Faculty Bibliography

Neutrophil apoptosis is impaired in neonates, and this contributes to prolonged inflammation and tissue injury in infants after infection or trauma. In the present studies, we investigated whether labor generates mediators that further suppress apoptosis. We found that neutrophil apoptosis was reduced in neonates exposed to labor, when compared with infants delivered by cesarean section before labor. This was not due to alterations in caspase-3 or inhibitor of apoptosis protein-2 (IAP-2). In contrast, labor primed neutrophils to express tumor necrosis factor alpha (TNF-alpha), suggesting that proinflammatory mediators contribute to reduced apoptosis after labor. Eicosanoids generated via cyclooxygenase-2 (Cox-2) and lipoxygenase (Lox) also regulate neutrophil apoptosis. 15-Lox, which generates proapoptotic lipoxins, but not Cox-2, was greater in neutrophils before labor, relative to cells exposed to labor. Anti-inflammatory eicosanoids exert their effects in part via peroxisome proliferator-activated receptor gamma (PPAR-gamma). Expression of gelatinase-associated lipocalin and catalase, two markers of PPAR-gamma activity, were increased in neonatal neutrophils before labor, relative to cells exposed to labor. These findings suggest that the anti-inflammatory environment is maintained before labor, in part, by eicosanoids. Although increased neutrophil longevity after labor is important for host defense in the immediate newborn period, it may contribute to inflammatory or oxidative injury in susceptible infants.

PROBLEM/OBJECTIVE:Interleukin-10 (IL-10) is thought to be a key cytokine for the maintenance of pregnancy. Here we examined the expression profiles of IL-10 and cyclo-oxygenase-2 (COX-2), and the effect of IL-10 on COX-2 expression and prostaglandin release in the human placenta from preterm labor deliveries associated with chorioamnionitis. METHOD OF STUDY/METHODS:Placental tissues from preterm labor and term labor deliveries were processed for ex vivo placental explant culture system. IL-10 expression was assessed by enzyme-linked immunosorbent assay (ELISA) and immunohistochemical (IHC) analysis. COX-2 expression was evaluated by IHC, Western blotting and reverse transcriptase-polymerase chain reaction. Prostaglandin E2 (PGE2) release was measured by ELISA. RESULTS:IL-10 was significantly reduced in chorioamnionitis-associated preterm labor as well as in term labor placental tissues compared with second trimester normal pregnancy samples obtained from elective terminations. Similar results were obtained with freshly isolated cytotrophoblasts from these deliveries. As expected, COX-2 mRNA was detected at significant levels in tissues from term and preterm labor deliveries compared with no labor term deliveries. Importantly, IL-10 inhibited COX-2 expression in cultured placental explants from preterm labor deliveries, but not from term labor samples. Inhibition of COX-2 expression coincided with reduced PGE2 release. CONCLUSION/CONCLUSIONS:These results demonstrate the importance of IL-10 in countering inflammation associated with preterm labor, and suggest that term and preterm parturition may, in part, represent different conditions.

Specialized NK cells are recruited in high numbers to the mammalian embryo implantation sites, yet remain pregnancy compatible. It is not well understood whether uterine NK (uNK) cells become adversely activated and mediate fetal demise, a common complication of early pregnancy. In this study we show that mating of IL-10(-/-) mice resulted in fetal resorption or intrauterine growth restriction in response to very low doses of LPS. Pregnancy in congenic wild-type mice was normal even at 10-fold higher LPS doses. Fetal resorption in IL-10(-/-) mice was associated with a significant increase in uNK cell cytotoxic activation and invasion into the placenta. Depletion of uNK cells, TNF-alpha neutralization, or IL-10 administration rescued pregnancy in LPS-treated IL-10(-/-) animals. Our results identify an immune mechanism of fetal demise involving IL-10 deficiency, NK cells, and inflammation. These results may provide insight into adverse pregnancy outcomes in humans.

The accumulation of neutrophils at sites of tissue injury or infection is mediated by chemotactic factors released as part of the inflammatory process. Some of these factors are generated as a direct consequence of tissue injury or infection, including degradation fragments of connective tissue collagen and bacterial- or viral-derived peptides containing collagen-related structural motifs. In these studies, we examined biochemical mechanisms mediating the biologic activity of synthetic polypeptides consisting of repeated units of proline (Pro), glycine (Gly), and hydroxyproline (Hyp), major amino acids found within mammalian and bacterial collagens. We found that the peptides were chemoattractants for neutrophils. Moreover, their chemotactic potency was directly related to their size and composition. Thus, the pentameric peptides (Pro-Pro-Gly)5 and (Pro-Hyp-Gly)5 were more active in inducing chemotaxis than the corresponding decameric peptides (Pro-Pro-Gly)10 and (Pro-Hyp-Gly)10. In addition, the presence of Hyp in peptides reduced chemotactic activity. The synthetic peptides were also found to reduce neutrophil apoptosis. In contrast to chemotaxis, this activity was independent of peptide size or composition. The effects of the peptides on both chemotaxis and apoptosis were blocked by inhibitors of phosphatidylinositol 3-kinase (PI3-K) and p38 mitogen-activated protein (MAP) kinase. However, only (Pro-Pro-Gly)5 and (Pro-Pro-Gly)10 induced expression of PI3-K and phosphorylation of p38 MAP kinase, suggesting a potential mechanism underlying reduced chemotactic activity of Hyp-containing peptides. Although none of the synthetic peptides tested had any effect on intracellular calcium mobilization, each induced nuclear binding activity of the transcription factor NF-kappa B. These findings indicate that polymeric polypeptides containing Gly-X-Y collagen-related structural motifs promote inflammation by inducing chemotaxis and blocking apoptosis. However, distinct calcium-independent signaling pathways appear to be involved in these activities.

Apoptosis, which leads to phagocytosis by mononuclear cells, represents the primary mechanism for removing neutrophils from inflamed tissues and minimizing injury. The present studies show that membrane phosphatidylserine turnover and permeability, as well as DNA fragmentation, were reduced in neutrophils from neonates when compared with adults. The activity of caspase 3 and expression of the proapoptotic proteins Bax, Bad, and Bak were also decreased in neonatal relative to adult neutrophils. These findings are consistent with impaired apoptosis in neonatal cells, which may contribute to prolonged inflammation in infants after oxidative stress or infection. Neutrophil apoptosis is induced by endogenous ligands such as Fas (FasL), which engage death receptors of the tumor necrosis factor/nerve growth factor superfamily, including Fas receptor (FasR). We found that expression of FasR was decreased in neonatal when compared with adult cells. Moreover, neonatal neutrophils did not undergo apoptosis in response to anti-FasR antibody and exhibited impaired chemotaxis to soluble FasL. However, in both adult and neonatal cells, p38 mitogen-activated protein kinase and phosphatidylinositol 3-kinase inhibitors blocked Fas-induced activity. These data suggest that prolonged survival of neonatal neutrophils at injured sites is due, in part, to reduced responsiveness to FasL. This may be related to decreased expression of both FasR and Bcl-2-family proteins that mediate neutrophil apoptosis.

BACKGROUND:Human milk, which contains compounds beneficial to infants, is often expressed and stored before use. Changes in its antioxidant activity with storage have not been studied. OBJECTIVES/OBJECTIVE:To measure antioxidant activity of fresh, refrigerated (4 degrees C), and frozen human milk (-20 degrees C), stored for two to seven days; to compare the antioxidant activity of milk from mothers delivering prematurely and at term; to compare the antioxidant activity of infant formulas and human milk. METHODS:Sixteen breast milk samples (term and preterm) were collected from mothers within 24 hours of delivery and divided into aliquots. Fresh samples were immediately tested for antioxidant activity, and the rest of the aliquots were stored at -20 degrees C or 4 degrees C to be analysed at 48 hours and seven days respectively. The assay used measures the ability of milk samples to inhibit the oxidation of 2,2'-azino-di-3-(ethylbenzthiazolinesulphonate) to its radical cation compared with Trolox. RESULTS:Antioxidant activity at both refrigeration and freezing temperatures was significantly decreased. Freezing resulted in a greater decrease than refrigeration, and storage for seven days resulted in lower antioxidant activity than storage for 48 hours. There was no difference in milk from mothers who delivered prematurely or at term. Significantly lower antioxidant activity was noted in formula milk than in fresh human milk. CONCLUSIONS:To preserve the antioxidant activity of human milk, storage time should be limited to 48 hours. Refrigeration is better than freezing and thawing.

Multidrug resistance conferring proteins (MDRCP) are ATP-binding cassette (ABC) transporters known to significantly influence the absorption, distribution, metabolism, and elimination (ADME) and toxic behavior of many therapeutic agents. Research in the pharmacogenomics area has suggested that mutations and variable expression patterns of these MDCRPs may exist in tissue samples from different ethnic groups. The goal of this study was to examine the expression of P-glycoprotein (PGP), sister of PGP (S-PGP), multidrug resistance protein 3 (Mdr3), multidrug resistance like proteins 1-5 (MRP 1-5), and lung resistance associated protein (LRP) in tissue slides and protein lysates derived from normal adult small or large intestines of Caucasian or Chinese origin. Our results demonstrated ubiquitous expression of PGP, MRP 1, MRP 4, and LRP in the small and large intestinal epitheliums originating from both Caucasian and Chinese origin. S-PGP, Mdr3, MRP 2, and MRP 3 exhibited variable expression in the tissue slides and protein lysates derived from the Chinese and Caucasian small and large intestines. MRP 5 was not observed in any of the samples studied. The results suggest that MDCRPs may have distinct expression profiles in the small and large intestines that potentially vary with genetic background. These studies provide a foundation for further investigations to verify these findings across a wider number of patients of different ethnic backgrounds.

The immunosuppressant rapamycin has been shown to inhibit G(1)/S transition of the cell cycle. This inhibition is thought to be mediated by maintenance of the threshold levels of cyclin-dependent kinase (CDK) inhibitor p27(Kip1) (p27) and inhibition of p70 s6 kinase (p70(s6k)). However, recent evidence suggests that cells still remain sensitive to rapamycin in the absence of functional p27 or p70(s6k). Here, we show that rapamycin represses cyclin D3 levels in activated human T lymphocytes with no inhibitory effects on cyclin D2. Furthermore, rapamycin elicits similar cyclin D3 modulatory effects in B lymphocytes. The overall effect of rapamycin on cyclin D3 leads to impaired formation of active complexes with Cdk4 or Cdk6 and subsequent inhibition of cyclin D3/CDK kinase activity. Decrease in cyclin D3 protein levels is due to translational repression and not due to attenuated transcription of the cyclin D3 gene. Importantly, stable overexpression of cyclin D3 (2-2.5 fold) in Jurkat T cell transfectants renders them resistant to lower doses (1-10 ng/ml) of rapamycin. These results point to a critical role of cyclin D3 in rapamycin-mediated immunosuppressive effects in T cells and cell cycle regulation in lymphocytes in general.

PROBLEM: The inflammatory-anti-inflammatory cytokine network is thought to play a critical role in regulated progression and termination of pregnancy. The aim of this study was to evaluate the effects of interferon (IFN)-gamma on the expression of Cyclooxygenase (COX)-2 and production of prostaglandin E(2) (PGE(2)) in the human placenta from term and preterm labor deliveries. METHOD OF STUDY: Placental explant culture system was used. COX-2 expression was determined by complementary techniques of immunohistochemistry and Western blotting. Released IFN-gamma and PGE(2) by placental explants were measured by enzyme-linked immunosorbent assay. Signal transducer and activator of transcription 1 (STAT1) phosphorylation was evaluated by Western blotting using a specific antibody. RESULTS: IFN-gamma was poorly detected in the placenta but was significantly expressed in decidual tissues from both term and preterm pregnancies as detected by immunohistochemistry. IFN-gamma significantly inhibited COX-2 expression and PGE(2) release in cultured placental explants from term and preterm labor deliveries. This effect most likely occurred in a STAT1-dependent manner as this regulatory protein was phosphorylated in response to IFN-gamma. IFN-gamma receptor (IFN-gammaR) was expressed in normal early pregnancy placental samples. However, its expression was significantly reduced in placental samples from term and preterm deliveries. Of interest, IFN-gammaR was expressed in placentas from term and preterm labor deliveries after 24 hr in culture. CONCLUSIONS: Our data suggest that the human placenta is an important site for IFN-gamma-mediated repression of COX-2 expression and PGE2 production, implying that functional withdrawal of IFN-gamma may be involved in the onset of term or preterm labor.