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| Significance of preterm birth: |
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Birth before 37 weeks of gestation is a major cause of fetal and neonatal morbidity and mortality. Problems due to prematurity or low birth weight (LBW) have been well documented and these factors account for 60% of all neonatal mortality.1 Gibbs and colleagues reported that premature rupture of the fetal membranes (PROM) is associated with one third of all preterm deliveries and is the third leading cause of perinatal death.2 The etiology of preterm birth and PROM is multifactorial and an exact cause in each case can rarely be identified. Recent evidence suggests a strong association between infection, preterm labor (PTL) and PROM. IAI and the host inflammatory response can lead to the accumulation of pro inflammatory cytokines, many of which are known to induce the release of uterotonins (substances that cause the uterus to contract) like prostaglandin from gestational tissues, thereby causing uterine contractions and labor.3 Role of inflammatory cytokines in preterm labor: Until recently, it had been accepted that microorganisms alone were responsible for the ill effects and metabolic derangements associated with infection. However, the concentrations of the toxins or the number of bacterial cells required to generate the ill effects leading to preterm labor are rarely achieved in the amniotic fluid of women or in the placental tissues.3 It is now well established that many of these ill effects are mediated by endogenous host products in response to an infectious process.3 In the late 80's Romero and Casey suggested that the host immune response could be a causative factor in the infection mediated preterm labor process.3,4 Romero and Casey observed that macrophages were ubiquitous in the maternal decidua, fetal membranes and in other reproductive tract compartments. Decidual cells are macrophage-like in nature, and upon activation both macrophages and decidual cells release inflammatory mediators (cytokines) such as IL-1, IL-6, IL-8 and TNF-".3,4 Romero and colleagues and several others have studied the levels of inflammatory cytokines in the amniotic fluid (AF) of women with PTL and IAI. They documented that the levels of all these cytokines are elevated during PTL and IAI compared to normal term labor. Decidua as a site of inflammatory cytokine production: Several studies have been conducted to localize the tissue origin of the inflammatory cytokines in the reproductive tract. Romero, 3 Casey 4 and Hunt 5 have suggested that resident macrophages throughout the reproductive tract, and macrophage-like decidual cells were responsible for the immune response in the uterine cavity. They were able to demonstrate that placental macrophages and decidual cells behave in the same fashion and each is able to release large amounts of inflammatory cytokines in response to bacterial toxins. Fetal membrane as a site of inflammatory cytokine production: In the early 90s our laboratory hypothesized that fetal membranes
could respond to microbial invasion of the intraamniotic cavity (MIAC) and IAI and were not passive in the inflammatory
response. We developed an amniochorion organ explant system to document the fetal membrane cytokine response to
an in vitro model of IAI.6 The organ explant system was developed to preserve the integrity of the amniochorion
and allow the investigation of this tissue as a functional unit. As it was evident the fetal membranes were active
participants in the labor process through prostaglandin production and release, we proposed that the membranes
might also be capable of cytokine synthesis. Table I.
After documenting fetal membranes as a source of some major cytokines associated with infection, we also examined the expression (by PCR) of IL-1, TNF, IL-6 and IL-8 in membranes collected from women with documented IAI. These cytokines were studied since their association with IAI and the outcome of labor has been established unequivocally.5 The results are given in table II. Table II
Cytokines
Non infected control membranes
Infected Membranes*
IL-1ß
+
+++
TNF
+
+++
IL-6
-
+++
IL-8
-
+++
* Experiments were semi-quantitative and the data were reproducible These findings agreed with previous reports using decidua and amniotic fluid and determining that IL-6 and IL-8 are two key marker cytokines associated with infection and complications of pregnancy. Additionally, these cytokines exhibit similar patterns of expression in amniochorion. We also found that all the inflammatory cytokines released
were increased in response to bacterial toxins (LPS and PGPS [from Gram positive cell wall]). The amniochorion
cytokine release pattern in response to LPS stimulation in vitro mirrored the inflammatory response noted in amniotic
fluid during IAI. IL-6 and IL-8 levels were in the nanogram ranges and IL-1 and TNF levels were in the picogram
range in both culture media and amniotic fluid.11 IL-6 levels were much higher compared to the levels of IL-8 confirming
the predicted association of IL-6 with PTL. However, the pattern of IL-6 and IL-8 response to PGPS was different
from that of LPS in that PGPS stimulated more IL-8 than IL-6.12 We propose that fetal membranes must be an essential
part of the host response to IAI or MIAC and that they are integral in determining the Inhibitory effects of Interleukin-10 on human fetal membranes: The absence of immunomodulatory cytokines in our system under normal as well as infected conditions led us to believe that these cytokines could be the limiting factors of immune response that otherwise would result in PTL. We tested this hypothesis by stimulating fetal membranes with various doses of IL-10. As expected IL-10 was found to regulate the expression of IL-6, IL-8 and TNF at the transcriptional level thus decreasing the bioavailability of these cytokines for action. A dose of 100 ng/ml of IL-10 was found to totally inhibit cytokine transcription in the presence of an active infectious stimulus (LPS).14-16 Interestingly Greig and colleagues documented that the levels of IL-10 required to inhibit fetal membrane cytokine production were never achieved in the amniotic fluid of women with PTL and IAI, 17 suggesting that IL-10 may in fact be the natural immuno regulatory cytokine required for preventing PTL induced by inflammatory cytokines. From these reports it has been evident that the fetal membrane immune response plays a vital role in the outcome of pregnancy related complications. Ongoing studies evaluating cytokine kinetics in our laboratory suggest that fetal membranes exhibit a cytokine network interaction during various pathophysiological conditions. This networking of cytokines (eg. IL-1 and TNF stimulation of IL-6 production, down regulation of IL-1 and TNF by IL-6, IL-1 stimulation of TNF etc.) once initiated, creates a vicious cycle of events. Once initiated this networking will proceed even in the absence of an apparent infection or after antibiotic and tocolytic administration. We hypothesize that these phenomenon can be controlled by a counter regulatory cytokine like IL-10 which inhibits the transcriptional activation of these genes as well as acts as a nuclease to destroy the specific cytokine mRNA. Our past nine years of work in this field suggests that the fetal immune response is as significant as the maternal response and the fetal membrane response is essential in understanding the phenomenon of prematurity. Preliminary animal experimentation has been conducted to evaluate the efficacy of immunoinhibitory cytokines such as IL-10 in stopping cytokine induced uterine contractions. IL-10 inhibits preterm contractions mediated by IL-1 $ in experimental animal models: Gravett and colleagues in their recent studies documented that IL-10 can reduce IL-1$ induced uterine contractility in rhesus monkeys by 85%. IL-1$ alone produced 11,650 contraction units (HCA; mm Hg x sec/hr) where as IL-1$ + IL-10 reduced the contraction units to 1,800 (HCA; mm Hg x sec/hr) and base line was 280 (HCA; mm Hg x sec/hr). The terminal phase half life of IL-10 was found to be 14 hours and no side effects were noticed. Hematological indices were found to be normal in IL-10 treated animals.18 Pharmacodynamics of subcutaneous recombinant human IL-10 in healthy volunteers: Studies conducted by Huhn et al19 have documented the pharmacokinetics of IL-10 in healthy human volunteers. The study goals included pharmacokinetics, tolerability, and immuno modulatory effects of IL-10 in healthy humans. Normal volunteers received a single doses of recombinant human IL-10 (range of doses 0.1 - 100 µg/kg body weight) or placebo by intravenous injection. The data from this study showed no major side effects other than dose related moderate flu like symptoms, (chills, head ache and myalgia) with higher doses of IL-10. The mean terminal phase t½ ranged from 2.3 ± 0.5 to 3.7 ± 0.8 hours. Dose related response also included transient neutrophilia, monocytosis and lymphopenia. In summary, single doses of IL-10 were tolerated by healthy volunteers. Reproducible and predictable pharmacokinetics were seen. In a time and dose dependent manner IL-10 suppressed inflammatory cytokine (IL-1$ and TNF-") production from whole blood from these patients stimulated ex vivo with bacterial lipopolysaccharide. They suggested a clinical usefulness of this compound as a modulator of specific components of inflammation. IL-10 is now under phase I trials for treatment of inflammatory bowel disease and chronic active Crohn's disease.
EXPERIMENTAL MODELS SHOWS INHIBITION OF
PRETERM CONTRACTIONS AFTER IL-10 ADMINISTRATION There has not been a new pharmacologic agent for the treatment of preterm labor approved by the FDA for two decades. Only ritordrine hydrochloride is approved by the FDA for the treatment of PTL although multiple other agents are used including MgSO4, terbutaline, indocin, nifedipine, salbutamol and nitroglycerine. All of these agents are directed at stopping the endpoint of the preterm labor pathway i.e. uterine contractions. The large majority of these drugs prevent calcium utilization by the myometrial cells (Mg++, calcium channel blockers, $ mimetic agents) while indocin blocks prostaglandin synthesis. We proposes the use of IL-10 in the treatment of PTL. We believe the failure of other tocolytic agents is due to their failure to address the self perpetuating pro-inflammatory cytokine network which leads to prostaglandin production and thereby labor. Supporting data for this belief exists both in vitro and in preliminary primate studies. We believe that IL-10, when used in concert with traditional tocolytic agents and, in some cases, appropriate antibiotic treatment, will prove more effective than traditional tocolysis alone. References:
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