INT-407 Cells Research Articles

Biological and chemical enhancements of Elaeocarpus sylvestris var. ellipticus through fermentation: Implications for therapeutic and industrial applications

This study aimed to evaluate the biological and chemical improvements that take place during the yeast fermentation of hot water-extracted Elaeocarpus sylvestris (HES). HES was diluted with sterile distilled water to a final concentration of 1:200 (v/v), followed by the inoculation of Pichia fermentans (PF) and Saccharomyces cerevisiae (SC) were inoculated as starters. The fermentation by yeast strains proceeded for five days, during which the viable cell count and pH were monitored. The radical scavenging activity was assessed using DPPH and ABTS assays. The impact of HES on proinflammatory cytokines in RAW 264.7 cells and cytotoxicity in HT-29, MCF-7, and INT407 cells was examined. Changes in metabolic profile during fermentation were analyzed using GC-TOF-MS and UPLC-Orbitrap-MS/MS. HES fermented with PF and SC maintained viable cell counts throughout the 5-day fermentation period. The pH levels were consistently within the range of 3.62 ± 0.03 to 3.65 ± 0.15. The expression of TNF-α and IL-6 significantly decreased at all concentrations (31–500 mg/mL) in RAW cells treated with lipopolysaccharide and HES, with cytokine expression levels returning to baseline levels. The cell viability of INT407 significantly increased following HES treatment (p < 0.05). Fermentation of HES with PF or SC led to significant changes in the composition ratios of carboxylic acids and derivatives, and organooxygen compounds. The HES extract demonstrates potential applicability as a raw material with antioxidative, anti-inflammatory, and anticancer properties for applications in the food and cosmetic industries.

Hemoglobin Hydrolyzate Promotes Iron Absorption in the Small Intestine through Iron Binding Peptides.

Hemoglobin is an excellent source of iron supplements, and its hydrolyzate spontaneously binds iron during digestion and promotes iron absorption in vivo. However, the underlying mechanisms of what peptides bind and how they bind iron ions remain unclear. This study prepared the porcine hemoglobin hydrolyzate through enzymatic hydrolysis and acid treatment and investigated the mechanisms of hemoglobin hydrolyzate on iron absorption through the determination of iron levels in dietary intervention mice, iron binding site analyses, peptide digestion analyses, molecular simulation docking, and INT407 cell validation. The results showed that ingestion of the hemoglobin hydrolyzate diets increased iron levels in the blood of mice, accompanied by the upregulation of duodenal iron circulation-related genes such as ferritin, PCBP1, and HP. Carboxyl, imidazole groups, and aromatic amino acid residues were iron binding sites of hemoglobin hydrolyzate during digestion. VDEVGGEA and VDEVGGE were found to involve the spontaneous and efficient binding of hemoglobin hydrolyzate to iron ions in the intestinal cavity. In particular, the DEVGGE peptide was the typical sequence for hemoglobin hydrolytic peptides to exert iron binding activity.

Plant-Derived Phenolic Acids Limit the Pathogenesis of Salmonella Typhimurium and Protect Intestinal Epithelial Cells during Their Interactions.

The incidence of gastrointestinal illness attributable to Salmonella enterica serovar Typhimurium (ST) remains a concern for public health worldwide, as it can progress into systemic infections mediated by the type-three secretion system (T3SS), which allows for adherence and invasion to intestinal epithelial cells. The current study evaluates the ability of gallic acid (GA), protocatechuic acid (PA), and vanillic acid (VA) to impair the adhesion and invasion abilities of ST to a human epithelial (INT-407) cell monolayer while also assessing their cytotoxicity. GA, PA, and VA inhibited detectable ST growth at specific concentrations but showed cytotoxicity against INT-407 cells (>20% reduction in viability) after 3 h of treatments. Adjusting the pH of the solutions had a neutralizing effect on cytotoxicity, though it did reduce their antimicrobial potency. Adhesion of ST was reduced significantly when the cells were treated with 4.0 mg/mL of VA, whereas invasion was reduced in all treatments, with GA requiring the lowest concentration (0.5 mg/mL). Relative gene expression of virulence genes after treatment with GA showed downregulation in the T3SS regulator and effector hilA and sipA, respectively. These findings suggest further use of phenolic acids in reducing the activity of key virulence factors critical during ST infection.

Molecular mechanism of Enteroaggregative Escherichia coli induced apoptosis in cultured human intestinal epithelial cells.

Background. Enteroaggregative Escherichia coli (EAEC) is an evolving etiological agent of acute and persistent diarrhoea worldwide. The previous study from our laboratory has reported the apoptosis-inducing activity of EAEC in human small intestinal and colonic epithelial cell lines. In the present investigation, we have explored the underlying mechanism of EAEC-induced apoptosis in human intestinal epithelial cell lines.Methods. INT-407 and HCT-15 cells were infected with EAEC-T8 and EAEC-pT8 (plasmid cured strain of EAEC-T8) separately. Cells cultured in the absence of bacteria served as a negative control in all the experiments. For the subsequent experiments, the molecular mechanism(s) of epithelial cell aposptosis was measured in EAEC infecting both the cell lines by flow cytometry, real-time PCR and Western blotting.Results and conclusions. EAEC was found to activate the intrinsic/mitochondrial apoptotic pathway in both the cell lines through upregulation of pro-apoptotic Bax and Bak, un-alteration/reduction in the level of anti-apoptotic Bcl-2 and Bcl-XL, decrease in mitochondrial transmembrane potential, accumulation of cytosolic cytochrome c leading to activation of procaspase-9 and procaspase-3, which ultimately resulted in DNA fragmentation and apoptosis. Further, an increased expression of Fas, activation of procaspase-8 and upregulation of pro-apoptotic Bid in the EAEC-infected cells indicated the involvement of extrinsic apoptotic pathway too in this process. Our finding has undoubtedly led to an increased understanding of EAEC pathogenesis, which may be helpful to develop an improved strategy to combat the infection.

Virulence Characteristics and Molecular Typing of Carbapenem-Resistant ST15 Klebsiella pneumoniae Clinical Isolates, Possessing the K24 Capsular Type

Klebsiella pneumoniae is an opportunistic pathogen that frequently causes nosocomial and community-acquired (CA) infections. Until now, a limited number of studies has been focused on the analyses of changes affecting the virulence attributes. Genotypic and phenotypic methods were used to characterise the 39 clinical K. pneumoniae isolates; all belonged to the pan-drug resistant, widespread clone ST 15 and expressed the K24 capsule. PFGE has revealed that the isolates could be divided into three distinct genomic clusters. All isolates possessed allS and uge genes, known to contribute to the virulence of K. pneumoniae and 10.25% of the isolates showed hypermucoviscosity, 94.87% produced type 1 fimbriae, 92.3% produced type 3 fimbriae, and 92.3% were able to produce biofilm. In vivo persistence could be supported by serum resistance 46.15%, enterobactin (94.87%) and aerobactin (5.12%) production and invasion of the INT407 and T24 cell lines. Sequence analysis of the whole genomes of the four representative strains 11/3, 50/1, 53/2 and 53/3 has revealed high sequence homology to the reference K. pneumoniae strain HS11286. Our results represent the divergence of virulence attributes among the isolates derived from a common ancestor clone ST 15, in an evolutionary process that occurred both in the hospital and in the community.

Role of ST6GAL1 and ST6GAL2 in subversion of cellular signaling during enteroaggregative Escherichia coli infection of human intestinal epithelial cell lines

Emerging evidence have suggested that aberrant sialylation on cell-surface carbohydrate architecture may influence host-pathogen interactions. The α2,6-sialyltransferase (ST) enzymes were found to alter the glycosylation pattern of the pathogen-infected host cell-surface proteins, which could facilitate its invasion. In this study, we assessed the role of specific α2,6-ST enzymes in the regulation of enteroaggregative E. coli (EAEC)-induced cell signaling pathways in human intestinal epithelial cells. EAEC-induced expression of α2,6-ST family genes in HCT-15 and INT-407 cell lines was assessed at mRNA level by qRT-PCR. Specific esi-RNA was used to silence the target ST-gene in each of the EAEC-infected cell type. Subsequently, the role of these enzymes in regulation of EAEC-induced cell signaling pathways was unraveled by analyzing the expression of MAPkinases (ERK1/2, p38, JNK) and transcription factors (NFκB, cJun, cFos, STAT) at mRNA and protein levels by qRT-PCR and western immunoblotting, respectively, expression of selected sialoglycoproteins by western immunoblotting along with the secretory IL-8 response using sandwich ELISA. ST6GAL-1 and ST6GAL-2 were efficiently silenced in EAEC-infected HCT-15 and INT-407 cells, respectively. Significant reduction in EAEC-induced activation of MAPKs, transcription factors, sialoglycoproteins, and IL-8 secretion was noted in ST-silenced cells in comparison to the respective control cells. We propose that ST6GAL-1 and ST6GAL-2 are quintessential for EAEC-induced stimulation of MAPK-mediated pathways, resulting in activation of transcription factors, leading to an inflammatory response in the human intestinal epithelial cells. Our study may be helpful to design better therapeutic strategies to control EAEC- infection. KEY POINTS: • EAEC induces α2,6-sialyltransferase (ST) upregulation in intestinal epithelial cells • Target STs (ST6GAL-1 & ST6GAL-2) were efficiently silenced using specific esiRNAs • Expression of MAPKs, transcription factors & IL-8 was reduced in ST silenced cells.

Role of enteroaggregative E.coli induced activation of epidermal growth factor receptor in cultured human intestinal epithelial cell lines

Enteric pathogens exploit the versatility of cytoskeletal system for internalization into non-phagocytic cells, as a crucial step in their pathogenic life cycle. Enteropathogenic Escherichia coli(EPEC) andEnterohemorrhagic Escherichia coli (EHEC) were shown to form actin pedestals in host cells using type-III secretion system. Earlier, we reported that EAEC induced increase in intracellular calcium ions might have crucial role in F-actin rearrangements in INT-407 cells leading to its invasion. It was suggested that EGFR might contribute in Rck-mediated adherence and invasion of Salmonella in host cells. In the present study, we assessed the role of EAEC induced activated EGFR in human intestinal epithelial cell lines (INT-407 &amp; HCT-15). The presence of activated EGFR was detected in membrane fractions ofeach cell line, infected with two different strains of EAEC (EAEC-T8 &amp; EAEC-O42) separatelyfor 3h in presence and absence of Tyrphostin AG1478 (EGFR-inhibitor), by western immunoblotting using p-EGFR (Y1068) antibody. Adherence and invasion of EAEC-T8 to each cell line were checked in presence of Tyrphostin AG1478. Further, the effect of Tyrphostin AG1478 on cytoskeletal F-actin rearrangement of EAEC-T8 infected cells was assessed under confocal microscope following staining with TRITC-phalloidin. EAEC-T8 induced maximum increase in EGFR autophosphorylation at Y1068. Adherence and invasion of EAEC-T8 as well as this organism induced cytoskeletal F-actin polymerization were found to be inhibited in presence of Tyrphostin AG1478. Our study revealed that EAEC induced activated EGFR might play a major role in host cell adherence and cytoskeletal rearrangements leading to invasion of the organism in these cells.

Cellular uptake of anthocyanins extracted from black soybean, grape, and purple sweet potato using INT-407 cells.

The online version contains supplementary material available at 10.1007/s10068-021-00976-y.

Probiotic characterization of bacterial strains from fermented South Indian tomato pickle and country chicken intestine having antioxidative and antiproliferative activities.

The present study aims to evaluate the potential antioxidant and antiproliferative properties of probiotic bacterial isolates Weissella cibaria p3B, Bacillus subtilis CS, and Bacillus tequilensis CL, isolated from South Indian fermented tomato pickle (homemade) and gut content of indigenous country chicken. The bacterial isolates exhibited antimicrobial activity against food-borne, human pathogenic bacteria, along with better survival under different bile and acidic conditions, hydrophobicity towards several hydrocarbons, and adherence to intestinal epithelial cells (INT-407 cells). Also, the intact cell (IC) mixture of the three species showed better DPPH, ABTS, and Fe2+ chelating activity as compared to the individual IC or cell extract (CE) activity. Among the three bacterial species, W. cibaria p3B revealed maximum antiproliferative activity against HeLa and Caco-2 cancer cells, all of which were nontoxic to INT-407 cells. Apart from being non-hemolytic, the bacterial isolates did not display any necrotic inhibition in HeLa and Caco-2 cells. The cell free supernatant (CFS) of the three bacterial isolates were tested for the production of antimicrobial peptides or bacteriocins. It found that the CFS of bacterial isolates was stable at various temperature, pH and sensitive to proteolytic enzymes confirms protenoius in nature of the antimicrobil peptides or bacteriocins. The bacterial isolates showed promising antimicrobial, antioxidant as well as antiproliferative activities with better survival ability at different pH and bile concentrations. The three bacterial isolates were able to produce potential antimicrobial peptides or bacteriocins. These results indicate better compatibility of our bacterial isolates against synthetic drugs to avoid adverse side effects and can be processed as dietary supplements against food and human pathogens. They can also provide antioxidative and antiproliferative benefits to humans and animals.

Cornelian Cherry Iridoid-Polyphenolic Extract Improves Mucosal Epithelial Barrier Integrity in Rat Experimental Colitis and Exerts Antimicrobial and Antiadhesive Activities In Vitro.

Background and Aims Inflammatory bowel disease pharmacotherapy, despite substantial progress, is still not satisfactory for both patients and clinicians. In view of the chronic and relapsing disease course and not always effective treatment with adverse effects, attempts to search for new, more efficient, and safer substances are essential and reasonable. This study was designed to elucidate the impact of cornelian cherry iridoid-polyphenolic extract (CE) and loganic acid (LA) on adherent-invasive E. coli growth and adhesion in vitro and to assess the effect of pretreatment with CE or LA on the course of intestinal inflammation in rat experimental colitis compared with sulfasalazine. Methods Antibacterial and antiadhesive activities of CE and LA were assessed using microdilution, Int407 cell adherence, and yeast agglutination assays. The colitis model was induced by 2,4,6-trinitrobenzenesulfonic acid. Studied substances were administered intragastrically for 16 days prior to colitis induction. Body weight loss; colon index; histological injuries; IL-23, IL-17, TNF-α, and chemerin levels; and STAT3, Muc2, and TFF3 mRNA expression were evaluated. Results Only CE exerted antimicrobial and antiadhesive activities in vitro and alleviated colonic symptoms. CE coadministrated with sulfasalazine was more effective than single compounds in reversing increased concentrations of TNF-α, IL-17, and chemerin and decreased Muc2 mRNA expression. Conclusions CE exerted a protective effect against experimental colitis via impaired mucosal epithelial barrier restoration and intestinal inflammatory response attenuation and given concomitantly with sulfasalazine counteracted colitis in a more effective way than sulfasalazine alone, which indicates their synergistic interaction. The beneficial effect of CE may also be due to its bacteriostatic and antiadhesive activities.

Phytoglycoprotein isolated from Dioscorea batatas Decne promotes intestinal epithelial wound healing.

Dioscorea batatas Decne (DBD) has been used to heal various illnesses of the kidney and intestine as an herbal medicine in Asia. As a source of therapeutic agents, many glycoproteins have been isolated from mushrooms and plants, but the functional role of glycoprotein in intestinal epithelial wound healing has not been understood yet. In the present study, we investigated the wound healing potentials of the 30 kDa glycoprotein (DBD glycoprotein) isolated from DBD in human intestinal epithelial (INT-407) cells. We found that DBD glycoprotein (100 μg·mL-1) significantly increased the motility of INT-407 cells for 24 h by activating protein kinase C (PKC). DBD glycoprotein stimulated the activation of p38 mitogen-activated protein kinase (MAPK), which is responsible for the phosphorylation of NF-κB inhibitor α (IκBα). DBD glycoprotein increased the level of profilin-1 (PFN1), α-actinin and F-actin expression via activation of transcription factor, nuclear factor-kappa B (NF-κB) during its promotion of cell migration. Experimental mouse colitis was induced by adding dextran sulfate sodium (DSS) to the drinking water at a concentration of 4% (W/V) for 7 days. We figured out that administration of DBD glycoprotein (10 and 20 mg·kg-1) lowers the levels of disease activity index and histological inflammation in DSS-treated ICR mice. In this regard, we suggest that DBD glycoprotein has ability to promote the F-actin-related migration signaling events via activation of PKC and NF-κB in intestinal epithelial cells and prevent inflammatory bowel disease.

Piperlongumine induces ROS mediated cell death and synergizes paclitaxel in human intestinal cancer cells

Piperlongumine (PL), a herbal drug extracted from long pepper (Piper longum L), is known for its anti-inflammatory and anti-cancer properties. Although, its anti-cancer potential has been evaluated in cancer models like breast, pancreatic, gastric, hepatocellular and lung carcinoma, there is no report on its bio-activity evaluation in intestinal cancers. Here, we report the anti-neoplastic potential of PL against human intestinal carcinoma in-vitro and its possible mechanisms of action. Cytotoxicity studies demonstrate that PL inhibits cell proliferation of INT-407 and HCT-116 cells in a concentration and time-dependent manner. Also, PL elevated the levels of intracellular reactive oxygen species, which may lead to lethal oxidative stress, mitochondrial dysfunction, and nuclear fragmentation. Remarkably, P53, P21, BAX, and SMAD4 were significantly upregulated after PL treatment whereas; BCL2 and SURVIVIN were down-regulated. Moreover, the combination study also shows the synergistic effect of PL with the current chemotherapeutic drug paclitaxel. These findings suggest that PL possesses anti-neoplastic properties in intestinal cancer cells.

Modulation of Campylobacter jejuni Motility, Adhesion to Polystyrene Surfaces, and Invasion of INT407 Cells by Quorum-Sensing Inhibition.

Campylobacter jejuni is a major foodborne pathogen, and the LuxS-mediated quorum-sensing (QS) system influences its motility, biofilm formation, invasion, host colonization, and virulence. QS therefore represents a target for the control of C. jejuni. The aim of this study was to investigate the correlation of QS inhibition with changes in C. jejuni motility, adhesion to polystyrene surfaces, and adhesion to and invasion of INT407 cells. This was achieved by studying (i) the luxS-deficient mutant and (ii) treatment of C. jejuni with 20 natural extracts as six essential oils, 11 ethanolic extracts, and three pure compounds. Compared to the wild-type, the ΔluxS mutant showed decreased motility, adhesion to polystyrene surfaces, and invasion of INT407 cells. The anti-QS effects of the treatments (n = 15/20) were assayed using Vibrio harveyi BB170 bioluminescence. Moderate positive correlation was shown between C. jejuni QS reduction and reduced motility (τ = 0.492, p = 0.024), adhesion to polystyrene surfaces (τ = 0.419, p = 0.008), and invasion (r = 0.394, p = 0.068). The best overall effect was achieved with a Sedum rosea (roseroot) extract, with 96% QS reduction, a 1.41 log (96%) decrease in adhesion to polystyrene surfaces, and an 82% decrease in invasion. We show that natural extracts can reduce motility, adhesion to polystyrene surfaces, and invasion of INT407 cells by C. jejuni through modulation of the LuxS (QS) system.

DNA damage and survival in bystander human intestinal cells treated with conditioned medium from tritium-labeled cells

Background: Tritium exposure could be one of the radiation hazards in case of accidental exposure with intestine as one of the major target organs. In the cells, low-energy beta emitted from tritium would traverse a very short distance (a few microns). Hence, the intestinal epithelial cells with nuclear localization of tritium would exert its radiobiological effect also through bystander mechanism. In the present study, the effect of conditioned medium obtained from tritiated thymidine-labeled human normal intestinal epithelial (INT407) cells was studied on respective bystander cells in terms of magnitude of survival and induction of DNA damage. Materials and Methods: The survival and proliferation of bystander INT407 cells treated with control/irradiated conditioned medium were studied using clonogenic and 5-bromo-2-deoxyuridine (BrdU)-labeling assays. The magnitude of DNA double-strand break was measured by immunofluorescence of η-H2AX by confocal microscopy. Intracellular nitric oxide (NO) in these cells was measured using 4,5-diaminofluorescein diacetate fluorescent dye. Results: Bystander cells treated with conditioned medium from tritiated thymidine-labeled cells showed increased clonogenic survival and BrdU labeling. Cells labeled with tritiated thymidine showed attenuation of η-H2AX foci at longer period (24 and 48 h) of labeling than at 15 h. Moreover, the bystander cells treated with irradiated conditioned medium showed a higher magnitude of η-H2AX foci at 24 h. However, compared to 24 h, 48-h treatment of irradiated conditioned medium resulted in a decrease in η-H2AX foci in the bystander cells. Increased level of intracellular NO was observed in the bystander cells treated with irradiated conditioned medium. Conclusions: Bystander cells treated with conditioned medium obtained from tritiated thymidine-labeled cells showed increased clonogenic survival and proliferation, which was correlated with an increase in DNA double-strand break and NO production in these cells.

Protective effects of camellia oil (Camellia brevistyla) against indomethacin-induced gastrointestinal mucosal damage in vitro and in vivo

Abstract Accumulating evidence reveals that nonsteroidal anti-inflammatory drugs (NSAIDs), such as indomethacin, cause oxidative stress and inflammation, which consequently cause gastrointestinal (GI) mucosal damage. Camellia oil, a common edible oil used in Asia, has excellent antioxidative and anti-inflammatory characteristics. Herein, we examined the benefits and protective effects of camellia oil in indomethacin-induced human intestinal Int-407 cells and a mouse model of indomethacin-induced gastric mucosal damage. Camellia oil pretreatment significantly increased cell viability and wound healing and reduced reactive oxygen species production in indomethacin-induced Int-407 cells. In vivo experiments revealed that camellia oil preadministration prevented gastric wound generation by decreasing inflammatory mediators interleukin-6, tumor necrosis factor-α, and cyclooxygenase-2 levels; increasing heme oxygenase-1 antioxidant protein level; and elevating transforming growth factor-β and vascular endothelial growth factor levels in indomethacin-induced BALB/c mice. Thus, camellia oil is a functional dietary oil that prevents oxidative damage and inflammation in NSAID-induced GI mucosal damage.

Synbiotic-like effect of linoleic acid overproducing Lactobacillus casei with berry phenolic extracts against pathogenesis of enterohemorrhagic Escherichia coli

BackgroundMajority of enteric infections are foodborne and antimicrobials including antibiotics have been used for their control and treatment. However, probiotics or prebiotics or their combination offer a potential alternative intervention strategy for improving the host health and preventing foodborne pathogen colonization/infections in reservoir. Further, bioengineered probiotics expressing bioactive products to achieve specific function is highly desirable. Recently, we over-expressed mcra (myosin cross-reactive antigen) gene in Lactobacillus casei (Lc) and developed a bioengineered probiotics Lc + CLA which produce higher amounts of metabolites including conjugated linoleic acid (CLA). Furthermore, we also reported that prebiotic like components such as berry pomace (byproduct) phenolic extracts (BPEs) can enhance the growth of probiotics and improved the beneficial effects of probiotics. In this study, we evaluated the antimicrobial effect of modified Lc + CLA in combination of BPEs on growth, survival and pathogenesis of enterohemorrhagic Escherichia coli (EHEC).ResultsIn mixed culture condition, the growth of EHEC was significantly reduced in the presence Lc + CLA and/or BPEs. Cell-free cultural supernatant (CFCS) collected from Lc or Lc + CLA strain also inhibited the growth and survival of EHEC and the inhibitory effects of CFCSs against EHEC were enhanced in the presence of BPEs in concentration dependent manner. Interaction between EHEC and intestinal epithelial INT-407 cells were also altered significantly in the presence of either Lc or Lc + CLA strain or their CFCSs with or without BPEs. The expression of multiple virulence genes and physicochemical properties of EHEC were also altered when the bacterial cells were pretreated with CFCSs and/or BPEs.ConclusionsThese results showed that diet containing bioactive Lc + CLA and natural prebiotic like component such as BPEs might be an effective way to prevent foodborne infections with EHEC.

Antimicrobial Effect and Probiotic Potential of Phage Resistant Lactobacillus plantarum and its Interactions with Zoonotic Bacterial Pathogens.

Development of phage-resistant probiotic particularly Lactobacillus is an alternative approach to enhance their beneficial effects as in animal feed supplements. In this study, we developed phage-resistant Lactobacillus plantarum (LP+PR) mutant and compared their antimicrobial effects and probiotic potential against zoonotic bacterial pathogens including Salmonella enterica serovar Typhimurium, enterohemorrhagic Escherichia coli (EHEC), Staphylococcus aureus, and Listeria monocytogenes with phage-sensitive L. plantarum (LP) strain. LP+PR strain showed markedly higher growth rate than wild-type LP strain. In co-culture with LP+PR and in the presence of cell-free cultural supernatants (CFCSs) of LP+PR, the growth of S. Typhimurium, EHEC, S. aureus, and L. monocytogenes were reduced significantly (P < 0.05). The adhesion ability of LP+PR was slightly higher than the LP on human epithelial INT-407 cells. Most importantly, LP+PR strain significantly inhibited the adhesive and invasive abilities of all four zoonotic pathogens to INT-407 cells (P < 0.05). Moreover, real-time qPCR revealed that in the presence of LP+PR strain or its CFCSs, expression of virulence genes of these zoonotic bacterial pathogens were suppressed significantly (P < 0.05). These findings suggest that the LP+PR strain is capable of inhibiting major zoonotic bacterial pathogens efficiently and would be a potential candidate for industrial usage in animal production or fermentation.

In vitro model of postoncosphere development, and in vivo infection abilities of Taenia solium and Taenia saginata.

Taenia solium is known to cause human cysticercosis while T. saginata does not. Comparative in vitro and in vivo studies on the oncosphere and the postoncospheral (PO) forms of T. solium and T. saginata may help to elucidate why cysticercosis can occur from one and not the other. The aim of this study was to use in vitro culture assays and in vivo models to study the differences in the development of the T. solium and T. saginata oncosphere. Furthermore, this study aimed to evaluate the expression of cytokines and metalloproteinases (MMPs) in human peripheral blood mononuclear cells (PBMCs), which were stimulated by these oncospheres and PO antigens. T. solium and T. saginata activated oncospheres (AO) were cultured in INT-407 and HCT-8 intestinal cells for 180 days. The T. solium began to die while the T. saginata grew for 180 days and developed to cysticerci in INT-407 cells. Rats were inoculated intracranially with AO and PO forms of either T. saginata or T. solium. Rats infected with T. solium AO and PO forms developed neurocysticercosis (NCC), while those infected with the T. saginata did not. Human PMBCs were stimulated with antigens of AO and PO forms of both species, and the production of cytokines and metalloproteinases (MMPs) was measured. The T. solium AO antigen stimulated a higher production of IL-4, IL-5, IL-13, IFN-γ, and IL-2 cytokines compared to T. saginata AO. In the PO form, the T. saginata PO antigen increased the production of IL-4, IL-5, IL-13, IFN-γ, IL-1β, IL-6, IL-10, TNF-α and IL-12 cytokines compared to T. solium, suggesting that this global immune response stimulated by different forms could permit survival or destruction of the parasite depending of their life-cycle stage. Regarding MMPs, T. solium AO antigen stimulated a higher production of MMP-9 compared to T. saginata AO antigen, which may be responsible for altering the permeability of intestinal cells and facilitating breakdown of the blood-brain barrier during the process of invasion of host tissue.

Intestinal colonization and acute immune response in commercial turkeys following inoculation with Campylobacter jejuni constructs encoding antibiotic-resistance markers

Consumption of contaminated poultry products is one of the main sources of human campylobacteriosis, of which Campylobacter jejuni subsp. jejuni (C. jejuni) is responsible for approximately 90% of the cases. At slaughter, the ceca of commercial chickens and turkeys are the main anatomical site where C. jejuni asymptomatically colonizes. We have previously colonized commercial turkey poults with different isolates of C. jejuni and evaluated different media to best enumerate Campylobacter from intestinal samples, but the host-response is unknown in turkeys. Enumeration of Campylobacter (colony forming units (cfu)/gram of intestinal contents) can be challenging, and can be confounded if animals are colonized with multiple species of Campylobacter. In order to precisely enumerate the C. jejuni isolate used to experimentally colonize turkeys, constructs of C. jejuni (NCTC 11,168) were tagged with different antibiotic resistance markers at the CmeF locus (chloramphenicol (CjCm) or kanamycin (CjK)). We sought to examine the kinetics of intestinal colonization using the antibiotic resistant constructs, and characterize the immune response in cecal tissue of turkeys. In vitro analysis of the tagged antibiotic-resistant constructs demonstrated no changes in motility, morphology, or adherence and invasion of INT-407 cells compared to the parent isolate NCTC 11,168. Two animal experiments were completed to evaluate intestinal colonization by the constructs. In experiment 1, three-week old poults were colonized after oral gavage for 14 days, and CjCm and CjK cfu were recovered from cecal, but not ileal contents. In experiment 2, nine-week old poults were orally inoculated with CjCm, and the abundance of CjCm cfu/g of cecal contents significantly decreased beyond 14 days after inoculation. Significant lesions were detected in CjCm colonized poults at day 2 post-colonization. Using immunohistochemistry, Campylobacter antigen was detected in between cecal villi by day 7 of CjCm colonized poults. Quantitative RT-PCR of CjCm-colonized cecal tissue demonstrated significant down-regulation of IL-1β, IL-10 and IL-13 mRNA, and significant up-regulation of IL-6, IL-8, IL-17 A, IL-22 and IFNγ mRNA on day 2, and for some on day 7 post-colonization. All differentially expressed genes were similar to mock-infected poults by day 14. These data suggest that C. jejuni induced a brief inflammatory response in the cecum of poults that quickly resolved. Results from this study provide valuable insight into host-response and persistent colonization of the turkey cecum. These findings will help to develop and test strategies to promote food safety in commercial turkeys.

Protocatechuic acid-mediated DJ-1/PARK7 activation followed by PI3K/mTOR signaling pathway activation as a novel mechanism for protection against ketoprofen-induced oxidative damage in the gastrointestinal mucosa

Oxidative stress contributes to the progression of non-steroidal anti-inflammatory drug (NSAID)-induced gastrointestinal (GI) cell apoptosis. In our previous study, we reported that nuclear factor erythroid 2-related factor 2 (Nrf2) plays a protective role against ketoprofen-induced GI mucosal oxidative injury. Recent reports suggest that Nrf2 could exhibit antioxidative and antiapoptosis responses through up-regulation of DJ-1 (PARK7). In the current study, we proposed that induction of DJ-1 expression by protocatechuic acid (PCA) might provide a potential therapeutic approach for treating oxidative stress-associated GI ulcer diseases. The results indicated that PCA increased mRNA expression of glutathione peroxidase and heme oxygenase-1 through up-regulation of DJ-1 followed by Nrf2 translocation. Furthermore, PCA protected Int-407 cells against ketoprofen-induced oxidative stress by regulating the DJ-1, PI3K, and mTOR pathways. Pretreatment with PCA inhibited mitochondrial ROS generation, up-regulated the mitochondrial membrane potential, and down-regulated pro-apoptotic Bax as well as downstream caspase-8, caspase-9, and caspase-3 activity, and reversed impaired DJ-1 and anti-apoptotic Bcl-2 protein expression in Int-407 cells induced by ketoprofen. Similar to the in vitro results, SD rats treated with PCA before administration of ketoprofen exhibited decreased caspase-3 protein expression as well as oxidative damage, and impairment of the antioxidant system and DJ-1 protein expression in the GI mucosa were reversed. The administration of lansoprazole, a type of proton pump inhibitor (PPI), strongly inhibited ketoprofen-induced GI mucosal injuries via up-regulation of DJ-1, indicating that DJ-1 is essential for the dietary antioxidant- and PPI drug-mediated mechanism of ulcer therapy. These results suggest that DJ-1 could be a novel target for protection against ketoprofen-induced GI ulcers due to its antioxidant and anti-apoptosis characteristics.