Chlamydia heat shock protein 60 induces trophoblast apoptosis through TLR4

Intrauterine infection affects placental development and function, and subsequently may lead to complications such as preterm delivery, intrauterine growth retardation, and preeclampsia; however, the molecular mechanisms are not clearly known. TLRs mediate innate immune responses in placenta, and recently, TLR2-induced trophoblast apoptosis has been suggested to play a role in infection-induced preterm delivery. Chlamydia trachomatis is the etiological agent of the most prevalent sexually transmitted bacterial infection in the United States. In this study, we show that in vitro chlamydial heat shock protein 60 induces apoptosis in primary human trophoblasts, placental fibroblasts, and the JEG3 trophoblast cell line, and that TLR4 mediates this event. We observed a host cell type-dependent apoptotic response. In primary placental fibroblasts, chlamydial heat shock protein 60-induced apoptosis was caspase dependent, whereas in JEG3 trophoblast cell lines it was caspase independent. These data suggest that TLR4 stimulation induces apoptosis in placenta, and this could provide a novel mechanism of pathogenesis for poor fertility and pregnancy outcome in women with persistent chlamydia infection.     

Elevated maternal cortisol early in pregnancy predicts third trimester levels of placental corticotropin releasing hormone (CRH): priming the placental clock

The purposes of this study were to determine the intervals when placental corticotrophic-releasing hormone (CRH) was most responsive to maternal cortisol. A sample of 203 women each were evaluated at 15, 19, 25 and 31 weeks gestation and followed to term. Placental CRH and maternal adrenocorticotropin hormone (ACTH), B-endorphin and cortisol were determined from plasma. CRH levels increased faster and were higher in women who delivered preterm compared with women who delivered at term (F3,603 = 5.73, p < .001). Simple effects indicated that CRH levels only at 31 weeks predicted preterm birth (F1,201 = 5.53, p = .02). Levels of cortisol were higher in women who delivered preterm at 15 weeks gestation (F1,201 = 4.45, p = .03) with a similar trend at 19 weeks gestation. Hierarchical regression suggested that the influence on birth outcome of maternal cortisol early in pregnancy was mediated by its influence on placental CRH at 31 weeks. Elevated cortisol at 15 weeks predicted the surge in placental CRH at 31 weeks (R = .49, d.f. = 1,199, Fchange = 61.78, p < .0001). Every unit of change in cortisol (microg/dl) at 15 weeks was associated with a 34 unit change of CRH (pg/ml) at 31 weeks. These findings suggested that early detection of stress signals by the placenta stimulated the subsequent release of CRH and resulted in increased risk for preterm delivery.     

A structural limitation on enzyme activity: the case of HMG-CoA synthase

Recent structural studies of the HMG-CoA synthase members of the thiolase superfamily have shown that the catalytic loop containing the nucleophilic cysteine follows the phi and psi angle pattern of a II' beta turn. However, the i + 1 residue is conserved as an alanine, which is quite unusual in this position as it must adopt a strained positive phi angle to accommodate the geometry of the turn. To assess the effect of the conserved strain in the catalytic loop, alanine 110 of Enterococcus faecalis 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase was mutated to a glycine. Subsequent enzymatic studies showed that the overall reaction rate of the enzyme was increased 140-fold. An X-ray crystallographic study of the Ala110Gly mutant enzyme demonstrated unanticipated adjustments in the active site that resulted in additional stabilization of all three steps of the reaction pathway. The rates of acetylation and hydrolysis of the mutant enzyme increased because the amide nitrogen of Ser308 shifts 0.4 A toward the catalytic cysteine residue. This motion positions the nitrogen to better stabilize the intermediate negative charge that develops on the carbonyl oxygen of the acetyl group during both the formation of the acyl-enzyme intermediate and its hydrolysis. In addition, the hydroxyl of Ser308 rotates 120 degrees to a position where it is able to stabilize the carbanion intermediate formed by the methyl group of the acetyl-S-enzyme during its condensation with acetoacetyl-CoA.     

VEGF modulates erythropoiesis through regulation of adult hepatic erythropoietin synthesis

Vascular endothelial growth factor (VEGF) exerts crucial functions during pathological angiogenesis and normal physiology. We observed increased hematocrit (60-75%) after high-grade inhibition of VEGF by diverse methods, including adenoviral expression of soluble VEGF receptor (VEGFR) ectodomains, recombinant VEGF Trap protein and the VEGFR2-selective antibody DC101. Increased production of red blood cells (erythrocytosis) occurred in both mouse and primate models, and was associated with near-complete neutralization of VEGF corneal micropocket angiogenesis. High-grade inhibition of VEGF induced hepatic synthesis of erythropoietin (Epo, encoded by Epo) >40-fold through a HIF-1alpha-independent mechanism, in parallel with suppression of renal Epo mRNA. Studies using hepatocyte-specific deletion of the Vegfa gene and hepatocyte-endothelial cell cocultures indicated that blockade of VEGF induced hepatic Epo by interfering with homeostatic VEGFR2-dependent paracrine signaling involving interactions between hepatocytes and endothelial cells. These data indicate that VEGF is a previously unsuspected negative regulator of hepatic Epo synthesis and erythropoiesis and suggest that levels of Epo and erythrocytosis could represent noninvasive surrogate markers for stringent blockade of VEGF in vivo.     

Traditional Chinese medicine as dual guardians against hypertension and cancer?

This study utilizes the comprehensive traditional Chinese medicine database TCM Database@Taiwan ( http://tcm.cmu.edu.tw/ ) in conjunction with structure-based and ligand-based drug design to identify multi-function Src inhibitors. The three potential TCM candidates identified as having suitable docking conformations and bioactivity profiles were Angeliferulate, (3R)-2'-hydroxy-3',4'-dimethoxyisoflavan-7-O-beta-D-glucoside (HMID), and 3-[2',6-dihydroxy-5'-(2-propenyl)[1,1'-biphenyl]3-yl]-(E)-2-propenoic acid (3PA). Molecular dynamics simulation demonstrated that the TCM candidates have more stable interactions with the cleft and in complex with Src kinase compared to Saracatinib. Angeliferulate and HMID, both originated from Angelica sinensis, not only interact with Lys298 and amino acids from different loops in the cleft, but also with Asp407 located on the activation loop. These interactions are important to reduce the opening of the activation loop due to phosphorylation, hence stabilize the Src kinase cleft structure and inhibit activation. The TCM candidates also exhibited high affinity to other cancer-related target proteins (EGFR, HER2, and HSP90). Our observations suggest that the TCM candidates might have multi-targeting effects in hypertension and cancer.     

CD8+ Foxp3+ regulatory T cells are induced during graft-versus-host disease and mitigate disease severity

Regulatory T cells (Tregs), in particular CD4(+) Foxp3(+) T cells, have been shown to play an important role in the maintenance of tolerance after allogeneic stem cell transplantation. In the current study, we have identified a population of CD8(+) Foxp3(+) T cells that are induced early during graft-versus-host disease (GVHD), constitute a significant percentage of the entire Treg population, and are present in all major GVHD target organs. These cells expressed many of the same cell surface molecules as found on CD4(+) Tregs and potently suppressed in vitro alloreactive T cell responses. Induction of these cells correlated positively with the degree of MHC disparity between donor and recipient and was significantly greater than that observed for CD4(+)-induced Tregs (iTregs) in nearly all tissue sites. Mice that lacked the ability to make both CD8(+) and CD4(+) iTregs had accelerated GVHD mortality compared with animals that were competent to make both iTreg populations. The absence of both iTreg populations was associated with significantly greater expansion of activated donor T cells and increased numbers of CD4(+) and CD8(+) T cells that secreted IFN-γ and IL-17. The presence of CD8(+) iTregs, however, was sufficient to prevent increased GVHD mortality in the complete absence of CD4(+) Tregs, indicating at least one functional iTreg population was sufficient to prevent an exacerbation in GVHD severity, and that CD8(+) iTregs could compensate for CD4(+) iTregs. These studies define a novel population of CD8(+) Tregs that play a role in mitigating the severity of GVHD after allogeneic stem cell transplantation.     

Antigen-independent induction of Tim-3 expression on human T cells by the common γ-chain cytokines IL-2, IL-7, IL-15, and IL-21 is associated with proliferation and is dependent on the phosphoinositide 3-kinase pathway

T cell Ig mucin domain-containing molecule 3 (Tim-3) is a glycoprotein found on the surface of a subset of CD8(+) and Th1 CD4(+) T cells. Elevated expression of Tim-3 on virus-specific T cells during chronic viral infections, such as HIV-1, hepatitis B virus, and hepatitis C virus, positively correlates with viral load. Tim-3(+) cytotoxic T cells are dysfunctional and are unable to secrete effector cytokines, such as IFN-γ and TNF-α. In this study, we examined potential inducers of Tim-3 on primary human T cells. Direct HIV-1 infection of CD4(+) T cells, or LPS, found to be elevated in HIV-1 infection, did not induce Tim-3 on T cells. Tim-3 was induced by the common γ-chain (γc) cytokines IL-2, IL-7, IL-15, and IL-21 but not IL-4, in an Ag-independent manner and was upregulated on primary T cells in response to TCR/CD28 costimulation, as well as γc cytokine stimulation with successive divisions. γc cytokine-induced Tim-3 was found on naive, effector, and memory subsets of T cells. Tim-3(+) primary T cells were more prone to apoptosis, particularly upon treatment with galectin-9, a Tim-3 ligand, after cytokine withdrawal. The upregulation of Tim-3 could be blocked by the addition of a PI3K inhibitor, LY 294002. Thus, Tim-3 can be induced via TCR/CD28 costimulation and/or γc cytokines, likely through the PI3K pathway.     

Variable Rate Application of Nematicides on Cotton Fields: A Promising Site-Specific Management Strategy

Field tests were conducted to determine if differences in response to nematicide application (i.e., root-knot nematode (RKN) populations, cotton yield, and profitability) occurred among RKN management zones (MZ). The MZ were delineated using fuzzy clustering of five terrain (TR) and edaphic (ED) field features related to soil texture: apparent soil electrical conductivity shallow (EC_a-shallow) and deep (EC_a-deep), elevation (EL), slope (SL), and changes in bare soil reflectance. Zones with lowest mean values of EC_{a- shallow}, EC_{a- deep}, NDVI, and SL were designated as at greater risk for high RKN levels. Nematicide-treated plots (4 rows wide and 30 m long) were established in a randomized complete block design within each zone, but the number of replications in each zone varied from four to six depending on the size of the zone.The nematicides aldicarb (Temik 15 G) and 1,3-dichloropropene (1,3-D,Telone II) were applied at two rates (0.51 and 1.0 kg a.i./ha for aldicarb, and 33.1 and 66.2 kg a.i./ha for 1,3-D) to RKN MZ in commercial fields between 2007 and 2009. A consolidated analysis over the entire season showed that regardless of the zone, there were not differences between aldicarb rates and 1,3-D rates. The result across zones showed that 1,3-D provided better RKN control than did aldicarb in zones with low EC_a values (high RKN risk zones exhibiting more coarse-textured sandy soils). In contrast, in low risk zones with relatively higher EC_a values (heavier textured soil), the effects of 1,3-D and aldicarb were equal and application of any of the treatments provided sufficient control. In low RKN risk zones, a farmer would often have lost money if a high rate of 1,3-D was applied. This study showed that the effect of nematicide type and rate on RKN control and cotton yield varied across management zones (MZ) with the most expensive treatment likely to provide economic benefit only in zones with coarser soil texture. This study demonstrates the value of site specific application of nematicides based on management zones, although this approach might not be economically beneficial in fields with little variability in soil texture.     

A nomenclature for intestinal in vitro cultures

Many advances have been reported in the long-term culture of intestinal mucosal cells in recent years. A significant number of publications have described new culture media, cell formations, and growth patterns. Furthermore, it is now possible to study, e.g., the capabilities of isolated stem cells or the interactions between stem cells and mesenchyme. However, at the moment there is significant variation in the way these structures are described and named. A standardized nomenclature would benefit the ability to communicate and compare findings from different laboratories using the different culture systems. To address this issue, members of the NIH Intestinal Stem Cell Consortium herein propose a systematic nomenclature for in vitro cultures of the small and large intestine. We begin by describing the structures that are generated by preparative steps. We then define and describe structures produced in vitro, specifically: enterosphere, enteroid, reconstituted intestinal organoid, induced intestinal organoid, colonosphere, colonoid, and colonic organoid.