THE NATURE AND ORIGIN OF THE SOLUBLE PROTEIN IN HUMAN AMNIOTIC FLUID

1. Amniotic fluid surrounds the human fetus and is separated from the uterus by the amnion, chorion and placenta. The ability to obtain samples of amniotic fluid from women by a simple procedure has encouraged studies on the nature and origin of the fluid, and on its use for the diagnosis of a variety of clinical conditions. The fluid contains cells, which are of fetal origin, and can be grown in a tissue culture. Cyto-genetic and biochemical analyses can therefore be used to detect chromosomal aberrations and inborn errors of metabolism in the fetus. 2. The supernatant of amniotic fluid contains many of the solutes typical of extracellular fluid. In particular, it contains a wide range of proteins and those which are of fetal origin are likely to be of use in the prenatal diagnosis of fetal disease. This review examines the nature and origin of the soluble protein in amniotic fluid, and discusses the diagnostic uses of the proteins which are of fetal origin. 3. In other mammals, the arrangement of the fetal membranes is different from that in man, and these differences are reflected by changes in the nature of the amniotic fluid. Thus data from other animals have little applicability to man. 4. Electrophoresis and immunoelectrophoresis have established that the major proteins in amniotic fluid are also present in maternal and fetal sera. Their concentrations in the fluid are influenced by their molecular weight and proteins larger than about 2.5 times 10⁶ may be excluded. Towards term, phenotyping studies show that a number of serum proteins in amniotic fluid are of maternal origin. In the case of group-specific component (Gc) this has been shown to be so throughout pregnancy. Such proteins must enter the fluid by diffusing across either the chorion or the chorionic plate and then the amnion. 5. It has been previously claimed that various serum proteins in amniotic fluid are of fetal origin. For albumin and IgG there are data that strongly support a maternal origin. The evidence on the origin of insulin is inconclusive. The concentration of β₂-microglobulin in amniotic fluid exceeds that in maternal serum and is probably too high also for fetal serum to be its major source. It has a wide tissue distribution and probably enters the fluid from surrounding structures. 6. Alpha-fetoprotein in amniotic fluid is of fetal origin as it is present in maternal serum at far lower concentrations. It is found in fetal serum, urine and yolk sac, but it is not clear how it enters the amniotic fluid of normal fetuses. The concentrations of Gc and alpha-fetoprotein have been measured in amniotic fluid and in their sera of origin. The relative concentration of Gc in amniotic fluid was found to be much greater than that of alpha-fetoprotein and the concentration gradients of these marker proteins can be compared with data for other proteins. In this way further evidence has been obtained that the albumin, α₁,-antitrypsin and transferrin in amniotic fluid are mainly of maternal origin throughout pregnancy. 7. Immunological studies have shown that at least three proteins of non-serum origin are present in amniotic fluid and they have also been located in the amnion and uterine decidua. 8. The enzymes present in amniotic fluid are summarized. Many lysosomal enzymes are clearly of fetal origin since they show altered specific activities in the appropriate cases where the fetus is affected with an inborn error of metabolism. For other enzymes, analysis of specific activity gradients can help to decide the extent to which an enzyme is of serum origin, although this will not exclude the possibility of a maternal (uterine) contribution. The results of such analyses suggest that, relative to the serum protein in amniotic fluid, the greatest concentrations of the minor non-serum proteins in the fluid occurs between thirteen and eighteen weeks of pregnancy and also towards term. 9. Some inborn errors of metabolism may be diagnosed prenatally by measuring the specific activity of the respective enzyme in amniotic fluid. However, the presence of different enzymes with similar substrate specificities has prevented this in Pompe's disease. 10. In cases where the fetus is affected with anencephaly or spina bifida there is an increase in the concentration of alpha-fetoprotein in the amniotic fluid. This has provided a way of detecting these diseases early enough to allow termination of pregnancy. 11. The discovery of new proteins in fetal serum and in the tissues surrounding the amniotic cavity would seem to provide the best chance of extending the uses of amniotic fluid into the other areas of prenatal medicine.     

Ontogeny of aquaporins in human fetal membranes

It has been proposed that four members of the aquaporin family (AQPs 1, 3, 8, and 9) are involved in the control of amniotic fluid (AF) homeostasis, as illustrated by their differential expression patterns in normal and pathological human term fetal membranes. However, there are no data available to date on their ontogeny throughout pregnancy. Our objective was to determine spatiotemporal expression profiles of the mRNA and proteins of all 13 members of this transmembrane channel family. For this purpose, we used healthy fetal membranes from the first, second, and third trimesters of pregnancy. Total mRNA and proteins were extracted from total membranes and from separated amnion and chorion. Quantitative PCR, Western blot, and immunohistochemistry experiments were carried out to determine the presence of AQPs and to quantify their spatiotemporal expression patterns throughout pregnancy. The WISH cell line was tested to propose a cellular model for the role of AQPs in the amnion compartment. AQP11 expression was established in amniotic membranes at term. Aquaporins 1, 3, 8, 9, and 11 mRNA and proteins were present in amnion and chorion throughout human gestation. Each AQP has a time-specific expression pattern, with AQP1 presenting the highest variation in terms of mRNA and protein levels. The WISH cell line also expressed the same five AQPs. Taken together, these results indicate that AQPs are expressed and potentially involved in the regulation of AF homeostasis throughout pregnancy. This also clearly supports the hypothesis that abnormal expression could occur at any time during pregnancy, ultimately leading to obstetrical pathologies such as polyhydramnios or oligohydramnios.     

Amniotic fluid exerts a neurotrophic influence on fetal neurodevelopment via the ERK/GSK-3 pathway

Background

The fetus is surrounded by the amniotic fluid (AF) contained by the amniotic sac of the pregnant female. The AF is directly conveyed to the fetus during pregnancy. Although AF has recently been reported as an untapped resource containing various substances, it remains unclear whether the AF could influence fetal neurodevelopment.

Results

We used AF that was extracted from embryos at 16 days in pregnant SD rat and exposed the AF to the neural cells derived from the embryos of same rat. We found that the treatment of AF to cortical neurons increased the phosphorylation in ERK1/2 that is necessary for fetal neurodevelopment, which was inhibited by the treatment of MEK inhibitors. Moreover, we found the subsequent inhibition of glycogen synthase kinase-3 (GSK-3), which is an important determinant of cell fate in neural cells. Indeed, AF increased the neural clustering of cortical neurons, which revealed that the clustered cells were proliferating neural progenitor cells. Accordingly, we confirmed the ability of AF to increase the neural progenitor cells through neurosphere formation. Furthermore, we showed that the ERK/GSK-3 pathway was involved in AF-mediated neurosphere enlargement.

Conclusions

Although the placenta mainly supplies oxygenated blood, nutrient substances for fetal development, these findings further suggest that circulating-AF into the fetus could affect fetal neurodevelopment via MAP kinases-derived GSK-3 pathway during pregnancy. Moreover, we suggest that AF could be utilized as a valuable resource in the field of regenerative medicine.     

Gastric inhibitory polypeptide (GIP) concentration in human amniotic fluid

Several studies report that placenta and amniotic fluid (AF) may be a source of many peptide hormones. Although the presence of gastric inhibitory polypeptide (GIP) in amniotic fluid has not been described, it is present in the fetal gut. In this study we report the presence of insulin and GIP in human AF of normal and diabetic pregnancies. GIP concentrations in the AF collected two hours after an arginine tolerance test (ATT), at 34-36 weeks of gestation, were evaluated in 8 normal and 53 diabetic pregnant women. GIP was found in all samples of AF. The mean AF-GIP concentrations were 133 +/- 19 pmol/l in controls and 111 +/- 6 pmol/l in the diabetics, being the GIP values of the diabetics belonging to White Class B significantly lower than those of normals (99 +/- 10 vs 133 +/- 19 pmol/l). The GIP/IRI molar ratio was significantly lower in the diabetics than in controls (1.2 +/- 0.2 vs 2.5 +/- 0.4); moreover the GIP/IRI molar ratio was significantly higher in AF collected from diabetic pregnant women who delivered overweight infants than in AF of normal weight infants or controls. This finding would suggest a negative feedback mechanism between GIP and insulin in fetus.     

Differentiation in human amniotic fluid cell cultures: chorionic gonadotropin production.

Two of the distinguishable cell classes subcultured from human amniotic fluid were examined for their capability to produce human chorionic gonadotropin (hCG) as determined by radioimmunoassay. The class that predominates in most cultures used for prenatal genetic diagnosis, previously termed AF (for amniotic fluid), secretes hCG into the culture medium. Dermal fibroblasts do not, nor does another type of cultured cell from amniotic fluid, previously termed F because of a resemblance to fibroblasts. Primary AF cultures produce more hCG than do subcultures. Evidence that this hormone is intact hCG is provided by its immunoreactivity with antisera raised against the beta-subunit and against the intact molecule of hCG. Furthermore, a dose-response curve for hormone in culture medium is parallel to that of highly purified intact hCG. It is postulated that AF cultures are derived from fetal membranes and retain properties of trophoblast.     

Glycoproteins that distinguish different cell types found in amniotic fluid

Summary Two concanavalin A-binding glycoproteins of approximate Mw 150 K and 140 K are described, which enable two of the main cell types found in amniotic fluid samples obtained by amniocentesis, AF and F cells, to be reliably distinguished. Further, the Mw 150 K glycoprotein has been used to trace the developmental origin of the AF cell type. AF cells, which generally form the majority of amniotic fluid cells found in culture, appear to arise in the extra-embryonic membranes, specifically the amnion.     

Differentiation in human amniotic fluid cell cultures: Chorionic gonadotropin production

Summary Two of the distinguishable cell classes subcultured from human amniotic fluid were examined for their capability to produce human chorionic gonadotropin (hCG) as determined by radioimmunoassay. The class that predominates in most cultures used for prenatal genetic diagnosis, previously termed AF (for amniotic fluid), secretes hCG into the culture medium. Dermal fibroblasts do not, nor does another type of cultured cell from amniotic fluid, previously termed F because of a resemblance to fibroblasts. Primary AF cultures produce more hCG than do subcultures. Evidence that this hormone is intact hCG is provided by its immunoreactivity with antisera raised against the β-subunit and against the intact molecule of hCG. Furthermore, a dose-response curve for hormone in culture medium is parallel to that of highly purified intact hCG. It is postulated that AF cultures are derived from fetal membranes and retain properties of trophoblast. Research supported by Grand HD 11379 from the National Institutes of Health.     

Association of Ipomoea carnea and BCG reduces birth defects caused by cyclophosphamide in rats

Immunosuppressive drugs can induce the development of malformations in fetuses of mothers exposed to them, possibly affecting the placental function directly or by crossing the placenta to enter fetal circulation. However, activation of the maternal immune system with well-known immunomodulator substances has been shown to produce a significant decrease in morphological defects caused by diverse teratogenic agents. All of these studies were performed on mice only, whereas the rat is the chosen species for developing teratological studies. Thus, the purpose of the present study was to investigate the possible protective effect of Bacillus Calmette Guérin (BCG) and/or the aqueous fraction (AF) of the plant Ipomoea carnea on the decrease of the teratogenic effect resulting from cyclophosphamide (CP), an antineoplastic and immunosuppressive drug, exposure in pregnant rats. It was verified that both BCG and/or AF attenuated the embryotoxic effects of CP in rats. All immune stimulated dams demonstrated an increase in placenta and fetus body weight. In conclusion, the present work showed that the rat is a good model for performing studies which aim for a clearer understanding of the mechanism by which maternal stimulation reduces malformations and how the association of I. carnea AF and BCG provided improved immunostimulation compared to BCG alone; however, additional studies are required to determine the specific mechanisms by which immune stimulant substances decrease malformation.     

Computer model of fetal-maternal heat exchange in sheep

We constructed and used a mathematical model of maternal-fetal heat exchange in the sheep to explore the effects of changes in certain parameters on steady-state fetal temperatures and to determine whether the fetus in the model has any potential to control its own temperature. The model took into account both fetal and placental heat production and exchange of heat in the placenta, across the fetal skin, via amniotic fluid, and through the uterine wall. The maternal ewe was assumed to be a constant temperature heat sink. Changes in placental or fetal heat production were calculated to change the ratio of heat exiting across the placenta or fetal skin significantly but to have little effect on fetal core temperature, e.g., a rise of only 0.8 degrees C was predicted after a twofold increase in fetal heat production. Fetal placental blood flow was calculated to affect fetal temperature the most of any flow, a reduction to zero causing fetal temperature to rise 5.0 degrees C. Changes in heat conductances between fetal skin and amniotic fluid, or between amniotic fluid and uterine wall, had minimal effect on fetal temperature. From the model calculations here and because heat exchange within the sheep placenta has previously been calculated to be extremely efficient, we conclude that the fetal sheep has little ability to control its temperature by changes in heat dissipated through extraplacental pathways. Thus the model predicts an effective heat clamp that closely links fetal to maternal temperature.     

Anandamide levels in human female reproductive tissues: Solid-phase extraction and measurement by ultraperformance liquid chromatography tandem mass spectrometry

Anandamide (N-arachidonoylethanolamide), a bioactive lipid, is reported to play a role in pregnancy maintenance and parturition. Our aims were to (1) evaluate AEA levels at the human maternal:fetal interface and (2) validate the use of solid-phase extraction of AEA from tissues. AEA was analyzed in cord and maternal blood, amniotic fluid, placenta, and fetal membranes collected during Caesarean section (n = 14). Extraction efficiencies were 42 and 36% for the placenta and the fetal membranes, respectively. Tissue AEA was quantified using an isotope-dilution method and UPLC-ESI-MS/MS giving intra- and inter-day variability for tissues spiked with 0.2, 1, and 5 pmol/g AEA of less than 12%. Accuracy for these spiked samples was between 95% and 103% for fetal membranes and between 99% and 114% for placenta. Mean AEA concentrations were 2.72 ± 1.04 pmol/g for placenta and 1.19 ± 0.68 pmol/g for fetal membranes, and 0.93 ± 0.28, 0.88 ± 0.33, 0.77 ± 0.30, and 0.06 ± 0.04 nM for maternal, umbilical vein, and umbilical artery plasma and amniotic fluid. Higher AEA concentrations were found in placenta compared to fetal membranes (P < 0.0001), in umbilical vein compared with umbilical artery (P = 0.0015), and in plasma from maternal circulation compared with umbilical artery (P = 0.0152). The relevance of these changes in AEA concentrations at the maternal:fetal interface requires further investigation.     

羊水菌群的研究进展

传统观念认为胎儿在宫内发育的过程是无菌的。随着研究方法的快速发展,针对于微生物的研究方法从传统的培养到现代分子生物学检测的进步,研究者已经认识到胎盘、羊水中具有微生物的存在。目前的研究尚不能明确胎儿宫内胎盘、羊水的菌群来源及菌群转移的具体途径,但已有的研究提示羊水菌群最可能来源为母亲肠道、生殖道以及口腔。本文综述了研究者对羊水菌群认识的转变、羊水菌群的来源以及羊水菌群与早产的相关性。     

Enzyme-linked immunosorbent assay of the D2-glycoprotein

Abstract: D2 is a glycoprotein enriched in neuronal membranes and probably involved in intercellular adhesion. An immunochemical relationship between D2 and the neuronal cell adhesion molecule from chick has been demonstrated. Changes in D2 concentration in human body fluids correlate to certain neurological diseases.We here report the purification of the D2 membrane proteins from fetal and adult human brain and the demonstration of physicochemical differences between the two proteins. Enrichments of 133 times (fetal D2) and 350 times (adult D2) were found. Specific rabbit antisera against the purified D2 proteins were produced, and this enabled the setting up of an enzyme-linked immunosorbent assay for D2 quantification in human brain extracts, cerebrospinal fluids, sera, and amniotic fluids.     

Intestinal and renal origin of trehalase activity in rabbit amniotic fluid

To utilize specific fetal markers in amniotic fluid for prenatal detection of fetal anomalies, it is necessary to determine the precise tissue origin of these markers. In rabbit fetuses, we distinguished between intestinal and renal forms of trehalase (alpha,alpha'-trehalose-1-D-glucohydrolase, EC 3.2.1.28) in amniotic fluid on the basis of differences in net electric charges. Trehalase was solubilized from purified brush-border membranes of fetal rabbit kidney and intestine by Triton X-100 treatment, whereas the trehalase activity in amniotic fluid was soluble. The kinetic properties of trehalase from intestine, kidney and amniotic fluid were very similar. The Mr of the soluble amniotic fluid trehalase was between 72,600 and 66,300 from hydrodynamic parameters, depending on the amount of sugar bound to the enzyme, and 48,500 by radiation inactivation, a method which detects only the protein part of the enzyme. For membrane-bound trehalase from kidney and intestine in situ the radiation inactivation method also gave a molecular size of around 49,000. Isoelectric focusing of freshly solubilized membranes allowed us to distinguish between renal and intestinal forms of trehalase in rabbit fetuses on the basis of different isoelectric points. Each trehalase form was also present in the amniotic fluid but in varying proportions depending on the gestational age at which the amniotic fluid was collected. The results suggest that early in gestation amniotic fluid trehalase activity originates exclusively from the fetal kidney but that more and more intestinal enzyme is released into the amniotic cavity as the fetus develops. Similar results were also obtained when ion-exchange chromatography was used to separate the various trehalase forms. The development of trehalase activity in rabbit fetal kidney and intestine correlates well with its occurrence in the amniotic fluid; trehalase activity in the kidney develops early in gestation whereas the intestinal trehalase activity develops just before term.     

IL-6 trans-signaling system in intra-amniotic inflammation, preterm birth, and preterm premature rupture of the membranes

Classic IL-6 signaling is conditioned by the transmembrane receptor (IL-6R) and homodimerization of gp130. During trans-signaling, IL-6 binds to soluble IL-6R (sIL-6R), enabling activation of cells expressing solely gp130. Soluble gp130 (sgp130) selectively inhibits IL-6 trans-signaling. To characterize amniotic fluid (AF) IL-6 trans-signaling molecules (IL-6, sIL-6R, sgp130) in normal gestations and pregnancies complicated by intra-amniotic inflammation (IAI), we studied 301 women during second trimester (n = 39), third trimester (n = 40), and preterm labor with intact (n = 131, 85 negative IAI and 46 positive IAI) or preterm premature rupture of membranes (PPROM; n = 91, 61 negative IAI and 30 positive IAI). ELISA, Western blotting, and real-time RT-PCR were used to investigate AF, placenta, and amniochorion for protein and mRNA expression of sIL-6R, sgp130, IL-6R, and gp130. Tissues were immunostained for IL-6R, gp130, CD15(+) (polymorphonuclear), and CD3(+) (T cell) inflammatory cells. The ability of sIL-6R and sgp130 to modulate basal and LPS-stimulated release of amniochorion matrix metalloprotease-9 was tested ex vivo. We showed that in physiologic gestations, AF sgp130 decreases toward term. AF IL-6 and sIL-6R were increased in IAI, whereas sgp130 was decreased in PPROM. Our results suggested that fetal membranes are the probable source of AF sIL-6R and sgp130. Immunohistochemistry and RT-PCR revealed increased IL-6R and decreased gp130 expression in amniochorion of women with IAI. Ex vivo, sIL-6R and LPS augmented amniochorion matrix metalloprotease-9 release, whereas sgp130 opposed this effect. We conclude that IL-6 trans-signaling molecules are physiologic constituents of the AF regulated by gestational age and inflammation. PPROM likely involves functional loss of sgp130.     

Increase in 15-hydroxyprostaglandin dehydrogenase activity in the ovine placentome at parturition and effect of oestrogen

Type 1 NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (PGDH) is the key enzyme for metabolism of active primary prostaglandins to inactive forms in gestational tissues. The present study examined the activity and immunolocalization of PGDH in the ovine placenta, fetal membranes and uterus over the latter half of pregnancy, and its potential regulation by oestradiol. Placenta, fetal membranes and myometrium were collected from sheep with known single insemination dates on days 70, 100 and 135 of gestation and in active labour demonstrated by electromyographic activity. In addition, chronically catheterized fetuses were infused with oestradiol (100 microgram kg(-1) per 24 h) (n = 5) or saline vehicle into the fetus from day 120 to day 125. PGDH activity measured in placental extracts remained constant from day 70 to day 135 of gestation, and then significantly (P < 0.05) increased by 300% in active labour. Immunoreactive PGDH was localized in the placentome at all stages and was present predominantly in the fetal component of the placentome in uninucleate, but not in binucleate, trophoblast cells. Similarly, in the fetal membranes PGDH immuno-reactivity was present in the uninucleate trophoblast but not in the binucleate cells of the chorion. PGDH immunostaining was also present in the endometrial luminal epithelium, in the smooth muscle of the myometrium, and the glandular epithelium of the cervix. Infusion of oestradiol into the fetal circulation from day 120 to day 125 of gestation had no effect on placental PGDH activity. Immunohistochemistry was used to localize oestrogen receptor alpha in intrauterine tissues to investigate further the failure of oestradiol to increase PGDH activity. Immunoreactive oestrogen receptor alpha was not present in the fetal component of the placenta, although it was expressed in adjacent maternal-derived cells. It is concluded that (1) PGDH activity increases in late gestation; (2) PGDH is expressed in uninucleate trophoblast cells in the ovine placenta and fetal membranes, and also in the maternal endometrial epithelium and stroma, myometrium and cervix; (3) oestrogen receptor alpha is not expressed in fetal cells in the placenta or fetal membranes; and (4) the increase in PGDH activity is not regulated by oestradiol administered to the fetus.     

Molecular characterization of a MSRV-like sequence identified by RDA from monozygotic twin pairs discordant for schizophrenia.

Retroviral-related amplicons were used in modified RDA to identify four sequences from affected members of three pairs of monozygotic twins discordant for schizophrenia. One sequence (schizophrenia associated retrovirus, SZRV-1, GenBank Accession No. AF135487) is characterized here. It is similar to two known sequences of retroviral origin: multiple sclerosis-associated retrovirus, MSRV (GenBank Accession No. AF009668), and ERV-9 (GenBank Accession No. S77575). It is present in multiple copies in the human genome and has been localized to six different chromosomal sites. A zooblot shows that this multicopy sequence is predominant in the primate lineage and present in rhesus monkeys and humans. SZRV-1 is expressed as a 9-kb RNA band in the placenta. This could offer support to the hypothesis that retroviral sequences transposing during fetal growth may alter neurodevelopmental genes and cause diseases, although its direct involvement in the causation of schizophrenia remains to be established.     

Application of fluorescein-labelled lectins with different glycan-binding specificities to the studies of cellular glycoconjugates in human full-term placenta

In the present study 13 lectins of plant, fungal and animal origin, characterised by different glycan-binding specificities were used to examine the structure and distribution of specific glycans in the tissues of human placenta. Histochemical analysis was focused on villi (villous syncytiotrophoblast and stroma), cytotrophoblast of the basal plate and amniotic epithelium. It was found that glycoconjugates containing mannose and N-acetyl glucosamine were widely expressed while external fucosyl residues were absent on all studied structures of placenta. Lectins GNA (revealing non-reducing terminal mannosyl residues) and LPA (revealing sialic acid) bound selectively to the villus structures. The presence of sialic acid residues was observed in the superficial plasmalemma of syncytiotrophoblast and on the surface of the foetal capillary endothelium. Lectins LCA and PSA showed specific affinity to the plasmalemma of the cytotrophoblast and to the amniotic epithelium basement membrane. N-acetyl lactosamine-specific fungal lectin PSL selectively labelled amniotic epithelial and cytotrophoblast cell membranes and superficial plasmalemma of the syncytiotrophoblast.     

Endotoxin-neutralizing antimicrobial proteins of the human placenta

Microbial colonization and infection of placental tissues often lead to adverse pregnancy outcomes such as preterm birth, a leading cause of neonatal morbidity and mortality. The fetal membranes of the placenta, a physical and active barrier to microbial invasion, encapsulate the fetus and secure its intrauterine environment. To examine the innate defense system of the human placenta, antimicrobial peptides were isolated from the fetal membranes of human placenta and characterized biochemically. Two salt-resistant antimicrobial host proteins were purified to homogeneity using heparin-affinity and reversed-phase HPLC. Characterization of these proteins revealed that they are identical to histones H2A and H2B. Histones H2A and H2B showed dose-dependent inhibition of the endotoxin activity of LPS and inhibited this activity by binding to and therefore blocking both the core and lipid A moieties of LPS. Consistent with a role for histones in the establishment of placental innate defense, histones H2A and H2B were highly expressed in the cytoplasm of syncytiotrophoblasts and amnion cells, where the histone proteins were localized mainly to the epithelial surface. Furthermore, culturing of amnion-derived WISH cells led to the constitutive release of histone H2B, and histones H2A and H2B contribute to bactericidal activity of amniotic fluid. Our studies suggest that histones H2A and H2B may endow the epithelium of the placenta with an antimicrobial and endotoxin-neutralizing barrier against microorganisms that invade this immune-privileged site.