scholarly journals Further delineation of the continuous human neoplastic enterochromaffin cell line, KRJ-I, and the inhibitory effects of lanreotide and rapamycin

2007 ◽  
Vol 38 (1) ◽  
pp. 181-192 ◽  
Author(s):  
Mark Kidd ◽  
Geeta N Eick ◽  
Irvin M Modlin ◽  
Roswitha Pfragner ◽  
Manish C Champaneria ◽  
...  
Keyword(s):  
Substance P ◽  
Cell Line ◽  
Tryptophan Hydroxylase ◽  
Intracellular Camp ◽  
Cell Of Origin ◽  
Rt Pcr ◽  
Cytoplasmic Vesicles ◽  
Ec Cells ◽  
Gastrointestinal Carcinoid

Small intestinal carcinoids (SICs) are the most prevalent gastrointestinal carcinoid and characterized by local invasion metastasis and protean symptomatology. The proliferative and secretory regulation of the cell of origin, the enterochromaffin (EC) cell has not been characterized. The absence of either a pure preparation of normal EC cells or human EC carcinoid cell lines has hindered the development of therapeutic agents. We therefore further characterized the neoplastic SIC cell line, KRJ-I by assessing its secretory (serotonin (5-HT)) and proliferative responses and defining its log growth phase transcriptome. Electron microscopy demonstrated oval, lobulated nuclei and substance P, and 5-HT-positive cytoplasmic vesicles. RT-PCR detected transcripts for chromogranin A (CHGA), VMAT1 (SLC18A1), tryptophan hydroxylase (TPH1), substance P (TAC1), guanylin (GUCA2A), and SERT (SLC6A4). By immunohistochemistry, all cells were positive for CHGA, SERT, VMAT1, and TPH1. Transcriptome analysis (Affymetrix U133 Plus chips) identified somatostatin SSTR2/3, adrenergic α1C and β1, dopamine D2, nicotinic-type cholinergic A5, A6, B1, muscarinic acetylcholine M4, and 5-HT-2A receptors. The presence of transcripts for SSTR1, SSTR2, and SSTR3 receptors was confirmed by RT-PCR and sequencing. Isoproterenol (ISO) resulted in a dose-dependent increase in intracellular cAMP (EC50=340 nM) and 5-HT (EC50=81 nM) which was completely inhibited by the cAMP antagonist 2′,5′-dideoxyadenosine (10 μM). Preincubation with a SSTR agonist, lanreotide, inhibited Ip-stimulated 5-HT secretion (IC50=420 nM). Both lanreotide (10 nM) and rapamycin (50 nM) inhibited proliferation (20±12 and 35±5% respectively) in serum-free medium whereas gefitinib (1 nM–10 μM) inhibited proliferation at micromolar concentrations. KRJ-I is a neoplastic EC cell line that can be used as an in vitro model of SICs as it will allow elucidation and clarification of the secretory and proliferative mechanism(s) of neoplastic EC cells and the molecular signatures that characterize each of these responses.

scholarly journals Activation of Type 1 CRH Receptor Isoforms Induces Serotonin Release from Human Carcinoid BON-1N Cells: An Enterochromaffin Cell Model

Endocrinology ◽  
2011 ◽  
Vol 152 (1) ◽  
pp. 126-137 ◽  
Author(s):  
S. Vincent Wu ◽  
Pu-Qing Yuan ◽  
Jim Lai ◽  
Kelvin Wong ◽  
Monica C. Chen ◽  
...  
Keyword(s):  
Tryptophan Hydroxylase ◽  
Cell Function ◽  
Splice Variants ◽  
Cell Model ◽  
Serotonin Release ◽  
Intracellular Camp ◽  
Mrna Levels ◽  
Maximal Increase ◽  
Ec Cells

Abstract CRH and 5-hydroxytryptamine (5-HT) are expressed in human colonic enterochromaffin (EC) cells, but their interactions at the cellular level remain largely unknown. The mechanistic and functional relationship between CRH and 5-HT systems in EC cells was investigated in a human carcinoid cloned BON cell line (BON-1N), widely used as an in vitro model of EC cell function. First, we identified multiple CRH1 splice variants, including CRH1a, CRH1c, CRH1f, and a novel form lacking exon 4, designated here as CRH1i, in the BON-1N cells. The expression of CRH1i was also confirmed in human brain cortex, pituitary gland, and ileum. Immunocytochemistry and immunoblot analysis confirmed that BON-1N cells were CRH1 and 5-HT positive. CRH, urocortin (Ucn)-1, and cortagine, a selective CRH1 agonist, all increased intracellular cAMP, and this concentration-dependent response was inhibited by CRH1-selective antagonist NBI-35965. CRH and Ucn-1, but not Ucn-2, stimulated significant ERK1/2 phosphorylation. In transfected human embryonic kidney-293 cells, CRH1i isoforms produced a significant increase in pERK1/2 in response to CRH1 agonists that was sensitive to NBI-35965. CRH and Ucn-1 stimulated 5-HT release that reached a maximal increase of 3.3- and 4-fold at 10−8m over the basal level, respectively. In addition, exposure to CRH for 24-h up-regulated tryptophan hydroxylase-1 mRNA levels in the BON-1N cells. These findings define the expression of EC cell-specific CRH1 isoforms and activation of CRH1-dependent pathways leading to 5-HT release and synthesis; thus, providing functional evidence of a link exists between CRH and 5-HT systems, which have implications in stress-induced CRH1 and 5-HT-mediated stimulation of lower intestinal function.

scholarly journals The role of mechanical forces and adenosine in the regulation of intestinal enterochromaffin cell serotonin secretion

2012 ◽  
Vol 302 (3) ◽  
pp. G397-G405 ◽  
Author(s):  
A. Chin ◽  
B. Svejda ◽  
B. I. Gustafsson ◽  
A. B. Granlund ◽  
A. K. Sandvik ◽  
...  
Keyword(s):  
Mechanical Stimulation ◽  
Tryptophan Hydroxylase ◽  
Cell Function ◽  
Cell System ◽  
Receptor Expression ◽  
Intracellular Camp ◽  
Serotonin Secretion ◽  
A2b Receptor ◽  
Ec Cells ◽  
Inflammatory Bowel

Enterochromaffin (EC) cells of the diffuse neuroendocrine cell system secrete serotonin (5-HT) with activation of gut motility, secretion, and pain. These cells express adenosine (ADORA) receptors and are considered to function as mechanosensors. Physiological pathways mediating mechanosensitivity and adenosine responsiveness remain to be fully elucidated, as do their roles in inflammatory bowel disease (IBD) and neoplasia. Pure (98–99%) FACS-sorted normal and IBD human EC cells and neoplastic EC cells (KRJ-I) were studied. IBD-EC cells and KRJ-I overexpressed ADORA2B. NECA, a general ADORA receptor agonist, stimulated, whereas the A2B receptor antagonist MRS1754 inhibited, 5-HT release (EC50 = 1.8 × 10−6 M; IC50 = 3.7 × 10−8 M), which was associated with corresponding alterations in intracellular cAMP levels and pCREB (Ser133). Mechanical stimulation using a rhythmic flex model induced transcription and activation of Tph1 (tryptophan hydroxylase) and VMAT1 (vesicular monoamine transporter 1) and the release of 5-HT, which could be inhibited by MRS1754 and amplified by NECA. Secretion was also inhibited by H-89 (PKA inhibitor) while Tph1 and VMAT1 transcription was regulated by PKA/MAPK and PI3K-mediated signaling. Normal and IBD-EC cells also responded to NECA and mechanical stimulation with PKA activation, cAMP production, and 5-HT release, effects reversible by MRS1754. EC cells express stimulatory ADORA2B, and rhythmic stretch induces A2B activation, PKA/MAPK/IP3-dependent transcription, and PKA-dependent secretion of 5-HT synthesis and secretion. Receptor expression is amplified in IBD and neoplasia, and 5-HT release is increased. Determination of factors that regulate EC cell function are necessary for understanding its role as a mechanosensory cell and to facilitate the development of agents that can selectively target cell function in EC cell-associated disease.

scholarly journals Jianpi Yangwei decoction promotes apoptosis and suppresses proliferation of 5-fluorouracil resistant gastric cancer cells in vitro and in vivo

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Huijuan Tang ◽  
Wenjie Huang ◽  
Qiang Yang ◽  
Ying Lin ◽  
Yihui Chen ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Drug Resistance ◽  
Cell Line ◽  
Real Time ◽  
Mtt Assay ◽  
Xenograft Tumor ◽  
Rt Pcr ◽  
Induced Apoptosis

Abstract Background The exploration of new therapeutic agents targeting 5-Fu resistance may open a new opportunity to gastric cancer treatment. The objective is to establish a 5-Fu resistant gastric cancer cell line and observe the effect of Jianpi Yangwei decoction (JPYW) on its apoptosis and drug-resistance related proteins. Methods MTT assay was used to measure the effect of JPYW on the BGC823 cells proliferation, and the apoptosis was observed by flow cytometry and Hoechst fluorescence staining. The BGC823 xenograft tumor nude mice models were established, the apoptosis was detected by Tunel method. BGC-823/5-Fu was established by repeated low-dose 5-Fu shocks, the drug resistance index and proliferation were detected by the MTT assay; MDR1 mRNA was detected by real-time RT-PCR; Western blot was used to detect the ratio of p-AKT to AKT; The BGC823/5-Fu xenograft tumor nude mice models were established and apoptosis was measured. The expressions of MRP1, MDR1, ABCG2, AKT, p-AKT, caspase-3 and bcl-2 were detected by immunohistochemistry and the AKT mRNA expression was detected by real-time RT-PCR. Results JPYW induced apoptosis in BGC823 cells; Drug-resistant cell line BGC-823/5-Fu was sucessfully established; JPYW induced apoptosis of BGC823/5-Fu cells, down-regulated the expression of MRP1, MDR1 and ABCG2 in vitro and in vivo, and further decreased MDR1 expression when combined with pathway inhibitor LY294002 (P < 0.05); JPYW down-regulated the ratio of p-AKT to AKT in vitro in a dose-dependent manner, the same as after the combination with LY294002 (P < 0.05). Conclusion JPYW can induce apoptosis of BGC823 and BGC823/5-Fu cells, and down-regulate the expression of MDR1, MRP1, ABCG2 in vitro and in vivo. Its in vitro effect is related to the PI3K/AKT signaling pathway.

Growth Suppressors IFI-204 and IFI-205 Inhibit Primitive Hematopoietic Cell Proliferation In Vitro and in Vivo.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1691-1691
Author(s):  
Kimberly Klarmann ◽  
Daniel Gough ◽  
Benyam Asefa ◽  
Chris Clarke ◽  
Katie Renn ◽  
...  
Keyword(s):  
Stem Cell ◽  
Cell Growth ◽  
Cell Line ◽  
Constitutive Expression ◽  
Rt Pcr ◽  
Hematopoietic Stem ◽  
Inhibit Cell Growth ◽  
Stem And Progenitor Cells

Abstract Members of the interferon inducible-200 (IFI-200) family of proteins inhibit cell growth and may be important mediators of differentiation. We examined IFI-204 and IFI-205 mRNA expression in purified populations of hematopoietic stem and progenitor cells at different stages of maturation using quantitative RT-PCR and found that their expression markedly increased during myeloid maturation. To evaluate the effect of IFI-205 and IFI-204 on hematopoietic stem cell (HSC) growth, we transduced these genes into mouse bone marrow cells (BMC) using retroviral vectors. The presence IFI-204 or IFI-205 resulted in a decrease in cell growth in response to hematopoietic growth factors. Further analysis revealed the infected cells were 98% c-Kit+ Sca-1+, indicative of the stem cell surface phenotype, suggesting they may be blocked in a primitive stage of maturation. When transplanted, BMC transduced with IFI-204 or IFI-205 failed to engraft lymphoid, myeloid, or erythroid lineages in both short and long term reconstitution assays, suggesting that constitutive expression of IFI-204 and IFI-205 inhibited HSC development both in vitro and in vivo. However, based on the quantitative RT-PCR results, which show that IFI-205 increased during myeloid differentiation, we know its endogenous, regulated expression must permit the cells to mature. Therefore, to study of the effects of these genes on differentiation we transduced the mulitpotential EML (erythroid, myeloid, lymphoid) cell line with IFI-204 and IFI-205 to circumvent severe growth inhibition caused by expression of IFI-204 and Ifi-205 in normal cells. Single cell analysis of EMLs transduced with IFI-205 demonstrated that expression of IFI-205 in this cell line did not significantly inhibit cell growth. We have isolated EML clones from the transduced cells and verified IFI-205 expression. In addition, we generated transgenic mice that express IFI-205 under control of the Vav and MRP8 promoters, and we identified transgenic lines that express IFI-205 at higher levels compared to wild type controls. Analysis of hematopoiesis in these animals is currently in progress. Altogether, our data demonstrate 3 findings: 1) IFI-204 and IFI-205 expression increases during myeloid development based on quantitative RT-PCR analysis, 2) constitutive expression of IFI-204 and -205 results in potent inhibition of growth and maturation of normal hematopoietic stem and progenitor cells in vivo and in vitro and 3) these genes did not significantly inhibit the proliferation of the EML cell line, which provides us with a means to study the mechanism by which these molecules regulate myeloid maturation. Finally, the considerable inhibitory effects of these family members on normal hematopoietic cell growth suggest their potential as therapeutic modalities for treatment of leukemia.

To Study Resistant Mechanisms and Reversal In An Imatinib Resistant Ph+ Positive Acute Lymphoblastic Leukemia Cell Line

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2135-2135
Author(s):  
Hongyun Xing ◽  
Yuping Gong ◽  
Ting Liu
Keyword(s):  
Cell Signaling ◽  
Cell Line ◽  
Lymphoblastic Leukemia ◽  
Kinase Domain ◽  
Rt Pcr ◽  
Imatinib Resistance ◽  
Abl Kinase ◽  
Imatinib Resistant ◽  
Cell Signaling Pathways

Abstract Abstract 2135 Objective To establish an imatinib resistant Bcr-Abl positive acute lymphoblastic leukemia (ALL) cell line in vitro and to study imatibin resistance in Ph+ ALL. The reversal of the imatinib resistance by rapamycin, the second generation tyrosine kinase inhibitor and proteasome inhibitor was studied. Methods Ph(+) ALL SUP-B15 cell line was cultured in gradually increasing concentrations of imatinib to generate the imatinib resistant cell line at 6 μM imatinib. The cytotoxic effect of imatinib and other drugs was analyzed by MTT assay. RT-PCR, flow cytometry, Western blot analyses of proteins, DNA sequence analysis of ABL kinase domain were used to clarify the possible mechanisms of the imatinib resistance in the SUP-B15/RI cell line. Results We established the imatinib resistant Ph+ ALL cell line. The fusion bcr-abl gene was 6.1 times as high as that of the parental sensitive cell, and the mdr1 gene also increased 1.7 times in SUP-B15/RI cell line by the RT-PCR detection. However, the expression of hoct1 Abcl–2 and topoIIα gene were no difference between two cell lines by the RT-PCR detection. A K362S point mutation in the Abl kinase domain of SUP-B15/RI was found. The detection of cell signaling pathway of PI3K/AKT/mTOR, RAS/RAF, NF-κBA JNK and STAT showed the expression of PTEN and 4EBP-1 was down-regulated, AKT, mTOR and P70S6K was up-regulated and the expression of other cell signaling pathways in SUP-B15/RI was similar to its parental sensitive cell line. Dasatinib, nilotinib, and bortezomib could inhibit proliferation of SUP-B15/RI cells at nM concentration. SUP-B15/RI cell line also showed partial resistance to dasatinib and nilotinib, but not bortezomib. The combination of imatinib with rapamycin had synergistic effect to the resistance cell line. Conclusion In vitro, we establish imatinib resistant Ph + ALL cell line. Overexpression of bcr-abl and mdr1 gene, K362S point mutation in ABL kinase domain and up-regulation of the cell signaling pathways of PI3K/AKT/mTOR, RAS/RAF in SUP-B15/RI cell line were involved in the resistance mechanisms. The SUP-B15/RI cell line was also resistant to the second generation tyrosine kinaeses dasatinib and nilotinib,not bortezomib in vitro. However, the combination of imatinib with rapamycin can partially overcome the resistance. Blockade of the ubiquitin-proteasome could be a promising pathway to overcome resistance to imatinib. Disclosures: No relevant conflicts of interest to declare.

Therapeutic Efficacy and Cure Of Sensitive and T315I Pan-Resistant Human Ph+ Leukemia In Mice Using a TCR-Like Antibody To WT1/HLA-A0201 Alone, Or In Combination With Tyrosine Kinase Inhibitors

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 855-855
Author(s):  
Leonid Dubrovsky ◽  
Elliott Brea ◽  
Dmitry Pankov ◽  
Nicholas Veomett ◽  
Tao Dao ◽  
...  
Keyword(s):  
Combination Therapy ◽  
Tyrosine Kinase ◽  
Tumor Growth ◽  
Cell Line ◽  
Wilms Tumor ◽  
High Dose ◽  
Human Leukemia ◽  
Rt Pcr

Abstract Acute and chronic leukemias, including CD34+ CML stem cells, overexpress the Wilms tumor gene 1 (WT1) protein, making WT1 an attractive therapeutic target. ESKM is a fully human IgG1 antibody that targets a 9 amino acid sequence (RMF) of the protein WT1 in the context of HLA-A0201, allowing it to target an undruggable, widely expressed, intracellular oncogene product. BV173 is an HLA-A0201+, human Ph+ ALL cell line that expresses WT1, and tagged by our lab with luciferase. We engineered a tyrosine kinase inhibitor (TKI) resistant BV173-R cell line by transducing BV173 with the resistant T315I Bcr-Abl plasmid. Antibody-dependent cellular cytotoxicity (ADCC) was evaluated in vitro by chromium release assay, utilizing human PBMC effectors. Tumor growth in vivo was assessed in NOD/SCID gamma (NSG) mice with bioluminescence imaging (BLI). RT-PCR was used to evaluate minimal residual disease in mice with negative BLI signal at the end of therapy. Imatinib, dasatinib, and ponatinib were used at up to maximally tolerated doses, given IP once daily. ESKM was administered at 100 µg twice weekly IP. ESKM mediated ADCC against both BV173 and BV173-R cell lines in vitro. In a BV173 engrafted human leukemia xenograft model, ESKM was more potent than imatinib, with median tumor growth reduction of 78% vs 52%. Combination of imatinib and ESKM therapy resulted in a 94% reduction in leukemic growth. High dose dasatinib (40 mg/kg daily) was more potent than ESKM, but discontinuation of therapy due to dasatinib toxicity resulted in relapse. Combination with ESKM therapy with dasatinib resulted in cure in 75% of mice, confirmed by bone marrow RT-PCR three weeks after termination of therapy. For mice cytoreduced with dasatinib followed by consolidation therapy with ESKM, delayed relapse was observed, but no cures. ESKM was highly superior to imatinib and dasatinib against the T315I BV173-R leukemia in vivo. Cures were not achieved with combination therapy of ESKM and either first or second generation TKIs against resistant T315I leukemia. Ponatinib at 10 mg/kg had higher efficacy than ESKM alone against BV173-R, but mice treated with combination of ESKM and ponatinib had superior tumor reduction. CONCLUSION: ESKM is an effective therapeutic antibody for sensitive and T315I Ph+ ALL. Resistant T315I Ph+ leukemic growth is inhibited more effectively by ESKM therapy compared to imatinib and dasatinib, and combination therapy with ESKM is superior to ponatinib. Supported by the Leukemia and Lymphoma Society, NIH R01CA55349, P01 23766 and T32CA62948-18. Disclosures: Yan: Eureka Therapeutics: Employment. Liu:Eureka Therapeutics: Employment, Equity Ownership.

Acidosis-Stimulated Neurons of the Medullary Raphe Are Serotonergic

2001 ◽  
Vol 85 (5) ◽  
pp. 2224-2235 ◽  
Author(s):  
Wengang Wang ◽  
Jyoti K. Tiwari ◽  
Stefania Risso Bradley ◽  
Rey V. Zaykin ◽  
George B. Richerson
Keyword(s):  
Cell Culture ◽  
Substance P ◽  
Tryptophan Hydroxylase ◽  
Brain Slices ◽  
Serotonergic Neurons ◽  
Electrophysiological Properties ◽  
Medullary Raphe ◽  
Raphe Neurons

Neurons of the medullary raphe project widely to respiratory and autonomic nuclei and contain co-localized serotonin, thyrotropin-releasing hormone (TRH), and substance P, three neurotransmitters known to stimulate ventilation. Some medullary raphe neurons are highly sensitive to pH and CO2 and have been proposed to be central chemoreceptors. Here it was determined whether these chemosensitive neurons are serotonergic. Cells were microdissected from the rat medullary raphe and maintained in primary cell culture for 13–70 days. Immunoreactivity for serotonin, substance P, and TRH was present in these cultures. All acidosis-stimulated neurons ( n = 22) were immunoreactive for tryptophan hydroxylase (TpOH-IR), the rate-limiting enzyme for serotonin biosynthesis, whereas all acidosis-inhibited neurons ( n= 16) were TpOH-immunonegative. The majority of TpOH-IR medullary raphe neurons (73%) were stimulated by acidosis. The electrophysiological properties of TpOH-IR neurons in culture were similar to those previously reported for serotonergic neurons in vivo and in brain slices. These properties included wide action potentials (4.55 ± 0.5 ms) with a low variability of the interspike interval, a postspike afterhyperpolarization (AHP) that reversed 25 mV more positive than the Nernst potential for K+, prominent A current, spike frequency adaptation and a prolonged AHP after a depolarizing pulse. Thus the intrinsic cellular properties of serotonergic neurons were preserved in cell culture, indicating that the results obtained using this in vitro approach are relevant to serotonergic neurons in vivo. These results demonstrate that acidosis-stimulated neurons of the medullary raphe contain serotonin. We propose that serotonergic neurons initiate a homeostatic response to changes in blood CO2 that includes increased ventilation and modulation of autonomic function.

scholarly journals Determination of the Infectivity of Cryopreserved Theileria annu-lata Sporozoites in Tick Derived Stabilates Iran Ak-93 Strain, by In Vivo and In Vitro Methods

2019 ◽  
Author(s):  
Hossein MODIRROUSTA ◽  
Gholamreza HABIBI ◽  
Parviz SHAYAN ◽  
Asghar AFSHARI ◽  
Ali MIRJALILI ◽  
...  
Keyword(s):  
Cell Culture ◽  
Infected Cell ◽  
Cell Line ◽  
Challenge Test ◽  
Infected Cell Line ◽  
Rt Pcr ◽  
In Vitro Cell Culture ◽  
Microscopic Inspection

Background: The protozoan parasite Theileria annulata is the causative agent of tropical theileriosis in cattle. Vaccination is recommended by administration of attenuated schizont-infected cell lines. The expected protective immunity post-vaccination can be demonstrated by challenge test through inoculation of highly virulent infective sporozoites. The aim of this study was to produce Hyalomma anatolicum anatolicum tick infected with T. annulata (local strain) for preparation of tick-derived sporozoite stabilates for molecular characterization and infectivity test assay. Methods: A local T. annulata strain was used for experimental infection of calves. A field isolate of H. a. anatolicum was isolated, laboratory-reared and infected by blood-feeding on Theileria infected above-mentioned calves. The infectivity of calf, tick and prepared stabilate were confirmed by clinical signs of theileriosis, microscopic inspection, RT-PCR and in vitro cell culture. Results: The tick stabilate was prepared and cryopreserved in liquid nitrogen. The infectivity of the tick stabilate was verified by in vivo bioassay, in vitro cell culture infection, microscopic inspection in salivary glands and RT-PCR assay. The in vitro produced cell line in this study was characterized by T. annulata Cytochrome b gene analyzing. Conclusion: The infectivity of a new prepared tick-derived sporozoite stabilate was confirmed in susceptible calves; by microscopically, post mortem, tick microscopic and molecular assays. Moreover, naïve PBMCs were transformed and proliferated by T. annulata infected tick stabilate to immortal T. annulata schizont infected cell line. The potent infective sporozoite tick derived stabilate could be used for vaccine efficacy and challenge test as well as in vaccine development.

scholarly journals In vitro immunomodulatory effects of thymol and cinnamaldehyde in a pig intestinal epithelial cell line (IPEC-J2)

2020 ◽  
Vol 8 (3) ◽  
pp. 127-134
Author(s):  
C. Shen ◽  
L.G. Christensen ◽  
P.B. Rasmussen ◽  
K.M. Kragh
Keyword(s):  
Epithelial Cell ◽  
Cell Line ◽  
Tight Junctions ◽  
Feed Additives ◽  
Cellular Damage ◽  
Feed Additive ◽  
Epithelial Cell Line ◽  
Rt Pcr ◽  
Wound Recovery

Thymol and cinnamaldehyde are phytogenic feed additives developed to improve gut health and growth performance in poultry and swine. This study evaluated the in vitro immune modulating effects of thymol and cinnamaldehyde blend (TCB) in a porcine gut epithelial cell line (IPEC-J2), with or without cellular damage caused by challenge with lipopolysaccharides. Cytotoxicity, permeability, wound-healing and bacteria adhesion assays were recorded. The expression of cytokines, tight junctions and polymeric immunoglobulin receptor (pIgR) were measured by RT-PCR. The IPEC-J2 cells were cultured in the presence of TCB at concentrations ranging from 1 ng/ml to 1 μg/ml and displayed high viability (>90%). TCB increased barrier integrity (13.8% less in lipopolysaccharide challenge which induced gut epithelial leakage, P<0.05) and accelerated the initial speed of wound recovery (day 1, 26% wound recovery in TCB treated vs 7% in control, P<0.05; day 2, 54 vs 39%, P<0.001). The RT-PCR analysis of cell culture showed that TCB upregulated anti-inflammatory cytokine interleukin (IL)-10 (73.3%, P<0.05) in non-stimulated IPEC-J2 cells, while, when stimulated, pIgR (9.7%, P<0.05) and tight junctions claudin-4 (9.4%, P<0.05) were upregulated by TCB. Furthermore, TCB significantly increased Lactobacillus acidophilus adherence to gut epithelial cells (285.0%, P<0.05). Overall, the current in vitro study showed that TCB can induce various immune responses, which may explain its in vivo benefits as feed additive.

Sign in / Sign up

Export Citation Format

Share Document