Yana Filipovich

The adaptor protein MyD88 is essential for E coli-induced preterm delivery in mice.

OBJECTIVE We used a mouse model of infection-induced preterm delivery to examine the roles of 2 adaptor proteins with central functions in Toll-like receptor signaling: MyD88 (myeloid differentiation primary-response gene 88) and TRIF (Toll/IL-1 receptor (TIR)-domain-containing adaptor protein-inducing IFN-beta). STUDY DESIGN Mice deficient (KO) for MyD88, TRIF, both (DKO) or neither (WT) were inoculated into the uterus with killed Escherichia coli. Delivery outcomes, fetal status, serum progesterone, and nuclear translocation of the transcription factor nuclear factor kappa B (NFkappaB) were determined. RESULTS Preterm birth (delivery in less than 48 hours) occurred in WT and TRIF-KO animals in a dose-dependent fashion, reaching 100% with 5-10 x 10(9) bacteria, while MyD88-KO and DKO animals were completely protected from delivery. Intrauterine fetal survival, maintenance of circulating progesterone levels, and nuclear translocation of NFkappaB were also dependent upon MyD88 but not TRIF. In contrast, induction of uterine interleukin (IL)-1beta and tumor necrosis factor alpha (TNF-alpha) depends upon actions of both MyD88 and TRIF. CONCLUSION E coli-induced preterm delivery in the mouse is completely dependent upon MyD88 but not TRIF.

Depletion of polymorphonuclear leukocytes has no effect on preterm delivery in a mouse model of Escherichia coli-induced labor

OBJECTIVE The objective of the study was to investigate the role of polymorphonuclear leukocytes (PMNs) in a mouse model of Escherichia coli-induced labor. STUDY DESIGN Intraperitoneal injection of rabbit antimouse PMN antiserum or control was performed in CD-1 mice 29 hours and 5 hours prior to laparotomy and intrauterine injection of either killed E coli or phosphate-buffered saline on day 14.5 of pregnancy. Preterm delivery was defined as delivery of at least 1 pup within 48 hours. Circulating leukocyte counts were determined manually or by flow cytometry at the time of surgery and 8, 24, and 48 hours afterward. Maternal and fetal tissues were analyzed in a separate group of animals 8 hours after surgery. RESULTS Pretreatment with anti-PMN antiserum significantly decreased the numbers of circulating leukocytes and the proportion of neutrophils among all leukocytes by 70-80% at surgery and at least 8 hours thereafter. Neutrophil depletion significantly reduced 2 markers of neutrophil activation in the uterus and placenta (neutrophil elastase and myeloperoxidase activity) and neutrophil infiltration into gestational tissues in bacterially treated animals to baseline (control) levels but did not affect preterm birth rates. The large E coli-induced increases in uterine inflammatory markers (interleukin-1β, tumor necrosis factor, chemokine ligand-5, cyclooxygenase-2) were not affected or were only minimally affected by neutrophil depletion. CONCLUSION Although PMN antiserum reduces both neutrophil number and activity, it does not diminish sensitivity to bacterially induced delivery or meaningfully alter the expression of inflammatory markers in the mouse model. Preterm birth and inflammation in this model are not likely to depend on neutrophil function.

Failure of E. coli bacteria to induce preterm delivery in the rat

BackgroundWe sought to develop a model of bacterially induced preterm delivery in rats to parallel similar models in mice.MethodsFemale Sprague-Dawley rats on day 17 of gestation (normal term = 21–22 days) were inoculated into the uterus with either 2 × 109 – 7 × 1010 killed E. coli organisms, 1 – 4 × 108 live E. coli or sterile solution. These inoculations were made either via trans-cervical catheter or by direct intrauterine injection at laparotomy. Animals were then observed for delivery for variable periods up to term. Necropsies were performed and fetal viability was assessed.ResultsNo rats delivered prematurely after bacterial exposure (27 animals observed for at least 48 hours), and all animals followed to term (n = 3) delivered live pups. No dams exhibited signs of systemic illness. There was a statistically significant but small negative effect of killed E. coli on fetal viability (100% of 80 fetuses from 6 control pregnancies and 93% of 182 fetuses from 14 bacterially-treated pregnancies were alive at necropsy, p = 0.014). Live bacteria had a larger effect on fetal viability, with only 64% of 14 fetuses, 47% of 28 fetuses and 32% of 31 fetuses surviving after trans-cervical administration of 7 × 107, 2 × 108 and 4 × 108E. coli, respectively.ConclusionUnlike mice, it has proven difficult to induce preterm labor in the rat using E. coli as a stimulating agent. The relevant literature is reviewed and hypotheses are offered to explain this phenomenon.

Maternal and fetal roles in bacterially induced preterm labor in the mouse

BACKGROUND The relative roles of the mother and fetus in signaling for labor remain poorly understood. OBJECTIVE We previously demonstrated using gene knockout (KO) mice that Escherichia coli-induced preterm delivery is completely dependent on MyD88, a toll-like receptor adaptor protein. Here we leveraged this finding to conduct a genetic experiment testing whether the mother, the fetus, or both signal for parturition in bacterially induced labor. STUDY DESIGN Six different maternal/fetal genotype combinations for MyD88 were studied: wild-type (WT) dams carrying one of the following: (1) WT or (2) MyD88 heterozygous (het) fetuses (generated by mating WT females with WT or MyD88-knockout [KO] males, respectively); (3) WT dams carrying MyD88-KO fetuses (generated by replacing the ovaries of WT females with MyD88-KO ovaries, followed by mating with MyD88-KO males); a similar strategy was used to generate MyD88-KO dams carrying (4) MyD88-KO, (5) MyD88 het, or (6) WT fetuses. On day 14.5 of gestation, mice received intrauterine injections of either 1 × 10(9) killed E coli or sterile medium. Delivery of ≥ 1 fetus within 48 hours was considered preterm. A separate group of similarly treated pregnant mice was euthanized 5 hours after surgery for gene expression and tissue analysis. RESULTS E coli-induced preterm delivery is dependent on maternal and not fetal genotype: > 95% of WT and < 5% of MyD88-KO dams deliver prematurely, regardless of fetal genotype (P = .0001). In contrast, fetal survival in utero is influenced by fetal genotype: in MyD88-KO dams, in which premature birth rarely occurs, only 81% of WT and 86% of MyD88-heterozygous fetuses were alive 48 hours after surgery compared with 100% of MyD88-KO fetuses (P < .01). Messenger ribonucleic acids for the inflammatory mediators interleukin-1β, tumor necrosis factor, interleukin-6, and cyclooxygenase-2 were elevated in uterine tissues only in WT mothers treated with E coli and were low or undetectable in the uteri of KO mothers or in animals treated with saline. Serum progesterone levels were lower in KO mothers with WT ovaries than in WT mothers with KO ovaries, but bacterial exposure did not have an impact on these levels. CONCLUSION In the murine E coli-induced labor model, preterm delivery and uterine expression of inflammatory mediators is determined by the mother and not the fetus and is not attributable to a decline in serum progesterone.

MP83-16 EXPLORING 2D AND 3D CULTURE ON RESPONSES TO COMBINATORIAL DRUG THERAPY IN HUMAN PROSTATE CELL LINES

a multifunctional protein found in the nucleocytoplasm of most human cells, and is abnormally translocated to the cell membrane in cancer. One of the mechanisms by which NCL exerts its oncogenic activity is through the biogenesis of miRs -21, -221, and -222. 4LB5 is a novel single-chain fragment variable (scFv) antibody that binds preferentially to cancer cell membranes via the RNA-binding domain of NCL and inhibits production of these oncogenic miRs. The therapeutic and diagnostic potential of 4LB5 has been described in breast and hepatocellular carcinoma cells in previous studies. Given the supporting evidence, we aim to characterize the effects of 4LB5 on CRPC and compare its efficacy among androgen-dependent and androgen-independent cells. METHODS: Levels of NCL expression in PCa cell lines DU145, PC3, and LNCaP were investigated with western blot (WB). Cell surface ELISA was performed to compare 4LB5 binding across PCa cell lines. Cell survival assay was performed using 50 nM 4LB5 or control buffer. Levels of mature and precursor forms of miRs in 4LB5-treated cells were assessed using quantitative real-time polymerase chain reaction. MDA-MB-231 cells were used a positive control in all experiments. RESULTS: All cell lines expressed NCL as evidenced by WB, and downregulation of NCL was observed with siNCL transfection. ELISA results showed a strong exponential association between 4LB5 concentration and binding activity in DU145 (R 1⁄4 0.9972), PC3 (R 1⁄4 0.9887), and LNCaP (R 1⁄4 0.9897) cells, with more binding units observed in PC3 and DU145 compared to LNCaP. 4LB5 significantly inhibited proliferation of DU145 (p 1⁄4 0.000016) and PC3 (p 1⁄4 0.0074) cells, but not LNCaP (p 1⁄4 0.45). Treatment with 4LB5 caused downregulation of mature forms of miRs 21, 221, and 222 with expected upregulation of precursor forms in PC3 cells, but LNCaP cells exhibited the opposite effect. CONCLUSIONS: 4LB5 binds CRPC cells and inhibits proliferation through abrogation of oncogenic miR production. 4LB5 could represent a novel therapeutic option in CRPC patients.

MP17-15 LONG-TERM LEPTIN TREATMENT RESULTS IN A REDUCTION IN PROSTATIC EPITHELIAL HYPERPLASIA IN THE OBOB MOUSE MODEL

INTRODUCTION AND OBJECTIVES: Benign prostatic hyperplasia (BPH) is a major public health problem with high morbidity and associated cost. BPH arises in the context well recognized comorbidities including metabolic syndrome (MetS), obesity and diabetes. Animal models do not reflect all aspects of the human disease. Recently, an animal model, leptin-deficient obese ObOb mice has been reported that mimics the features of MetS and prostatic hyperplasia. However, details of the linkage between MetS/obesity and prostatic hyperplasia remain unknown in this model. To assess the relationship between MetS/obesity and prostatic hyperplasia in ObOb mice, we evaluated the morphological phenotype of control and leptin-treated ObOb mice prostate. METHODS: We started with 10-week-old male ObOb and strain-matched control mice. Three groups were examined: lean C57/Bl/ 6J (strain-matched control), non-treatedObOb, and leptin-treated ObOb mice. Leptin was delivered using a subcutaneous Alzet micro-osmotic pump. Leptin was delivered 5 mg/day for the initial 12 weeks of the study, and at 10 mg/day for the final 12 weeks. The liver, pancreas, spleen, skin, femoral muscle, subcutaneous fat, visceral fat, periprostatic fat, brown fat, and urogenital organs were harvested for analysis. RESULTS: In ObOb mice, total body weight and liver weight were reduced 25%-35% by leptin treatment. Leptin supplementation dramatically reduced body weight during the first month, but weight then increased slowly. Non-fasting serum blood glucose levels were normal range ( 250mg/dl) in all ObOb mice, but a glucose tolerance test revealed significantly longer recoveries in non-treated vs. leptin-treated ObObmice. Compared to non-treated ObObmice, liver weight of leptintreated ObOb mice is significant lighter but still heavier than lean C57/ Bl/6J mice. Anterior, ventral, and dorsolateral prostate glands in nontreated ObOb mice exhibited epithelial hyperplasia compared to lean C57/Bl/6J mice. In the leptin-treated mice, the number of hyperplastic prostate glands in each lobe was significant lower than in non-treated ObOb mice. CONCLUSIONS: Our results demonstrated that long-term leptin treatment results in a reduction in prostatic epithelial hyperplasia in the ObOb mouse model. Full characterization of this animal model may elucidate molecular mechanisms linking MetS/obesity and prostatic hyperplasia.