Drug delivery to the human and mouse uterus using immunoliposomes targeted to the oxytocin receptor.

BACKGROUND: The ability to provide safe and effective pharmacotherapy during obstetric complications, such as preterm labor or postpartum hemorrhage, is hampered by the systemic toxicity of therapeutic agents leading to adverse side effects in the mother and fetus. Development of novel strategies to target tocolytic and uterotonic agents specifically to uterine myocytes would improve therapeutic efficacy while minimizing the risk of side effects. Ligand-targeted liposomes have emerged as a reliable and versatile platform for targeted drug delivery to specific cell types, tissues or organs. OBJECTIVE: Our objective was to develop a targeted drug delivery system for the uterus utilizing an immunoliposome platform targeting the oxytocin receptor. STUDY DESIGN: We conjugated liposomes to an antibody that recognizes an extracellular domain of the oxytocin receptor. We then examined the ability of oxytocin receptor-targeted liposomes to deliver contraction-blocking (nifedipine, salbutamol and rolipram) or contraction-enhancing (dofetilide) agents to strips of spontaneously contracting myometrial tissue in vitro (human and mouse). We evaluated the ability of oxytocin receptor-targeted liposomes to localize to uterine tissue in vivo, and assessed if targeted liposomes loaded with indomethacin were capable of preventing lipopolysaccharide-induced preterm birth in mice. RESULTS: Oxytocin receptor-targeted liposomes loaded with nifedipine, salbutamol or rolipram consistently abolished human myometrial contractions in vitro, while oxytocin receptor-targeted liposomes loaded with dofetilide increased contraction duration. Nontargeted control liposomes loaded with these agents had no effect. Similar results were observed in mouse uterine strips. Following in vivo administration to pregnant mice, oxytocin receptor-targeted liposomes localized specifically to the uterine horns and mammary tissue. Targeting increased localization to the uterus 7-fold. Localization was not detected in the maternal brain or fetus. Targeted and nontargeted liposomes also localized to the liver. Oxytocin receptor-targeted liposomes loaded with indomethacin were effective in reducing rates of preterm birth in mice, whereas nontargeted liposomes loaded with indomethacin had no effect. CONCLUSION: Our results demonstrate that oxytocin receptor-targeted liposomes can be used to either inhibit or enhance human uterine contractions in vitro. In vivo, the liposomes localized to the uterine tissue of pregnant mice and were effective in delivering agents for the prevention of inflammation-induced preterm labor. The potential clinical advantage of targeted liposomal drug delivery to the myometrium is reduced dose and reduced toxicity to both mother and fetus.

Cyclic stretch augments production of neutrophil chemokines, matrix metalloproteinases, and activin a in human endometrial stromal cells.

PROBLEM: The aim of this study was to test our hypothesis: Contractile activity that occurs in the uterus during menstruation induces biochemical factors that enhance remodeling of the endometrium. METHOD OF STUDY: Cyclic stretch, mimicking contractile activity during menstruation, was applied to human endometrial stromal cells (ESC) using the Flexercell Tension system. The concentration and activity of CXCL8, CXCL1, MMPs, and activin A were measured using ELISAs and specific assays. Neutrophil chemotactic activity was evaluated using migration assays. RESULTS: Cyclic stretch significantly induced ESC secretion of CXCL8 and CXCL1 and neutrophil chemotaxis. Stretch also increased MMP-1, MMP-2, and MMP-3 activity, activin A secretion, and activity in ESC. CONCLUSION: These results indicate that the contractile activities of the uterus during menstruation contribute to the remodeling of the endometrium, by inducing chemokine secretion, MMP expression, activity, and neutrophil chemotaxis.

Cyclic stretch augments production of neutrophil chemokines and matrix metalloproteinases-1 (MMP-1) from human decidual cells, and the production was reduced by progesterone.

PROBLEM: The purpose of this study was to evaluate the impact of mechanical stretch caused by uterine contraction and progesterone (P4) on decidual cells (DC), neutrophil chemokines, and MMP-1 expression. METHOD OF STUDY: DC were cultured, and cyclic stretch was applied using a computer-operated cell stretch system. Interleukin (IL)-8, growth-regulated oncogene (GRO) α, matrix metalloproteinase (MMP)-1, and mRNA and/or protein expression/activity was measured using RT-PCR and ELISA. Neutrophil chemotactic activity in conditioned media was evaluated using migration assays. The effect of P4 was also studied. RESULTS: Cyclic mechanical stretch increased IL-8, GROα, mRNA and protein, and MMP-1 production and activity level. Supernatant from stretched cells induced neutrophil chemotactic activity significantly. P4 suppressed the effect of stretch. CONCLUSION: The current study demonstrates that cyclic mechanical stretch stimulates the production of neutrophil chemokines and MMP-1 from human decidual cells, and the production was reduced by progesterone. These findings suggest that decidual cells are responding to mechanical and endocrine signals and induce biochemical factors and thereby contribute to the regulation of human labor.

Cyclic stretch augments production of neutrophil chemokines and matrix metalloproteinase-1 in human uterine smooth muscle cells.

PROBLEM: The aim of this study was to investigate the impact of uterine contraction on the immune environment within the uterus during parturition. METHOD OF STUDY: Uterine smooth muscle cells (USMC) were isolated from uterine myometrial tissues and cultured. The effects of cyclic stretch on mRNA and/or protein expression of IL-8, Groα, and pro-MMP-1 by USMC were measured using RT-PCR and ELISA. Neutrophil chemotactic activity in conditioned media was evaluated using migration assays. To evaluate the effect of progesterone (P(4) ), USMC were pretreated with P(4) for 24 hr. RESULTS: Cyclic stretch increased IL-8 and Groα mRNA and protein and pro-MMP-1 production significantly. Supernatants from stretched cells induced neutrophil chemotactic activity significantly; these effects were abrogated by anti-IL-8 or Groα neutralizing antibodies. Stretch effects were reduced by P(4) . CONCLUSION: These results suggest that uterine contraction may induce neutrophil infiltration and MMP-1 production, which may contribute to cervical ripening and rupture of membrane. The inhibitory effects of P(4) may explain the mechanism by which progestin prevents preterm labor.

N-acetylcysteine prevents preterm birth by attenuating the LPS-induced expression of contractile associated proteins in an animal model.

OBJECTIVE: Intrauterine infection is associated with maternal immune activation (MIA) leading to preterm birth through upregulation of contractile associated proteins (CAPs). We hypothesized that N-acetylcysteine would decrease NF-κB activation and CAP expression in a MIA model for preterm birth. METHODS: Pregnant CD-1 mice were given intrauterine LPS or saline on day 15/20. They received NAC or saline prior to injection and were monitored until delivery. The rate of preterm birth in the control, LPS, and LPS + NAC animals was determined. In another group, animals were sacrificed 6 h after treatment and myometrium was collected. COX-2, connexin 43, and oxytocin receptor expression was determined. RESULTS: LPS administration resulted in preterm birth and this effect was attenuated by NAC. LPS increased COX-2, connexin 43, and oxytocin receptor expression. NAC significantly decreased COX-2 expression. LPS increased NF-κB activation; this was attenuated by NAC. CONCLUSION: NAC may be beneficial in prevention of MIA-related preterm birth through attenuation of NF-κB activation and COX-2 upregulation.

The frequency and clinical significance of intra-amniotic inflammation in women with preterm uterine contractility but without cervical change: do the diagnostic criteria for preterm labor need to be changed?

OBJECTIVE: The objective of this study was to determine the frequency and clinical significance of intra-amniotic inflammation in patients with preterm increased uterine contractility with intact membranes but without cervical change. METHODS: Amniocentesis was performed in 132 patients with regular uterine contractions and intact membranes without cervical change. Amniotic fluid was cultured for bacteria and mycoplasmas and assayed for matrix metalloproteinase-8 (MMP-8). Intra-amniotic inflammation was defined as an elevated amniotic fluid MMP-8 concentration (>23 ng/mL). RESULTS: (1) Intra-amniotic inflammation was present in 12.1% (16/132); (2) Culture-proven intra-amniotic infection was diagnosed in 3% (4/132) of patients without demonstrable cervical change on admission or during the period of observation; and (3) Patients with intra-amniotic inflammation had significantly higher rates of preterm delivery and adverse outcomes, and shorter amniocentesis-to-delivery intervals than those without intra-amniotic inflammation (P < 0.05 for each). Adverse outcomes included chorioamnionitis, funisitis, and neonatal death. CONCLUSION: Intra-amniotic inflammation was present in 12% of patients with regular uterine contractions without cervical change, while culture-proven intra-amniotic infection was present in 3%. The presence of intra-amniotic inflammation was a significant risk factor for adverse neonatal outcomes. These observations question whether cervical changes should be required for the diagnosis of preterm labor, because patients without modifications in cervical status on admission or during a period of observation are at risk for adverse pregnancy outcomes.

Challenge with ovalbumin antigen increases uterine and cervical contractile activity in sensitized guinea pigs.

OBJECTIVE: The aims of this study were to investigate the effects of ovalbumin challenge on uterine and cervical contractility, intrauterine pressure, and uterine electromyography activity in sensitized guinea pigs. STUDY DESIGN: Guinea pigs were sensitized by injection of ovalbumin-aluminum hydroxide suspension. Control animals were injected with the aluminum hydroxide suspension only. On days 55-57 of pregnancy, longitudinal uterine and cervical strips from guinea pigs were prepared for isometric tension recording. Nonpregnant guinea pigs were outfitted with telemetric transducers to record intrauterine pressure and uterine electromyography. RESULTS: Ovalbumin significantly increased contractility of uterine and cervical strips from sensitized versus nonsensitized animals. These effects were abolished by histamine H(1) receptor antagonist in uterine strips and by histamine H(1) receptor antagonist and a mast cell stabilizer in cervical strips from sensitized animals. Cyclooxygenase and 5-lipoxygenase inhibitors had no significant effect on the response to ovalbumin. Treatment with ovalbumin in vivo significantly increased intrauterine pressure and uterine electromyography activity in sensitized but not in nonsensitized, animals. CONCLUSION: Our findings indicate that type I hypersensitivity reactions may be important in mediating uterine contractility in pregnant and nonpregnant states.

Pretreatment with toll-like receptor 4 antagonist inhibits lipopolysaccharide-induced preterm uterine contractility, cytokines, and prostaglandins in rhesus monkeys.

Intrauterine infection, which occurs in most early preterm births, triggers an immune response culminating in preterm labor. The authors hypothesize that blockade of lipopolysaccharide (LPS)-induced immune responses by a toll-like receptor 4 antagonist (TLR4A) would prevent elevations in amniotic fluid (AF) cytokines, prostaglandins, and uterine contractility. Chronically catheterized rhesus monkeys at 128 to 147 days' gestation received intra-amniotic infusions of either (1) saline (n = 6), (2) LPS (0.15-10 microg; n = 4), or (3) TLR4A pretreatment with LPS (10 microg) 1 hour later (n = 4). AF cytokines, prostaglandins, and uterine contractility were compared using 1-way ANOVA with Bonferroni-adjusted pairwise comparisons. Compared with saline controls, LPS induced significant elevations in AF interleukin-8 (IL-8), tumor necrosis factor (TNF)- alpha, PGE(2), PGF(2)(alpha), and uterine contractility (P < .05). In contrast, TLR4A pretreatment inhibited LPS-induced uterine activity and was associated with significantly lower AF IL-8, TNF-alpha, PGE(2), and PGF(2)( alpha) versus LPS alone (P < .05). Toll-like receptor antagonists, together with antibiotics, may delay or prevent infection-associated preterm birth.

Effects of chronic specific urogenital infections on contractility of the human isolated pregnant myometrium.

The contractile activity of the isolated myometrium of pregnant women with mycoplasma, chlamydia and mixed infections has been studied by pharmacological organ bath method. We found that mycoplasma infection decreased while chlamydia or mixed infection increased myometrium contraction evoked by oxytocin or prostaglandin F2alpha. The results of this study could be important for the prediction of possible complications during pregnancy and labour in women with chronic specific urogenital infections.

[Premature delivery and inflammation]. / Accouchement prématuré et inflammation.

Premature delivery results from multiple closely interdependent factors. Inflammation is most generally caused by cervicovaginal infection that may progress to intra-uterine infection or inflammation. Severe chorioamniotitis is found in 75% of all premature deliveries compared with 15% in term deliveries. Premature rupture of the membranes is the cause of premature delivery in 30-40% of premature deliveries although the diagnosis of chorioamniotitis can also be established with intact membranes, sometimes on the basis of histological findings alone. The degree of prematurity is correlated with the severity of the histological chorioamniotitis. The severity and the duration of the lesions is often the cause of antibiotic failure for the treatment of threatening premature delivery. Inflammation mediators, mainly proinflammatory cytokines (IL1, TNF-alpha), chemokines (IL6, IL8 and MIP-1alpha) and immunomodulator cytokines (IL6) and immunosuppressive cytokines (IL10, IL4) are produced by the amniotic and decidual membranes and are found in the fetal circulation and amniotic fluid. This reaction triggers a cascade of events leading to the production of prostaglandins and cyclooxygenase (COX2) activity, that cause uterine contractions. The inflammation may be initiated locally, even from an extrapelvic location, This leads to a fetal and/or maternal systemic inflammatory reaction. Systemic fetal expression of deregulated inflammatory phenomena can lead to neonatal lesions of lung and brain white matter tissue. This explains the failure of tocolysis and antibiotics in uncontrolled situations and suggests new avenues for therapy using selective inhibitors of COX2.

[Appearance of inflammatory cytokines interleukin-1 beta and interleukin-6 in amniotic fluid during labor and in intrauterine pathogen colonization]. / Das Auftreten der inflammatorischen Zytokine Interleukin-1 beta und Interleukin-6 im Fruchtwasser bei Wehentätigkeit sowie bei intrauteriner Keimbesiedlung.

Subclinical intrauterine infections have been proposed to be one of the leading causes of preterm labor. The determination of inflammatory cytokines IL-1 beta and IL-6 in the amniotic fluid may be useful to identify women who have infection-associated preterm labor. Amniotic fluid was collected from 99 women during amniocentesis, during cesarean section or at the time of amniotomy using sterile technique. IL-1 beta and IL-6 were determined by a specific ELISA. Fluid of each sample was cultured for aerobic and anaerobic bacteria and for Mycoplasma hominis and Ureaplasma urealyticum. Different populations were identified according to the criteria "gestational age", "active labor", "positive amniotic fluid cultures". Interleukin-6 was detectable in all samples of amniotic fluid. The second-trimester (weeks 14-27) amniotic fluid concentration of IL-6 (18-2270 pg/ml) was higher than in the third trimester (weeks 28-42, 4-329 pg/ml). The difference was significant. Women in active labor had higher levels of IL-6 in their amniotic fluid than women not in labor (p < 0.01). There is no significant difference between women with preterm labor and delivery (weeks 28-37, 597-8670 pg/ml) and with term labor and delivery (weeks 38-42, 24-8300 pg/ml). Only culture negative samples were included in this population. Interleukin-1 beta was not detectable in the majority (90%) of these samples. 30% of the women in labor had positive amniotic fluid cultures. The IL-6 concentration of this population was not elevated in comparison to women in labor with negative cultures. Interleukin-1 beta was present in high concentrations (5-1100 pg/ml) in all fluid samples with detectable bacteria. Our data suggest that IL-1 beta may indicate subclinical uterine infection. IL-6 is elevated in all fluid samples of women in active labor.

[The role of cytokines in the induction of labor, cervical ripening and rupture of the fetal membranes]. / Zur Rolle von Zytokinen bei Weheninduktion, Zervixreifung und Blasensprung.

Even today prematurity is the major cause of perinatal mortality. Prematurity has multiple causes. There is a growing body of evidence supporting the association between silent intrauterine infection and preterm birth. Bacterial products may activate macrophages ubiquitous present in the decidua, placenta and fetal membranes. These cells after activation secrete a large variety of mediators including tumour necrosis factor alpha (TNFalpha) and interleukin (IL)-1. Besides these cytokines IL-2, IL-3, IL-4, IL-6, IL-8, IL-10, epidermal growth factor, granulocyte-colony stimulating factor and transforming growth factor beta have been identified in intrauterine tissues and in the amniotic fluid. The majority of these substances (TNFalpha, IL-1, IL-2, IL-3, IL-6) can stimulate the prostaglandin-biosynthesis by intrauterine tissues (amnion, chorion, decidua), some of them have antiinflammatory effects (IL-10, transforming growth factor alpha). These effects are mediated by receptors on the target cells; specific receptor antagonists (for example for IL-1) were found in high concentrations in amniotic fluid during normal pregnancy. This cytokine network is in a sensitive balance and probably associated with an uncomplicated course of pregnancy. Systemic or localized infections as well as tissue injury initiate the induction of the prostaglandin synthesis cascade thus leading to pregnancy loss via augmented cytokine secretion. Furthermore, cytokines may be involved in the regulation of preterm and term cervical ripening. The changes in mechanical properties of the cervix are associated with a reduction of collagen content and alterations in the glycosaminoglycan pattern within the cervical extracellular matrix. IL-1 can stimulate the synthesis of collagenases, and IL-8 may play an important role in the regulation of the invasion of neutrophilic granulocytes into the cervical stroma with subsequent degranulation and release of proteases. The cytokine-stimulated collagenase production in the fetal membranes is responsible for the reduction of their tensile strength and may be associated with rupture of the membranes. The cytokine network seems to be a sensitive regulation system. Disturbances of its balance by environmental (e.g. infection) or intrauterine influences (e. g. extension by the fetus) may lead to termination of pregnancy.

Interleukin-6 does not stimulate rat myometrial contractions in an in vitro model.

PROBLEM: Interleukin-6 (IL-6) increases in culture-positive amniotic fluid in women with preterm labor. IL-6 stimulates the production of prostaglandins leading to increased uterine activity. METHODS: We tested the hypothesis that IL-6 increases myometrial activity through release of uterotonic mediators. We studied the effect of IL-6 on uterine contractions in the absence and presence of fetal membranes to determine if the effect was on myometrium alone or was mediated through fetal membranes/decidua. IL-6 in concentrations of 100, 10, 0.1 or 0 ng/ml was added to the maternal side of the dual chamber-fetal membrane-uterine muscle in vitro model. RESULTS: We found that 10 ng/ml of IL-6 alone, without fetal membranes, caused a significant decrease in uterine contractions over time (P < or = 0.01). This decrease was not observed with the addition of term, nonlabored fetal membranes. CONCLUSIONS: IL-6 in the presence or absence of membranes, over a four log fold dose range, did not stimulate uterine contractions.

Endotoxin, interleukin-1 beta, interleukin-6, or tumor necrosis factor-alpha do not acutely stimulate isolated murine myometrial contractile activity.

OBJECTIVES: Endotoxin, interleukin-1 beta, interleukin-6, and tumor necrosis factor-alpha have been implicated in the pathogenesis of preterm labor, but their acute effect on myometrial contractile activity is unknown. The objective of this study was to determine their effect on isolated pregnant murine myometrial contractile activity. STUDY DESIGN: Isometric contractions were measured in myometrium isolated from pregnancy day 18 Swiss-Webster mice. Frequency, duration, amplitude, and integrated area were compared before and after the addition of endotoxin (10(3) and 10(4) ng/ml) (n = 6), interleukin-1 beta (10 and 10 ng/ml) (n = 6), interleukin-6 (1 and 10 ng/ml) (n = 6), and tumor necrosis factor-alpha (1 and 10 ng/ml) (n = 6). Results were analyzed with the Wilcoxon rank-sum test. RESULTS: The addition of endotoxin, interleukin-1 beta, interleukin-6, or tumor necrosis factor-alpha did not result in a change in the contractile activity of isolated pregnant murine myometrium compared with control. CONCLUSION: Endotoxin, interleukin-1 beta, interleukin-6, and tumor necrosis factor-alpha do not acutely increase isolated murine myometrial contractile activity.