Systemic anticoagulation in ECMO.

While unfractionated heparin (UFH) remains the mainstay of anticoagulation during pediatric extracorporeal life support, direct thrombin inhibitors (DTIs) are increasingly used. In this article, we will review most recent evidence regarding utilization of both UFH and DTIs and compare their known advantages and disadvantages. We will present anticoagulation monitoring strategies during ECMO and outline the most recent Extracorporeal Life Support Organization's anticoagulation guidelines, however with the caveat that there are no true consensus recommendations for anticoagulation management in pediatric ECMO. With these updates, we will serve as the bedside clinician's refresher on common practices for anticoagulation during "routine" ECMO. We will additionally highlight special circumstances, including high risk surgical procedures during ECMO, in which adjustments in anticoagulation and/or addition of antifibrinolytic therapy might mitigate risk.

Muscarinic receptor agonist-induced ßPix binding to ß-catenin promotes colon neoplasia.

M3 muscarinic receptors (M3R) modulate ß-catenin signaling and colon neoplasia. CDC42/RAC guanine nucleotide exchange factor, ßPix, binds to ß-catenin in colon cancer cells, augmenting ß-catenin transcriptional activity. Using in silico, in vitro, and in vivo approaches, we explored whether these actions are regulated by M3R. At the invasive fronts of murine and human colon cancers, we detected co-localized nuclear expression of ßPix and ß-catenin in stem cells overexpressing M3R. Using immunohistochemistry, immunoprecipitation, proximity ligand, and fluorescent cell sorting assays in human tissues and established and primary human colon cancer cell cultures, we detected time-dependent M3R agonist-induced cytoplasmic and nuclear association of ßPix with ß-catenin. ßPix knockdown attenuated M3R agonist-induced human colon cancer cell proliferation, migration, invasion, and expression of PTGS2, the gene encoding cyclooxygenase-2, a key player in colon neoplasia. Overexpressing ßPix dose-dependently augmented ß-catenin binding to the transcription factor TCF4. In a murine model of sporadic colon cancer, advanced neoplasia was attenuated in conditional knockout mice with intestinal epithelial cell deficiency of ßPix. Expression levels of ß-catenin target genes and proteins relevant to colon neoplasia, including c-Myc and Ptgs2, were reduced in colon tumors from ßPix-deficient conditional knockout mice. Targeting the M3R/ßPix/ß-catenin axis may have therapeutic potential.

Muscarinic receptor activation in colon cancer selectively augments pro-proliferative microRNA-21, microRNA-221 and microRNA-222 expression.

Overexpression of M3 subtype muscarinic receptors (M3R) hastens colon cancer progression. As microRNA (miRNA) expression is commonly dysregulated in cancer, we used microarrays to examine miRNA profiles in muscarinic receptor agonist-treated human colon cancer cells. We used quantitative RT-PCR (qPCR) to validate microarray results and examine miRNA expression in colon cancers and adjacent normal colon. These assays revealed that acetylcholine (ACh) treatment robustly induced miR-222 expression; miR-222 levels were three-fold higher in cancer compared to normal colon. In kinetic studies, ACh induced a 4.6-fold increase in pri-miR-222 levels within 1 h, while mature miR-222 increased gradually to 1.8-fold within 4 h. To identify post-M3R signaling mediating these actions, we used chemical inhibitors and agonists. ACh-induced increases in pri-miR-222 were attenuated by pre-incubating cells with atropine and inhibitors of protein kinase C (PKC) and p38 MAPK. Treatment with a PKC agonist, phorbol 12-myristate 13-acetate, increased pri-miR-222 levels, an effect blocked by PKC and p38 MAPK inhibitors, but not by atropine. Notably, treatment with ACh or transfection with miR-222 mimics increased cell proliferation; atropine blocked the effects of ACh but not miR-222. These findings identify a novel mechanism whereby post-M3R PKC/p38 MAPK signaling stimulates miR-222 expression and colon cancer cell proliferation.

Zinc finger protein 277 is an intestinal transit-amplifying cell marker and colon cancer oncogene.

Sustained proliferative signaling and resisting cell death are hallmarks of cancer. Zinc finger protein 277 (ZNF277; murine Zfp277), a transcription factor regulating cellular senescence, is overexpressed in colon cancer, but its actions in intestinal homeostasis and neoplasia are unclear. Using human and murine intestine, human colon cancer cells, and ApcMin/+ mice with dysregulated ß-catenin signaling and exuberant intestinal neoplasia, we explored the actions of ZNF277/Zfp277 and defined the underlying mechanisms. In normal human and murine intestine, ZNF277/Zfp277 was expressed uniquely in early stem cell progenitors, undifferentiated transit-amplifying cells (TACs). Zfp277 was overexpressed in the ApcMin/+ mouse colon, implicating ZNF277/Zfp277 as a transcriptional target of ß-catenin signaling. We confirmed this by showing ß-catenin knockdown reduced ZNF277 expression and, using chromatin IP, identified 2 ß-catenin binding sites in the ZNF277 promoter. Zfp277 deficiency attenuated intestinal epithelial cell proliferation and tumor formation, and it strikingly prolonged ApcMin/+ mouse survival. RNA-Seq and PCR analyses revealed that Zfp277 modulates expression of genes in key cancer pathways, including ß-catenin signaling, the HOXD family that regulates development, and p21WAF1, a cell cycle inhibitor and tumor suppressor. In both human colon cancer cells and the murine colon, ZNF277/Zfp277 deficiency induced p21WAF1 expression and promoted senescence. Our findings identify ZNF277/Zfp277 as both a TAC marker and colon cancer oncogene that regulates cellular proliferation and senescence, in part by repressing p21WAF1 expression.

Targeted intestinal deletion of Rho guanine nucleotide exchange factor 7, ßPIX, impairs enterocyte proliferation, villus maturation, and mucosal defenses in mice.

Rho guanine nucleotide exchange factors (RhoGEFs) regulate Rho GTPase activity and cytoskeletal and cell adhesion dynamics. ßPix, a CDC42/RAC family RhoGEF encoded by ARHGEF7, is reported to modulate human colon cancer cell proliferation and postwounding restitution of rat intestinal epithelial monolayers. We hypothesized that ßPix plays a role in maintaining intestinal epithelial homeostasis. To test this hypothesis, we examined ßPix distribution in the human and murine intestine and created mice with intestinal epithelial-selective ßPix deletion [ßPixflox/flox/Tg(villin-Cre); Arhgef7 CKO mice]. Using Arhgef7 conditional knockout (CKO) and control mice, we investigated the consequences of ßPix deficiency in vivo on intestinal epithelial and enteroid development, dextran sodium sulfate-induced mucosal injury, and gut permeability. In normal human and murine intestines, we observed diffuse cytoplasmic and moderate nuclear ßPix immunostaining in enterocytes. Arhgef7 CKO mice were viable and fertile, with normal gross intestinal architecture but reduced small intestinal villus height, villus-to-crypt ratio, and goblet cells; small intestinal crypt cells had reduced Ki67 staining, compatible with impaired cell proliferation. Enteroids derived from control mouse small intestine were viable for more than 20 passages, but those from Arhgef7 CKO mice did not survive beyond 24 h despite addition of Wnt proteins or conditioned media from normal enteroids. Adding a Rho kinase (ROCK) inhibitor partially rescued CKO enteroid development. Compared with littermate control mice, dextran sodium sulfate-treated ßPix-deficient mice lost more weight and had greater impairment of intestinal barrier function, and more severe colonic mucosal injury. These findings reveal ßPix expression is important for enterocyte development, intestinal homeostasis, and resistance to toxic injury.NEW & NOTEWORTHY To explore the role of ßPix, a guanine nucleotide exchange factor encoded by ARHGEF7, in intestinal development and physiology, we created mice with intestinal epithelial cell Arhgef7/ßPix deficiency. We found ßPix essential for normal small intestinal epithelial cell proliferation, villus development, and mucosal resistance to injury. Moreover, Rho kinase signaling mediated developmental arrest observed in enteroids derived from ßPix-deficient small intestinal crypts. Our studies provide insights into the role Arhgef7/ßPix plays in intestinal epithelial homeostasis.

The Role of M3 Muscarinic Receptor Ligand-Induced Kinase Signaling in Colon Cancer Progression.

Despite a reduction in incidence over the past decade, colon cancer remains the second most common cause of cancer death in the United States; recent demographics suggest this disease is now afflicting younger persons. M3 muscarinic receptor (M3R) mRNA and protein are over-expressed in colon cancer, and M3R can be activated by both traditional (e.g., acetylcholine) and non-traditional (e.g., bile acids) muscarinic ligands. In this review, we weigh the data supporting a prominent role for key protein kinases downstream of M3R activation in promoting colon cancer progression and dissemination. Specifically, we explore the roles that downstream activation of the mitogen activated protein kinase/extracellular signal-related kinase (MAPK/ERK), protein kinase C, p38 MAPK, and phosphatidylinositol 3-kinase/Akt (PI3K/Akt) pathways play in mediating colon cancer cell proliferation, survival, migration and invasion. We assess the impact of M3R-stimulated induction of selected matrix metalloproteinases germane to these hallmarks of colon cancer progression. In this context, we also critically review the reproducibility of findings derived from a variety of in vivo and in vitro colon cancer models, and their fidelity to human disease. Finally, we summarize the therapeutic potential of targeting various steps from ligand-M3R interaction to the activation of key downstream molecules.

Two sides to colon cancer: mice mimic human anatomical region disparity in colon cancer development and progression.

AIM: Strong evidence reveals important differences between cancers in the proximal vs. distal colon. Animal models of metastatic colon cancer are available but with varying degrees of reproducibility and several important limitations. We explored whether there were regional differences in the location of murine colon cancers and assessed the utility of murine models to explore the biological basis for such differences. METHODS: We re-analyzed data from our previous studies to assess the regional distribution of murine colon cancer. In survival surgery experiments, we injected HT-29 human colon cancer cells into the wall of the cecum or distal colon of Nu(NCr)-Foxn1nu or NOD.Cg-PrkdcscidIl2rgTim1Wji/SzJ mice and compared the development of primary tumors and metastases. RESULTS: Within 7-17 weeks after intramural cecal injection of HT-29 cells, eight mice failed to develop solid primary tumors or metastases. In contrast, within four weeks after cell injection into the distal colon, 13 mice developed metastases - 12 mice developed subcutaneous metastases; of these, four developed liver metastases and one developed both liver and lung metastases. One mouse developed liver metastases only. Histological examination confirmed these lesions were adenocarcinomas. CONCLUSION: Our findings reveal the preferential growth of murine colon neoplasia and invasive human orthotopic xenografts in the distal mouse colon. The new approach of injecting cells into the distal colon wall results in a pattern of colon cancer development that closely mimics the progression of metastatic colon cancer in humans. This novel model of colon neoplasia has great potential for exploring anatomical differences in colon cancer and testing novel therapeutics.

Anomalous mesenteric vessel-a rare etiology of intermittent partial small bowel obstruction.

We report a case of an 11-year with recurrent episodes of abdominal pain and vomiting. Diagnostic laparoscopy during a severe episode demonstrated an anomalous blood vessel incompletely encircling the bowel, leading to transient partial small bowel obstruction. This likely represents a remnant vessel from the omphalomesenteric duct. Recurrent abdominal pain and vomiting in the pediatric population requires careful history and physical exam, and a willingness to entertain unusual diagnoses.

miRNA-17 members that target Bmpr2 influence signaling mechanisms important for embryonic stem cell differentiation in vitro and gastrulation in embryos.

Body axes and germ layers evolve at gastrulation, and in mammals are driven by many genes; however, what orchestrates the genetic pathways during gastrulation remains elusive. Previously, we presented evidence that microRNA-17 (miRNA-17) family members, miR-17-5p, miR-20a, miR-93, and miR-106a were differentially expressed in mouse embryos and functioned to control differentiation of the stem cell population. Here, we identify function(s) that these miRNAs have during gastrulation. Fluorescent in situ hybridization miRNA probes reveal that these miRNAs are localized at the mid/posterior primitive streak (ps) in distinct populations of primitive ectoderm, mesendoderm, and mesoderm. Seven different miRNA prediction algorithms are identified in silico bone morphogenic protein receptor 2 (Bmpr2) as a target of these miRNAs. Bmpr2 is a member of the TGFß pathway and invokes stage-specific changes during gastrulation. Recently, Bmpr2 was shown regulating cytoskeletal dynamics, cell movement, and invasion. Our previous and current data led to a hypothesis by which members of the miR-17 family influence gastrulation by suppressing Bmpr2 expression at the primitive streak. This suppression influences fate decisions of cells by affecting genes downstream of BMPR2 as well as mesoderm invasion through regulation of actin dynamics.

Absence of STAT1 in donor-derived plasmacytoid dendritic cells results in increased STAT3 and attenuates murine GVHD.

Selective targeting of non-T cells, including antigen-presenting cells (APCs), is a potential strategy to prevent graft-versus-host-disease (GVHD) but to maintain graft-versus-tumor (GVT) effects. Because type I and II interferons signal through signal transducer and activator of transcription-1 (STAT1), and contribute to activation of APCs after allogeneic bone marrow transplant (alloBMT), we examined whether the absence of STAT1 in donor APCs could prevent GVHD while preserving immune competence. Transplantation of STAT1(-/-) bone marrow (BM) prevented GVHD induced by STAT1(+/+) T cells, leading to expansion of B220(+) cells and regulatory T cells. STAT1(-/-) BM also preserved GVT activity and enhanced overall survival of tumor-challenged mice in the setting of GVHD. Furthermore, recipients of allogeneic STAT1(-/-) BM demonstrated increased CD9(-)Siglec H(hi) plasmacytoid dendritic cells (pDCs), and depletion of pDCs after STAT1(-/-) BM transplantation prevented GVHD resistance. STAT1(-/-) pDCs were found to produce decreased free radicals, IFNα, and interleukin (IL)-12, and increased IL-10. Additionally, STAT1(-/-) pDCs that were isolated after alloBMT showed increased gene expression of S100A8 and S100A9, and transplantation of S100A9(-/-) BM reduced GVHD-free survival. Finally, elevated STAT3 was found in STAT1(-/-) pDCs isolated after alloBMT. We conclude that interfering with interferon signaling in APCs such as pDCs provides a novel approach to regulate the GVHD/GVT axis.

A pan-inhibitor of DASH family enzymes induces immune-mediated regression of murine sarcoma and is a potent adjuvant to dendritic cell vaccination and adoptive T-cell therapy.

Multimodality therapy consisting of surgery, chemotherapy, and radiation will fail in approximately 40% of patients with pediatric sarcomas and result in substantial long-term morbidity in those who are cured. Immunotherapeutic regimens for the treatment of solid tumors typically generate antigen-specific responses too weak to overcome considerable tumor burden and tumor suppressive mechanisms and are in need of adjuvant assistance. Previous work suggests that inhibitors of DASH (dipeptidyl peptidase IV activity and/or structural homologs) enzymes can mediate tumor regression by immune-mediated mechanisms. Herein, we demonstrate that the DASH inhibitor, ARI-4175, can induce regression and eradication of well-established solid tumors, both as a single agent and as an adjuvant to a dendritic cell (DC) vaccine and adoptive cell therapy (ACT) in mice implanted with the M3-9-M rhabdomyosarcoma cell line. Treatment with effective doses of ARI-4175 correlated with recruitment of myeloid (CD11b) cells, particularly myeloid DCs, to secondary lymphoid tissues and with reduced frequency of intratumoral monocytic (CD11bLy6-CLy6-G) myeloid-derived suppressor cells. In immunocompetent mice, combining ARI-4175 with a DC vaccine or ACT with tumor-primed T cells produced significant improvements in tumor responses against well-established M3-9-M tumors. In M3-9-M-bearing immunodeficient (Rag1) mice, ACT combined with ARI-4175 produced greater tumor responses and significantly improved survival compared with either treatment alone. These studies warrant the clinical investigation of ARI-4175 for treatment of sarcomas and other malignancies, particularly as an adjuvant to tumor vaccines and ACT.

Val-boroPro accelerates T cell priming via modulation of dendritic cell trafficking resulting in complete regression of established murine tumors.

Although tumors naturally prime adaptive immune responses, tolerance may limit the capacity to control progression and can compromise effectiveness of immune-based therapies for cancer. Post-proline cleaving enzymes (PPCE) modulate protein function through N-terminal dipeptide cleavage and inhibition of these enzymes has been shown to have anti-tumor activity. We investigated the mechanism by which Val-boroPro, a boronic dipeptide that inhibits post-proline cleaving enzymes, mediates tumor regression and tested whether this agent could serve as a novel immune adjuvant to dendritic cell vaccines in two different murine syngeneic murine tumors. In mice challenged with MB49, which expresses the HY antigen complex, T cell responses primed by the tumor with and without Val-boroPro were measured using interferon gamma ELISPOT. Antibody depletion and gene-deficient mice were used to establish the immune cell subsets required for tumor regression. We demonstrate that Val-boroPro mediates tumor eradication by accelerating the expansion of tumor-specific T cells. Interestingly, T cells primed by tumor during Val-boroPro treatment demonstrate increased capacity to reject tumors following adoptive transfer without further treatment of the recipient. Val-boroPro -mediated tumor regression requires dendritic cells and is associated with enhanced trafficking of dendritic cells to tumor draining lymph nodes. Finally, dendritic cell vaccination combined with Val-boroPro treatment results in complete regression of established tumors. Our findings demonstrate that Val-boroPro has antitumor activity and a novel mechanism of action that involves more robust DC trafficking with earlier priming of T cells. Finally, we show that Val-boroPro has potent adjuvant properties resulting in an effective therapeutic vaccine.

Novel 5'TOPmRNAs regulated by ribosomal S6 kinase are important for cardiomyocyte development: S6 kinase suppression limits cardiac differentiation and promotes pluripotent cells toward a neural lineage.

Moving stem cells from bench to bedside has been a challenging task. Undermining this task is comprehending and optimizing the underlying regulatory mechanisms that drive differentiation of stem cells into desired cell and tissue types. Here we present evidence that ribosomal S6 kinase (S6K) is among the proteins upregulated as embryonic stem cells (ESCs) and human induced pluripotent stem cells differentiate into beating cardiomyocytes. We hypothesized that S6K plays a pivotal role in cardiomyogenesis, primarily because it regulates the translation of 3 cardiac-involved genes recently shown to have 5' terminal oligopyrimidine (5'TOP) sequences: connexin 43 (Cx43), desmoplakin (Dsp), and phosphatase and tensin homolog (PTEN). Along with another independent laboratory, we confirmed that S6K is indeed upregulated in beating ESC-derived cardiomyocytes compared to the surrounding nonbeating, differentiated cells. S6K short interfering RNA-transfected stem cell cultures indicate that inhibition of S6K strongly hinders development of cardiomyocyte beating and translation of Cx43, Dsp, and PTEN; these cardiac 5'TOP mRNAs were only properly translated in cells with S6K, supporting our hypothesis. An unexpected discovery took the role of S6K one step further: S6K-knockdown stem cell cultures developed significantly more neurons than seen in embryoid bodies subjected to a typical cardiac differentiation protocol. These results introduced the novel idea that in addition to its critical cardiac roles, S6K may be a significant factor that prevents stem cells from pursuing a neuronal pathway. Overall, results have indicated the necessity of S6K for normal stem cell cardiomyogenesis, as well as lowered S6K expression for stem cell neurogenesis.

Extracorporeal photopheresis attenuates murine graft-versus-host disease via bone marrow-derived interleukin-10 and preserves responses to dendritic cell vaccination.

Extracorporeal photopheresis (ECP) is emerging as a therapy for graft-versus-host-disease (GVHD), but the full mechanism of action and the impact on immunity have not been fully established. After murine minor histocompatibility antigen-mismatched bone marrow (BM) transplantation (allo-BMT), coinfusion of ECP-treated splenocytes with T cell-replete BM attenuated GVHD irrespective of the donor strain of the ECP-treated splenocytes, and was associated with increased numbers of regulatory T cells. Coculture of myeloid dendritic cells (DCs) with ECP-treated splenocytes resulted in increased interleukin (IL)-10 production after submaximal stimulation with lipopolysaccharide. Furthermore, male myeloid DCs exposed to ECP-treated splenocytes were less potent at inducing CD8(+) HY responses when used as a vaccine in vivo. The efficacy of ECP-treated splenocytes was enhanced when administered just before delayed donor lymphocyte infusion following T cell-depleted allo-BMT, allowing for the administration of sufficient numbers of T cells to respond to myeloid DC vaccination in the absence of a thymus. Finally, the therapeutic effect of ECP-treated splenocytes was lost in recipients of IL-10-deficient BM. We demonstrate that ECP-treated splenocytes attenuate GVHD irrespective of the source of ECP-treated cells via a mechanism that likely involves modulation of DCs and requires IL-10 produced by BM-derived cells. Importantly, the attenuation of GVHD by ECP-treated splenocytes permits donor lymphocyte infusion-dependent responses to DC vaccines after allo-BMT.