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The spread of coronavirus disease 2019 (COVID-19) has had a significant impact on business and economies globally. This paper aims to investigate the impact of COVID-19 on firm performance in China...

Lung fibrosis is increasingly detected with aging and has been associated with poor outcomes in acute lung injury or infection. However, the molecular programs driving this pro-fibrotic evolution are unclear. Here we profile distal lung samples from healthy human donors across the lifespan. Gene expression profiling by bulk RNAseq reveals both increasing cellular senescence and pro-fibrotic pathway activation with age. Quantitation of telomere length shows progressive shortening with age, which is associated with DNA damage foci and cellular senescence. Cell type deconvolution analysis of the RNAseq data indicates a progressive loss of lung epithelial cells and an increasing proportion of fibroblasts with age. Consistent with this pro-fibrotic profile, second harmonic imaging of aged lungs demonstrates increased density of interstitial collagen as well as decreased alveolar expansion and surfactant secretion. In this work, we reveal the transcriptional and structural features of fibrosis and associated functional impairment in normal lung aging., Age is associated with increasing vulnerability to both acute and chronic lung diseases. Employing genomic analysis and live lung imaging, this study reveals a profile of increased cellular senescence, telomere shortening, and fibrosis-induced impaired alveolar function in the natural history of human lung aging.

The objective of this paper is to examine the relationship between intellectual capital (IC) and firms’ financial leverage by exploring whether firm profitability mediates this relationship, using a dataset of Chinese agricultural listed companies during the period of 2014–2020. Financial leverage is measured by the debt-to-asset ratio, and IC is measured via the modified value-added intellectual coefficient (MVAIC) model. The results reveal that financial leverage is lower in firms with higher levels of IC, and IC positively affects firm profitability. In addition, firm profitability partially mediates the relationship between IC and financial leverage. When MVAIC is disaggregated into its four components, firm profitability has a partially mediating effect on the relationship between physical and human capitals and financial leverage. This paper might provide corporate managers with a clear understanding of IC’s impact on firm indebtedness.

Acid aspiration, which can result from several etiologies, including postoperative complications, leads to direct contact of concentrated hydrochloric acid (HCl) with the alveolar epithelium. As a result, rapid endothelial activation induces alveolar inflammation, leading to life-threatening pulmonary edema. Because mechanisms underlying the rapid endothelial activation are not understood, here we determined responses in real time through optical imaging of alveoli of live mouse lungs. By alveolar micropuncture, we microinfused concentrated HCl in the alveolar lumen. As expected, acid contact with the epithelium caused rapid, but transient, apical injury. However, there was no concomitant membrane injury to the endothelium. Nevertheless, H(2)O(2)-mediated epithelial-endothelial paracrine signaling induced endothelial barrier failure, as detected by microvascular dextran leakage and lung water quantification. Remarkably, endothelial mitochondria regulated the barrier failure by activating uncoupling protein 2 (UCP2), thereby inducing transient mitochondrial depolarization that led to cofilin-induced actin depolymerization. Knockdown, or endothelium-targeted deletion of UCP2 expression, blocked these responses, including pulmonary edema. To our knowledge, these findings are the first to mechanistically implicate endothelial mitochondria in acid-induced barrier deterioration and pulmonary edema. We suggest endothelial UCP2 may be a therapeutic target for acid-induced acute lung injury.

Gastrin was initially identified as the hormone primarily responsible for gastric acid secretion, but was subsequently shown to be a growth factor for the proximal stomach, acting through the gastrin receptor CCK2R. Studies in the past several decades have explored the role of gastrin, along with its incompletely processed precursors, in cancer development. The growth in long-term PPI use has frequently led to elevations in serum gastrin levels in patients with upper GI disease, including GERD, peptic ulcers, and chronic gastritis. However, while accumulated evidence has shown that gastrin likely does not promote-and may even suppress-distal antral gastric cancer, questions have now arisen regarding possible effects of gastrin on the development of gastric cardia cancer or esophageal adenocarcinoma at gastroesophageal junction. Here, we provide an overview of the possible roles of these gastrin peptides in upper GI cancer.

// Yoomi Lee 1, * , Aleksandra M. Urbanska 2, * , Yoku Hayakawa 2 , Hongshan Wang 2, 3 , Andrew S. Au 2 , Aesis M. Luna 4 , Wenju Chang 2, 3 , Guangchun Jin 2 , Govind Bhagat 4 , Julian A. Abrams 2 , Richard A. Friedman 5, 6 , Andrea Varro 7 , Kenneth K. Wang 8 , Malcolm Boyce 9 , Anil K. Rustgi 10 , Antonia R. Sepulveda 4 , Michael Quante 11 , Timothy C. Wang 2 1 Division of Hematology and Oncology, Department of Medicine, Columbia University Medical Center, New York, NY, USA 2 Division of Digestive and Liver Diseases, Department of Medicine, Columbia University Medical Center, New York, NY, USA 3 Department of General Surgery, Zhongshan Hospital, Fudan University, Shanghai, China 4 Department of Pathology and Cell Biology, Columbia University Medical Center, New York, NY, USA 5 Biomedical Informatics Shared Resource, Herbert Irving Comprehensive Cancer Center, New York, NY, USA 6 Department of Biomedical Informatics, Columbia University Medical Center, New York, NY, USA 7 Department of Physiology, Institute of Translational Medicine, University of Liverpool, Liverpool, England 8 Division of Gastroenterology and Hepatology, Mayo Clinic, Rochester, MN, USA 9 Hammersmith Medicines Research, Central Middlesex Hospital, London, UK 10 Division of Gastroenterology, Departments of Medicine and Genetics, Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA 11 Medical Clinic II, Clinic of the Right Bank, Technical University of Munich, Munich, Germany * These authors contributed equally to this work Correspondence to: Timothy C. Wang, email: tcw21@columbia.edu Keywords: Barrett’s esophagus, esophageal cancer, gastrin, gastrin receptors, stem cells Received: March 15, 2016 Accepted: June 13, 2016 Published: July 18, 2016 ABSTRACT Objective: The incidence of esophageal adenocarcinoma (EAC) is increasing, but factors contributing to malignant progression of its precursor lesion, Barrett’s esophagus (BE), have not been defined. Hypergastrinemia caused by long-term use of proton pump inhibitors (PPIs), has been suggested as one possible risk factor. The gastrin receptor, CCK2R, is expressed in the cardia and upregulated in BE, suggesting the involvement of the gastrin-CCK2R pathway in progression. In the L2-IL-1β mouse model, Barrett’s-like esophagus arises from the gastric cardia. Therefore, we aimed to analyze the effect of hypergastrinemia on CCK2R+ progenitor cells in L2-IL-1β mice. Design: L2-IL-1β mice were mated with hypergastrinemic ( INS-GAS ) mice or treated with PPIs to examine the effect of hypergastrinemia in BE progression. CCK2R-CreERT crossed with L2-IL-1β mice were used to analyze the lineage progenitor potential of CCK2R+ cells. Cardia glands were cultured in vitro , and the effect of gastrin treatment analyzed. L2-IL-1β mice were treated with a CCK2R antagonist YF476 as a potential chemopreventive drug. Results: Hypergastrinemia resulted in increased proliferation and expansion of Barrett’s-like esophagus. Lineage tracing experiments revealed that CCK2R+ cells are long-lived progenitors that can give rise to such lesions under chronic inflammation. Gastrin stimulated organoid growth in cardia culture, while CCK2R inhibition prevented Barrett’s-like esophagus and dysplasia. Conclusions: Our data suggest a progression model for BE to EAC in which CCK2R+ progenitor cells, stimulated by hypergastrinemia, proliferate to give rise to metaplasia and dysplasia. Hypergastrinemia can result from PPI use, and the effects of hypergastrinemia in human BE should be studied further.

The stability parameters of implants (ITV, ISQ & PTV) according to different sizes of controlled bone defects made in implant osteotomies were analyzed and the correlation among the three kinds of implant stability parameters was tested in this study. 45 tapped screw-type dental implants were inserted in three types of implant osteotomies made in 8 fresh-frozen pig femoral bones: Type1 — without coronal bone defect, Type2 — with 3 mm coronal bone defects, and Type3 — with 6 mm coronal bone defects. The insertion torque values, ISQ & PTV of implants were measured and analyzed statistically. It is concluded that the circumferential coronal bone defects statistically influence the primary stability of implants; ITV, ISQ and PTV are suitable and available to detect the peri-implant coronal bone defects in 3 mm increments, and ITV and PTV are more sensitive to coronal cortical bone loss. There was a strong correlation between ITV and ISQ.

The stem cells that maintain and repair the postnatal skeleton remain undefined. One model suggests that perisinusoidal mesenchymal stem cells (MSCs) give rise to osteoblasts, chondrocytes, marrow stromal cells, and adipocytes, although the existence of these cells has not been proven through fate-mapping experiments. We demonstrate here that expression of the bone morphogenetic protein (BMP) antagonist gremlin 1 defines a population of osteochondroreticular (OCR) stem cells in the bone marrow. OCR stem cells self-renew and generate osteoblasts, chondrocytes, and reticular marrow stromal cells, but not adipocytes. OCR stem cells are concentrated within the metaphysis of long bones not in the perisinusoidal space and are needed for bone development, bone remodeling, and fracture repair. Grem1 expression also identifies intestinal reticular stem cells (iRSCs) that are cells of origin for the periepithelial intestinal mesenchymal sheath. Grem1 expression identifies distinct connective tissue stem cells in both the bone (OCR stem cells) and the intestine (iRSCs)., National Institutes of Health (U.S.) (Grant R01 RHL115145A)

On the basis of core descriptions, examinations of rock thin sections, and EMI logging, a fluvial-dominated fan delta depositional system consisting of the fan delta plain and front subfacies from the lower Urho Formation of the Upper Permian in the Karamay oilfield of Xinjiang, NW China, was recognized in this research. The typical characteristics of this kind of fan delta is the depositional processes that were dominated by the fluvial and tractive current structure that developed very well, while pro-fan delta and gravity deposition occurred very less. The key microfacies associations in the fan delta plain subfacies are braided channel, sheeted flow, mud flow, and sieve deposits, while the fan delta front subfacies commonly contains subaqueous channels, interdistributary channels, debris flow, grain flow, and subaqueous levee microfacies. A study of image logging, grain size, and compositions of rocks indicate that provenance directions are different from the early to the last depositional periods of the Urho Formation, i.e. it changed from the southwest initially to the northwest in the latest among the fifth period to the first period. In addition, with the corresponding provenance direction changing, the sedimentary facies also transform regularly. This paper illuminated the sedimentary facies, subfacies and microfacies and also discussed deeply the depositional distribution and evolution of the lower Urho Formation. A sedimentary model of the fluvial-dominated fan delta was presented in this paper.

Mussel adhesive proteins are non-toxic, biodegradable and have low immunogenic qualities that make them highly attractive for medical purposes compared with man-made materials. 3,4-DOPA, which is a dopamine precursor, is a critical element for achieving mussel-like adhesive properties. In present study, polydopamine was coated onto the titanium surfaces by dipping in a 2 mg/mL dopamine solution in 10 mM Tris-HCl buffer at pH 8.5. EDX element analysis and Raman spectroscopic analysis of the surface were conducted to verify the formation of polydopamine. The surface characteristics of polydopamine-coated titanium plates were observed by SEM and AFM. Hydrophilicity and corrosion-resistance were also evaluated by static contact angle measurements and potentiodynamic polarization corrosion tests. The absorption intensity for pH-induced polymerization rapidly increased for the initial four hours and thereafter smoothly increased. EDX element analyses revealed that the content of carbon and oxygen increased and the content of titanium decreased after the coating process. In the Raman spectra, polydopamine resulted in two new peaks at approximately 1 370 and 1 570 cm−1 resulting from the stretching and deformation of catechol. Microstructural features revealed that nanometer-sized bright granules were randomly distributed after coating, and these nanoparticles grew with increased coating time. The Ra values of polydopamine-coated titanium plates were showed to increase with coating time. Compared to the pure titanium curve, the inflection point of polydopamine-coated titanium was located at a higher corrosion potential and lower corrosion current density. Findings from our research suggested that polydopamine coatings offer a versatile approach for titanium surface modification.

Bone marrow-derived epithelial cells (BMDECs) are recruited to sites of chronic inflammation; however, their clinical significance has not been determined. Here, we used gender-mismatched allogeneic bone marrow transplantation (BMT) in the context of the classical multistage model for murine cutaneous carcinogenesis to probe the recruitment of BMDECs in skin tumors initiated with the carcinogen, dimethylbenz[a]anthracene (DMBA), and promoted with the phorbol ester, 12-O-tetradecanolyphorbol-13-acetate (TPA). We detected clusters of bone marrow-derived cells (BMDCs) in over 40% of papillomas, where they occupied 25% or more of the epithelial lesional area. In the dysplastic ulcers which arose in some irradiated DMBA/TPA treated mice, the magnitude of the recruitment was greater. The BMDCs clustered in the cutaneous epithelium where they became immunoreactive to epidermal keratins, and proliferated and stratified, thereby contributing to the lesions comparably with the progeny of hair follicle stem cells in engrafted Krt1-15Cre;R26R mice. Moreover, cytokeratins were detected by immunostaining and Q-PCR in plastic adherent bone marrow cells (BMCs) cultured in the presence of filter-separated epidermal keratinocytes (KCs). Cytokeratin production was enhanced by bone morphogenetic protein 5 (BMP5). Moreover, BMCs migrated towards the alarmin protein, High Mobility Group Box 1 (HMGB1), as well as towards epidermal KCs in ex vivo invasion assays. Furthermore, when naïve female mice received BMTs from donors previously treated with DMBA, several papillomas and a squamous cell carcinoma were observed after TPA promotion alone. We conclude that surprisingly large numbers of BMDECs are recruited to a subset of cutaneous papillomas and dysplastic ulcers and reflect a previously unrecognized systemic contribution to these lesions. We also conclude that carcinogen-exposed cells originating in the BMCs are sufficient to initiate a subset of benign and malignant lesions upon tumor promotion. Ultimately, these findings may provide novel targets for treatment of non-melanoma skin cancers as well as other cancers. Citation Format: Heuijoon Park, Sonali Lad, Kelsey Boland, Kelly Johnson, Nyssa Readio, Guangchun Jin, Samuel A sfaha, Kelly S. Patterson, Ashok Singh, Xiangdong Yang, Douglas Londono, Anupama Singh, Carol Trempus, Derek Gordon, Timothy C. Wang, Rebecca J. Morris. Chronic inflammation-mediated contribution of bone marrow-derived epithelial cells and hair follicle stem cells to development of cutaneous neoplasms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-039.

// Guangchun Jin 1, 2 , Kosuke Sakitani 1 , Hongshan Wang 1, 3 , Ying Jin 1 , Alexander Dubeykovskiy 1 , Daniel L. Worthley 4 , Yagnesh Tailor 1 and Timothy C. Wang 1 1 Division of Digestive and Liver Diseases, Department of Medicine, Columbia University Medical Center, New York, NY, USA 2 The Research Institute, Yanbian University Hospital, Jilin, China 3 Department of General surgery, Zhongshan Hospital, Fudan University, Shanghai, China 4 South Australian Health and Medical Research Institute, University of Adelaide, Adelaide, South Australia, Australia Correspondence to: Timothy C. Wang, email: tcw21@columbia.edu Keywords: GPR56, progastrin, proliferation, stem cell, colorectal cancer Abbreviations: Human progastrin (hGAS), G-protein coupled receptor 56 (GPR56), 5-bromo-2’-deoxyuridine (BrdU), Bone morphogenetic protein (BMP). Received: November 02, 2016 Accepted: February 22, 2017 Published: March 23, 2017 ABSTRACT Overexpression of human progastrin increases colonic mucosal proliferation and colorectal cancer progression in mice. The G-protein coupled receptor 56 (GPR56) is known to regulate cell adhesion, migration, proliferation and stem cell biology, but its expression in the gut has not been studied. We hypothesized that the promotion of colorectal cancer by progastrin may be mediated in part through GPR56. Here, we found that GPR56 expresses in rare colonic crypt cells that lineage trace colonic glands consistent with GPR56 marking long-lived colonic stem-progenitor cells. GPR56 was upregulated in transgenic mice overexpressing human progastrin. While recombinant human progastrin promoted the growth and survival of wild-type colonic organoids in vitro , colonic organoids cultured from GPR56 −/− mice were resistant to progastrin. We found that progastrin directly bound to, and increased the proliferation of, GPR56-expressing colon cancer cells in vitro , and proliferation was increased in cells that expressed both GPR56 and the cholecystokinin-2 receptor (CCK2R). In vivo , deletion of GPR56 in the mouse germline abrogated progastrin-dependent colonic mucosal proliferation and increased apoptosis. Loss of GPR56 also inhibited progastrin-dependent colonic crypt fission and colorectal carcinogenesis in the azoxymethane (AOM) mouse model of colorectal cancer. Overall, we found that progastrin binds to GPR56 expressing colonic stem cells, which in turn promotes their expansion, and that this GPR56-dependent pathway is an important driver and potential new target in colorectal carcinogenesis.

Histidine decarboxylase (HDC), the unique enzyme responsible for histamine generation, is highly expressed in myeloid cells, but its function in these cells is poorly understood. Here we show that Hdc-knockout mice show a high rate of colon and skin carcinogenesis. Using Hdc-EGFP bacterial artificial chromosome (BAC) transgenic mice in which EGFP expression is controlled by the Hdc promoter, we show that Hdc is expressed primarily in CD11b +Ly6G+ immature myeloid cells (IMCs) that are recruited early on in chemical carcinogenesis. Transplant of Hdc-deficient bone marrow to wild-type recipients results in increased CD11b + Ly6G + cell mobilization and reproduces the cancer susceptibility phenotype of Hdc-knockout mice. In addition, Hdc-deficient IMCs promote the growth of tumor allografts, whereas mouse CT26 colon cancer cells downregulate Hdc expression through promoter hypermethylation and inhibit myeloid cell maturation. Exogenous histamine induces the differentiation of IMCs and suppresses their ability to support the growth of tumor allografts. These data indicate key roles for Hdc and histamine in myeloid cell differentiation and CD11b+Ly6G+IMCs in early cancer development. © 2011 Nature America, Inc. All rights reserved.

Bone marrow mesenchymal stem cells (MSCs) have been shown to have immune modulatory effects. Despite efforts to identify these cells in vivo, to date, MSCs have been defined mainly by their in vitro cell characteristics. Here, we show that Lin−CD44hiSca1−cKit+CD34− cells make up ∼0.5%–1% of murine whole bone marrow cells and yield nearly an equal amount of fibroblastic colony-forming units (CFU-F) as whole bone marrow. After transplantation into lethally irradiated recipients, Lin−CD44hiSca1−cKit+CD34− cells engrafted in the bone marrow long-term and demonstrated characteristics of MSCs, including capacity to differentiate into osteoblasts and adipocytes. To examine whether Lin−CD44hiSca1−cKit+CD34− cells have immune modulatory effects, in vitro coculture with activated CD4+ T-cells resulted in decreased Th17 cell differentiation by Lin−CD44hiSca1−cKit+CD34− cells. Furthermore, serial infusions with Lin−CD44hiSca1−cKit+CD34− cells reduced the progression to low-grade gastric dysplasia in mice infected with chronic Helicobacter felis (p = .038). This correlated with reduced gastric interleukin (IL)-17F, IL-22, and ROR-γt gene expression in responding mice (p < .05). These data suggest that bone marrow derived Lin−CD44hiSca1−cKit+CD34− cells have characteristics of MSCs and reduce progression of early gastric tumorigenesis induced by chronic H. felis infection. The prevention of dysplastic changes may occur through inhibition of Th17-dependent pathways.Disclosure of potential conflicts of interest is found at the end of this article.

Molar tooth (briefly called MT) carbonate is one of the Proterozoic carbonates with enigmatic sedimentary structure. According to the morphology of MT, it can be divided into two main genesis-morphology types, i.e., autochthonous and allochthonous, and each type can be further divided into a series of subtypes. The autochthonous MT can be divided into filamentous (MF1), ribbon, nodular and dotted type (MF4), in which the ribbon one can be subdivided into simple vertical (MF2) and complex ribbon (MF3), broken ribbon by storm in situ (MF5) and MT within nodular limestone (MF6). Allochthonous MT includes conglomerate clasts and horizontal detritus. Studying on the links between MT and the host rocks on five stratigraphic sections in the southeastern Jilin and eastern Liaoning indicates that the morphology of MT is closely related to host rocks, and seven genetic types by relationships between MT and the host rocks with facies interpretations are classified and discussed in this paper. The sedimentary environments of MT formation will be diverse if their morphologies are different.

Objective Progastrin is the incompletely cleaved precursor of gastrin that is secreted by G-cells in the gastric antrum. Both gastrin and progastrin bind to the CCK2 receptor ( Cckbr or CCK2R) expressed on a subset of gastric epithelial cells. Little is known about how gastrin peptides and CCK2R regulate gastric stem cells and carcinogenesis. Interconversion among progenitors in the intestine is documented, but the mechanisms by which this occurs are poorly defined. Design We generated CCK2R-CreERT mice and performed inducible lineage tracing experiments. CCK2R+ antral cells and Lgr5+ antral stem cells were cultured in a three-dimensional in vitro system. We crossed progastrin-overexpressing mice with Lgr5-GFP-CreERT mice and examined the role of progastrin and CCK2R in Lgr5+ stem cells during MNU-induced carcinogenesis. Results Through lineage tracing experiments, we found that CCK2R defines antral stem cells at position +4, which overlapped with an Lgr5 neg or low cell population but was distinct from typical antral Lgr5 high stem cells. Treatment with progastrin interconverts Lgr5 neg or low CCK2R+ cells into Lgr5 high cells, increases CCK2R+ cell numbers and promotes gland fission and carcinogenesis in response to the chemical carcinogen MNU. Pharmacological inhibition or genetic ablation of CCK2R attenuated progastrin-dependent stem cell expansion and carcinogenesis. Conclusions CCK2R labels +4 antral stem cells that can be activated and expanded by progastrin, thus identifying one hormonal trigger for gastric stem cell interconversion and a potential target for gastric cancer chemoprevention and therapy.

We used allogeneic bone marrow transplantation (BMT) and a mouse multistage cutaneous carcinogenesis model to probe recruitment of bone marrow-derived epithelial cells (BMDECs) in skin tumors initiated with the carcinogen, dimethylbenz[a]anthracene (DMBA), and promoted with 12-O-tetradecanolyphorbol-13-acetate (TPA). BMDECs clustered in the lesional epithelium, expressed cytokeratins, proliferated, and stratified. We detected cytokeratin induction in plastic-adherent bone marrow cells (BMCs) cultured in the presence of filter-separated keratinocytes (KCs) and bone morphogenetic protein 5 (BMP5). Lineage-depleted BMCs migrated towards High Mobility Group Box 1 (HMGB1) protein and epidermal KCs in ex vivo invasion assays. Naive female mice receiving BMTs from DMBA-treated donors developed benign and malignant lesions after TPA promotion alone. We conclude that BMDECs contribute to the development of papillomas and dysplasia, demonstrating a systemic contribution to these lesions. Furthermore, carcinogen-exposed BMCs can initiate benign and malignant lesions upon tumor promotion. Ultimately, these findings may suggest targets for treatment of non-melanoma skin cancers., Bone marrow-derived epithelial cells can be recruited to sites of chronic inflammation. Here, the authors using allogenic bone marrow transplantation in a multistage murine cutaneous carcinogenesis model show that bone marrow-derived epithelial cells and hair follicle stem cells are recruited to cutaneous neoplasms during tumor promotion of carcinogen-exposed skin and bone marrow.

Transgenic mice overexpressing human progastrin (hGAS) show colonic crypt hyper-proliferation and elevated susceptibility to colon carcinogenesis. We aimed to investigate effects of p53 mutation on colon carcinogenesis in hGAS mice. We show that introducing a p53 gene mutation further increases progastrin dependent BrdU labeling and results in markedly elevated number of aberrant crypt foci (ACF) and colonic tumors. We demonstrate that hGAS/Lgr5-GFP mice have higher number of Lgr5+ colonic stem cells per crypt when compared to Lgr5-GFP mice indicating that progastrin changes crypt biology through increased stem cell numbers and additional p53 mutation leads to more aggressive phenotype in this murine colon cancer model.

Gastrin is secreted from a subset of neuroendocrine cells residing in the gastric antrum known as G cells, but low levels are also expressed in fetal pancreas and intestine and in many solid malignancies. Although past studies have suggested that antral gastrin is transcriptionally regulated by inflammation, gastric pH, somatostatin, and neoplastic transformation, the transcriptional regulation of gastrin has not previously been demonstrated in vivo. Here, we describe the creation of an enhanced green fluorescent protein reporter (mGAS-EGFP) mouse using a bacterial artificial chromosome that contains the entire mouse gastrin gene. Three founder lines expressed GFP signals in the gastric antrum and the transitional zone to the corpus. In addition, GFP(+) cells could be detected in the fetal pancreatic islets and small intestinal villi, but not in these organs of the adult mice. The administration of acid-suppressive reagents such as proton pump inhibitor omeprazole and gastrin/CCK-2 receptor antagonist YF476 significantly increased GFP signal intensity and GFP(+) cell numbers in the antrum, whereas these parameters were decreased by overnight fasting, octreotide (longlasting somatostatin ortholog) infusion, and Helicobacter felis infection. GFP(+) cells were also detected in the anterior lobe of the pituitary gland and importantly in the colonic tumor cells induced by administration with azoxymethane and dextran sulfate sodium salt. This transgenic mouse provides a useful tool to study the regulation of mouse gastrin gene in vivo, thus contributing to our understanding of the mechanisms involved in transcriptional control of the gastrin gene. Copyright © 2011 the American Physiological Society.

Hyperproliferation of the colonic epithelium, leading to expansion of colonic crypt progenitors, is a recognized risk factor for colorectal cancer. Overexpression of progastrin, a nonamidated and incompletely processed product of the gastrin gene, has been shown to induce colonic hyperproliferation and promote colorectal cancer in mice, but the mechanism of pathogenesis has not been defined. Cholecystokinin-2 receptor (CCK2R) is the primary receptor for cholecystokinin (CCK) and amidated gastrin. Here, we show that Cck2r was expressed in murine colonic crypts and upregulated in the transgenic mice that overexpress human progastrin. Murine deletion of Cck2r abrogated progastrin-dependent increases in colonic proliferation, mucosal thickness, and beta-catenin and CD44 expression in the colon tumor. In addition, either deletion or antagonism of Cck2r resulted in the inhibition of progastrin-dependent increases in progenitors expressing doublecortin and CaM kinase-like-1 (DCAMKL1), stem cells expressing leucine rich repeat-containing G protein-coupled receptor 5 (LgR5), and colonic crypt fission. Furthermore, in the azoxymethane mouse model of colorectal carcinogenesis, Cck2r deletion in human progastrin-overexpressing mice resulted in markedly decreased aberrant crypt foci formation and substantially reduced tumor size and multiplicity. Taken together, these observations indicate that progastrin induces proliferative effects, primarily in colonic progenitor cells, through a CCK2R-dependent pathway. Moreover, our data suggest that CCK2R may be a potential target in the treatment or prevention of colorectal cancer.

von Willebrand disease (VWD), the most common inherited bleeding disorder in the U.S. population, is caused by defects in the expression and processing of von Willebrand factor (VWF), a blood glycoprotein required for normal hemostasis that mediates the adhesion of platelets to sites of vascular damage by binding to specific platelet glycoproteins and to constituents of exposed connective tissue. To assess whether VWF deficiency can be corrected by gene transfer, a plasmid expressing the intact 8.4-kb murine VWF coding sequence, directed by the cyto-megalovirus immediate/early promoter/enhancer, was delivered through hydrodynamic tail vein injection into VWF knockout mice (VWF–/–) that exhibit defects in hemostasis, including highly prolonged bleeding time and spontaneous bleeding events, closely mimicking severe human VWD. VWF antigen levels in plasma from animals receiving VWF cDNA, but not control animals, revealed normalized levels of circulating VWF that persisted for at least 1 week after injection. Western blot analysis of plasma from animals receiving VWF cDNA, but not control animals, revealed high molecular–weight multimers with patterns similar to those observed in wild-type mice. Reverse transcription–polymerase chain reaction (RT-PCR) on RNA isolated from the livers of animals receiving VWF cDNA, but not control animals, demonstrated that VWF was expressed in the liver, and immunohistochemical analysis of the livers of treated VWF–/– mice revealed VWF-specific staining throughout the liver parenchyma but not in endothelial cells. Plasma from treated VWF–/– mice, but not control VWF–/– mice, supported the hypothesis that murine platelets aggregate in the presence of botrocetin. Although levels of circulating factor VIII in untreated VWF–/– mice were less than 10% those in wild-type mice, levels of factor VIII in VWF–/– animals treated with VWF cDNA, but not in control animals, were normalized to values in wild-type mice, indicating the restoration of factor VIII carrier function for VWF in treated mice that persisted for at least 1 week at higher doses of VWF cDNA. Most important, bleeding time was normalized by 48 hours after the delivery of VWF cDNA, but not by the control plasmid. These data suggest that with the use of gene transfer of VWF cDNA, VWF protein can be expressed, processed, and secreted in a physiologically active form; thus, it may be possible to correct VWD using gene transfer.

Background & Aims Progastrin stimulates colonic mucosal proliferation and carcinogenesis through the cholecystokinin 2 receptor (CCK2R) - partly by increasing the number of colonic progenitor cells. However, little is known about the mechanisms by which progastrin stimulates colonic cell proliferation. We investigated the role of bone morphogenetic proteins (BMPs) in progastrin induction of colonic cell proliferation via CCK2R. Methods We performed microarray analysis to compare changes in gene expression in the colonic mucosa of mice that express a human progastrin transgene, gastrin knockout mice, and C57BL/6 mice (controls); the effects of progastrin were also determined on in vitro colonic crypt cultures from cholecystokinin 2 receptor knockout and wild-type mice. Human colorectal and gastric cancer cells that expressed CCK2R were incubated with progastrin or Bmp2; levels of β-arrestin 1 and 2 were knocked down using small interfering RNAs. Cells were analyzed for progastrin binding, proliferation, changes in gene expression, and symmetric cell division. Results The BMP pathway was down-regulated in the colons of human progastrin mice compared with controls. Progastrin suppressed transcription of Bmp2 through a pathway that required CCK2R and was mediated by β-arrestin 1 and 2. In mouse colonic epithelial cells, down-regulation of Bmp2 led to decreased phosphorylation of Smads1/5/8 and suppression of inhibitor of DNA binding 4. In human gastric and colorectal cancer cell lines, CCK2R was necessary and sufficient for progastrin binding and induction of proliferation; these effects were blocked when cells were incubated with recombinant Bmp2. Incubation with progastrin increased the number of CD44+, bromodeoxyuridine+, and NUMB+ cells, indicating an increase in symmetric divisions of putative cancer stem cells. Conclusions Progastrin stimulates proliferation in colons of mice and cultured human cells via CCK2R- and β-arrestin 1 and 2-dependent suppression of Bmp2 signaling. This process promotes symmetric cell division. © 2013 by the AGA Institute.

overexpressed Plexin B1 compared to normal pancreas tissues. Endogenous Plexin B1 and Met protein were detected in eight pancreatic cancer cell lines, with a variety of expression levels. Sema 4D stimulation activated Met protein, and significantly induced cell migration. Discussion: Here we show that Sema 4D involves in the regulation of cell motility through its receptor Plexin B1. And the results of immunostaining suggest that Sema 4D could play a role in the interaction between tumor cells and tumor microenvironment in pancreatic cancer tissues. As many attempts are now ongoing to target the tumor stroma, these findings provide a new insight into molecular targeted therapies for pancreatic cancer.

Background/aim: The anterior and posterior vagal trunks supply the each side of the gastric mucosa separately, and the denervation of either side does not impair the functional capacity of the other, leaving gastric acid output and circulating gastrin levels unchanged. In this study, we investigated the importance of the neural innervation and the brain-gut axis in cancer initiation by performing vagotomy in hypergastrinemic transgenic (INS-GAS) mice with a phenotype of spontaneous gastric cancer. Methods: Age/sex matched wild-type (WT) and INS-GAS mice were killed at 1, 2, 4, 6, 8, 10 and 12 months of age (at least 10 mice/ group) for histological examination of gastric cancer. INS-GAS mice at 6 months of age were randomized into 4 groups of surgery: unilateral anterior truncal vagotomy (29 mice), bilateral truncal vagotomy with pyloroplasty (24), pyloroplasty (27), and sham operation (25). Both the anterior and posterior sides of the stomachs were removed at 6 months postoperatively (i.e., 12 months of age) for pathological and immunohistochemical analyses. Results: Gastric cancer was found neither in WT mice regardless of age nor in INS-GAS mice that were

Background: The tumor microenvironment plays a pivotal role in carcinogenesis. Myofibroblasts, a major component of tumor stroma, have been demonstrated to be derived in a large part from bone marrow. However, the specific contributions of bone marrow-derived myofibroblasts to cancer growth and progression are not completely understood. In this study, we established bone marrow-derived (BMD) inflammation-related gastric myofibroblasts (MF) and investigated the role of BMD-MF in tumor growth. Methods: Gastric myofibroblast were isolated from EGFP bone marrow-transplanted IL-1β transgenic mice and wild type mice. Gene expression was assessed by real-time RT-PCR, immunocytochemistry staining and Western blot. The levels of cytokines and chemokines were measured by ELISA. Cell migration and invasion were determined by transwell migration assay and 3-D organic co-culture system, respectively. Tumor growth in vivo was determined in a SCID xenograft model. Results: We isolated and cultured successfully gastric myofibroblasts from BMT-IL-1β mice and WT mice. The pure BMD-MF (EGFP+) expressed myofibroblast markers (a-SMA and vimentin), but did not expressed markers of epithelial cells (E-cadherin). The mRNA expression of IL-1β, TNF-α, IL-6, SDF-1, MMP-1, TGF-β and COX-2 was significantly higher in the BMD-IL-1β-MF than WT-MF. Furthermore, BMD-IL-1β-MF secreted much higher levels IL-6 and SDF1 peptides in the culture medium than did WF-MF. The BMD-IL-1β MF significantly increased gastric cancer cell migration and invasion compared to WT-MF, and CXCR4 inhibitor (AMD3100) reduced cell migration induced by both myofibroblasts, indicating that the SDF1/CXCR4 axis is responsible for cell migration and invasion. Furthermore, in xenograft studies SCID mice co-injected gastric cancer cells (105 MKN45) and BMD-IL-1β MF formed larger size tumor compared those co-injected with WT-MF and those injected alone with gastric cancer cells. Mice injected with MKN45 alone on the left side and BMD-IL-1β-MF alone on the right side, surprisingly formed larger size tumors on the left side compared to those non-injected with BMD-IL-1β-MF on the right side. Furthermore, the EGFP+ MF could be detected in the xenograft tumor on the left side, suggesting that MF can migrate to the contralateral tumor and promote tumor growth. Moreover, treatment of CXCR4 inhibition (AMD3100) reduced tumor growth and the migration of MF to the contralateral tumor, indicating that SDF1/CXCR4 axis plays an important in the migration of myofibroblast and tumor growth. Conclusion: Our results for the first time show that tumor-associated myofibroblasts in the IL-1β gastric cancer model are bone marrow derived and have a stronger capacity to promote growth and invasion. The project was support by NIH grant 5U54CA126513, R01CA093405 and R01CA120979 (Wang TC) and NIH grant R01A120915 (Yang CS). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1412.

Background & Aims: We have previously reported that overexpression of amidated gastrin in transgenic (INS-GAS) mice with Helicobacter felis (H. felis) infection accelerated gastric corpus carcinogenesis, while overexpression of non-amidated glycine-extended gastrin in transgenic (MTI-G-gly) mice inhibited parietal cell loss and atrophy in stomach. We have also reported that overexpression of the full length progastrin in transgenic (hGAS) mice stimulated colon carcinogenesis in azoxymethane-treated mice. In this study, we investigated the role of progastrin for Helicobacter-associated gastric carcinogenesis in mice. Methods: hGAS, INS-GAS and non-transgenic wild type (B6 wt) mice on a C57BL/6 background were infected with H. felis for 12 or 18 months (m). We also analyzed male hGAS, INS-GAS and wild type (FVB wt) mice on a FVB/N background with H. felis infection for 7 m. Infection status was assessed by realtime PCR, microscopic evaluation and serum titer of H. felis-specific IgG antibodies. Proliferating cells and parietal cells were investigated by immunohistochemistry (IHC) with Ki-67 and HK-ATP-beta antibodies, respectively. Expression of stem cell-related genes such as CD44 & DCAMKL-1 was also analyzed by IHC. Results: At 12 m post infection (p.i.), INS-GAS mice had mild corpus dysplasia and B6 wt mice had severe gastritis or metaplasia, while hGAS mice had only mild to moderate gastritis, respectively. At 18 m p.i., both INS-GAS and B6 wt mice had severe atrophic gastritis and corpus dysplasia, while hGAS mice showed only moderate gastritis with mild gastric atrophy but no corpus dysplasia. In contrast, hGAS mice had moderate to severe antral or pyloric dysplasia, while INS-GAS and B6 wt mice did not. H. felis colonization remained stable over time among 3 groups of mice. The serum titer of H. felis-specific IgG antibody as well as Th1-Th2 polarization calculated by IgG1/IgG2c subclass ratio showed no significant differences among 3 groups. At 18 m p.i., the number of Ki-67(+) cells per gastric corpus gland was the highest in INS-GAS and the lowest in hGAS mice. Parietal cell loss was remarkable in INS-GAS and B6 wt, but minimal in hGAS mice. CD44(+) and DCAMKL-1(+) cell numbers were significantly increased in INS-GAS and B6 wt, while essentially unchanged in hGAS mice. On a FVB/N background, INS-GAS mice with H. felis infection for 7 m showed gastric corpus adenocarcinoma, and infected FVB wt mice showed severe gastric atrophy with intestinal metaplasia, while infected hGAS mice showed only moderate gastritis without metaplasia or dysplasia. Conclusions: These results point to a protective effect of progastrin on Helicobacter-associated gastric corpus carcinogenesis perhaps by inhibiting the loss of parietal cells. In contrast antral pathology is increased in hGAS mice. We conclude that the different forms of gastrin have contrasting effects on Helicobacter-associated gastric corpus carcinogenesis in mice. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4159.

G A A b st ra ct s by ELISA. The colon cancer cell line MC38 was used for wound healing and cell proliferation assays using MSC conditioned (50% MSC / 50% with fresh) or control (50% MC38/ 50% fresh) media, examined at intervals in triplicate. For In Vivo studies, 8 week old APC min mice were injected intravenously with 5 million RFP-MSC. At four weeks, small intestine and colons were removed, adenomas enumerated, examined by direct fluorescent microscopy and standard H&E. RESULTS: WT mice receiving sham injections or RFP-MSC did not develop tumors. APC min mice receiving sham injection did not develop colon tumors. APC mice receiving MSCs developed 3 colonic tumors (n=4 each group). One mouse had one tumor, one had 2 tumors and 2 mice did not have any tumors. There was no statistical difference between small bowel adenomas in the APC min mice +/-MSC. Tumors were adenomas, and adenocarcinoma. Colonic epithelium was host derived, with CD44+, alpha smooth muscle actin + RFP-MSC incorporated into the stroma as myofibroblasts. MSC conditioned media contains TNF-alpha (216+/-48 pg/ml), MCP-1 (153+/-75pg/ml), CCL-5 (12804+/-9750pg/mml, IL6(3971+/-98 pg/ml) and undetectable levels of IFN, IL2, IL4 or IL10. Conditioned media increased proliferation of MC38 monolayers at all time points and increased monolayer wound healing compared to control media at 18 and 21 hours. Addition of anti-TNF antibody decreased proliferation and wound healing to control levels stressing the importance of this cytokine. Conclusion: Culture derived MSC migrate to at-risk tissue and initiate colon neoplasia by incorporating into stroma. MSCs produce factors (such as TNF-alpha) that provide colon adenocarcinoma cells (MC38) with migration and growth advantage. A model in which we can manipulate stromal-tumor interactions will be useful for identifying new targets for colon cancer prevention and therapy.