CD177+ cells produce neutrophil extracellular traps that promote biliary atresia.

BACKGROUND & AIMS: We have previously reported on the potential pathogenic role of neutrophils in biliary atresia (BA). Herein, we aimed to delineate the role of CD177+ neutrophils in the pathogenesis of BA. METHODS: Immune cells from the livers of mice with rhesus rotavirus-induced BA were analysed. Single-cell RNA-sequencing was performed to specifically analyse Gr-1+ (Ly6C/Ly6G+) cells in the liver. Gene expression profiles of CD177+ cells were analysed using the Smart-Seq RNA-sequencing method, and the pathogenesis of BA was examined in Cd177-/- mice. Neutrophil extracellular trap (NET) inhibitors were used to determine the role of CD177+ cell-derived NETs in BA-associated bile duct damage, and a pilot clinical study evaluated the potential effects of N-acetylcysteine on NET release in BA. RESULTS: Increased levels of Gr-1+ cells were observed in the livers of mice with rhesus rotavirus-induced BA. RNA-sequencing analysis revealed that CD177+ cells were the main population of Gr-1+ cells and expressed elevated levels of both interferon-stimulated and neutrophil degranulation genes. Cd177-/- BALB/c mice exhibited delayed disease onset and reduced morbidity and mortality. High numbers of mitochondria were detected in CD177+ cells derived from mice with BA; these cells were associated with increased levels of reactive oxygen species and increased NET formation, which induced the apoptosis of biliary epithelial cells in cocultures. In a pilot clinical study, the administration of N-acetylcysteine to patients with BA reduced CD177+ cell numbers and reactive oxygen species levels, indicating a potential beneficial effect. CONCLUSIONS: Our data indicate that CD177+ cells play an important role in the initiation of BA pathogenesis via NET formation. CLINICAL TRIAL REGISTRATION: The pilot study of N-acetylcysteine treatment in patients with BA was registered on the Chinese Clinical Trial Registry (ChiCTR2000040505). LAY SUMMARY: Neutrophils (a type of innate immune cell, i.e. an immune cell that doesn't target a specific antigen) are thought to play a role in the development of biliary atresia (a rare but potentially lethal condition of the bile ducts that occurs in infants). Herein, we found that neutrophils expressing a particular protein (CD177) played an important role in bile duct damage by releasing a special structure (NET) that can trap and kill pathogens but that can also cause severe tissue damage. A pilot study in patients with biliary atresia showed that inhibiting NETs could have a beneficial effect.

Down-regulation of STAT3 enhanced chemokine expression and neutrophil recruitment in biliary atresia.

Biliary atresia (BA) is an immune-related disorder and signal transducer and activator of transcription 3 (STAT3) is a key signalling molecule in inflammation. The present study was designed to clarify the function of STAT3 in BA. STAT3 expression was examined in patients and a mouse BA model in which STAT3 levels were further altered with a specific inhibitor or activator. Neutrophil accumulation and the levels of the neutrophil chemoattractants (C-X-C motif) ligand 1 (CXCL1) and IL-8 were determined. The effects of STAT3 inhibition on IL-8 expression were examined in human biliary epithelial cell (BEC) cultures. Functional changes in liver STAT3+ neutrophils in the mouse model were analysed with 10× single cell RNA-seq methods. Results showed STAT3 and p-STAT3 expression was reduced in BA liver tissue compared with control samples. Administration of a STAT3 inhibitor increased jaundice and mortality and reduced body weight in BA mice. In contrast, the STAT3 activator ameliorated BA symptoms. Extensive neutrophil accumulation together with CXCL1 up-regulation, both of which were suppressed by an anti-CXCL1 antibody, were observed in the STAT3 inhibitor-treated group. Recombinant IL-8 administration increased disease severity in BA mice, and the STAT3 activator had the reverse effect. Inhibiting STAT3 increased apoptosis of human BECs together with up-regulated IL-8 expression. RNA-seq analysis revealed reduced the numbers of STAT3 expressing neutrophil in BA which was accompanied by marked enhanced interferon-related antiviral activities. In conclusion, STAT3 reduction, enhanced IL-8 and CXCL1 expression and promoted the accumulation of interferon-responsive neutrophils resulting in BEC damage in BA.

De novo mutations in Caudal Type Homeo Box transcription Factor 2 (CDX2) in patients with persistent cloaca.

The cloaca is an embryonic cavity that is divided into the urogenital sinus and rectum upon differentiation of the cloacal epithelium triggered by tissue-specific transcription factors including CDX2. Defective differentiation leads to persistent cloaca in humans (PC), a phenotype recapitulated in Cdx2 mutant mice. PC is linked to hypo/hyper-vitaminosis A. Although no gene has ever been identified, there is a strong evidence for a genetic contribution to PC. We applied whole-exome sequencing and copy-number-variants analyses to 21 PC patients and their unaffected parents. The damaging p.Cys132* and p.Arg237His de novo CDX2 variants were identified in two patients. These variants altered the expression of CYP26A1, a direct CDX2 target encoding the major retinoic acid (RA)-degrading enzyme. Other RA genes, including the RA-receptor alpha, were also mutated. Genes governing the development of cloaca-derived structures were recurrently mutated and over-represented in the basement-membrane components set (q-value < 1.65 × 10-6). Joint analysis of the patients' profile highlighted the extracellular matrix-receptor interaction pathway (MsigDBID: M7098, FDR: q-value < 7.16 × 10-9). This is the first evidence that PC is genetic, with genes involved in the RA metabolism at the lead. Given the CDX2 de novo variants and the role of RA, our observations could potentiate preventive measures. For the first time, a gene recapitulating PC in mouse models is found mutated in humans.

The Role of Neonatal Gr-1+ Myeloid Cells in a Murine Model of Rhesus-Rotavirus-Induced Biliary Atresia.

Activation of innate immunity together with cholangiocyte damage occurs in biliary atresia (BA). However, detailed information on the inflammatory cells involved is lacking. This study investigates both the pathophysiology of CD11b+Gr-1+ cells in a mouse model of BA and their presence in BA patients. CD11b+Gr-1+ cells were targeted by an anti-Ly6G antibody in murine BA induced by inoculation with rhesus rotavirus. Expression of the Ly6G homolog CD177+ was examined in biopsies from BA patients. The symptoms of BA were ameliorated, and survival was prolonged in those mice receiving 5 to 10 µg of antibody per mouse every 3 days for four times compared with the mice treated with virus alone. However, the mice later developed chronic BA with persistent low body weight and jaundice. Hepatic inflammatory cells were reduced compared with acute BA. Blockade of extrahepatic bile ducts occurred, whereas intrahepatic ductules were partially preserved, and a progressive increase in liver fibrosis was observed. High levels of CD11b+Gr-1+ cells were present in these mice. The administration of an anti-Ly6G antibody again in those chronic BA mice reduced jaundice and restored body weight. In BA patients CD177+ cells were highly expressed in the liver. Our data suggest that the chronic mouse BA model shares key characteristics with clinical BA and indicates the importance of CD11b+Gr-1+ cells in the initiation and progression of BA.

Silver nanoparticle treatment ameliorates biliary atresia syndrome in rhesus rotavirus inoculated mice.

Biliary atresia (BA) is a neonatal biliary system disease closely associated with viral infection and bile duct inflammation. Silver nanoparticles (AgNps) have previously revealed antiviral and anti-inflammatory properties. In this study, we have investigated the effects of AgNps in the treatment of the Rhesus rotavirus inoculation induced BA in mice. The morphology, liver histopathology, clinical biochemistry examination, and inflammatory cells were analyzed in BA mice. Results indicated that AgNps could significantly increase the survival rate of BA mice, and reduce jaundice and weight lost and the liver enzymes and bilirubin metabolism clinical parameters were close to the normal levels. Diminished numbers of NK cells were observed by flow cytometry analysis and immunohistochemical staining. Furthermore, the viral load was reduced and transcripts for TGF-ß mRNA were augmented after AgNps treatment. Collectively, our results suggest that AgNps treatment has beneficial effects on the BA mouse model partially through upregulation of TGF-ß.

Roles of Hoxb5 in the development of vagal and trunk neural crest cells.

Neural crest cells (NC) are a group of multipotent stem cells uniquely present in vertebrates. They are destined to form various organs according to their anterior-posterior (A-P) levels of origin in the neural tube (NT). They develop into a wide spectrum of cell lineages under the influence of signaling cascades, neural plate border genes and NC specifier genes. Although this complex gene regulatory network (GRN) specifies the fate of NC and the combinatory action of Hox genes executed at the time of NC induction governs the patterning of NC for the formation of specific structures along the A-P axis, not much information on how GRN and Hox genes directly interact and orchestrate is available. This review summarizes recent findings on the multiple roles of Hoxb5 on the survival and cell lineage differentiation of vagal and trunk NC cells during early development, by direct transcriptional regulation of NC specifier genes (Sox9 and Foxd3) of the GRN. We will also review findings on the transcriptional regulation of Ret by Hoxb5 in the population of the vagal NC that are committed to the enteric neuron and glia lineages. Functional redundancy between Hox proteins (Hoxa5 and Hoxc5) from the same paralogue group as Hoxb5, and the cooperative effects of Hox cofactors, collaborators and transcription factors in the Hoxb5 transcriptional regulation of target genes will also be discussed.

Silver nanoparticles promote osteogenesis of mesenchymal stem cells and improve bone fracture healing in osteogenesis mechanism mouse model.

The potential use of osteo-conducive biomaterials in the promotion of bone fracture healing has attracted wide attention. This study investigated if silver nanoparticles (AgNps) could promote the proliferation and osteogenesis of mesenchymal stem cells (MSCs), and improve bone fracture healing. We showed that AgNps promoted MSCs' proliferation and osteogenic differentiation in vitro. Using a mouse femoral facture model, AgNps encapsulated in collagen promoted the formation of fracture callus, and induced early closure of the fracture gap. AgNps may promote the formation of the callus and the subsequent end joining of the fracture bone via multiple routes: (i) chemo-attraction of MSCs and fibroblasts to migrate to the fracture site; (ii) induction of the proliferation of MSCs; (iii) induction of osteogenic differentiation of MSCs via induction/activation of TGF-ß/BMP signaling in MSCs. We concluded that AgNps might be beneficial as an adjunct treatment for bone fracture healing clinically. FROM THE CLINICAL EDITOR: Silver nanoparticles are widely used in wound management in the clinical setting. In this article, the authors demonstrated a novel application in that these nanoparticles were efficient in promoting osteoblastic differentiation in both in-vitro and in-vivo studies. The findings may provide a new treatment direction for bone fracture in the future.

G1 checkpoint establishment in vivo during embryonic liver development.

BACKGROUND: The DNA damage-mediated cell cycle checkpoint is an essential mechanism in the DNA damage response (DDR). During embryonic development, the characteristics of cell cycle and DNA damage checkpoint evolve from an extremely short G1 cell phase and lacking G1 checkpoint to lengthening G1 phase and the establishment of the G1 checkpoint. However, the regulatory mechanisms governing these transitions are not well understood. In this study, pregnant mice were exposed to ionizing radiation (IR) to induce DNA damage at different embryonic stages; the kinetics and mechanisms of the establishment of DNA damage-mediated G1 checkpoint in embryonic liver were investigated. RESULTS: We found that the G2 cell cycle arrest was the first response to DNA damage in early developmental stages. Starting at E13.5/E15.5, IR mediated inhibition of the G1 to S phase transition became evident. Concomitantly, IR induced the robust expression of p21 and suppressed Cdk2/cyclin E activity, which might involve in the initiation of G1 checkpoint. The established G1 cell cycle checkpoint, in combination with an enhanced DNA repair capacity at E15.5, displayed biologically protective effects of repairing DNA double-strand breaks (DSBs) and reducing apoptosis in the short term as well as reducing chromosome deletion and breakage in the long term. CONCLUSION: Our study is the first to demonstrate the establishment of the DNA damage-mediated G1 cell cycle checkpoint in liver cells during embryogenesis and its in vivo biological effects during embryonic liver development.

Biliary atresia.

Biliary atresia (BA) is a progressive inflammatory fibrosclerosing disease of the biliary system and a major cause of neonatal cholestasis. It affects 1:5,000-20,000 live births, with the highest incidence in Asia. The pathogenesis is still unknown, but emerging research suggests a role for ciliary dysfunction, redox stress and hypoxia. The study of the underlying mechanisms can be conceptualized along the likely prenatal timing of an initial insult and the distinction between the injury and prenatal and postnatal responses to injury. Although still speculative, these emerging concepts, new diagnostic tools and early diagnosis might enable neoadjuvant therapy (possibly aimed at oxidative stress) before a Kasai portoenterostomy (KPE). This is particularly important, as timely KPE restores bile flow in only 50-75% of patients of whom many subsequently develop cholangitis, portal hypertension and progressive fibrosis; 60-75% of patients require liver transplantation by the age of 18 years. Early diagnosis, multidisciplinary management, centralization of surgery and optimized interventions for complications after KPE lead to better survival. Postoperative corticosteroid use has shown benefits, whereas the role of other adjuvant therapies remains to be evaluated. Continued research to better understand disease mechanisms is necessary to develop innovative treatments, including adjuvant therapies targeting the immune response, regenerative medicine approaches and new clinical tests to improve patient outcomes.

Functional analyses of RET mutations in Chinese Hirschsprung disease patients.

BACKGROUND: Hirschsprung disease (HSCR) is a congenital disease characterized by the absence of ganglion cells in various length of distal digestive tract. The rearranged during transfection gene (RET) is considered the major gene in HSCR. Although an increasing number of HSCR-associated RET coding sequence (CDS) mutations have been identified in recent years, not many have been investigated for functional consequence on the RET protein. METHODS AND RESULTS: We examined the functional implications of the de novo RET-CDS mutations V145G, Y483X, V636fsX1, and F961L that we first identified in sporadic Chinese patients with HSCR. The V145G disrupted RET glycosylation and F961L RET phosphorylation. Presumably, the truncation mutations would affect the translocation or the anchoring of the RET protein onto the cellular membrane. CONCLUSION: The study of RET-CDS mutations that appear de novo is essential not only for understanding the mechanistic of the disease but also for penetrance and recurrence risk estimations, being the ultimate goal for the improvement in disease management and counseling.

Biliary Atresia - emerging diagnostic and therapy opportunities.

Biliary Atresia is a devastating pediatric cholangiopathy affecting the bile ducts of the liver. In this review, we describe recent progress in the understanding of liver development with a focus on cholangiocyte differentiation and how use of technical platforms, including rodent, zebrafish and organoid models, advances our understanding of Biliary Atresia. This is followed by a description of potential pathomechanisms, such as autoimmune responses, inflammation, disturbed apical-basal cell polarity, primary cilia dysfunction as well as beta-amyloid accumulation. Finally, we describe current and emerging diagnostic opportunities and recent translation breakthroughs for Biliary Atresia in the area of emerging therapy development, including immunomodulation and organoid-based systems for liver and bile duct repair.

DNA damage mediated s and g(2) checkpoints in human embryonal carcinoma cells.

For mouse embryonic stem (ES) cells, the importance of the S and G(2) cell cycle checkpoints for genomic integrity is increased by the absence of the G(1) checkpoint. We have investigated ionizing radiation (IR)-mediated cell cycle checkpoints in undifferentiated and retinoic acid-differentiated human embryonal carcinoma (EC) cells. Like mouse ES cells, human EC cells did not undergo G(1) arrest after IR but displayed a prominent S-phase delay followed by a G(2)-phase delay. In contrast, although differentiated EC cells also failed to arrest at G(1)-phase after IR, they quickly exited S-phase and arrested in G(2)-phase. In differentiated EC cells, the G(2)-M-phase cyclin B1/CDC2 complex was upregulated after IR, but the G(1)-S-phase cyclin E and the cyclin E/CDK2 complex were expressed at constitutively low levels, which could be an important factor distinguishing DNA damage responses between undifferentiated and differentiated EC cells. S-phase arrest and expression of p21 could be inhibited by 7-hydroxystaurosporine, suggesting that the ataxia-telangiectasia and Rad-3-related-checkpoint kinase 1 (ATR-CHK1), and p21 pathways might play a role in the IR-mediated S-phase checkpoint in EC cells. IR-mediated phosphorylation of ataxia-telangiectasia mutated, (CHK1), and checkpoint kinase 2 were distinctly higher in undifferentiated EC cells compared with differentiated EC cells. Combined with the prominent S and G(2) checkpoints and a more efficient DNA damage repair system, these mechanisms operate together in the maintenance of genome stability for EC cells.

Delayed application of silver nanoparticles reveals the role of early inflammation in burn wound healing.

Burn injury is common, and antimicrobial agents are often applied immediately to prevent wound infection and excessive inflammatory response. Although inflammation is essential for clearing bacteria and creating an environment conducive to the healing process, it is unclear what time-frame inflammation should be present for optimal wound healing. This study critically investigated the role of early inflammation in burn wound healing, and also revealed the molecular mechanisms underlying the pro-healing effects of silver nanoparticles (AgNPs). We created a burn injury mouse model using wild-type and Smad3-/- mice, which were topically treated with AgNPs at different post-burn days, and examined the healing processes of the various groups. We also delineated the molecular pathways underlying the anti-inflammation and pro-healing effects of AgNPs by morphological and histological analysis, immuno-histochemistry, and western blotting. Our results showed that (1) AgNPs regulated pro-inflammatory cytokine IL-6 production of keratinocytes and neutrophils infiltration through KGF-2/p38 signaling pathway, (2) Topical AgNPs treatment immediately after burn injury significantly supressed early inflammation but resulted in delayed healing, (3) A short delay in AgNPs application (post-burn day 3 in our model) allowed early inflammation in a controlled manner, and led to optimal burn wound healing. Thus, our current study showed that some degree of early inflammation was beneficial, but prolonged inflammation was detrimental for burn wound healing. Further evaluation and clinical translation of this finding is warranted.

Prokineticin-1 (Prok-1) works coordinately with glial cell line-derived neurotrophic factor (GDNF) to mediate proliferation and differentiation of enteric neural crest cells.

Enteric neural crest cells (NCC) are multipotent progenitors which give rise to neurons and glia of the enteric nervous system (ENS) during fetal development. Glial cell line-derived neurotrophic factor (GDNF)/RET receptor tyrosine kinase (Ret) signaling is indispensable for their survival, migration and differentiation. Using microarray analysis and isolated NCCs, we found that 45 genes were differentially expressed after GDNF treatment (16 h), 29 of them were up-regulated including 8 previously undescribed genes. Prokineticin receptor 1 (PK-R1), a receptor for Prokineticins (Prok), was identified in our screen and shown to be consistently up-regulated by GDNF in enteric NCCs. Further, PK-R1 was persistently expressed at a lower level in the enteric ganglions of the c-Ret deficient mice when compared to that of the wild-type littermates. Subsequent functional analysis showed that GDNF potentiated the proliferative and differentiation effects of Prok-1 by up-regulating PK-R1 expression in enteric NCCs. In addition, expression analysis and gene knock-down experiments indicated that Prok-1 and GDNF signalings shared some common downstream targets. More importantly, Prok-1 could induce both proliferation and expression of differentiation markers of c-Ret deficient NCCs, suggesting that Prok-1 may also provide a complementary pathway to GDNF signaling. Taken together, these findings provide evidence that Prok-1 crosstalks with GDNF/Ret signaling and probably provides an additional layer of signaling refinement to maintain proliferation and differentiation of enteric NCCs.

Perturbation of hoxb5 signaling in vagal neural crests down-regulates ret leading to intestinal hypoganglionosis in mice.

BACKGROUND & AIMS: The enteric nervous system (ENS) controls intestinal peristalsis, and defective development of this system results in hypo/aganglionosis, as seen in Hirschsprung's disease. In the embryo, vagal neural crest cells (NCC) migrate and colonize the intestine rostrocaudally then differentiate into the ganglia of the ENS. Vagal NCC express the homeobox gene Hoxb5, a transcriptional activator, in human and mouse, so we used transgenic mice to investigate the function of Hoxb5 and the receptor tyrosine kinase gene Ret, which is affected in many patients with Hirschsprung's disease, in ENS development. METHODS: We perturbed the Hoxb5 pathway by expressing a chimeric protein enb5, in which the transcription activation domain of Hoxb5 was replaced with the repressor domain of the Drosophila engrailed protein (en), in vagal NCC. This enb5 transcriptional repressor competes with wild-type Hoxb5 for binding to target genes, exerting a dominant negative effect. RESULTS: We observed that 30.6% +/- 2.3% of NCC expressed enb5 and that these enb5-expressing NCC failed to migrate to the distal intestine. A 34%-37% reduction of ganglia (hypoganglionosis) and slow peristalsis and, occasionally, absence of ganglia and intestinal obstruction were observed in enb5-expressing mice. Ret expression was markedly reduced or absent in NCC and ganglia, and enb5 blocked Hoxb5 induction of Ret in neuroblastoma cells. CONCLUSIONS: Our data indicate that Ret is a downstream target of Hoxb5 whose perturbation causes Ret haploinsufficiency, impaired NCC migration, and hypo/aganglionosis, suggesting that Hoxb5 may contribute to the etiology of Hirschsprung's disease.

LPS-induced up-regulation of TGF-beta receptor 1 is associated with TNF-alpha expression in human monocyte-derived macrophages.

The immunosuppressive activity of TGF-beta-mediated signaling is well documented, but in contrast, its ability to promote proinflammatory responses is less clear. In this study, we report that blockade of TGF-beta signaling by a specific inhibitor of the TGF-beta receptor I [activin receptor-like kinase 5 (ALK5)] SB431542 significantly reduces the production of TNF-alpha, a key proinflammatory cytokine, by LPS-stimulated human monocyte-derived macrophages. ALK5 protein was only detectable after LPS stimulation, and the failure of treatment with SB431542 to alter TNF-alpha mRNA expression indicates that regulation is post-transcriptional. The additive effect of blocking TGF-beta and p38 MAPK signaling on reducing TNF-alpha but not IL-6 production suggests that there is selectivity in pathway signaling. SB431542 had similar inhibitory effects on TNF-alpha production by human monocytes and endothelial cells as well as macrophages. Furthermore, treatment with SB431542 reduced plasma TNF-alpha levels and tissue damage and thereby, prevented the lethal effects of LPS in a mouse model of septic shock. Our data demonstrate a direct effect of TGF-beta signaling via ALK5 on the regulation of TNF-alpha synthesis.

Prokineticin-1 modulates proliferation and differentiation of enteric neural crest cells.

Prokineticins (Prok-1 and Prok-2) belong to a newly identified AVIT protein family. They are involved in variety of activities in various tissues, including smooth muscle contraction of the gastrointestinal tract and promoting proliferation of endothelial cells derived from adrenal gland. Importantly, they also act as the survival factors to modulate growth and survival of neurons and hematopoietic stem cells. In this study we demonstrated that Prok-1 (but not Prok-2) protein is expressed in the mucosa and mesenchyme of the mouse embryonic gut during enteric nervous system development. Its receptor, PK-R1 is expressed in the enteric neural crest cells (NCCs). To elucidate the physiological role(s) of Prok-1 in NCCs, we isolated the NCCs from the mouse embryonic gut (E11.5) and cultured them in the form of neurospheres. In an in vitro NCC culture, Prok-1 was able to activate both Akt and MAPK pathways and induce the proliferation and differentiation (but not migration) of NCCs via PK-R1. Knock-down of PK-R1 using siRNA resulted in a complete abolishment of Prok-1 induced proliferation. Taken together, it is the first report demonstrating that Prok-1 acts as a gut mucosa/mesenchyme-derived factor and maintains proliferation and differentiation of enteric NCCs.

Implications of endocrine gland-derived vascular endothelial growth factor/prokineticin-1 signaling in human neuroblastoma progression.

PURPOSE: Neuroblastoma is a common pediatric tumor that is derived from improperly differentiated neural crest cells (NCC). We recently revealed that endocrine gland-derived vascular endothelial growth factor/prokineticin-1 (EG-VEGF/Prok-1) is a key factor mediating the growth and differentiation of enteric NCCs during development. In this report, we further elucidate its role in neuroblastoma progression. EXPERIMENTAL DESIGN: We studied the expression and copy number of EG-VEGF/Prok-1 receptors (PK-R1 and PK-R2) in 26 neuroblastoma tumors by real-time reverse transcription-PCR and immunohistochemical analysis. Implication of EG-VEGF/Prok-1 signaling in neuroblastoma progression was further shown in a neuroblastoma cell line (SK-N-SH). RESULTS: We found that all neuroblastoma samples from stages II to IV expressed both PK-R1 and PK-R2. Kruskall-Wallis signed rank tests revealed that the expression level of PK-R1 transcript is associated with the stages and metastasis of the neuroblastoma (P<0.05), and PK-R2 is persistently higher in advanced-stage neuroblastoma samples. About 38% of the neuroblastoma tumors (10:26) possessed MYCN amplification, whereas no PK-R1 and PK-R2 amplifications were detected, suggesting that the overexpression of the receptors was not due to gene amplification. Subsequent functional studies showed that EG-VEGF/Prok-1 activates the Akt pathway to induce the proliferation of neuroblastoma cells. Targeted down-regulation studies revealed that EG-VEGF/Prok-1-mediated proliferation requires the presence of these two receptors, and that PK-R2 is essential for inhibiting apoptosis. In vitro migration and invasion assays also indicated that EG-VEGF/Prok-1 significantly enhances the cell migration/invasion of SK-N-SH. CONCLUSIONS: Our study has shown for the first time that aberrant EG-VEGF/Prok-1 signaling favors neuroblastoma progression and could be a potential target for future neuroblastoma treatment.

Inhibition of sonic hedgehog signaling reduces chronic rejection and prolongs allograft survival in a rat orthotopic small bowel transplantation model.

BACKGROUND: Although acute graft rejection can be successfully controlled by immunosuppressive agents, chronic rejection (CR), which is characterized by arteriosclerosis in the donor organ vessels, is a major hurdle to long-term allograft survival. Sonic hedgehog (Shh), a morphogen critical in embryogenesis, also promotes peripheral immunity, which prompted us to investigate if inhibition of Shh signaling could reduce CR and thereby enhance allograft survival. METHODS: In a rat orthotopic small bowel transplantation model, FK506 prevented acute rejection; however, recipients eventually lost their grafts by CR. Anti-Shh antibody or isotype control were administered to animals at day 30 postoperatively. Graft survival, tissue fibrosis, vascular occlusion, and expression of vascular endothelial growth factor (VEGF) were investigated. RESULTS: Immunostaining revealed that Shh and the Hedgehog receptor Patched 1 (Ptc1) are strongly expressed in CR grafts and that Ptc1 expression partially overlapped with that of ED-1, a macrophage marker. In contrast, only minimal expression of Shh and Ptc1 was detected in syngeneic grafts. Grafts survival was significantly prolonged after anti-Shh antibody treatment compared with the immunoglobulin G control (116 vs. 77.5 days). Collagen deposition and vascular occlusion in the mesentery were markedly reduced in recipients of the anti-Shh antibody. Specific transcripts and protein expression for VEGF, which was present mainly in the blood vessels, were reduced. CONCLUSION: In a rat small bowel transplantation model, anti-Shh antibody treatment reduced CR and prolonged graft survival. These beneficial effects of Shh treatment may occur partly by reducing VEGF expression in the blood vessels of the allografts.

Whole exome sequencing coupled with unbiased functional analysis reveals new Hirschsprung disease genes.

BACKGROUND: Hirschsprung disease (HSCR), which is congenital obstruction of the bowel, results from a failure of enteric nervous system (ENS) progenitors to migrate, proliferate, differentiate, or survive within the distal intestine. Previous studies that have searched for genes underlying HSCR have focused on ENS-related pathways and genes not fitting the current knowledge have thus often been ignored. We identify and validate novel HSCR genes using whole exome sequencing (WES), burden tests, in silico prediction, unbiased in vivo analyses of the mutated genes in zebrafish, and expression analyses in zebrafish, mouse, and human. RESULTS: We performed de novo mutation (DNM) screening on 24 HSCR trios. We identify 28 DNMs in 21 different genes. Eight of the DNMs we identified occur in RET, the main HSCR gene, and the remaining 20 DNMs reside in genes not reported in the ENS. Knockdown of all 12 genes with missense or loss-of-function DNMs showed that the orthologs of four genes (DENND3, NCLN, NUP98, and TBATA) are indispensable for ENS development in zebrafish, and these results were confirmed by CRISPR knockout. These genes are also expressed in human and mouse gut and/or ENS progenitors. Importantly, the encoded proteins are linked to neuronal processes shared by the central nervous system and the ENS. CONCLUSIONS: Our data open new fields of investigation into HSCR pathology and provide novel insights into the development of the ENS. Moreover, the study demonstrates that functional analyses of genes carrying DNMs are warranted to delineate the full genetic architecture of rare complex diseases.