John W. Hotra

Emergence of hormonal and redox regulation of galectin-1 in placental mammals: Implication in maternal-fetal immune tolerance

Galectin-1 is an anti-inflammatory lectin with pleiotropic regulatory functions at the crossroads of innate and adaptive immunity. It is expressed in immune privileged sites and is implicated in establishing maternal–fetal immune tolerance, which is essential for successful pregnancy in eutherian mammals. Here, we show conserved placental localization of galectin-1 in primates and its predominant expression in maternal decidua. Phylogenetic footprinting and shadowing unveil conserved cis motifs, including an estrogen responsive element in the 5′ promoter of LGALS1, that were gained during the emergence of placental mammals and could account for sex steroid regulation of LGALS1 expression, thus providing additional evidence for the role of galectin-1 in immune–endocrine cross-talk. Maximum parsimony and maximum likelihood analyses of 27 publicly available vertebrate and seven newly sequenced primate LGALS1 coding sequences reveal that intense purifying selection has been acting on residues in the carbohydrate recognition domain and dimerization interface that are involved in immune functions. Parsimony- and codon model-based phylogenetic analysis of coding sequences show that amino acid replacements occurred in early mammalian evolution on key residues, including gain of cysteines, which regulate immune functions by redox status-mediated conformational changes that disable sugar binding and dimerization, and that the acquired immunoregulatory functions of galectin-1 then became highly conserved in eutherian lineages, suggesting the emergence of hormonal and redox regulation of galectin-1 in placental mammals may be implicated in maternal–fetal immune tolerance.

Excess and deficient omega-3 fatty acid during pregnancy and lactation cause impaired neural transmission in rat pups

Omega-3 fatty acids (omega-3 FA) consumption during pregnancy and lactation is beneficial to fetal and infant growth and may reduce the severity of preterm births. Thus, scientists and clinicians are recommending increasingly higher omega-3 FA doses for pregnant women and nursing babies for advancing the health of preterm, low birth weight, and normal babies. In contrast, some studies report that over-supplementation with omega-3 FA can have adverse effects on fetal and infant development by causing a form of nutritional toxicity. Our goal was to assess the effects of omega-3 FA excess and deficiency during pregnancy and lactation on the offsprings neural transmission as evidenced by their auditory brainstem responses (ABR). Female Wistar rats were given one of three diets from day 1 of pregnancy through lactation. The three diets were the Control omega-3 FA condition (omega-3/omega-6 ratio approximately 0.14), the Deficient omega-3 FA condition (omega-3/omega-6 ratio approximately 0%) and the Excess omega-3 FA condition (omega-3/omega-6 ratio approximately 14.0). The Control diet contained 7% soybean oil, whereas the Deficient diet contained 7% safflower oil and the Excess diet contained 7% fish oil. The offspring were ABR-tested on postnatal day 24. The rat pups in the Excess group had prolonged ABR latencies in comparison to the Control group, indicating slowed neural transmission times. The pups in the Excess group also showed postnatal growth restriction. The Deficient group showed adverse effects that were milder than those seen in the Excess group. Milk fatty acid profiles reflected the fatty acid profiles of the maternal diets. In conclusion, excess or deficient amounts of omega-3 FA during pregnancy and lactation adversely affected the offsprings neural transmission times and postnatal thriving. Consuming either large or inadequate amounts of omega-3 FA during pregnancy and lactation seems inadvisable because of the potential for adverse effects on infant development.

Severe preeclampsia is characterized by increased placental expression of galectin-1.

Objective. Galectin-1 is a major anti-inflammatory protein expressed by the placenta and immune cells that can bias the character of inflammatory responses toward the Th2 type. Galectin-1 is expressed in immune privileged sites, it can facilitate immune tolerance and tumor immune escape, and it has been successfully used for the suppression of experimental autoimmune diseases as well as graft-versus-host disease in murine models. We propose that an abnormal immune response in some pregnancy complications may be associated with changes in placental expression of galectin-1. To test this hypothesis, we studied placental galectin-1 mRNA and protein expression and localization in women with preeclampsia (PE) and in those who delivered a small-for-gestational age (SGA) neonate. Study design. This cross-sectional study included pregnant women matched for gestational age at delivery in the following groups: (1) severe PE (n = 10), (2) severe PE complicated with SGA (n = 10), (3) SGA without PE (n = 10), and (4) controls (n = 10). Galectin-1 mRNA and protein were localized in placentas by in situ hybridization and immunofluorescence microscopy. Galectin-1 mRNA expression was determined by quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR), and galectin-1 protein content by Western blot. Non-parametric statistics were used for analysis. Results. (1) In normal term placentas, galectin-1 mRNA or immunofluorescence signals were detected in the trophoblasts, villous stromal cells, Hofbauer cells, endothelial cells of the villous blood vessels, and the villous stroma. (2) Placental galectin-1 mRNA expression was significantly higher in severe PE (with or without SGA) than in controls (1.47-fold, p = 0.004; 1.44-fold, p = 0.003, respectively) and in SGA (1.68-fold, p = 0.001; 1.64-fold, p = 0.001, respectively). (3) Trophoblasts in placentas of patients with severe PE had the most intense galectin-1 immunostaining. Conclusions. (1) We report for the first time the placental expression and localization of galectin-1 mRNA and demonstrate that the protein is abundantly present in third trimester human placentas. (2) Placental galectin-1 expression is higher in severe PE than in normal pregnancy regardless of the presence of SGA. (3) However, it is not altered in SGA without PE. We propose that the increased placental expression of galectin-1 in patients with severe PE may represent a fetal response to an exaggerated systemic maternal inflammation; thus, galectin-1 may be implicated in maternal–fetal immune tolerance in humans.

Magnesium sulfate protection of fetal rat brain from severe maternal hypoxia

Objective To determine whether severe maternal hypoxia affects fetal rat physical characteristics and causes neuronal damage, and whether magnesium sulfate can decrease these effects. Methods At 17 days gestation, rats were randomly assigned to one of four groups that received saline injections and room air (n = 6), magnesium sulfate and room air (n = 5), saline and hypoxia (n = 5) or magnesium sulfate and hypoxia (n = 5). Maternal magnesium sulfate or saline injections were given for 4 hours. In groups 3 and 4 this was followed by a hypoxia chamber protocol that included a gas mixture of 9% oxygen and 3% carbon dioxide for 2 hours. After 72 hours of recovery, fetuses were delivered abdominally, perfused transcardially, and brains removed intact. Fetal body and brain weight and size were measured. Brains were embedded in paraffin, sectioned, and stained. A neuropathologist masked to the protocol performed histologic grading of brain regions. Results Exposure to the hypoxia chamber resulted in decreased maternal oxygen tension and pH (from 82.8 ± 20.0 to 49.2 ± 14.4 mmHg, and from 7.37 ± 0.05 to 7.20 ± 0.04, respectively; P < .005). Magnesium sulfate administration resulted in higher magnesium levels in blood (from 1.52 ± 0.2 to 3.77 ± 0.7 mg/dL; P < .001). The hypoxia protocol resulted in a significant decrease in fetal body and brain size, but not weight. Hypoxia also caused an increase in the proportion of fetal rats that had brain injury, including shrinkage of cells and karyorrhexis in the hippocampus and thalamus (from 0% to 38.1% and 38.9%, respectively; P < .05). Magnesium sulfate reduced these effects on fetal brain histopathology and size. Conclusion Severe maternal rat hypoxia resulted in significant fetal neuronal damage and decreased fetal body and brain size. Maternal magnesium sulfate administration reduced the effect of hypoxia on fetal brain histopathology and size without affecting body size.

Characterization of the transcriptome of chorioamniotic membranes at the site of rupture in spontaneous labor at term

OBJECTIVE The purpose of this study was to compare the transcriptome between the site of membrane rupture and the chorioamniotic membranes away from the site of rupture. STUDY DESIGN The transcriptome of amnion and chorion (n=20 each) from and distal to the site of rupture from women with spontaneous labor and vaginal delivery at term after spontaneous rupture of membranes was profiled with Illumina HumanHT-12 microarrays. Selected genes were validated with the use of quantitative reverse transcription-polymerase chain reaction. RESULTS Six hundred seventy-seven genes were differentially expressed in the chorion between the rupture and nonrupture sites (false discovery rate<0.1; fold change>1.5). Quantitative reverse transcription-polymerase chain reaction confirmed the differential expression in 10 of 14 genes. Enriched biological processes included anatomic structure development, cell adhesion and signal transduction. Extracellular matrix-receptor interaction was the most impacted signaling pathway. CONCLUSION The transcriptome of fetal membranes after spontaneous rupture of membranes in term labor is characterized by region- and tissue-specific differential expression of genes that are involved in signature pathways, which include extracellular matrix-receptor interactions.

Auditory Brainstem Response (ABR) Abnormalities across the Life Span of Rats Prenatally Exposed to Alcohol

BACKGROUND Fetal alcohol syndrome (FAS) is a leading cause of neurodevelopmental impairments (NDIs) in developed countries. Sensory deficits can play a major role in NDI, yet few studies have investigated the effects of prenatal alcohol exposure on sensory function. In addition, there is a paucity of information on the lifelong effects of prenatal alcohol exposure. Thus, we sought to investigate the effects of prenatal alcohol exposure on auditory function across the life span in an animal model. Based on prior findings with prenatal alcohol exposure and other forms of adverse prenatal environments, we hypothesized that animals prenatally exposed to alcohol would show an age-dependent pattern of (i) hearing and neurological abnormalities as postweanling pups, (ii) a substantial dissipation of such abnormalities in young adulthood, and (iii) a resurgence of such abnormalities in middle-aged adulthood. METHODS Pregnant rats were randomly assigned to an untreated control (CON), a pair-fed control (PFC), or an alcohol-treated (ALC) group. The ALC dams were gavaged with 6 mg/kg alcohol daily from gestation day (GD) 6 to 21. The PFC dams were gavaged daily from GD6 to GD21 with an isocaloric and isovolumetric water-based solution of maltose-dextrins and pair-fed to the ALC dams. The CON dams were the untreated group to which the ALC and CON groups were compared. Hearing and neurological functions in the offspring were assessed with the auditory brainstem response (ABR) at the postnatal ages of 22, 220, and 520 days. RESULTS In accord with our hypothesis, ABR abnormalities were first observed in the postweanling pups, largely dissipated in young adulthood, and then resurged in middle-aged adulthood. This age-related pattern suggests that the ALC pups had a developmental delay that dissipated in young adulthood and an enhanced age-related deterioration that occurred in middle-aged adulthood. Such a pattern is consistent with the fetal programming hypothesis of adult-onset diseases (the Barker hypothesis). CONCLUSIONS Our findings have important clinical implications for the assessment and management of (i) childhood hearing disorders and their comorbidities (i.e., speech-and-language, learning, and attention deficit disorders) and (ii) enhanced age-related hearing and neurological degeneration in middle-aged adulthood that can result from prenatal alcohol exposure. We recommend hearing evaluation be a part of any long-term follow-up for FAS patients and patients exposed to any adverse prenatal environment.

Sildenafil Citrate and Fetal Outcome in Pregnant Rats

Objective: To determine the effects of sildenafil citrate on fetal growth in maternal rats exposed to hypoxia. Methods: Timed pregnant rats were randomized to either hypoxia or control on gestational days (GD) 18–20, and received either sildenafil (45 mg/kg) orally every 12 h on GD 18–21 or an equal volume of sterile water. Fetal pups were retrieved by laparotomy on GD 21. Pup weight and length were evaluated and cGMP measured in maternal and fetal blood. Results: In the non-hypoxic rats, sildenafil exposure was associated with a decrease in size(4.75 ± 0.43 vs. 5.11 ± 0.34 g, p = 0.00). In contrast, in the hypoxic rat model, sildenafil exposure was associated with increased size of the offspring (5.48 ± 0.45 vs. 5.16 ± 0.36 g, p = 0.016). Maternal cGMP levels were increased in the presence of both sildenafil and hypoxia (23.0 ± 10.5 vs. 15.6 ± 2.7 pmol/ml, p = 0.001). Conclusion: Exposure to sildenafil in a non-hypoxic setting results in a decrease in fetal size. Sildenafil in the presence of a stimulus, hypoxia, will lead to increased fetal size. These results suggest that sildenafil may have some influence on fetal growth. How these effects occur and by what mechanism remain to be determined.

Chorioamnionitis and increased galectin-1 expression in PPROM - An anti-inflammatory response in the fetal membranes?

Problem Galectin‐1 can regulate immune responses upon infection and inflammation. We determined galectin‐1 expression in the chorioamniotic membranes and its changes during histological chorioamnionitis.

Timing of Fetal Nucleated Red Blood Cell Count Elevation in Response to Acute Hypoxia

We determined the effect of an acute episode of severe hypoxia on peripheral nucleated red blood cell (RBC) counts in the fetal rat. Timed pregnant rats were randomized to a 2-hour exposure to hypoxia (placement in a chamber containing a gas mixture with 9% O2 +3% CO2 + balanced N2) or to a 2-hour exposure to a sham chamber containing room air. Two maternal animals per group underwent cesarean section immediately after the 2-hour period and then 4, 12, 24, 36, 48, and 60 h after exposure. Fetal nucleated RBC counts were compared between groups at each time interval. The nucleated RBC counts were not significantly different in the hypoxia group until 12 h (mean ± SEM 158.0 ± 22.4 RBC/10 high-power fields vs. 90.6 ± 11.0; p = 0.03) and 24 h (133.2 ± 16.0 vs. 84.1 ± 9.0; p = 0.04) after exposure. There were no differences between groups 36, 48, and 60 h after exposure. In the near-term rat fetus, acute hypoxia was associated with a delayed but transient increase in peripheral nucleated RBC counts.

A molecular signature of an arrest of descent in human parturition

OBJECTIVE This study was undertaken to identify the molecular basis of an arrest of descent. STUDY DESIGN Human myometrium was obtained from women in term labor (TL; n = 29) and arrest of descent (AODes; n = 21). Gene expression was characterized using Illumina HumanHT-12 microarrays. A moderated Student t test and false discovery rate adjustment were applied for analysis. Confirmatory quantitative reverse transcription-polymerase chain reaction and immunoblot were performed in an independent sample set. RESULTS Four hundred genes were differentially expressed between women with an AODes compared with those with TL. Gene Ontology analysis indicated enrichment of biological processes and molecular functions related to inflammation and muscle function. Impacted pathways included inflammation and the actin cytoskeleton. Overexpression of hypoxia inducible factor-1a, interleukin -6, and prostaglandin-endoperoxide synthase 2 in AODes was confirmed. CONCLUSION We have identified a stereotypic pattern of gene expression in the myometrium of women with an arrest of descent. This represents the first study examining the molecular basis of an arrest of descent using a genome-wide approach.