Rescue from lethal Shiga toxin 2-induced renal failure with a cell-permeable peptide.

Intestinal infection with Shiga toxin (Stx)-producing E.coli is a leading cause of hemolytic uremic syndrome and acute renal injury in otherwise healthy children in the US. Antibiotics are contraindicated and a therapeutic priority is agents that act intracellularly against the bacterial toxins that drive kidney injury. Our aim was to evaluate whether intravenous administration of a cell-permeable peptide (TVP) that binds to Stx2 will reduce disease severity and rescue juvenile baboons from a lethal Stx2 dose (50 ng/kg). TVP (5 mg/kg) was delivered i.v. simultaneously with toxin (prevention protocol) or at 6 or 24 h after toxin with daily 1 mg/kg supplements up to day 4 (rescue protocols). Biomarkers were monitored in blood and urine up to 28 days. TVP therapy resulted in either absence of clinical signs of acute kidney injury and normal urine output (prevention), or delayed and reduced BUN and creatinine levels (rescue) with concomitant survival. Delayed peptide administration significantly reduced thrombocytopenia, but surprisingly did not alter anemia even when monitored for 28 days in rescued survivors. This is the first successful cell-permeable therapeutic that counteracts Stx2 lethality in an animal model, which recapitulates many of the human responses to enteric infection.

Distinct physiologic and inflammatory responses elicited in baboons after challenge with Shiga toxin type 1 or 2 from enterohemorrhagic Escherichia coli.

Shiga toxin-producing Escherichia coli is a principal source of regional outbreaks of bloody diarrhea and hemolytic-uremic syndrome in the United States and worldwide. Primary bacterial virulence factors are Shiga toxin types 1 and 2 (Stx1 and Stx2), and we performed parallel analyses of the pathophysiologies elicited by the toxins in nonhuman primate models to identify shared and unique consequences of the toxemias. After a single intravenous challenge with purified Stx1 or Stx2, baboons (Papio) developed thrombocytopenia, anemia, and acute renal failure with loss of glomerular function, in a dose-dependent manner. Differences in the timing and magnitude of physiologic responses were observed between the toxins. The animals were more sensitive to Stx2, with mortality at lower doses, but Stx2-induced renal injury and mortality were delayed 2 to 3 days compared to those after Stx1 challenge. Multiplex analyses of plasma inflammatory cytokines revealed similarities (macrophage chemoattractant protein 1 [MCP-1] and tumor necrosis factor alpha [TNF-alpha]) and differences (interleukin-6 [IL-6] and granulocyte colony-stimulating factor [G-CSF]) elicited by the toxins with respect to the mediator induced and timing of the responses. Neither toxin induced detectable levels of plasma TNF-alpha. To our knowledge, this is the first time that the in vivo consequences of the toxins have been compared in a parallel and reproducible manner in nonhuman primates, and the data show similarities to patient observations. The availability of experimental nonhuman primate models for Stx toxemias provides a reproducible platform for testing antitoxin compounds and immunotherapeutics with outcome criteria that have clinical meaning.

Regulation of the prostaglandin enzymatic system by estradiol and progesterone in nonpregnant sheep cervix.

In the present study, we examined the in vivo effects of estradiol (E(2)) and progesterone on cyclooxygenase (COX) 2, prostaglandin F synthase (PTGFS, also known as PGFS), and membrane-associated prostaglandin E synthase 1 (mPTGES1) expression at both mRNA and protein levels using a nonpregnant ovariectomized (OVX) sheep model. Sixteen ewes were OVX shortly after ovulation. After 40 days, ewes were treated with saline (Cont, n=5), or E(2) infused intravenously for 2 days (50 microg/day, n=5) or intravaginal progesterone (P) sponges for 10 days (0.3 g P, n=6). Cervical COX2, PTGFS, and mPTGES1 mRNA and protein were quantified by northern and western blot analyses respectively. In situ hybridization and/or immunocytochemistry were used to localize the cellular distribution of COX2, PTGFS, and mPTGES1 mRNAs and proteins. COX2 mRNA abundance increased significantly in the cervix after E(2) treatment (P<0.05). However, progesterone was a more potent stimulator than E(2) of COX2 mRNA and protein abundance in the cervix (P<0.01). In contrast, PTGFS and mPTGES1 mRNA and protein concentrations did not change after E(2) or progesterone treatment (P>0.05). COX2, PTGFS, and mPTGES1 mRNA and protein were only localized in cervical glandular epithelial cells. This study shows that increased cervical COX2 mRNA and protein, but not PTGFS and mPTGES1 mRNA and protein, were associated with E(2) and progesterone treatment in nonpregnant sheep. More strikingly, progesterone was a more potent stimulator of cervical COX2 expression than E(2). The expression of COX2, PTGFS, and mPTGES1 mRNA and/or protein was confined in the cervical glandular epithelial cells of nonpregnant sheep.

Regulation of membrane-associated prostaglandin E2 synthase 1 in pregnant sheep intrauterine tissues by glucocorticoid and estradiol.

This study was designed to determine the regulatory effect of glucocorticoid and estradiol on expression of ovine intrauterine membrane-associated prostaglandin E(2) synthase 1 (mPTGES1) in late gestation and at labor. For gestational and labor groups, 16 pregnant ewes from 95-147 d gestational age (dGA) and four pregnant ewes at spontaneous term labor were used. The fetal glucocorticoid group, 14 pregnant ewes at 123-125 dGA with fetuses, was divided into the following groups: after sham adrenalectomy (n = 5), adrenalectomy (n = 4), and adrenalectomy with fetal cortisol replacement to late gestation levels (n = 5). For the maternal glucocorticoid group, nine pregnant ewes were treated with saline (n = 4) and three courses of maternal dexamethasone (n = 5). For the estradiol group, 10 pregnant ewes at 119-121 dGA were treated with sesame oil (n = 5) or estradiol (n = 5) to produce labor levels of estradiol in maternal plasma. Endometrial, myometrial, and placental mRNA and proteins were analyzed by Northern and Western blot and immunocytochemistry for mPTGES1. Data were analyzed by Student's t test and ANOVA. There was a significant increase of placental mPTGES1 in late gestation. Glucocorticoids, given to the mother or fetus, significantly stimulated mPTGES1 in placenta. mPTGES1 was elevated only in the endometrium during spontaneous term labor and after estradiol treatment. The mPTGES1 was localized in the myometrial smooth muscle cells, endometrial stromal cells, and placental trophoblast cells. Our study suggested that increased expression of placental mPTGES1 throughout late gestation might result from the increased fetal and maternal circulating glucocorticoids, whereas elevated maternal plasma estradiol concentration might be responsible for the induced mPTGES1 expression in the endometrium during labor.

Sufficient progesterone-priming prior to estradiol stimulation is required for optimal induction of the cervical prostaglandin system in pregnant sheep at 0.7 gestations.

The purposes of this study were to determine the separate and interactive functions of progesterone and estradiol in regulating the cervical prostaglandin (PG) system in pregnant sheep at 0.7 gestations. At 106-108 days of gestational age (dGA), ewes were treated with vehicle for 14 days (n = 5) or vehicle for 12 days followed by estradiol 5 mg twice a day, intramuscularly for 2 days (n = 5) or progesterone 100 mg, twice a day, intramuscularly for 14 days (n = 5) or progesterone 100 mg twice a day, intramuscularly for 10 days and then 2 days vehicle followed by estradiol 5 mg twice a day intramuscularly for 2 days (n = 5). At 121-123 dGA, cervical tissues were obtained under halothane anesthesia. Cervical RNA and protein were extracted and analyzed for prostaglandin-endoperoxide synthase 2 (COX2), two PGE(2) receptors, PTGER2 and PTGER4, and estrogen receptor alpha (ESR1) by Northern and Western blot analysis. Immunocytochemistry and in situ hybridization were applied to localize cellular distribution of COX2, PTGER2, and PTGER4 in the cervix. Data were analyzed by ANOVA. COX2 and PTGER4 mRNAs and proteins were increased (P < 0.05) in ewes treated with combined estradiol and progesterone but not in ewes treated with estradiol or progesterone alone compared with controls. ESR1 mRNA was increased in ewes treated with progesterone and estradiol plus progesterone. In contrast, PTGER2 mRNA and protein remained the same after all treatments. COX2 mRNA and protein were localized only in cervical glandular epithelial cells, whereas PTGER2 and PTGER4 were localized in both cervical glandular epithelial and smooth muscle cells. In conclusion, these data suggest that additional progesterone priming at 0.7 gestations synergizes with estradiol to induce cervical COX2, PTGER4, and ESR1 and support our hypothesis that stimulation of the cervical PG system by estradiol is optimized by sufficient progesterone priming in the pregnant sheep cervix.

Prostaglandin mediates premature delivery in pregnant sheep induced by estradiol at 121 days of gestational age.

The experiments reported here were designed for both in vivo and in vitro approaches in the same animals to obtain a better picture of the role of estrogen in the control of parturition. Chronically catheterized pregnant ewes were treated with vehicle (n = 5) or estradiol (n = 6), 5 mg twice a day, im for 2 d starting at d 119 of gestation. Maternal and fetal plasma estradiol, progesterone, and cortisol were measured by RIA and maternal plasma prostaglandin (PG) F2alpha was measured by enzyme immunoassay. Intrauterine PG H synthase 2 mRNA and protein and placental P450(c17)alpha hydroxylase mRNA were determined by Northern, in situ hybridization, Western blot analysis, and immunocytochemistry. Data were analyzed by ANOVA. Five of six estradiol-treated ewes delivered their fetuses within 48 h; however, the placenta was still retained 5-6 h after fetal delivery. Both maternal plasma estradiol and PGF2 alpha increased significantly in the estradiol-treated group. Maternal and fetal plasma progesterone and cortisol were not altered in either group. There were significant increases of PGH synthase 2 mRNA and protein in myometrium, endometrium, and maternal placenta but not in fetal placenta in estradiol-treated ewes. Placental P450(c17)alpha hydroxylase mRNA was not detectable in vehicle or estradiol-treated groups. Estradiol can, in the absence of increase in plasma cortisol, stimulate uterine PG production and induce labor, resulting in fetal delivery in the sheep. Failure of placental delivery after estradiol treatment suggests that estradiol alone is insufficient to stimulate some of the key changes required to complete delivery at the stage of gestation studied.