Utilization of endoscopic inoculation in a mouse model of intrauterine infection-induced preterm birth: role of interleukin 1beta

A novel murine model of intrauterine infection/inflammation-induced preterm birth based on direct endoscopic intracervical inoculation is described. Using this model, we investigated infection-induced premature pregnancy loss in normal and interleukin (IL) 1beta-deficient mice. Seventy-four CD-1, HS, C57BL/6J wild type (IL-1beta+/+), and C57BL/6J IL-1beta-deficient (IL-1beta-/-) mice were inoculated intracervically using a micro-endoscope, at a time corresponding to 70% of average gestation. Intracervical injection of lipopolysaccharide (LPS) or Escherichia coli reliably induced premature birth: 100% of mice intracervically injected with LPS and 92% of mice with a positive endometrial E. coli culture delivered prematurely within 36 h after inoculation. No losses were observed in mice inoculated with saline. Pregnancy loss was associated with increased uterine tissue cyclooxygenase-2 gene expression and uterine content of IL-1beta, tumor necrosis factor alpha, macrophage inflammatory protein-1alpha, and IL-6, as well as elevation of nuclear factor-kappaB activity in uterine tissues. Although IL-1beta-/- mice exhibited decreased uterine cytokine production in response to bacteria and LPS, IL-1beta deficiency did not affect the rate of pregnancy loss. This model using direct intracervical bacterial or LPS inoculation is useful for studying preterm pregnancy loss in genetically altered mice in order to develop novel interventions for infection-associated preterm labor.     

Requirement for progesterone priming and its long-term effects on implantation in the mouse

At least 48 hr of progesterone (P4) priming has been documented to be essential for P4 and estrogen to initiate implantation in the rat. However, the length of this P4 priming requirement for implantation in the mouse has not been experimentally defined. Therefore, our first objective was to determine the length of P4-priming requirement for implantation in the mouse. Day 4 blastocysts were transferred into the uteri of Day 5 or Day 6 pseudopregnant mice that were ovariectomized on Day 1 (= vaginal plug) and treated with a single injection of P4 and 17 beta-estradiol (E2) only on Day 5, or a single injection of P4 on Day 5 followed by a second injection of P4 plus E2 on Day 6, respectively. Although none of the transferred blastocysts implanted in the uteri of P4-unprimed recipients, 46% of the transferred blastocysts implanted into the uteri of all recipients that were first primed with P4 24 hour prior to a second injection of P4 and E2. These results suggest that in contrast to the rat, the mouse uterus requires at most 24 hr of P4 priming before P4 and estrogen can initiate implantation. Our second objective was to determine whether P4 priming has a long-term effect on implantation in the mouse. Our present results and those of others suggest that the mouse uterus is exposed to rising P4 levels for 24 hr prior to implantation on Day 4 of pregnancy. Therefore, in the present investigation, induction of implantation by an injection of P4 and E2 following 5 days of ovariectomy performed on Day 4 of pregnancy clearly suggests that once exposed to P4 for 24 hr, the mouse uterus retains a long-term effect, i.e., following P4 withdrawal for several days, 24 hr of initial P4 priming is no longer required for P4 and estrogen to initiate implantation. Our next objective was to explore whether this long-term effect of P4 priming on implantation can be prolonged and potentiated by increasing the length of initial P4 priming. Thus, when the mice were ovariectomized on Day 4 of pregnancy and treated with P4 beginning on Day 5 for 4 days, the long-term effect on implantation was prolonged (8 days vs 5 days following P4 withdrawal) and potentiated (94% vs 0% mice with implantation following 8 days of P4 withdrawal) as compared with those with no P4 priming after ovariectomy.(ABSTRACT TRUNCATED AT 400 WORDS)     

Alteration of Response of Bean Leaves to Compatible and Incompatible Bacteria

Injection of Red Mexican bean leaves with Pseudomonas phascolicolaRace 2 (compatible, 18 h before P. mors-prunorum or P. phaseolicolaRace 1 (incompatible), or simultaneous inoculation with compatibleand incompatible bacteria (3:1) greatly delayed the appearanceof hypersensitive responses. When compatible bacteria were inoculated12 h or less before incompatible bacteria, or when the ratioof these bacteria was 1:1 or 0.3:1 in simultaneous inoculation,hypersensitive responses did develop. Earlier inoculation withincompatible bacteria delayed the appearance and severity ofdisease symptoms following later inoculation with compatiblebacteria. When selected areas of leaves were inoculated with compatiblebacteria, effects on hypersensitive responses were confinedto these areas when the whole leaf was inoculated later withincompatible bacteria. Inoculation through the upper leaf surfacewith incompatible bacteria did not affect susceptible responseswhen compatible bacteria were inoculated 24 h later throughthe lower surface. Treatment of leaves with heat-killed bacteria or live bacteriain numbers insufficient to cause hypersensitive responses didnot prevent development of these responses following later inoculationwith incompatible bacteria. Use of heat-killed bacteria didsuppress hypersensitive responses in tobacco leaves. Injectionof leaves with cycloheximide (20 p.p.m.) 30 min before inoculationwith incompatible bacteria suppressed leakage of electrolytesand browning of tissues associated with hypersensitive responses.Cycloheximide had little effect on leakage of electrolytes fromleaves inoculated with compatible bacteria or with the developmentof susceptible responses. Exposure of leaves to chloroform vapourdelayed hypersensitive responses by 24 h; treatment with peroxidasehad no effect.     

Folic Acid Protects against Lipopolysaccharide-Induced Preterm Delivery and Intrauterine Growth Restriction through Its Anti-Inflammatory Effect in Mice

Increasing evidence demonstrates that maternal folic acid (FA) supplementation during pregnancy reduces the risk of neural tube defects, but whether FA prevents preterm delivery and intrauterine growth restriction (IUGR) remains obscure. Previous studies showed that maternal lipopolysaccharide (LPS) exposure induces preterm delivery, fetal death and IUGR in rodent animals. The aim of this study was to investigate the effects of FA on LPS-induced preterm delivery, fetal death and IUGR in mice. Some pregnant mice were orally administered with FA (0.6, 3 or 15 mg/kg) 1 h before LPS injection. As expected, a high dose of LPS (300 μg/kg, i.p.) on gestational day 15 (GD15) caused 100% of dams to deliver before GD18 and 89.3% of fetuses dead. A low dose of LPS (75 μg/kg, i.p.) daily from GD15 to GD17 resulted in IUGR. Interestingly, pretreatment with FA prevented LPS-induced preterm delivery and fetal death. In addition, FA significantly attenuated LPS-induced IUGR. Further experiments showed that FA inhibited LPS-induced activation of nuclear factor kappa B (NF-κB) in mouse placentas. Moreover, FA suppressed LPS-induced NF-κB activation in human trophoblast cell line JEG-3. Correspondingly, FA significantly attenuated LPS-induced upregulation of cyclooxygenase (COX)-2 in mouse placentas. In addition, FA significantly reduced the levels of interleukin (IL)-6 and keratinocyte-derived cytokine (KC) in amniotic fluid of LPS-treated mice. Collectively, maternal FA supplementation during pregnancy protects against LPS-induced preterm delivery, fetal death and IUGR through its anti-inflammatory effects.     

Neutrophil migration into the bovine uterine lumen following intrauterine inoculation with killed Haemophilus somnus.

Polymorphonuclear neutrophils (PMN) in bovine uterine flushings following intrauterine deposition of killed bacteria were measured and the effect of immune status on the influx of PMN into the uterine lumen during oestrus was determined. Holstein heifers were immunized with a 270-kDa outer-membrane protein (omp-270) from Haemophilus somnus. During oestrus, immunized heifers (n = 21) received an intrauterine inoculum of either a heat-killed suspension of a homologous strain of H. somnus containing omp-270 (n = 7), a heterologous strain of H. somnus lacking omp-270 (n = 7), or phosphate-buffered saline (n = 7). Five additional heifers were inseminated with extended bovine semen. Uterine contents were collected in saline lavage immediately before inoculation (t0) and at 6, 24, 48, 72, 96, and 120 h after inoculation. The semen-inoculated heifers were lavaged only at t120. All groups experienced PMN infiltration which peaked 6 h after inoculation and tended to decline thereafter. Differences were not observed between treatment groups, indicating that neither bacterial inoculation nor immune status was as important in eliciting PMN effusion as the flushing procedure itself.     

Effects of bovine follicular fluid inhibin on serum gonadotrophin concentrations in ewes during oestrus

Experiments were conducted with ewes to investigate the effects of an enriched bovine follicular fluid inhibin preparation (INH) on gonadotrophin secretion after the onset of oestrus. Administration of INH (10 mg) 1 h after the onset of oestrus did not significantly alter the preovulatory FSH and LH surges or the second FSH peak. To determine the effects of INH on the second FSH surge, ewes were treated with saline (N = 7) or INH (N = 10) at 4 h (10 mg) and 24 h (5 mg) after the peak of the preovulatory LH surge. The second FSH surge was delayed about 24 h (P less than 0.05) in ewes treated with INH; however, the delay did not alter the interval to the next oestrus. In a third experiment, 16 ewes were assigned to 4 groups in a 2 x 2 factorial with the main effects being ovariectomy at 4 h and INH treatment (10 mg) at 4, 20 and 36 h after the peak of the LH surge. Controls received sham ovariectomy and saline injection as appropriate. Ovariectomy resulted in a rapid increase in serum FSH but not LH and this was delayed (P less than 0.05) by INH treatment. These results indicate that inhibin has a selective inhibitory action on FSH secretion in ewes and suggests that the second FSH surge results from increased basal FSH secretion due to decreased endogenous inhibin levels.     

Hormonal changes accompanying cigarette smoke-induced preterm births in a mouse model

Epidemiologic evidence indicates that maternal smoking increases the risk of preterm birth. While a number of plausible mechanisms for early delivery have been offered, the role of gestational hormones in this smoke-induced outcome is uncertain. Thus, a toxicologic study was performed to examine the effects and underlying hormonal mechanisms of mainstream cigarette smoke (MCS) exposure on gestational duration. Pregnant B6C3F1 mice were exposed by inhalation to MCS for 5 days/week (4 hrs/day) from Gestational Day (GD) 4 to parturition. Smoke-induced effects on gestational length, interpubic ligament length, maternal hormone secretion patterns (estradiol-17beta, progesterone, prolactin, and relaxin), body weight gain, postimplantation loss, litter size, and offspring sex ratio were examined. Dams exposed to MCS at a concentration equivalent to smoking less than one pack of cigarettes/day (carbon monoxide = 25 parts per million, total suspended particulates = 16 mg/m3) demonstrated a significant (P < 0.05) shortening of gestational duration (compared with pregnant, air-exposed mice). In addition, MCS-exposed mice sacrificed on GD 18 had significantly (P < 0.05) increased interpubic ligament length, elevated serum estrogen levels, and a reduced progesterone to estradiol-17beta ratio (compared with air-exposed controls); levels of progesterone and prolactin were only modestly decreased and increased, respectively, in the MCS-exposed mice. Smoke exposure had no significant effects on maternal relaxin levels, body weight gain, postimplantation loss, litter size, or sex ratio. Results of this study demonstrate that inhalation exposure of pregnant mice to a low dose of MCS shortens gestation and alters hormone secretory patterns, which are important for maintaining pregnancy and inducing parturition. These findings support the view that pregnant women who smoke (even modestly) may be at increased risk for preterm birth, and that early delivery may be related (at least partly) to MCS-induced.     

Surfactant protein A modulates the lipopolysaccharide-induced inflammatory response related to preterm birth

Surfactant protein A (SP-A) functions in homeostasis of lung surfactant and in innate immunity. SP-A is secreted by the fetal lung into amniotic fluid. Additionally it has been detected in gestational tissues. We propose that SP-A influences intrauterine inflammation that is commonly associated with preterm birth, the main underlying cause of neonatal mortality and morbidity. We used our previously established mouse model of LPS-induced preterm birth of live-born pups to investigate the role of SP-A in preterm birth. Mice overexpressing rat SP-A (rSP-A) under the control of human SP-C promoter were used. Cytokine concentrations in maternal and fetal serum and in amniotic fluid and mRNA levels of several inflammatory mediators in lungs and in intrauterine tissues were quantified using Cytometric Bead Array and RNase Protection Assay, respectively. Higher levels of SP-A mRNA were observed in fetal lungs and intrauterine tissues of rSP-A mice compared with wild-type. Using Western blot we detected excess of SP-A protein in fetal lung and in amniotic fluid of rSP-A animals. Despite some differences in the basal levels of TNF-α and IL-10 between rSP-A and wild-type animals, there were no differences in the duration of pregnancy. However, the levels of TNF-α, IL-10 and some other inflammatory mediators in intrauterine tissues and in amniotic fluid differed significantly between the mouse lines after maternal LPS given at 17 dpc. We conclude that SP-A modulates the levels of intrauterine inflammatory mediators involved in preterm birth and may contribute to inflammatory processes related to spontaneous preterm labor.     

Cervical remodeling/ripening at term and preterm delivery: the same mechanism initiated by different mediators and different effector cells

Background

Premature cervical remodeling/ripening is believed to contribute to preterm delivery (PTD), the leading cause of perinatal morbidity and mortality. Despite considerable research, the causes of term and PTD remain unclear, and there is no effective treatment for PTD. We previously demonstrated that complement activation plays a causative role in cervical remodeling that leads to PTD in mice.

Methodology/Principal Findings

Here we found that complement activation is not required for the physiological process that leads to term delivery in mice. Neither increased C3 cervical deposition nor increased C3a and C5a serum levels were observed at term. In addition, macrophages infiltration was found in PTD in contrast to term delivery were no leukocytes were found. Despite the different role of complement and different cellular effector cells, PTD and term delivery share a common dowsntream pathway characterized by increased metalloproteinases (MMPs) release and increased collagen degradation. However, different sources of MMPs were identified. Macrophages are the source of MMPs in PTD while cervical fibroblasts and columnar epithelial cells synthesize MMPs at term delivery. A dramatic diminution in serum progesterone levels precedes parturition at term but not in PTD, suggesting that progesterone withdrawal initiates cervical remodeling at term. On the other hand, MMPs release in PTD is triggered by C5a.

Conclusion and Significance

In conclusion, preterm and term cervical remodeling occur through the same mechanism but they are initiated by different mediators and effector cells. That complement activation is required for PTD but not for the physiological process that leads to term delivery, suggests that complement is a potential specific biomarker and selective target to prevent PTD and thus avert neonatal mortality and morbidity.     

Maternal serum lipids during pregnancy and infant birth weight: the influence of prepregnancy BMI

Maternal obesity may be associated with metabolic factors that affect the intrauterine environment, fetal growth, and the offspring's long-term risk for chronic disease. Among these factors, maternal serum lipids play a particularly important role. Our objective was to estimate the influence of variation in maternal serum lipid levels on variation in infant birth weight (BW) in overweight/obese and normal weight women. In a prospective cohort of 143 gravidas, we measured maternal serum levels of total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and triglycerides (TG) at 6-10, 10-14, 16-20, 22-26, and 32-36 weeks gestation. Effects of maternal serum lipid levels on infant BW adjusted for gestational age at delivery (aBW) were analyzed using linear regression models. In analyses stratified by maternal prepregnancy BMI categorized as normal (≤25.0 kg/m(2)) and overweight/obese (>25.0 kg/m(2)), we found a significant (P < 0.05) inverse association between aBW and HDL-C at all time points starting at 10 weeks gestation in overweight/obese women. No significant effect was found in normal weight women. In contrast, increased maternal serum TG was significantly associated with increased aBW only for normal weight women at 10-14 and 22-26 weeks gestation. Variation in aBW is not associated with variation in maternal serum TC or LDL-C for either stratum at any time point. We postulate that such differences may be involved in the "physiological programming" that influences later risk of chronic disease in the infants of overweight/obese mothers.     

Immune effect of heat-killed multistrain of Lactobacillus acidophilus against Salmonella typhimurium invasion to mice

AIMS: This study attempted to determine whether lactic acid bacteria (LAB) could have a better probiotic function when used as a multistrain mixture, i.e. Mix-LAB, than when used as a monostrain. To this end, three strains of Lactobacillus acidophilus, specifically strain LAP5, LAF1 and LAH7, were heat-killed and mixed. This heat-killed Mix-LAB was used to evaluate the effectiveness of multistrain in inhibiting Salmonella invasion into cultured cells and into organs (spleen and liver) of live mice. METHODS AND RESULTS: BALB/c mice were orally administered with heat-killed Mix-LAB or sterile normal saline (control) for seven consecutive days and then challenged with orally administered Salmonella typhimurium on day 8. Results showed that, at day 6 after the challenge, the mice which had received Mix-LAB exhibited lower rates (P < 0.05) of Salmonella invasion into liver and spleen than did the control mice. Also, before the Salmonella challenge, the serum tumour necrosis factor-alpha (TNF-alpha) levels were not significantly different (P > 0.05) between these two groups of mice. After the challenge, however, the serum TNF-alpha level was significantly elevated (P < 0.05) in the control group, but not significantly changed in the Mix-LAB fed mice. To investigate possible factors involved in heat-killed Mix-LABs antagonistic effect on Salmonella invasion of mouse organs, heat-killed single strain and Mix-LAB were evaluated for ability to inhibit Salmonella invasion into cultured human intestinal Int-407 and Caco-2 cells. Results showed that none of the heat-killed strains were able to protect these cultured cells from Salmonella invasion, even though strains of LAP5 and Mix-LAB were adherent to them. However, study of the activation of murine macrophage Raw 264.7 cells showed that heat-killed Mix-LAB stimulated TNF-alpha production, nitric oxide release, and increased phagocytic activity in macrophages. CONCLUSIONS: Our findings suggest that heat-killed Mix-LAB can inhibit Salmonella invasion of mouse organs through the immunomodulating role of activated macrophage. SIGNIFICANCE AND IMPACT OF THE STUDY: The ability of heat-killed Mix-LAB to prevent bacterial infection in mice was found to be more significant than that of viable monostrain. This effect may be due to the activation of the immune system rather than to the adherence of LAB to the intestine epithelium.     

Effects of Lactobacillus casei on Pseudomonas aeruginosa infection in normal and dexamethasone-treated mice

A single intraperitoneal injection of Lactobacillus casei YIT 0003 into normal or dexamethasone-treated mice led to nonspecific resistance against intraperitoneal challenge with lethal doses of Pseudomonas aeruginosa PAO 3047. The enhanced resistance was retained for 14 days (P less than 0.05) after injection with living L. casei. In contrast, the statistically significant duration of the enhanced resistance in mice treated intraperitoneally with living L. acidophilus YIT 0075 was only 5 days. The in vivo killing activity of peritoneal exudate cells (PECs) against P. aeruginosa 5 and 7 days after intraperitoneal injection of living L. casei was significantly higher than in the case of PECs elicited by L. acidophilus. In the case of intravenous injection of heat-killed L. casei before intraperitoneal challenge with P. aeruginosa, there were no survivors in the late period after administration of L. casei. A high correlation existed between the patterns of in vivo killing of P. aeruginosa by PECs and survival rate of mice injected intravenously with heat-killed L. casei. The reduced in vivo killing activity of PECs from dexamethasone-treated mice against P. aeruginosa infection was also augmented by the intraperitoneal injection of heat-killed L. casei. These results indicate that L. casei possesses a resistance-enhancing capacity against P. aeruginosa infection in vivo. Differences in the duration of the enhanced resistance caused by L. casei and by L. acidophilus may be due to differences in chemical composition and/or physicochemical properties of the cell walls of the two kinds of bacteria.     

Influence of pregnancy on the course of malaria in mice infected with Plasmodium berghei

The course of malarial infection was compared in pregnant mice inoculated with Plasmodium berghei at different stages of gestation. When 12-14 wk old, pregnant BALB/c mice were inoculated with 1 x 10(6) of P. berghei NK65-infected red cells at gestation day 0, 2, 4, 6, 8, 10, 12, 14 or 16, the mice inoculated on gestation days 6-12 expired 6.5 days after inoculation compared to 9.5 days in non-pregnant mice. Parasitemia in these pregnant mice increased rapidly on day 4 after inoculation and anemia also developed earlier on day 5. However, the degree of parasitemia and anemia in the terminal stage of infection in these pregnant mice was milder than that of non-pregnant controls. Blood urea nitrogen increased at the terminal stage although the degree of increase in mice inoculated on gestation days 6-10 was comparatively small. Pregnant malarial mice died earlier with less physiological changes than non-pregnant controls. It was concluded that pregnancy makes the host susceptible to physiological changes caused by malaria.     

Experimentally induced elevations in acid phosphatase activity in hemolymph of Biomphalaria glabrata (Mollusca).

The levels of acid phosphatase activity in the hemocytes and serum of the following four groups of the pulmonate gastropod Biomphalaria glabrata were ascertained: (1) those injected with heat-killed Bacillus megaterium, (2) those challenged with sterile distilled water, (3) those shaminjected, and (4) those left untampered. It was determined that challenge with heat-killed bacteria sesulted in significant elevations in acid phosphatase activity in both cells and serum at 1, 2, and 4 hr postinjection, with the level being highest at 2 hr. Injection with water resulted in a significant elevation in cellular enzyme level at 1 hr postinjection but not at 2 and 4 hr; however, there were significant elevations at 2 and 4 hr in serum. This is interpreted to indicate that the elevated intracellular enzyme was subsequently released into serum. Sham injection resulted in significant elevations in acid phosphatase levels in hemocytes at 2 and 4 hr postinjection but no increase of enzyme activity in serum during the course of the experiment. This is interpreted to mean that this hydrolase was not released as a result of sham injection. The source of acid phosphatase was apparently the cytoplasmic granules of granulocytes, which are true lysosomes.     

Effects of microbial challenge on sleep in rabbits

Rabbits challenged with viable Staphylococcus aureus exhibit marked time-dependent changes in sleep patterns. To examine the generality of this observation, we monitored sleep patterns for 24 h before and for 48 h after intravenous inoculation of rabbits with Streptococcus pyogenes, Escherichia coli, or Candida albicans. All three agents produced complex time-dependent changes in sleep. Inoculation with S. pyogenes or C. albicans increased the time spent in slow-wave sleep (SWS) during h 4-20 after challenge. Electroencephalographic delta wave amplitudes (DWA) increased during h 4-8 after injection, but decreased during h 24-38 after inoculation. Altered sleep patterns were not observed when similar doses of heat-killed organisms were administered. In contrast, inoculation with E. coli produced a large increase in both SWS time and DWA for the first 2-4 h after inoculation. DWA then decreased from 6 to 32 h after inoculation. Similar effects occurred when heat-killed E. coli were administered. Rapid eye movement sleep was reduced by all three agents. These data demonstrate that altered sleep patterns occur in response to infectious challenge in rabbits, and that these changes are related to the type of infectious organism involved.     

Prostaglandins mediate the fetal pulmonary response to intrauterine inflammation

Intra-amniotic (IA) lipopolysaccharide (LPS) induces intrauterine and fetal lung inflammation and increases lung surfactant and compliance in preterm sheep; however, the mechanisms are unknown. Prostaglandins (PGs) are inflammatory mediators, and PGE(2) has established roles in fetal lung surfactant production. The aim of our first study was to determine PGE(2) concentrations in response to IA LPS and pulmonary gene expression for PG synthetic [prostaglandin H synthase-2 (PGHS-2) and PGE synthase (PGES)] and PG-metabolizing [prostaglandin dehydrogenase (PGDH)] enzymes and PGE(2) receptors. Our second study aimed to block LPS-induced increases in PGE(2) with a PGHS-2 inhibitor (nimesulide) and determine lung inflammation and surfactant protein mRNA expression. Pregnant ewes received an IA saline or LPS injection at 118 days of gestation. In study 1, fetal plasma and amniotic fluid were sampled before and at 2, 4, 6, 12, and 24 h after injection and then daily, and fetuses were delivered 2 or 7 days later. Amniotic fluid PGE(2) concentrations increased (P < 0.05) 12 h and 3-6 days after LPS. Fetal lung PGHS-2 mRNA and PGES mRNA increased 2 (P = 0.0084) and 7 (P = 0.014) days after LPS, respectively. In study 2, maternal intravenous nimesulide or vehicle infusion began immediately before LPS or saline injection and continued until delivery 2 days later. Nimesulide inhibited LPS-induced increases in PGE(2) and decreased fetal lung IL-1β and IL-8 mRNA (P ≤ 0.002) without altering lung inflammatory cell infiltration. Nimesulide decreased surfactant protein (SP)-A (P = 0.05), -B (P = 0.05), and -D (P = 0.0015) but increased SP-C mRNA (P = 0.023). Thus PGHS-2 mediates, at least in part, fetal pulmonary responses to inflammation.