Uterine Nodal expression supports maternal immunotolerance and establishment of the FOXP3+ regulatory T cell population during the preimplantation period.

Pregnancy success is dependent on the establishment of maternal tolerance during the preimplantation period. The immunosuppressive function of regulatory T cells is critical to limit inflammation arising from implantation of the semi-allogeneic blastocyst. Insufficient maternal immune adaptations to pregnancy have been frequently associated with cases of female infertility and recurrent implantation failure. The role of Nodal, a secreted morphogen of the TGFß superfamily, was recently implicated during murine pregnancy as its conditional deletion (NodalΔ/Δ) in the female reproductive tract resulted in severe subfertility. Here, it was determined that despite normal preimplantation processes and healthy, viable embryos, NodalΔ/Δ females had a 50% implantation failure rate compared to NodalloxP/loxP controls. Prior to implantation, the expression of inflammatory cytokines MCP-1, G-CSF, IFN-γ and IL-10 was dysregulated in the NodalΔ/Δ uterus. Further analysis of the preimplantation leukocyte populations in NodalΔ/Δ uteri showed an overabundance of infiltrating, pro-inflammatory CD11bhigh Ly6C+ macrophages coupled with the absence of CD4+ FOXP3+ regulatory T cells. Therefore, it is proposed that uterine Nodal expression during the preimplantation period has a novel role in the establishment of maternal immunotolerance, and its dysregulation should be considered as a potential contributor to cases of female infertility and recurrent implantation failure.

Mechanism of Action and Pharmacology of Patiromer, a Nonabsorbed Cross-Linked Polymer That Lowers Serum Potassium Concentration in Patients With Hyperkalemia.

Hyperkalemia is a potentially life-threatening condition, and patients who have chronic kidney disease, who are diabetic, or who are taking renin-angiotensin-aldosterone system inhibitors are at increased risk. Therapeutic options for hyperkalemia tend to have limited effectiveness and can be associated with serious side effects. Colonic potassium secretion can increase to compensate when urinary potassium excretion decreases in patients with renal impairment, but this adaptation is insufficient and hyperkalemia still results. Patiromer is a novel, spherical, nonabsorbed polymer designed to bind and remove potassium, primarily in the colon, thereby decreasing serum potassium in patients with hyperkalemia. Patiromer has been found to decrease serum potassium in patients with hyperkalemia having chronic kidney disease who were on renin-angiotensin-aldosterone system inhibitors. Results of nonclinical studies and an early phase clinical study are reported here. Two studies with radiolabeled drug, one in rats and the other in dogs, confirmed that patiromer was not absorbed into the systemic circulation. Results of an in vitro study showed that patiromer was able to bind 8.5 to 8.8 mEq of potassium per gram of polymer at a pH similar to that found in the colon and had a much higher potassium-binding capacity compared with other resins, including polystyrene sulfonate. In a study in hyperkalemic rats, a decrease in serum potassium was observed via an increase in fecal potassium excretion. In a clinical study in healthy adult volunteers, a significant increase in fecal potassium excretion and a significant decrease in urinary potassium excretion were observed. Overall, patiromer is a high-capacity potassium binder, and the chemical and physical characteristics of patiromer may lead to good clinical efficacy, tolerability, and patient acceptance.

Uterine inactivation of muscle segment homeobox (Msx) genes alters epithelial cell junction proteins during embryo implantation.

Embryo implantation requires that the uterus differentiate into the receptive state. Failure to attain uterine receptivity will impede blastocyst attachment and result in a compromised pregnancy. The molecular mechanism by which the uterus transitions from the prereceptive to the receptive stage is complex, involving an intricate interplay of various molecules. We recently found that mice with uterine deletion ofMsxgenes (Msx1(d/d)/Msx2(d/d)) are infertile because of implantation failure associated with heightened apicobasal polarity of luminal epithelial cells during the receptive period. However, information on Msx's roles in regulating epithelial polarity remains limited. To gain further insight, we analyzed cell-type-specific gene expression by RNA sequencing of separated luminal epithelial and stromal cells by laser capture microdissection fromMsx1(d/d)/Msx2(d/d)and floxed mouse uteri on d 4 of pseudopregnancy. We found that claudin-1, a tight junction protein, and small proline-rich (Sprr2) protein, a major component of cornified envelopes in keratinized epidermis, were substantially up-regulated inMsx1(d/d)/Msx2(d/d)uterine epithelia. These factors also exhibited unique epithelial expression patterns at the implantation chamber (crypt) inMsx1(f/f)/Msx2(f/f)females; the patterns were lost inMsx1(d/d)/Msx2(d/d)epithelia on d 5, suggesting important roles during implantation. The results suggest thatMsxgenes play important roles during uterine receptivity including modulation of epithelial junctional activity.-Sun, X., Park, C. B., Deng, W., Potter, S. S., Dey, S. K. Uterine inactivation of muscle segment homeobox (Msx) genes alters epithelial cell junction proteins during embryo implantation.

Appropriate crypt formation in the uterus for embryo homing and implantation requires Wnt5a-ROR signaling.

Embryo homing and implantation occur within a crypt (implantation chamber) at the antimesometrial (AM) pole along the uterus. The mechanism by which this is achieved is not known. Here, we show that villi-like epithelial projections from the main uterine lumen toward the AM pole at regularly spaced intervals that form crypts for embryo implantation were disrupted in mice with uterine loss or gain of function of Wnt5a, or loss of function of both Ror1 and Ror2. This disruption of Wnt5a-ROR signaling resulted in disorderly epithelial projections, crypt formation, embryo spacing, and impaired implantation. These early disturbances under abnormal Wnt5a-ROR signaling were reflected in adverse late pregnancy events, including defective decidualization and placentation, ultimately leading to compromised pregnancy outcomes. This study presents deeper insight regarding the formation of organized epithelial projections for crypt formation and embryo implantation for pregnancy success.

Maternal Nodal inversely affects NODAL and STOX1 expression in the fetal placenta.

Nodal, a secreted signaling protein from the transforming growth factor beta (TGF-ß)-super family plays a vital role during early embryonic development. Recently, it was found that maternal decidua-specific Nodal knockout mice show intrauterine growth restriction (IUGR) and preterm birth. The chromosomal location of NODAL is in the same linkage area as the placental (fetal) pre-eclampsia (PE) susceptibility gene STOX1, which is associated with the familial form of early-onset, IUGR-complicated PE. As the STOX1 linkage was originally identified in women being born from a pre-eclamptic pregnancy as well as suffering from PE themselves, the linkage could in part be caused by NODAL, which is why the potential maternal-fetal interaction between STOX1 and NODAL was investigated. In the PE families with the STOX1 susceptibility allele carried by the children born from pre-eclamptic pregnancies, it was found that the pre-eclamptic mothers themselves all carried the NODAL H165R SNP, which causes a 50% reduced activity. Surprisingly, in decidua-specific Nodal knockout mice the fetal placenta showed up-regulation of STOX1 and NODAL expression. Conditioned media of human first trimester decidua and a human endometrial stromal cell line (T-HESC) treated with siRNAs against NODAL or carrying the H165R SNP were also able to induce NODAL and STOX1 expression when added to SGHPL-5 first trimester extravillous trophoblast cells. Finally, a human TGF-ß/BMP signaling pathway PCR-array on decidua and the T-HESC cell line with Nodal knockdown revealed upregulation of Activin-A, which was confirmed in conditioned media by ELISA. We show that maternal decidua Nodal knockdown gives upregulation of NODAL and STOX1 mRNA expression in fetal extravillous trophoblast cells, potentially via upregulation of Activin-A in the maternal decidua. As both Activin-A and Nodal have been implicated in PE, being increased in serum of pre-eclamptic women and upregulated in pre-eclamptic placentas respectively, this interaction at the maternal-fetal interface might play a substantial role in the development of PE.

NODAL signaling components regulate essential events in the establishment of pregnancy.

Successful mammalian reproduction is dependent on a receptive and nurturing uterine environment. In order to establish pregnancy in humans, the uterus must i) be adequately prepared to receive the blastocyst, ii) engage in a coordinated molecular dialog with the embryo to facilitate implantation, and iii) undergo endometrial decidualization. Although numerous factors have been implicated in these essential processes, the precise network of molecular interactions that govern receptivity, embryo implantation, and decidualization remain unclear. NODAL, a morphogen in the transforming growth factor ß superfamily, is well known for its critical functions during embryogenesis; however, recent studies have demonstrated an emerging role for NODAL signaling during early mammalian reproduction. Here, we review the established data and a recent wave of new studies implicating NODAL signaling components in uterine cycling, embryo implantation, and endometrial decidualization in humans and mice.

NODAL in the uterus is necessary for proper placental development and maintenance of pregnancy.

Preterm birth is the single leading cause of perinatal mortality in developed countries, affecting approximately 12% of pregnancies and accounting for 75% of neonatal loss in the United States. Despite the prevalence and severity of premature delivery, the causes and mechanisms that underlie spontaneous and idiopathic preterm birth remain unknown. Our inability to elucidate these fundamental causes has been attributed to a poor understanding of the signaling pathways associated with the premature induction of parturition and a lack of suitable animal models available for preterm birth research. In this study, we describe the generation and analysis of a novel conditional knockout of the transforming growth factor beta (TGFB) superfamily member, Nodal, from the maternal reproductive tract of mice. Strikingly, uterine Nodal knockout females exhibited a severe malformation of the maternal decidua basalis during placentation, leading to significant intrauterine growth restriction, and ultimately preterm birth and fetal loss on Day 17.5 of gestation. Using several approaches, we characterized aberrant placental development and demonstrated that reduced proliferation combined with increased apoptosis resulted in a diminished decidua basalis and compromised maternal-fetal interface. Last, we evaluated various components of the established parturition cascade and determined that preterm birth derived from the maternal Nodal knockout occurs prior to PTGS2 (COX-2) upregulation at the placental interface. Taken together, the results presented in this study highlight an in vivo role for maternal NODAL during placentation, present an interesting link between disrupted decidua basalis formation and premature parturition, and describe a potentially valuable model toward elucidating the complex processes that underlie preterm birth.

Nodal expression in the uterus of the mouse is regulated by the embryo and correlates with implantation.

Nodal, a transforming growth factor beta (TGFB) superfamily member, plays a critical role during early embryonic development. Recently, components of the Nodal signaling pathway were characterized in the human uterus and implicated in the tissue remodeling events during menstruation. Furthermore, the Nodal inhibitor, Lefty, was identified in the mouse endometrium during pregnancy, and its overexpression led to implantation failure. Nonetheless, the precise function and mechanism of Nodal signaling during pregnancy remains unclear. In order to elucidate the potential roles Nodal plays in these processes, we have generated a detailed profile of maternal Nodal expression in the mouse uterus throughout pregnancy. NODAL, although undetectable during the nonpregnant estrus cycle, was localized throughout the glandular epithelium of the endometrium during the peri-implantation period. Interestingly, Nodal expression generated a banding pattern along the proximal-distal axis of the uterine horn on Day 4.5 that directly correlated with blastocyst implantation. Embryo transfer experiments indicate the embryo regulates Nodal expression in the uterus and directs its expression at the time of implantation, restricting NODAL to the sites between implantation crypts. During the later stages of pregnancy, Nodal exhibits a dynamic expression profile that suggests a role in regulating the endometrial response to decidualization and associated trophoblast invasion.

Novel antibacterial azetidine lincosamides.

The synthesis and evaluation of novel azetidine lincosamides 1 are described. Eleven new (3-trans-alkyl)azetidine-2-carboxylic acids were synthesized via alkylation of N-TBS-4-oxo-azetidine-2-carboxylic acid and subsequent elaboration then coupled to 7-chloro-1-methylthio-lincosamine. The resulting lincosamides differ from the drug clindamycin in both the size of the ring and the position/structure of the alkyl side-chain. SAR within the series was explored with attention to alkyl variants in positions 1 and 3 of the azetidine ring.

RWJ-54428 (MC-02,479), a new cephalosporin with high affinity for penicillin-binding proteins, including PBP 2a, and stability to staphylococcal beta-lactamases.

RWJ-54428 (MC-02,479) is a new cephalosporin active against gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA). The potency of this new cephalosporin against MRSA is related to a high affinity for penicillin-binding protein 2a (PBP 2a), as assessed in a competition assay using biotinylated ampicillin as the reporter molecule. RWJ-54428 had high activity against MRSA strains COL and 67-0 (MIC of 1 micro g/ml) and also showed affinity for PBP 2a, with a 50% inhibitory concentration (IC(50)) of 0.7 micro g/ml. RWJ-54428 also displayed excellent affinity for PBP 5 from Enterococcus hirae R40, with an IC(50) of 0.8 micro g/ml and a MIC of 0.5 micro g/ml. The affinity of RWJ-54428 for PBPs of beta-lactam-susceptible S. aureus (MSSA), enterococci (E. hirae), and Streptococcus pneumoniae showed that the good affinity of RWJ-54428 for MRSA PBP 2a and E. hirae PBP 5 does not compromise its binding to susceptible PBPs. RWJ-54428 showed stability to hydrolysis by purified type A beta-lactamase isolated from S. aureus PC1. In addition, RWJ-54428 displayed low MICs against strains of S. aureus bearing the four classes of staphylococcal beta-lactamases, including beta-lactamase hyperproducers. The frequency of isolation of resistant mutants to RWJ-54428 from MRSA strains was very low. In summary, RWJ-54428 has high affinity to multiple PBPs and is stable to beta-lactamase, properties that may explain our inability to find resistance by standard methods. These data are consistent with its excellent activity against beta-lactam-resistant gram-positive bacteria.