LPS=1 microg/kg perfused placental tissue, (n=4)] in the maternal reservoir. Perfusate samples from the fetal and the maternal circulations were collected every 30 min and examined for PGE(2) levels by radio-immunoassay. PGE(2) levels in the fetal circulation were gradually increased reaching significant peak value of 479+/-159 pg/ml, as compared to PGE(2) levels in the maternal circulation (140+/-146 pg/ml) (p<0.05). After 10 hours of perfusion with control medium, PGE(2) levels in the maternal circulation (347+/-144 pg/ml) were significantly higher as compared to the fetal circulation (150+/-57 pg/ml) (p<0.05). In presence of LPS, PGE(2) levels in the fetal circulation increased reaching a peak value of 1028+/-663 pg/ml after 240 min of perfusion. The levels of PGE(2) in the control group after 240 min of perfusion were significantly lower (156+/-77 pg/ml) (p<0.05). No significant differences were detected in the levels of PGE(2) in the perfusate of the maternal compartment in presence of LPS, as compared to control. Our results suggest that the placenta may play an important role in maintaining high levels of PGE(2) in the fetal circulation and low PGE(2) levels in the maternal circulation during normal pregnancy. Moreover, placental PGE(2) release into the fetal and the maternal circulations may be differently affected in presence of intra-uterine infection/inflammation.

[Preeclampsia as a maternal vascular disease].

Preeclampsia, one of the main complications in pregnancy, affects 5-7% of all pregnancies, and is a leading cause of maternal and perinatal mortality. The placenta plays a pivotal role in the etiology of preeclampsia, and particularly, the trophoblast cells of the placenta. It is now believed that preeclampsia is a two stage disease. In the first stage, a defective implantation and placentation, causes a reduction in uteroplacental perfusion and placental ischemia/hypoxia. Placental ischemia may promote the release of a variety of factors to the maternal circulation. In the second stage, these factors initiate a cascade of cellular and molecular events leading to endothelial and vascular dysfunction. The endothelial dysfunction leads to the clinically recognized symptoms of the syndrome, which include hypertension, proteinuria, thrombocytopenia and impaired liver function. Hypertension is mediated by various endothelial and non-endothelial regulatory factors that are altered in preeclampsia. This review aims to summarize the recent knowledge on the implication of the placenta and various angiogenic factors in the pathogenesis of preeclampsia.

The expression of interleukin-15 and interleukin-18 by human term placenta is not affected by lipopolysaccharide.

The aim of the study was to examine the stimulatory effect of the inflammatory agent lipopolysaccharide (LPS) on the capacity of human term placenta to secrete interleukin (IL)-15 and IL-18. Isolated placental cotyledons from normal human term deliveries were dually perfused for ten hours with perfusion medium alone (n=5) or with perfusion medium containing LPS (1 microg/kg perfused placental tissue) (n=5). Placental tissue was collected from three different placental compartments (amnion, chorion, and placenta) before and after perfusion. The placental tissues collected were homogenized and examined for IL-15 and IL-18 by ELISA. In addition, formalin-fixed and paraffin-embedded sections from term placentas before perfusion were stained by immunohistochemistry to characterize the cellular origin of placental IL-15 and IL-18. Statistical significance was determined using paired/unpaired t-test. p<0.05 was considered significant. Our results show that IL-15 and IL-18 are produced more by chorionic tissue, as compared to the amnion and placental tissues. Moreover, we show that IL-15 and IL-18 are expressed by epithelial cells of the amnion, chorionic cells of the chorion and decidual cells of the decidua. However, IL-15, but not IL-18, was expressed also by syncytiotrophoblasts of the villi. Perfusion of LPS did not affect the capacity of amnion, chorion and placental tissues to secrete IL-15 and IL-18, as compared to control. The expression of IL-15 and IL-18 in the different compartments of the human placenta suggests a possible role for these two cytokines in normal placental development, pregnancy and labor. Moreover, our results indicate that IL-15 and IL-18 are not part of the mechanism of the response of human placenta to LPS.

Perfusion with lipopolysaccharide differently affects the secretion of tumor necrosis factor-alpha and interleukin-6 by term and preterm human placenta.

This study has compared the functional capacity of term and preterm placentas in terms of production of pro-inflammatory cytokines in a perfusion system reflecting their ability to react to inflammatory agents, such as lipopolysaccharide (LPS), mimicking the situation of chorioamnionitis. We have demonstrated that term placentas secrete higher levels of tumor necrosis factor (TNF)-alpha compared with preterm placentas. Moreover, TNF-alpha secretion was significantly higher after exposure to LPS in the maternal and fetal sides of term placentas. In contrast, in preterm placentas, only the fetal side responded with a significant increase in secretion of TNF-alpha after exposure to LPS. The maternal side of term placentas secreted significantly higher amounts of interleukin (IL)-6 compared with preterm placentas. Exposure to LPS significantly decreased IL-6 secretion from the maternal side in both term and preterm placentas. Moreover, the fetal side of term placentas secreted significantly lower amounts of IL-6 compared with preterm placentas. In summary, this study has indicated that term and preterm placental tissues have a differing responsiveness to LPS stimulation, with term placentas disposed to a higher TNF-alpha:IL-6 ratio. Release of cytokines into fetal circulation is less than into the maternal side. However, TNF-alpha is released into fetal circulation after LPS stimulation and this may be relevant to the etiology of chorioamnionitis.

Serum inhibin A, VEGF and TNFalpha levels after triggering oocyte maturation with GnRH agonist compared with HCG in women with polycystic ovaries undergoing IVF treatment: a prospective randomized trial.

BACKGROUND: We aimed to examine the serum levels of inhibin A, vascular endothelial growth factor (VEGF), tumour necrosis factor alpha (TNFalpha), estradiol (E2) and progesterone levels after triggering of final oocyte maturation with GnRH agonist compared with HCG in patients with polycystic ovaries (PCO) and to investigate the relationship between these markers and ovarian hyperstimulation syndrome (OHSS). METHODS: Twenty-eight patients with PCO, undergoing controlled ovarian hyperstimulation with FSH and GnRH antagonist for IVF-embryo transfer treatment, were randomized for triggering of final oocyte maturation with GnRH agonist (GnRH agonist group, n = 15) or HCG (HCG group, n = 13). Blood samples were obtained on the day of randomization and thereafter every 2-7 days. Serum levels of inhibin A, VEGF, TNFalpha, E2 and progesterone, the incidence of OHSS, ovarian size and pelvic fluid accumulation were evaluated. RESULTS: Serum inhibin A, E2 and progesterone levels were significantly lower in the GnRH agonist group compared with the HCG group, particularly on the day of embryo transfer (P < 0.0001). Serum VEGF and TNFalpha levels were similar between the two groups. Four patients in the HCG group developed severe OHSS, whereas no patient had any symptoms or signs of OHSS in the GnRH-agonist group (P < 0.05). CONCLUSIONS: In patients with PCO treated with FSH/GnRH antagonist, final oocyte maturation with GnRH agonist instead of HCG reduces significantly inhibin A, E2 and progesterone levels during the luteal phase. This phenomenon reflects the inhibition of the corpus luteum function and may explain, at least in part, the mechanism of OHSS prevention in high-risk patients. Our results do not support a crucial role for VEGF or TNFalpha in OHSS.