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Search Results (16)

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Keywords = CagA antigen

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11 pages, 257 KiB  
Article
Association of Antibody Responses to Helicobacter pylori Proteins with Colorectal Adenoma and Colorectal Cancer
by Flavia Genua, Julia Butt, Harsha Ganesan, Tim Waterboer and David J. Hughes
Pathogens 2024, 13(10), 897; https://doi.org/10.3390/pathogens13100897 - 14 Oct 2024
Viewed by 335
Abstract
Helicobacter pylori (H. pylori) has been implicated in colorectal carcinogenesis. Here, the association of immune responses to bacterial exposure with advancing stages of colorectal neoplasia was assessed by multiplex serology. Immunoglobulin (Ig) A and G antibody responses to thirteen proteins of [...] Read more.
Helicobacter pylori (H. pylori) has been implicated in colorectal carcinogenesis. Here, the association of immune responses to bacterial exposure with advancing stages of colorectal neoplasia was assessed by multiplex serology. Immunoglobulin (Ig) A and G antibody responses to thirteen proteins of H. pylori were measured by a Luminex-based multiplex assay in plasma from patients with colorectal cancer (CRC, n = 25), advanced adenoma (n = 82), or small polyps (n = 85) and controls (n = 100). Multivariable logistic regression was used to assess the association of bacterial seropositivity with colorectal neoplasia. The threshold for overall seropositivity required subjects to be positive for at least 4 out of the 13 tested antigens. In a cohort subset with matched data (n = 34), H. pylori seropositivity was correlated with bacterial abundance in both neoplastic and matched normal tissue. While no association was found between H. pylori seropositivity and the presence of CRC, IgA seropositivity to CagA was associated with a decreased risk of advanced adenoma (odds ratio, OR = 0.48, 95% confidence intervals, CIs: 0.24–0.96). Regarding IgG, higher antibody responses to HpaA was associated with advanced adenoma occurrence (OR = 2.46, 95% CI: 1.00–6.01), while responses to HP0395, CagA and Catalase were associated with polyp development (OR = 2.65, 95%, CI: 1.31–5.36, OR = 1.83, 95% CI: 1.01–3.32, and OR = 2.16, CI: 1.09–4.29, respectively). Positive correlations were found between H. pylori abundance in the normal mucosa and levels of both the IgA and IgG antibody response to Catalase and VacA antigens (r = 0.48, p < 0.01; r = 0.37, p = 0.04; r = 0.51, p < 0.01; and r = 0.71, p = 0.04, respectively). Conversely, H. pylori abundance was negatively correlated with levels of IgA antibody response to HpaA and with IgG antibody response to HP0231 in the diseased tissue (r = −0.34, p = 0.04 and r = −0.41, p = 0.01, respectively). The association between levels of H. pylori antigens and colorectal neoplasia risk gradually decreased with the adenoma progression, implicating the early activation of the immune response at the polyp stage. Thus, the evaluation of antibody response to certain bacterial antigens may indicate the presence of early-stage colorectal neoplasia. Further studies are needed to clarify the role H. pylori or the immune response to its antigens may have in colorectal carcinogenesis stages. Full article
(This article belongs to the Section Bacterial Pathogens)
13 pages, 1635 KiB  
Article
Optical Biosensor Based on Porous Silicon and Tamm Plasmon Polariton for Detection of CagA Antigen of Helicobacter pylori
by Guoguang Rong, Alexey Kavokin and Mohamad Sawan
Sensors 2024, 24(16), 5153; https://doi.org/10.3390/s24165153 - 9 Aug 2024
Viewed by 716
Abstract
Helicobacter pylori (H. pylori) is a common pathogen with a high prevalence of infection in human populations. The diagnosis of H. pylori infection is critical for its treatment, eradication, and prognosis. Biosensors have been demonstrated to be powerful for the rapid [...] Read more.
Helicobacter pylori (H. pylori) is a common pathogen with a high prevalence of infection in human populations. The diagnosis of H. pylori infection is critical for its treatment, eradication, and prognosis. Biosensors have been demonstrated to be powerful for the rapid onsite detection of pathogens, particularly for point-of-care test (POCT) scenarios. In this work, we propose a novel optical biosensor, based on nanomaterial porous silicon (PSi) and photonic surface state Tamm Plasmon Polariton (TPP), for the detection of cytotoxin-associated antigen A (CagA) of H. pylori bacterium. We fabricated the PSi TPP biosensor, analyzed its optical characteristics, and demonstrated through experiments, with the sensing of the CagA antigen, that the TPP biosensor has a sensitivity of 100 pm/(ng/mL), a limit of detection of 0.05 ng/mL, and specificity in terms of positive-to-negative ratio that is greater than six. From these performance factors, it can be concluded that the TPP biosensor can serve as an effective tool for the diagnosis of H. pylori infection, either in analytical labs or in POCT applications. Full article
(This article belongs to the Special Issue Optical Biosensors and Applications)
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Figure 1

Figure 1
<p>The PSi TPP biosensor structure and its optical measurement configuration. The Au NPs embedded in the first LP PSi layer means that Au NP can infiltrate into the nanopores of porous silicon. The picture on the bottom left shows the setup of the 12 × 8 biosensor array and its sequential measurement by an in-house developed equipment mainly consisting of a two-dimensional moving stage to carry and position the sensor array and a fiber spectrometer to take the optical measurement.</p> Full article ">Figure 2
<p>(<b>a</b>) Cross-sectional scanning electron microscopy image of the TPP biosensor structure. The periodic layered structure of the porous silicon DBR is clearly visible. The gold thin film is the bright layer on top of the porous silicon DBR with the thickness not to scale due to a focusing issue; (<b>b</b>) atomic force microscopy image of a Au thin film surface morphology, showing the nanoporous structure of Au due to conformal deposition onto porous silicon; (<b>c</b>) Color map of electrical field strength distribution profile of TPP biosensor simulated by COMSOL V5.5. The field peak resides in the first (or top) PSi layer close to the thin metal film. Light is incident from the left side of the TPP device in air with a power of 1 W/m; (<b>d</b>) An example of the reflection spectrum of the TPP device is that of where the resonant state manifests as the reflection minimum at a wavelength of around 730 nm; (<b>e</b>) Example of the redshift of the TPP resonance wavelength upon the specific biomolecular binding of 3 ng/mL CagA antigen with the CagA antibody immobilized on the biosensor surface beforehand. The spectrum of the TPP biosensor both before (solid curve) and after (dashed curve) binding is shown. The redshift of the resonance minimum, indicated as arrows, is around 360 pm. (<b>f</b>) Example TPP resonance spectra upon exposure to PBS buffer with CagA antibody immobilized on the biosensor surface beforehand. The spectrum of the TPP biosensor both before (solid curve) and after (dashed curve) PBS is shown. The shift of resonance minimum, indicated as arrows, is 0 pm.</p> Full article ">Figure 3
<p>Response characterization of CagA antigen detection for detecting varying concentrations of CagA antigen in the PBS buffer, with both PSi TPP (black circles) and PSi DBR (grey circles) biosensors. Error bars on the experimental data points (solid circle) show a standard error from five experiments, with each experiment using a different biosensor. The linear fittings (dashed curves) are performed to match the data points. The linear equations go through the origin and the quality of the fitting is also given in the figure.</p> Full article ">Figure 4
<p>Specificity test and competitivity test of the PSi TPP biosensor with the objective of CagA antigen detection. CagA has a concentration of 1 ng/mL and all other nonspecific species concentrations of 5 ng/mL. Error bars on the experimental data bars (solid rectangle) show the standard error from five experiments, with each experiment using a different biosensor.</p> Full article ">
17 pages, 6741 KiB  
Article
Nanoscale CAR Organization at the Immune Synapse Correlates with CAR-T Effector Functions
by Julia Sajman, Oren Yakovian, Naamit Unger Deshet, Shaked Almog, Galit Horn, Tova Waks, Anat Globerson Levin and Eilon Sherman
Cells 2023, 12(18), 2261; https://doi.org/10.3390/cells12182261 - 12 Sep 2023
Cited by 4 | Viewed by 1714
Abstract
T cells expressing chimeric antigen receptors (CARs) are at the forefront of clinical treatment of cancers. Still, the nanoscale organization of CARs at the interface of CAR-Ts with target cells, which is essential for TCR-mediated T cell activation, remains poorly understood. Here, we [...] Read more.
T cells expressing chimeric antigen receptors (CARs) are at the forefront of clinical treatment of cancers. Still, the nanoscale organization of CARs at the interface of CAR-Ts with target cells, which is essential for TCR-mediated T cell activation, remains poorly understood. Here, we studied the nanoscale organization of CARs targeting CD138 proteoglycans in such fixed and live interfaces, generated optimally for single-molecule localization microscopy. CARs showed significant self-association in nanoclusters that was enhanced in interfaces with on-target cells (SKOV-3, CAG, FaDu) relative to negative cells (OVCAR-3). CARs also segregated more efficiently from the abundant membrane phosphatase CD45 in CAR-T cells forming such interfaces. CAR clustering and segregation from CD45 correlated with the effector functions of Ca++ influx and target cell killing. Our results shed new light on the nanoscale organization of CARs on the surfaces of CAR-Ts engaging on- and off-target cells, and its potential significance for CAR-Ts’ efficacy and safety. Full article
(This article belongs to the Special Issue T Cells: Differentiation and Function in Immunity and Disease)
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Figure 1

Figure 1
<p>CAR design and cell-on-cell interaction method. (<b>A</b>) A schematic description of the CD138-CAR design and CD45. (<b>B</b>) FACS analysis for CD138 expression in different cell lines (CAG, FaDu, SKOV-3 and OVCAR-3). (<b>C</b>) A schematic representation of the cell-on-cell imaging approach. (<b>D</b>–<b>F</b>) Confocal imaging of two cells labelled with membrane dyes: CD138-CAR-T cell with DiO (green) on a SKOV-3 adherent cell, labelled with DiD (red). (<b>D</b>) Z-stack image of cell on cell. (<b>E</b>) Side view of the 3D confocal image in panel (<b>D</b>) of the two cells. (<b>F</b>) 2D plane of interaction between the two cells in panels (<b>D</b>,<b>E</b>). Scale-bars in panels (<b>D</b>,<b>F</b>) 5 µm or in panel (<b>F</b>) 3 µm.</p> Full article ">Figure 2
<p>Two-color super resolution imaging on CD138-CAR-T upon interaction with target cell line. (<b>A</b>–<b>H</b>) Fluorescently labelled CD138-CAR-T cell on FaDu target cell. (<b>A</b>,<b>B</b>) Direct STORM (dSTORM) imaging of CD138-CAR-T cell on FaDu target cells. CD138 CAR-T was labelled with αSTREP tag-FITC (green) and αCD45-Alexa647 (red). Shown is an overlay of bright-field imaging merged with fluorescence two-color dSTORM imaging. (<b>B</b>) Zoom in on the fluorescent image of the CAR-T cell in panel (<b>A</b>). (<b>D</b>,<b>G</b>) Zoom in on the fluorescent image in two small areas (~4 µm<sup>2</sup>) inside the CAR-T cell in panel (<b>B</b>). (<b>E</b>,<b>F</b>) Pair-correlation function of CD138-CAR receptor (green) and CD45 molecules (red) within zoom regions in panel (<b>C</b>) (N = 4 regions), as represented in panels (<b>D</b>,<b>G</b>). (<b>G</b>,<b>H</b>) The extent of mixing (EOM) between CD138-CAR and CD45 within the zoom regions in panel (<b>C</b>) (N = 4 regions), as represented in panels (<b>D</b>,<b>G</b>). Scale-bars in panels A5 µm, in panel (<b>B</b>) 2 µm and in panels (<b>D</b>,<b>G</b>)—0.2 µm. Error bars are SEM. (<b>I</b>–<b>L</b>). Fluorescently labelled CD138-CAR-T intact cell, without a target cell. (<b>I</b>) Direct STORM (dSTORM) imaging of CD138-CAR-T intact cell, without a target cell on the bottom coverslip. CD138-CAR-T was labelled with αSTREP tag-FITC (green) and αCD45-Alexa647 (red). Bright-field imaging was merged with fluorescent two-color dSTORM imaging. (<b>J</b>) Fluorescent image only of the CAR-T cell in panel (<b>I</b>). (<b>K</b>) Pair-correlation function of CD138-CAR receptor (green) and CD45 molecules (red). (<b>L</b>) The extent of mixing (EOM) between CD138-CAR and CD45.</p> Full article ">Figure 3
<p>Segregation of CAR and CD45 on target cell lines (Fixed cells). (<b>A</b>–<b>D</b>) Fixed two-color dSTORM imaging of CD138-CAR-T cell on different cell lines: (<b>A</b>) OVCAR-3; (<b>B</b>) SKOV-3; (<b>C</b>) CAG; (<b>D</b>) FaDu. Visualizing CAR receptor (FITC-labeled, green) and CD45 molecules (Alexa647-labeled, red). Images from left to right: BF and fluorescence (left); fluorescence zoom image (right). (<b>E</b>–<b>H</b>) The average pair-correlation function of CD138-CAR molecules (green line) and CD45 molecules (red line) in T cell upon interaction with different cell lines: (<b>E</b>) OVCAR-3 (N = 36 cells); (<b>F</b>) SKOV-3 (N = 36); (<b>G</b>) CAG (N = 38); (<b>H</b>) FaDu (N = 27). (<b>I</b>–<b>L</b>) The extent of mixing (EOM) between CD138-CAR and CD45 upon interaction with different cell lines: (<b>I</b>) OVCAR-3 (N = 36); (<b>J</b>) SKOV-3 (N = 36); (<b>K</b>) CAG (N = 38); (<b>L</b>) FaDu (N = 27). For reference, purple arrowheads indicate EOM (20 nm) values of OVCAR-3. Scale-bars in panels (<b>A</b>–<b>D</b>)—5 µm. Error bars are SEM.</p> Full article ">Figure 4
<p>Segregation of CAR and CD45 on target cell lines (Live cells). (<b>A</b>–<b>D</b>) Live two-color dSTORM imaging of CD138-CAR-T cell on different cell lines: (<b>A</b>) OVCAR-3; (<b>B</b>) SKOV-3; (<b>C</b>) CAG; (<b>D</b>) FaDu. Visualizing CAR receptor (FITC-labeled, green) and CD45 molecules (Alexa647-labeled, red). Images from left to right: BF and fluorescence (left); fluorescence zoom image (right). (<b>E</b>–<b>H</b>) The average pair-correlation function of CD138-CAR molecules (green line) and CD45 molecules (red line) in T cell upon interaction with different cell lines: (<b>E</b>) OVCAR-3 (N = 22); (<b>F</b>) SKOV-3 (N = 13); (<b>G</b>) CAG (N = 25); (<b>H</b>) FaDu (N = 21). (<b>I</b>–<b>L</b>) The extent of mixing (EOM) between CD138-CAR and CD45 upon interaction with different cell lines: (<b>I</b>) OVCAR-3 (N = 22); (<b>J</b>) SKOV-3 (N = 13); (<b>K</b>) CAG (N = 25); (<b>L</b>) FaDu (N = 21). For reference, purple arrowheads indicate EOM (20 nm) values of OVCAR-3. Scale-bars in panels (<b>A</b>–<b>D</b>)—5 µm. Error bars are SEM.</p> Full article ">Figure 5
<p>Extent of mixing and CAR self-clustering comparing in target vs. non-target cell lines. (<b>A</b>) A comparison of CD138-CAR self-clustering upon cell-on-cell interaction. Table on right summarizes <span class="html-italic">p</span>-values between target and non-target cells. (<b>B</b>,<b>C</b>) A comparison of EOM values on different cell lines at 20 nm and 200 nm in images of (<b>A</b>) fixed cells and (<b>B</b>) live cells. Error bars are SEM. Tables on right summarize <span class="html-italic">p</span>-values between target and non-target cells. Stars indicate <span class="html-italic">p</span>-values * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001. NS—non-significant.</p> Full article ">Figure 6
<p>Early and late effector functions of CAR-T cell interaction with target cell lines. (<b>A</b>) Ca<sup>++</sup> influx in CAR-T cells meeting different cell lines. Upper panel: Frames from movie of CD138-CAR-T round cells meeting CAG target adherent cell. In green Ca<sup>++</sup> indicator. Lower panel: Mean fluorescence intensity 4 min after introduction CAR-T cells to target cell lines. Tables at right summarize <span class="html-italic">p</span>-values between target and non-target cells. (<b>B</b>) Bright-field images of cell lines’ viability after CD138-CAR-T cell introduction for 24 h and 48 h. White arrowheads point to representative cell aggregates. (<b>C</b>) Analysis of the fraction of cells in aggregates for all cells and conditions in panel (<b>B</b>). Cell aggregation indicates CAR-T-mediated killing of the specified cell lines. (<b>D</b>) MTT cytotoxicity assay of CAR-Ts engaging target/non-target cell lines. Scale-bars in panel (<b>A</b>)—5 µm, in panel (<b>B</b>)—10 µm. Error bars are SEM. Three stars indicate <span class="html-italic">p</span>-values &lt; 0.001.</p> Full article ">Figure 7
<p>Correlation between normalized cell parameters and relative ligand expression. A plot of normalized cell parameters as a function of relative ligand expression (percent positive cells, measured by FACS). The parameters include: Segregation (i.e., 1-EOM) at 20 nm (calculated from <a href="#cells-12-02261-f005" class="html-fig">Figure 5</a>B); Segregation at 200 nm (calculated from <a href="#cells-12-02261-f005" class="html-fig">Figure 5</a>B); Self-clustering at 20 nm (<a href="#cells-12-02261-f005" class="html-fig">Figure 5</a>A); Ca<sup>++</sup> influx (<a href="#cells-12-02261-f006" class="html-fig">Figure 6</a>A); Excess killing (i.e., killing extent of cell line—killing of OVCAR-3, calculated from the MTT assay in <a href="#cells-12-02261-f006" class="html-fig">Figure 6</a>D). Error bars are SEM.</p> Full article ">
20 pages, 6770 KiB  
Article
Fabrication of a Molecularly Imprinted Nano-Interface-Based Electrochemical Biosensor for the Detection of CagA Virulence Factors of H. pylori
by Kirti Saxena, Bayu Tri Murti, Po-Kang Yang, Bansi Dhar Malhotra, Nidhi Chauhan and Utkarsh Jain
Biosensors 2022, 12(12), 1066; https://doi.org/10.3390/bios12121066 - 23 Nov 2022
Cited by 9 | Viewed by 2836
Abstract
H. pylori is responsible for several stomach-related diseases including gastric cancer. The main virulence factor responsible for its establishment in human gastric cells is known as CagA. Therefore, in this study, we have fabricated a highly sensitive MIP-based electrochemical biosensor for the detection [...] Read more.
H. pylori is responsible for several stomach-related diseases including gastric cancer. The main virulence factor responsible for its establishment in human gastric cells is known as CagA. Therefore, in this study, we have fabricated a highly sensitive MIP-based electrochemical biosensor for the detection of CagA. For this, an rGO and gold-coated, screen-printed electrode sensing platform was designed to provide a surface for the immobilization of a CagA-specific, molecularly imprinted polymer; then it was characterized electrochemically. Interestingly, molecular dynamics simulations were studied to optimize the MIP prepolymerization system, resulting in a well-matched, optimized molar ratio within the experiment. A low binding energy upon template removal indicates the capability of MIP to recognize the CagA antigen through a strong binding affinity. Under the optimized electrochemical experimental conditions, the fabricated CagA-MIP/Au/rGO@SPE sensor exhibited high sensitivity (0.275 µA ng−1 mL−1) and a very low limit of detection (0.05 ng mL−1) in a linear range of 0.05–50 ng mL−1. The influence of other possible interferents in analytical response has also been observed with the successful determination of the CagA antigen. Full article
(This article belongs to the Special Issue Advanced Functional Materials for Electrochemical Sensor and Biosensor Applications)
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Figure 1

Figure 1
<p>Chemical structures of MIP optimization components including (<b>a</b>) CagA, (<b>b</b>) MMA, (<b>c</b>) EGDMA, and (<b>d</b>) ACN. CagA was represented in the ribbon diagram, while other molecules were displayed in the ball and stick model.</p> Full article ">Figure 2
<p>(<b>a</b>) Determination of the size of gold nanoparticles via the DLS technique. (<b>b</b>) Surface characterization of synthesized MIP with CagA template. (<b>c</b>) Surface characterization of synthesized MIP after removal of the CagA template. (<b>d</b>) BET isotherm; adsorption and desorption curve. (<b>e</b>) Analysis of pore size distribution for MIP by BET.</p> Full article ">Figure 2 Cont.
<p>(<b>a</b>) Determination of the size of gold nanoparticles via the DLS technique. (<b>b</b>) Surface characterization of synthesized MIP with CagA template. (<b>c</b>) Surface characterization of synthesized MIP after removal of the CagA template. (<b>d</b>) BET isotherm; adsorption and desorption curve. (<b>e</b>) Analysis of pore size distribution for MIP by BET.</p> Full article ">Figure 2 Cont.
<p>(<b>a</b>) Determination of the size of gold nanoparticles via the DLS technique. (<b>b</b>) Surface characterization of synthesized MIP with CagA template. (<b>c</b>) Surface characterization of synthesized MIP after removal of the CagA template. (<b>d</b>) BET isotherm; adsorption and desorption curve. (<b>e</b>) Analysis of pore size distribution for MIP by BET.</p> Full article ">Figure 3
<p>(<b>a</b>) Electrostatic potential, (<b>b</b>) HOMO, and (<b>c</b>) LUMO isosurfaces of MMA after geometry optimization using the B3LYP hybrid functional. (<b>d</b>) Optimized structure, (<b>e</b>) LUMO, and (<b>f</b>) HOMO isosurfaces of CagA after geometry optimization with DFTB+ with 3ob Slater−Koster library.</p> Full article ">Figure 4
<p>Chemical structures of (<b>a</b>) MMA and (<b>b</b>) tryptophan of CagA along with their labelled atoms for RDF analysis.</p> Full article ">Figure 5
<p>RDFs of (<b>a</b>) TH−MO1 and (<b>b</b>) TH−MO2. g(r) shows the RDF values, while r(Å) represents the distance from a solute atom and the average bulk atom.</p> Full article ">Figure 6
<p>(<b>a</b>) Electrochemical response studies via the CV technique for Bare SPE (black), rGO@SPE (red), AuNPs/rGO@SPE (blue), MIP unwashed/AuNPs/rGO@SPE (green), and MIP washed/AuNPs/rGO@SPE (magenta) obtained in the presence of [Fe(CN)<sub>6</sub>]<sup>3−/4−</sup> electrolyte at 50 mV/s. (<b>b</b>) The EIS study of Bare SPE (black), rGO@SPE (red), AuNPs/rGO@SPE (blue), MIP unwashed/AuNPs/rGO@SPE (magenta), and MIP washed/AuNPs/rGO@SPE (green) at the frequency range of 10–2000 Hz.</p> Full article ">Figure 7
<p>Effect of various pH on the analytical performance of the MIP-based electrochemical biosensor.</p> Full article ">Figure 8
<p>Determination of optimum temperature for the fabricated sensing platform.</p> Full article ">Figure 9
<p>(<b>a</b>) DPV response study of MIP-CagA/Au/rGO@SPE with different concentrations of CagA antigen in the presence of [Fe(CN)<sub>6</sub>]<sup>3−/4−</sup> electrolytes. (<b>b</b>) Representation of the standard calibration curve of MIP-CagA/Au/rGO@SPE with different concentrations of target analytes with reference to DPV results.</p> Full article ">Figure 10
<p>(<b>a</b>) CV recorded for MIP-CagA/Au/rGO@SPE at different scan rates in the presence of [Fe(CN)<sub>6</sub>]<sup>3−/4−</sup> electrolytes. (<b>b</b>) Representation of variations in the peak oxidation current (Ipa) and reduction peak current (Ipc) at different scan rates than the linear graph.</p> Full article ">Figure 10 Cont.
<p>(<b>a</b>) CV recorded for MIP-CagA/Au/rGO@SPE at different scan rates in the presence of [Fe(CN)<sub>6</sub>]<sup>3−/4−</sup> electrolytes. (<b>b</b>) Representation of variations in the peak oxidation current (Ipa) and reduction peak current (Ipc) at different scan rates than the linear graph.</p> Full article ">Figure 11
<p>Plot of interferent studies of MIP-CagA/Au/rGO@SPE with 0.05 ng/mL CagA antigens.</p> Full article ">Figure 12
<p>Demonstration of the fabricated sensing electrode’s stability over the period of 60 days.</p> Full article ">Scheme 1
<p>Graphical representation of the fabrication steps involved in the development of the MIP-based biosensor for the detection of the CagA virulence factor of <span class="html-italic">H. pylori</span>.</p> Full article ">
12 pages, 2767 KiB  
Article
Potential Antigenic Candidates for the Development of Peptide-Based Vaccines to Induce Immunization against Helicobacter pylori Infection in BALB/c Mice
by Doaa M. AlEraky, Hatem M. Abuohashish, Amr S. Bugshan, Maha M. Abdelsalam, Hussain A. AlHawaj, Taleb T. AlKhamis, Fatimah A. AlDossary, Nabras M. Alrayes, Yasser M. Ragab, Zeinab AbdelKhalek, Omneya M. Helmy and Mohammed A. Ramadan
Int. J. Mol. Sci. 2022, 23(21), 12824; https://doi.org/10.3390/ijms232112824 - 24 Oct 2022
Cited by 2 | Viewed by 2496
Abstract
Helicobacter pylori (H. pylori) has been identified as a group-1 definite carcinogen. As of yet, there is no available vaccine for this microorganism. Our study aimed to identify antigenic peptides in H. pylori using an in silico proteomic approach, and to [...] Read more.
Helicobacter pylori (H. pylori) has been identified as a group-1 definite carcinogen. As of yet, there is no available vaccine for this microorganism. Our study aimed to identify antigenic peptides in H. pylori using an in silico proteomic approach, and to evaluate their effectiveness as potential vaccine candidates. Four different peptide sequences were prioritized using the reverse vaccinology, namely, CagA1, CagA2, VacA, and SabA. Peptides emulsified with Freunde’s adjuvant were used to immunize BALB/C mice. Subcutaneously immunized mice were challenged by oral administration of H. pylori. IgG, IgA, IL4, and IL17 were detected in mice sera. Histopathology of the dissected stomach of vaccinated and control mice were assessed using H&E stain. IgG was significantly higher in mice vaccinated with SabA. IL-4 was significantly increased in CagA1, CagA2, VacA, and SabA vaccinated mice compared to the adjuvant group. Additionally, histopathological examination of gastric tissue showed a protective effect in the vaccinated groups compared to adjuvant and PBS groups. Our findings indicate a promising effect of the tested epitopes, particularly the SabA antigen, to induce an immune response against H. pylori. Full article
(This article belongs to the Special Issue Advances in Antibody Design and Antigenic Peptide Targeting 2.0)
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Figure 1
<p>The results of subcellular localization of the predicted proteins of <span class="html-italic">H. pylori</span> strain 266595 by the protein localization prediction PSORTb server. Out of a total of 1445 proteins, the exact data reported at the PSORTdb server were 766 cytoplasmic (54.6%), 284 cytoplasmic membrane (20.2%), 21 extracellular (1.5%), 38 outer membrane (2.7%), 15 periplasmic membrane (1.1%), and 280 unknown proteins (19.9%). The predicted extracellular and outer membrane proteins were included in the prioritized dataset.</p> Full article ">Figure 2
<p>Effects of different vaccines (V1, V2, V3, and V4) on serum levels of immunoglobulin-G (IgG) and immunoglobulin-A (IgA) in BALB/c mice challenged with <span class="html-italic">H. pylori</span>. Data are presented as mean ± SD (<span class="html-italic">n</span> = 6) and statistically analyzed by one way ANOVA, followed by the Tukey–Kramer post hoc test. Statistical significance was considered when * <span class="html-italic">p</span> ≤ 0.05 and ** <span class="html-italic">p</span> ≤ 0.01. “a” vs. PBS group and “b” vs. adjuvant group.</p> Full article ">Figure 3
<p>Effects of different vaccines (V1, V2, V3, and V4) on serum levels of interleukin-4 (IL-4), interleukin-17 (IL-17), and their ratio in BALB/c mice challenged with <span class="html-italic">H. pylori</span>. Data are presented as mean ± SD (<span class="html-italic">n</span> = 6) and statistically analyzed by one way ANOVA, followed by the Tukey–Kramer post hoc test. Statistical significance was considered when * <span class="html-italic">p</span> ≤ 0.05 and ** <span class="html-italic">p</span> ≤ 0.01. “a” vs. PBS group and “b” vs. adjuvant group.</p> Full article ">Figure 4
<p>Photomicrographs of hematoxylin and eosin (H&amp;E)-stained sections of stomach and proximal duodenum of mice challenged with <span class="html-italic">H. pylori</span>. Images were taken at 20× (non-bordered images) and 40× (black bordered images) magnifications. Histopathology of stomach tissues from the vaccinated groups (V1, V2, V3, and V4) exhibited less inflammation to patchy infiltration of mixed leukocytes in the mucosa and submucosa (black arrows). Histopathology of gastric tissues of the adjuvant and PBS groups showed a marked increase in leukocyte infiltration extending below the submucosa (black arrows).</p> Full article ">Figure 5
<p>Histological scores of specimens from different vaccinated (V1, V2, V3, and V4) and unvaccinated (adjuvant and PBS) groups. Data are presented as mean ± SD and statistically analyzed by one way ANOVA, followed by the Tukey–Kramer post hoc test. Statistical significance was considered when * <span class="html-italic">p</span> ≤ 0.05 and ** <span class="html-italic">p</span> ≤ 0.01. “a” vs. PBS group and “b” vs. adjuvant group.</p> Full article ">Figure 6
<p>The main software used in the immunoinformatic analysis approach to shortlist the potential extracellular/outer membrane proteins, followed by an epitope-based analysis to predict antigenicity and to identify potential B-cell and T-cell epitope candidates.</p> Full article ">
17 pages, 2729 KiB  
Article
Accumulation of Deleterious Effects in Gastric Epithelial Cells and Vascular Endothelial Cells In Vitro in the Milieu of Helicobacter pylori Components, 7-Ketocholesterol and Acetylsalicylic Acid
by Adrian Ł. Gajewski, Mateusz Gawrysiak, Agnieszka Krupa, Tomasz Rechciński, Maciej Chałubiński, Weronika Gonciarz and Magdalena Chmiela
Int. J. Mol. Sci. 2022, 23(11), 6355; https://doi.org/10.3390/ijms23116355 - 6 Jun 2022
Cited by 8 | Viewed by 2811
Abstract
The Gastric pathogen Helicobacter pylori (HP) may influence the development of coronary heart disease (CHD). H. pylori induce reactive oxygen species (ROS), which transform cholesterol to 7-ketocholesterol (7-kCh), a CHD risk factor. Acetylsalicylic acid (ASA)—an Anti-aggregation drug used in CHD patients—may [...] Read more.
The Gastric pathogen Helicobacter pylori (HP) may influence the development of coronary heart disease (CHD). H. pylori induce reactive oxygen species (ROS), which transform cholesterol to 7-ketocholesterol (7-kCh), a CHD risk factor. Acetylsalicylic acid (ASA)—an Anti-aggregation drug used in CHD patients—may increase gastric bleeding and inflammation. We examined whether H. pylori driven ROS effects in the cell cultures of gastric epithelial cells (AGS) and vascular endothelial cells (HUVEC) progress in the milieu of 7-kCh and ASA. Cell cultures, exposed to 7-kCh or ASA alone or pulsed with the H. pylori antigenic complex—Glycine acid extract (GE), urease (UreA), cytotoxin associated gene A (CagA) protein or lipopolysaccharide (LPS), alone or with 7-kCh and ASA—were examined for ROS, apoptosis, cell integrity, interleukin (IL)-8, the activation of signal transducer, the activator of transcription 3 (STAT3), and wound healing. ASA and 7-kCh alone, and particularly in conjunction with H. pylori components, increased the ROS level and the rate of apoptosis, which was followed by cell disintegration, the activation of STAT3, and IL-8 elevation. AGS cells were unable to undergo wound healing. The cell ROS response to H. pylori components may be elevated by 7-kCh and ASA. Full article
(This article belongs to the Special Issue Helicobacter: Infection, Diagnosis and Treatment 2.0)
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<p>Reactive oxygen species. For the estimation of reactive oxygen species (ROS), the cell suspensions of gastric epithelial AGS cells (<b>A</b>) and vascular endothelial HUVEC cells (<b>B</b>) were treated for 24 h with <span class="html-italic">H. pylori</span> compounds: glycine acid extract—GE, subunit A of urease—UreA, cytotoxin associated gene A (CagA) protein, <span class="html-italic">H. pylori</span> lipopolysaccharide (LPS) or <span class="html-italic">E. coli</span> LPS, alone or in combination with acetylsalicylic acid—ASA, and 7-ketocholesterol—7-kCh, or in medium alone. The fluorescent probe—Dihydroetidine (DHE) was added to the wells, and fluorescence was measured. The ROS ratio was calculated based on relative fluorescence units (RFU) of stimulated cells vs. RFU of control cells in culture medium alone. Results are shown as means with standard deviations (SD) of five experiments performed in triplicates for each experimental variant. Statistical significance for * <span class="html-italic">p</span> &lt; 0.05.</p> Full article ">Figure 2
<p>Assessment of cell viability. Gastric epithelial AGS cells (<b>A</b>) or vascular endothelial HUVEC cells (<b>B</b>) were sub-cultured for 24 h in the culture medium alone or exposed to <span class="html-italic">H. pylori</span> components: glycine acid extract—GE, subunit A of urease—UreA, cytotoxin associated gene A (CagA) protein, <span class="html-italic">H. pylori</span> lipopolysaccharide (LPS) or <span class="html-italic">E. coli</span> LPS alone or with acetylsalicylic acid—ASA, and 7-ketocholesterol—7-kCh. Cell viability was evaluated using the tetrazolium yellow dye MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide], which was reduced by living cells to yield soluble purple formazan crystals that were solubilized and detected colorimetrically. Results are presented as the percentage means ± standard deviation (SD) relative to untreated cells of at least four independent experiments performed in triplicates for each experimental variant. Statistical significance for * <span class="html-italic">p</span> &lt; 0.05.</p> Full article ">Figure 3
<p>Assessment of cell apoptosis. Gastric epithelial AGS cells (<b>A</b>,<b>B</b>) or vascular endothelial HUVEC cells (<b>C</b>,<b>D</b>)) were sub-cultured in the culture medium alone or in the milieu of <span class="html-italic">H. pylori</span> components: glycine acid extract—GE, subunit A of urease—UreA, cytotoxin associated gene A (CagA) protein, <span class="html-italic">H. pylori</span> lipopolysaccharide (LPS) or <span class="html-italic">E. coli</span> LPS alone or with acetylsalicylic acid—ASA and 7-ketocholesterol—7-kCh. The intensity of AGS and HUVEC cell apoptosis was evaluated using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Cell nuclei were counterstained with Hoechst. Cells with apoptotic changes (red) were imaged in the fluorescence microscope at magnification ×20. (<b>A</b>,<b>C</b>) The graphs indicate the percentage of apoptotic cells. (<b>B</b>,<b>D</b>) Representative images of cells stained in the TUNEL assay (red), at magnification ×20. The results of four independent experiments performed in triplicates for each experimental variant are presented. Statistical significance for * <span class="html-italic">p</span> &lt; 0.05.</p> Full article ">Figure 4
<p>Permeability of cell monolayers—Paracellular flux assay. Gastric epithelial AGS cells (<b>A</b>) and vascular endothelial HUVEC cells (<b>B</b>) were cultured in the transwell system until they reached the confluence and then were treated with <span class="html-italic">H. pylori</span> components: glycine acid extract—GE, subunit A of urease—UreA, cytotoxin associated gene A (CagA) protein, <span class="html-italic">H. pylori</span> lipopolysaccharide (LPS) or <span class="html-italic">E. coli</span> LPS alone or with acetylsalicylic acid-ASA and 7-ketocholesterol—7-kCh. Thereafter, isothiocyanate fluorescein (FITC) dextran was added to the medium in the insert. The fluorescence of the FITC-dextran in the lower compartment was measured. The fluorescence intensity is shown as relative fluorescence units (RFU) after 120 min incubation of cells with dextran-FITC (<b>A</b>,<b>B</b> upper graphs) or at time 0 and after 30, 60, 90, 120, and 150 min of incubation (<b>C</b>,<b>D</b> lower graphs). Results are shown as means with standard deviation (SD) of four experiments performed in triplicates for each experimental variant. Statistical significance for * <span class="html-italic">p</span> &lt; 0.05.</p> Full article ">Figure 5
<p>Effects of ASA and/or 7-kCh on gastric epithelial cells (AGS). AGS cells were treated for 24 h with acetylsalicylic acid (ASA) and/or 7-ketocholesterol (7-kCh), and then examined for: (<b>A</b>) reactive oxygen species (ROS); (<b>B</b>) cell viability; (<b>C</b>) permeability of cell monolayers for fluorescein isothiocyanate (FITC) bound dextran, expressed as relative fluorescence units (RFU); (<b>D</b>) the production of interleukin (IL)-8; (<b>E</b>) phosphorylation of signal transducer and activator of transcription 3 (STAT3). Progress of wound healing (<b>F</b>,<b>G</b>), was assessed in scratch assay. Results are shown as means with standard deviation (SD) of four experiments performed in triplicates for each experimental variant. Statistical significance for * <span class="html-italic">p</span> &lt; 0.005. Cells in complete medium were used as control for natural cells. Cells treated with H<sub>2</sub>O<sub>2</sub> (0.06%) were used as negative control in cell viability assay and paracellular flux assay.</p> Full article ">Figure 6
<p>Effects of ASA and/or 7-kCh on vascular endothelial cells (HUVEC). HUVEC cells were treated 24 h with acetylsalicylic acid (ASA) and/or 7-ketocholesterol (7-kCh), and then examined for: (<b>A</b>) reactive oxygen species (ROS); (<b>B</b>) cell viability; (<b>C</b>) permeability of cell monolayers for fluorescein isothiocyanate (FITC) bound dextran, expressed as relative fluorescence units (RFU); (<b>D</b>) the production of interleukin (IL)-8; (<b>E</b>) phosphorylation of signal transducer and activator of transcription 3 (STAT3). Results are shown as means with standard deviation (SD) of four experiments performed in triplicates for each experimental variant. Statistical significance for * <span class="html-italic">p</span> &lt; 0.005. RFU—Relative fluorescence units. Cells in complete culture medium (medium) were used as control for natural cells. Cells treated with H<sub>2</sub>O<sub>2</sub> (0.06%) were used as negative control in cell viability assay and paracellular flux assay.</p> Full article ">
13 pages, 7453 KiB  
Article
Targeting Cytotoxin-Associated Antigen A, a Virulent Factor of Helicobacter pylori-Associated Gastric Cancer: Structure-Based In Silico Screening of Natural Compounds
by Shan He, Abdulraheem Ali Almalki, Misbahuddin M. Rafeeq, Ziaullah M. Sain, Amany I. Alqosaibi, Mashael M. Alnamshan, Ibtesam S. Al-Dhuayan, Abdul Rahaman, Yang Zhang, Hamsa Jameel Banjer, Farah Anjum, Haitham Ali M. Alzghaibi, Ali H. Alharbi and Qazi Mohammad Sajid Jamal
Molecules 2022, 27(3), 732; https://doi.org/10.3390/molecules27030732 - 23 Jan 2022
Cited by 5 | Viewed by 3718
Abstract
Gastric cancer is the fifth most frequent cancer and the third major cause of mortality worldwide. Helicobacter pylori, a bacterial infection linked with GC, injects the cytotoxin-associated antigen A (CagA; an oncoprotein) into host cells. When the phosphorylated CagA protein enters the [...] Read more.
Gastric cancer is the fifth most frequent cancer and the third major cause of mortality worldwide. Helicobacter pylori, a bacterial infection linked with GC, injects the cytotoxin-associated antigen A (CagA; an oncoprotein) into host cells. When the phosphorylated CagA protein enters the cell, it attaches to other cellular components, interfering with normal cellular signaling pathways. CagA plays an important role in the progression of GC by interacting with phosphatidylserine of the host cell membrane. Therefore, disrupting the CagA–phosphatidylserine connection using small molecules appears to be a promising therapeutic approach. In this report, we screened the natural compounds from ZINC database against the CagA protein using the bioinformatics tools. Hits were initially chosen based on their physicochemical, absorption, distribution, metabolism, excretion, and toxicity (ADMET) characteristics, as well as other drug-like characteristics. To locate safe and effective hits, the PAINS filter, binding affinities estimation, and interaction analysis were used. Three compounds with high binding affinity and specificity for the CagA binding pocket were discovered. The final hits, ZINC153731, ZINC69482055, and ZINC164387, were found to bind strongly with CagA protein, with binding energies of −11.53, −10.67, and −9.21 kcal/mol, respectively, which were higher than that of the control compound (−7.25 kcal/mol). Further, based on binding affinity and interaction pattern, two leads (ZINC153731, ZINC69482055) were chosen for molecular dynamics (MD) simulation analysis. MD results showed that they displayed stability in their vicinity at 100 ns. This study suggested that these compounds could be used as possible inhibitors of CagA protein in the fight against GC. However, additional benchwork tests are required to validate them as CagA protein inhibitors. Full article
(This article belongs to the Special Issue Bioactivities and In Silico Study of Phytochemicals)
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<p>Visualization and surface view of ZINC153731 in the active site of CagA protein (<b>a</b>,<b>b</b>). Three-dimensional (<b>c</b>) and 2D (<b>d</b>) view of CagA residue interacting with ZINC153731.</p> Full article ">Figure 2
<p>Visualization and surface view of ZINC69482055 in active site of CagA protein (<b>a</b>,<b>b</b>). Three-dimensional (<b>c</b>) and 2D (<b>d</b>) view of CagA residue interacting with ZINC69482055.</p> Full article ">Figure 3
<p>Visualization and surface view of ZINC164387 in active site of CagA protein (<b>a</b>,<b>b</b>). Three-dimensional (<b>c</b>) and 2D (<b>d</b>) view of CagA residue interacting with ZINC164387.</p> Full article ">Figure 4
<p>Visualization and surface view of DFMO in active site of CagA protein (<b>a</b>,<b>b</b>). Three-dimensional (<b>c</b>) and 2D (<b>d</b>) view of CagA residue interacting with DFMO.</p> Full article ">Figure 5
<p>(<b>a</b>) RMSD of CagA, (<b>b</b>) RMSD of ligand in the pocket, (<b>c</b>) RMSF.</p> Full article ">Figure 6
<p>(<b>a</b>) Rg, (<b>b</b>) SASA, and (<b>c</b>) solvent free energy.</p> Full article ">Figure 7
<p>(<b>a</b>) Secondary structure changes upon ligand binding, (<b>b</b>–<b>d</b>) hydrogen bond analysis of complexes.</p> Full article ">Figure 8
<p>The 2D projection of complexes. (<b>a</b>) CagA-free, CagA-ZINC153731, and CagA-ZINC69482055, (<b>b</b>) Eigenvector components, (<b>c</b>) RMS fluctuation.</p> Full article ">Figure 9
<p>GFE landscape of complexes.</p> Full article ">
17 pages, 2479 KiB  
Article
Hesperetin Inhibits Expression of Virulence Factors and Growth of Helicobacter pylori
by Hyun Woo Kim, Hyun Jun Woo, Ji Yeong Yang, Jong-Bae Kim and Sa-Hyun Kim
Int. J. Mol. Sci. 2021, 22(18), 10035; https://doi.org/10.3390/ijms221810035 - 17 Sep 2021
Cited by 17 | Viewed by 3338
Abstract
Helicobacter pylori (H. pylori) is a bacterium known to infect the human stomach. It can cause various gastrointestinal diseases including gastritis and gastric cancer. Hesperetin is a major flavanone component contained in citrus fruits. It has been reported to possess antibacterial, [...] Read more.
Helicobacter pylori (H. pylori) is a bacterium known to infect the human stomach. It can cause various gastrointestinal diseases including gastritis and gastric cancer. Hesperetin is a major flavanone component contained in citrus fruits. It has been reported to possess antibacterial, antioxidant, and anticancer effects. However, the antibacterial mechanism of hesperetin against H. pylori has not been reported yet. Therefore, the objective of this study was to determine the inhibitory effects of hesperetin on H. pylori growth and its inhibitory mechanisms. The results of this study showed that hesperetin inhibits the growth of H. pylori reference strains and clinical isolates. Hesperetin inhibits the expression of genes in replication (dnaE, dnaN, dnaQ, and holB) and transcription (rpoA, rpoB, rpoD, and rpoN) machineries of H. pylori. Hesperetin also inhibits the expression of genes related to H. pylori motility (flhA, flaA, and flgE) and adhesion (sabA, alpA, alpB, hpaA, and hopZ). It also inhibits the expression of urease. Hespereti n downregulates major virulence factors such as cytotoxin-associated antigen A (CagA) and vacuolating cytotoxin A (VacA) and decreases the translocation of CagA and VacA proteins into gastric adenocarcinoma (AGS) cells. These results might be due to decreased expression of the type IV secretion system (T4SS) and type V secretion system (T5SS) involved in translocation of CagA and VacA, respectively. The results of this study indicate that hesperetin has antibacterial effects against H. pylori. Thus, hesperetin might be an effective natural product for the eradication of H. pylori. Full article
(This article belongs to the Section Bioactives and Nutraceuticals)
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<p>Antibacterial activity of hesperetin against <span class="html-italic">H. pylori</span>. (<b>A</b>) Chemical structure of hesperetin. (<b>B</b>) Minimal inhibitory concentrations of hesperetin against five <span class="html-italic">H. pylori</span> reference strains (ATCC 49503, ATCC 43504, ATCC 51932, ATCC 700392, and SS1) were determined by the agar dilution method. (<b>C</b>) The minimal inhibitory concentration of hesperetin against <span class="html-italic">H. pylori</span> ATCC 49503 strain was confirmed by the broth dilution method. Data are presented as mean ± SEM. Results from triplicate experiments were analyzed by Student’s <span class="html-italic">t</span>-test (* <span class="html-italic">p</span> &lt; 0.05 and *** <span class="html-italic">p</span> &lt; 0.001).</p> Full article ">Figure 2
<p>Downregulation of replication and transcription genes of <span class="html-italic">H. pylori</span>. RNA was subjected to RT-PCR to detect mRNA expression levels of genes of (<b>A</b>) the replication machinery (<span class="html-italic">dna</span>A, <span class="html-italic">dna</span>B, <span class="html-italic">dna</span>E, <span class="html-italic">dna</span>N, <span class="html-italic">dna</span>Q, <span class="html-italic">pol</span>A, and <span class="html-italic">hol</span>B) and (<b>B</b>) the transcription machinery (<span class="html-italic">rpo</span>A, <span class="html-italic">rpo</span>B, <span class="html-italic">rpo</span>D, and <span class="html-italic">rpo</span>N). The expression of <span class="html-italic">efp</span> was used as an internal control. (<b>C</b>) Each band intensity was normalized to <span class="html-italic">efp</span>. Data are presented as mean ± SEM of triplicate experiments and were analyzed by Student’s <span class="html-italic">t</span>-test (* <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, and *** <span class="html-italic">p</span> &lt; 0.001).</p> Full article ">Figure 3
<p>Downregulation of the inhibitory effect of hesperetin on the expression and activity of urease in <span class="html-italic">H. pylori</span>. (<b>A</b>) <span class="html-italic">H. pylori</span> was treated with the indicated concentrations (0, 6.25, 12.5, 25 and 50 μM) of hesperetin. Collected RNA was subjected to RT-PCR to detect mRNA expression levels of urease subunits (<span class="html-italic">ure</span>A and <span class="html-italic">ure</span>B). The expression of <span class="html-italic">efp</span> was used as an internal control. (<b>B</b>) Each band intensity was normalized to <span class="html-italic">efp</span>. (<b>C</b>) Bacterial lysates were subjected to Western blotting to detect UreA and UreB proteins. A rabbit anti-<span class="html-italic">H. pylori</span> polyclonal antibody was used as an internal control. (<b>D</b>) After 72 h, the supernatant was collected and urease activity was determined by measuring the amount of ammonia. Data are presented as mean ± SEM of triplicate experiments and were analyzed by Student’s <span class="html-italic">t</span>-test (*** <span class="html-italic">p</span> &lt; 0.001).</p> Full article ">Figure 4
<p>Downregulation of the inhibitory effect of hesperetin on the expression of flagella genes and motility of <span class="html-italic">H. pylori</span>. (<b>A</b>) <span class="html-italic">H. pylori</span> was treated with hesperetin at indicated concentrations (0, 6.25, 12.5, 25, and 50 μM). The collected RNA was subjected to RT-PCR to detect mRNA expression levels of flagella genes (<span class="html-italic">flh</span>A, <span class="html-italic">fla</span>A, <span class="html-italic">fla</span>B, and <span class="html-italic">flg</span>E). The expression of <span class="html-italic">efp</span> was used as an internal control. (<b>B</b>) Each band intensity was normalized to <span class="html-italic">efp</span>. (<b>C</b>) Mueller–Hinton semi-solid agar containing 0.4% agar and hesperetin at the indicated concentrations (0, 6.25, 12.5, 25, and 50 μM). The negative control was a motility medium not inoculated with <span class="html-italic">H. pylori</span>. <span class="html-italic">H. pylori</span> was inoculated using a needle and cultured in a CO<sub>2</sub> incubator at 37 °C for 5 days. After incubation, the radius of <span class="html-italic">H. pylori</span> growth spread was measured. (<b>D</b>) Data are presented as mean ± SEM of triplicate experiments and were analyzed by Student’s <span class="html-italic">t</span>-test (*** <span class="html-italic">p</span> &lt; 0.001).</p> Full article ">Figure 5
<p>Downregulation of the inhibitory effect of hesperetin on the expression of adherence genes and adhesion of <span class="html-italic">H. pylori</span>. (<b>A</b>) <span class="html-italic">H. pylori</span> was treated with hesperetin at indicated concentrations (0, 6.25, 12.5, 25, and 50 μM). The collected RNA was subjected to RT-PCR to detect mRNA expression levels of adherence genes (<span class="html-italic">sab</span>A, <span class="html-italic">alp</span>A, <span class="html-italic">alp</span>B, <span class="html-italic">hpa</span>A, and <span class="html-italic">hop</span>Z). The expression of <span class="html-italic">efp</span> was used as an internal control. (<b>B</b>) Each band intensity was normalized to <span class="html-italic">efp</span>. (<b>C</b>) <span class="html-italic">H. pylori</span> was pretreated with hesperetin at the indicated concentrations (0, 6.25, 12.5, 25, and 50 μM). AGS cells were infected with <span class="html-italic">H. pylori</span> (100 MOI) for 30 min in a shaking incubator. After incubation, the cell complexity was analyzed using a flow cytometer. Data are presented as mean ± SEM of triplicate experiments and were analyzed by Student’s <span class="html-italic">t</span>-test (*** <span class="html-italic">p</span> &lt; 0.001).</p> Full article ">Figure 6
<p>Inhibitory effect of hesperetin on CagA and VacA translocation to the gastric cell line. (<b>A</b>) AGS gastric cancer cells were treated with hesperetin at indicated concentrations (0, 3.13, 6.25, 12.5, 25, 50, 100, and 200 μM) for 24 h. Cell viability was then measured by the WST assay. AGS cells were infected with <span class="html-italic">H. pylori</span> (100 MOI) and treated with hesperetin at indicated concentrations (0, 6.25, 12.5, 25 and 50 μM) for 24 h. (<b>B</b>) After 24 h of incubation, cell lysates were subjected to Western blotting to detect translocated CagA and VacA proteins. β-Actin was used as an internal control. <span class="html-italic">H. pylori</span> was treated with hesperetin at the indicated concentrations (0, 6.25, 12.5, 25, and 50 μM) for 72 h. (<b>C</b>) Bacterial lysates were subjected to Western blotting to detect CagA, VacA, and SecA. A rabbit anti-<span class="html-italic">H. pylori</span> polyclonal antibody was used as an internal control. (<b>D</b>) The collected RNA was subjected to RT-PCR to detect mRNA expression levels of <span class="html-italic">cag</span>A, T4SS components, <span class="html-italic">vac</span>A, and <span class="html-italic">sec</span>A genes. The expression of <span class="html-italic">efp</span> was used as an internal control. (<b>E</b>) Each band intensity was normalized to <span class="html-italic">efp</span>. Data are presented as mean ± SEM from three independent experiments and were analyzed by unpaired Student’s <span class="html-italic">t</span>-test (* <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, and *** <span class="html-italic">p</span> &lt; 0.001).</p> Full article ">
10 pages, 957 KiB  
Article
The Helicobacter pylori CagY Protein Drives Gastric Th1 and Th17 Inflammation and B Cell Proliferation in Gastric MALT Lymphoma
by Chiara Della Bella, Maria Felicia Soluri, Simone Puccio, Marisa Benagiano, Alessia Grassi, Jacopo Bitetti, Fabio Cianchi, Daniele Sblattero, Clelia Peano and Mario Milco D’Elios
Int. J. Mol. Sci. 2021, 22(17), 9459; https://doi.org/10.3390/ijms22179459 - 31 Aug 2021
Cited by 14 | Viewed by 2474
Abstract
Background: the neoplastic B cells of the Helicobacter pylori-related low-grade gastric mucosa-associated lymphoid tissue (MALT) lymphoma proliferate in response to H. pylori, however, the nature of the H. pylori antigen responsible for proliferation is still unknown. The purpose of the study [...] Read more.
Background: the neoplastic B cells of the Helicobacter pylori-related low-grade gastric mucosa-associated lymphoid tissue (MALT) lymphoma proliferate in response to H. pylori, however, the nature of the H. pylori antigen responsible for proliferation is still unknown. The purpose of the study was to dissect whether CagY might be the H. pylori antigen able to drive B cell proliferation. Methods: the B cells and the clonal progeny of T cells from the gastric mucosa of five patients with MALT lymphoma were compared with those of T cell clones obtained from five H. pylori–infected patients with chronic gastritis. The T cell clones were assessed for their specificity to H. pylori CagY, cytokine profile and helper function for B cell proliferation. Results: 22 of 158 CD4+ (13.9%) gastric clones from MALT lymphoma and three of 179 CD4+ (1.7%) clones from chronic gastritis recognized CagY. CagY predominantly drives Interferon-gamma (IFN-γ) and Interleukin-17 (IL-17) secretion by gastric CD4+ T cells from H. pylori-infected patients with low-grade gastric MALT lymphoma. All MALT lymphoma-derived clones dose dependently increased their B cell help, whereas clones from chronic gastritis lost helper activity at T-to-B-cell ratios greater than 1. Conclusion: the results obtained indicate that CagY drives both B cell proliferation and T cell activation in gastric MALT lymphomas. Full article
(This article belongs to the Special Issue Tumor Microenvironment: Interactions and Therapeutic Response)
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<p>Antigen specificity of <span class="html-italic">H. pylori</span>-reactive T cell clones derived from the gastric mucosa of <span class="html-italic">H. pylori</span>-infected patients with low-grade MALT B cell lymphoma (MALT) or uncomplicated chronic gastritis (CG). In vivo activated T cells were recovered from biopsy specimens of gastric mucosa and cloned by limiting dilution. T cell blasts from each clone were seeded in triplicate cultures with irradiated autologous peripheral blood mononuclear cells in the presence of medium alone or optimal doses of <span class="html-italic">H. pylori</span> lysate (10 μg/mL), or CagY (1 μg/mL). After 60 h, [<sup>3</sup>H] thymidine uptake was measured and expressed as mitogenic index. A significant difference (*) was found between the mitogenic index of CagY-specific MALT lymphoma-derived T clones and CG-derived ones. A highly significant (***) proliferation to CagY than to H.pylori lysate was found in MALT lymphoma-derived T clones.</p> Full article ">Figure 2
<p>Cytokine profile of gastric mucosa CagY-specific CD4<sup>+</sup> T cell clones obtained from <span class="html-italic">H. pylori</span>-infected patients with gastric low-grade MALT lymphoma. Th clones were tested for cytokine production (<b>A</b>,<b>B</b>). CagY-specific Th clones were stimulated with CagY and TNF-α and IL-4, IFN-γ and IL-17 production was measured in culture supernatants. In unstimulated cultures, levels of TNF-α, IL-4, IFN-γ and IL-17 were consistently &lt; 20 pg/mL. CD4<sup>+</sup> T cell clones producing IFN-γ, but not IL-17 nor IL-4, were coded as Th1. CD4<sup>+</sup> T cell clones producing IL-17, but not IFN-γ nor IL-4, were coded as Th17. CD4<sup>+</sup> T cell clones producing IFN-γ, and IL-17, but not IL-4, were coded as Th17/Th1. CD4<sup>+</sup> T cell clones producing TNF-α and IL-4, but not IL-17, were coded as Th0.</p> Full article ">Figure 3
<p>B cell proliferation to CagY. CagY-stimulated T cell clones derived from the gastric mucosa of patients with MALT lymphoma provide huge help for proliferation to autologous B cells (<b>A</b>). Irradiated T cell blasts of each CagY-reactive clone derived from patients with gastric MALT lymphoma or chronic gastritis were co-cultured for four days with peripheral blood autologous B cells (3 × 10<sup>4</sup>) at 0.2, 1, and 5 to 1 T-to-B-cell ratios in the presence of medium alone or CagY. Sixteen hours before harvesting, 0.5 μCi of [<sup>3</sup>H] thymidine was added, and its uptake was measured as mitogenic index. B cells cultured with or without CagY did not show any proliferation both in gastric low-grade MALT lymphoma and chronic gastritis patients at any CagY concentration used (0.1, 1, 10 μg/mL) (<b>B</b>). B cells cultured without CagY, with autologous irradiated gastric T cells did not show any proliferation both in gastric low-grade MALT lymphoma and in chronic gastritis patients at any T-to-B-cell ratios (0.2, 1, and 5 to 1) (<b>C</b>). * <span class="html-italic">p</span> &lt; 0.05; *** <span class="html-italic">p</span> &lt; 0.001.</p> Full article ">
12 pages, 1893 KiB  
Article
Select Porcine Elongation Factor 1α Sequences Mediate Stable High-Level and Upregulated Expression of Heterologous Genes in Porcine Cells in Response to Primate Serum
by Wu-Sheng Sun, Hyeon Yang, Jin Gu No, Haesun Lee, Nahyun Lee, Minguk Lee, Man-Jong Kang and Keon Bong Oh
Genes 2021, 12(7), 1046; https://doi.org/10.3390/genes12071046 - 7 Jul 2021
Cited by 2 | Viewed by 2385
Abstract
Genetically engineered (GE) pigs with various combinations of genetic profiles have been developed using heterologous promoters. This study aimed to identify autologous promoters for high and ubiquitous expression of xenotransplantation relevant genes in GE pigs. A 1.4 kb upstream regulatory sequence of porcine [...] Read more.
Genetically engineered (GE) pigs with various combinations of genetic profiles have been developed using heterologous promoters. This study aimed to identify autologous promoters for high and ubiquitous expression of xenotransplantation relevant genes in GE pigs. A 1.4 kb upstream regulatory sequence of porcine elongation factor 1α (pEF1α) gene was selected and isolated for use as a promoter. Activity of the pEF1α promoter was subsequently compared with that of the cytomegalovirus (CMV) promoter, CMV enhancer/chicken β-actin (CAG) promoter, and human EF1α (hEF1α) promoter in different types of pig-derived cells. Comparative analysis of luciferase and mutant human leukocyte antigen class E-F2A-β-2 microglobulin (HLA-E) expression driven by pEF1α, CMV, CAG, and hEF1α promoters revealed the pEF1α promoter mediated comparable expression levels with those of the CAG promoter in porcine ear skin fibroblasts (PEFs) and porcine kidney-15 (PK-15) cells, but lower than those of the CAG promoter in porcine aortic endothelial cells (PAECs). The pEF1α promoter provided long-term stable HLA-E expression in PEFs, but the CAG promoter failed to sustain those levels of expression. For xenogeneic serum-induced cytotoxicity assays, the cells were cultured for several hours in growth medium supplemented with primate serum. Notably, the pEF1α promoter induced significant increases in luciferase and HLA-E expression in response to primate serum in PAECs compared with those driven by the CAG promoter, suggesting the pEF1α promoter could regulate temporal expression of heterologous genes under xenogeneic-cytotoxic conditions. These results suggest the pEF1α promoter may be valuable for development of GE pigs spatiotemporally and stably expressing immunomodulatory genes for xenotransplantation. Full article
(This article belongs to the Section Animal Genetics and Genomics)
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<p>Sequence alignment of <span class="html-italic">hEF1α</span> and <span class="html-italic">pEF1α</span>, including the URS. (<b>A</b>) Structure comparison of the selected URS (red lines) of <span class="html-italic">hEF1α</span> and <span class="html-italic">pEF1α</span>. Exons are shown in black boxes. (<b>B</b>) Pairwise alignment of the URS of <span class="html-italic">hEF1α</span> and <span class="html-italic">pEF1α</span>. Three predicted CpG islands are filled with blue and indicated as numbers on top of the graph. Location of the CpG dinucleotides is indicated by vertical red bars under the x-axis. Ortholog gene sequences of <span class="html-italic">pEF1α</span> and <span class="html-italic">hEF1α</span> are connected on the map with dotted blue lines. Green lines indicate the locations of the promoters used in the current study. <span class="html-italic">hEF1α</span>, human elongation factor 1α; <span class="html-italic">pEF1α</span>, porcine elongation factor 1α; URS, upstream regulatory sequence.</p> Full article ">Figure 2
<p>Relative activity of various promoters in regulating luciferase expression in different types of porcine cells. (<b>A</b>) Schematic diagram of the luciferase reporter constructs used in the study. Numbers on the left indicate the approximate length of the respective promoters. (<b>B</b>) Promoter activity in PEFs. (<b>C</b>) Promoter activity in PK-15 cells. (<b>D</b>) Promoter activity in PAECs. All values are normalized relative to values for the CAG promoter. CAG, cytomegalovirus enhancer/chicken β-actin promoter; CMV, cytomegalovirus promoter; <span class="html-italic">hEF1α</span>, human elongation factor 1α; <span class="html-italic">pEF1α</span>, porcine elongation factor 1α; PEFs, porcine ear skin fibroblasts; PAECs, porcine aortic endothelial cells. Values are means ± SEM calculated from three replicates; ns, no significant difference; **, <span class="html-italic">p</span> &lt; 0.01; ***, <span class="html-italic">p</span> &lt; 0.001.</p> Full article ">Figure 3
<p>Comparative activity of the <span class="html-italic">pEF1α</span> promoter with that of the CAG promoter in regulating an immunomodulatory gene in different types of porcine cells. (<b>A</b>) Schematic of the cassettes for HLA-Ecw0304 and B2M (HLA-E) co-expression. Comparative expression of mRNA levels (<b>B</b>) and protein levels (<b>C</b>) of HLA-E in PEFs, -15 cells, and PAECs. <span class="html-italic">pEF1α</span>, porcine elongation factor 1α; HLA-E, human leukocyte antigen E; F2A, 2A self-cleaving peptides; B2M, β-2 microglobulin; p(A), poly A; Neo<sup>®®</sup>, neomycin resistance. PEFs, porcine ear skin fibroblasts; PAECs, porcine aortic endothelial cells. ***, <span class="html-italic">p</span> &lt; 0.001.</p> Full article ">Figure 4
<p>The <span class="html-italic">pEF1α</span> promoter mediates significant increases in heterologous gene expression in response to primate serum in PAECs. (<b>A</b>) Luciferase activity, mRNA expression levels, and protein expression levels of HLA-E transgenes in PAECs transiently transfected with the expression cassettes presented in <a href="#genes-12-01046-f003" class="html-fig">Figure 3</a>A, followed by incubation in culture medium supplemented with pig serum. (<b>B</b>) Luciferase activity, mRNA expression levels, and protein expression levels of HLA-E transgenes in transfected PAECs incubated in culture medium supplemented with primate serum. (<b>C</b>–<b>E</b>) Expression pattern of endogenous <span class="html-italic">pEF1α</span> (<b>C</b>), <span class="html-italic">TF</span> (<b>D</b>), and <span class="html-italic">ICAM2</span> (<b>E</b>) in PAECs cultured with porcine serum and primate serum for 1, 2, 3, 5, and 7 h. CAG, cytomegalovirus enhancer/chicken β-actin promoter; <span class="html-italic">pEF1α</span>, porcine elongation factor 1α; HLA, human leukocyte antigen E; <span class="html-italic">ICAM2</span>, intercellular adhesion molecule 2; <span class="html-italic">TF</span>, tissue factor. Values are means ± SEM calculated from three replicates. *, <span class="html-italic">p</span> &lt; 0.05; **, <span class="html-italic">p</span> &lt; 0.01; ***, <span class="html-italic">p</span> &lt; 0.001.</p> Full article ">Figure 5
<p>The <span class="html-italic">pEF1α</span> promoter mediates stable HLA-E expression during long-term culture. (<b>A</b>) CAG- and the <span class="html-italic">pEF1α</span>-HLA-E expression cassettes were transfected into PEFs and then selected as described in detail in the Materials and Methods. Genotype analysis revealed that the transfectants carried the cassettes on day 4 (D4), day 8 (D8), day 12 (D12), day 16 (D16), and day 20 (D20) after 10 days of primary selection. (<b>B</b>) Comparative analysis of HLA-E mRNA expression at D20 compared with that at D4. Values are means ±SEM calculated from three replicates. (<b>C</b>) Western blot analysis of HLA-E expression on different days of selection. (<b>D</b>) Flow cytometry analysis for HLA-E at D20 compared with that at D4. “+”, positive control; “−”, negative control; CAG, cytomegalovirus enhancer/chicken β-actin promoter; <span class="html-italic">pEF1α</span>, porcine elongation factor 1α; HLA, human leukocyte antigen E; MFI, mean fluorescence intensity.</p> Full article ">
10 pages, 3170 KiB  
Article
Serodiagnosis and Bacterial Genome of Helicobacter pylori Infection
by Aina Ichihara, Hinako Ojima, Kazuyoshi Gotoh, Osamu Matsushita, Susumu Take, Hiroyuki Okada, Akari Watanabe and Kenji Yokota
Toxins 2021, 13(7), 467; https://doi.org/10.3390/toxins13070467 - 5 Jul 2021
Cited by 2 | Viewed by 2800
Abstract
The infection caused by Helicobacter pylori is associated with several diseases, including gastric cancer. Several methods for the diagnosis of H. pylori infection exist, including endoscopy, the urea breath test, and the fecal antigen test, which is the serum antibody titer test that [...] Read more.
The infection caused by Helicobacter pylori is associated with several diseases, including gastric cancer. Several methods for the diagnosis of H. pylori infection exist, including endoscopy, the urea breath test, and the fecal antigen test, which is the serum antibody titer test that is often used since it is a simple and highly sensitive test. In this context, this study aims to find the association between different antibody reactivities and the organization of bacterial genomes. Next-generation sequences were performed to determine the genome sequences of four strains of antigens with different reactivity. The search was performed on the common genes, with the homology analysis conducted using a genome ring and dot plot analysis. The two antigens of the highly reactive strains showed a high gene homology, and Western blots for CagA and VacA also showed high expression levels of proteins. In the poorly responsive antigen strains, it was found that the inversion occurred around the vacA gene in the genome. The structure of bacterial genomes might contribute to the poor reactivity exhibited by the antibodies of patients. In the future, an accurate serodiagnosis could be performed by using a strain with few gene mutations of the antigen used for the antibody titer test of H. pylori. Full article
(This article belongs to the Special Issue Helicobacter pylori Toxins and Pathogenesis)
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<p>ROC curve of five strains. Four antigens from clinically isolated strains (#3, #5, #6 and #8) and kit original antigen (SL-Wako) were reacted with <span class="html-italic">H. pylori</span> negative and positive sera. ROC analysis was performed.</p> Full article ">Figure 2
<p>Genome rings. A genome ring was created to visualize the characteristics of each strain. From the outside, gray is the number of all ORFs, red is the forward ORF, blue is the reverse ORF, orange is the number of tRNAs, green is the number of rRNAs, and the innermost is the GC content.</p> Full article ">Figure 3
<p>Dot plot analysis. Dot plot analysis was performed for #3 and #5 (<b>A</b>), #3 and #6 (<b>B</b>), and #3 and #8 (<b>C</b>). Dot plot analysis is a graph for comparing the amino acid sequence of a protein and the base sequence of a nucleic acid to clarify the homology. The vertical axis is arranged from bottom to top and the horizontal axis is arranged from left to right and is plotted at the matching points.</p> Full article ">Figure 4
<p>Comparison around <span class="html-italic">cagA</span> genes of 4 strains. The site that encodes about 30 genes including <span class="html-italic">cagA</span> is called cag pathogenicity island (CagPAI). No major mutation was found in 4 strains of CagPAI. However, it was found that the position of CagPAI started from about 1 million bp in the #8 strain and from about 600,000 bp in the other three strains.</p> Full article ">Figure 5
<p>Comparison of <span class="html-italic">vacA</span> genes of 4 strains. The arrows of <span class="html-italic">vacA</span> and its surrounding genes are shown. The <span class="html-italic">vacA</span> genes of #3 and #5 were located at 1 million positions in the forward direction. In contrast, the <span class="html-italic">vacA</span> genes of #6 and #8 were present at 600,000 positions in reverse directions.</p> Full article ">Figure 6
<p>Protein productions of CagA (<b>A</b>) and VacA (<b>B</b>). The protein expression levels of CagA and VacA were examined using Western blotting (mean ± SE). The expression levels of CagA were significantly reduced at #6 and #8 compared to #3 and at #8 compared with #5. Although there was no significant difference in VacA expression level, there was, however, a tendency for the expression level to be low in #6 and #8.</p> Full article ">Figure 7
<p>Chromosome structure. From the results of the dot plot analysis and the map around the genes of <span class="html-italic">cagPAI</span> and <span class="html-italic">vacA</span>, we considered the possibility that mutations, such as inversion, occurred among the 4 strains in the genome. Most of the sequences are the same for #3 and #5. It was found that the first half of the sequence of #3 was reversed in the second half of the sequence in #6, and the second half of the sequence including <span class="html-italic">vacA</span> in #6 was reversed in the first half of the sequence in #6. The middle sequence containing <span class="html-italic">cagA</span> should be approximately homologous. There are many mutations in #8 compared to #3, #5 and #6. The positions of <span class="html-italic">cagA</span> and <span class="html-italic">vacA</span> are also completely different in #8.</p> Full article ">
15 pages, 2170 KiB  
Article
Evodiamine Inhibits Helicobacter pylori Growth and Helicobacter pylori-Induced Inflammation
by Ji Yeong Yang, Jong-Bae Kim, Pyeongjae Lee and Sa-Hyun Kim
Int. J. Mol. Sci. 2021, 22(7), 3385; https://doi.org/10.3390/ijms22073385 - 25 Mar 2021
Cited by 28 | Viewed by 3837
Abstract
Helicobacter pylori (H. pylori) classified as a class I carcinogen by the World Health Organization (WHO) plays an important role in the progression of chronic gastritis and the development of gastric cancer. A major bioactive component of Evodia rutaecarpa, evodiamine, [...] Read more.
Helicobacter pylori (H. pylori) classified as a class I carcinogen by the World Health Organization (WHO) plays an important role in the progression of chronic gastritis and the development of gastric cancer. A major bioactive component of Evodia rutaecarpa, evodiamine, has been known for its anti-bacterial effect and anti-cancer effects. However, the inhibitory effect of evodiamine against H. pylori is not yet known and the inhibitory mechanisms of evodiamine against gastric cancer cells are yet to be elucidated concretely. In this study, therefore, anti-bacterial effect of evodiamine on H. pylori growth and its inhibitory mechanisms as well as anti-inflammatory effects and its mechanisms of evodiamine on H. pylori-induced inflammation were investigated in vitr. Results of this study showed the growth of the H. pylori reference strains and clinical isolates were inhibited by evodiamine. It was considered one of the inhibitory mechanisms that evodiamine downregulated both gene expressions of replication and transcription machineries of H. pylori. Treatment of evodiamine also induced downregulation of urease and diminished translocation of cytotoxin-associated antigen A (CagA) and vacuolating cytotoxin A (VacA) proteins into gastric adenocarcinoma (AGS) cells. This may be resulted from the reduction of CagA and VacA expressions as well as the type IV secretion system (T4SS) components and secretion system subunit protein A (SecA) protein which are involved in translocation of CagA and VacA into host cells, respectively. In particular, evodiamine inhibited the activation of signaling proteins such as the nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) and the mitogen-activated protein kinase (MAPK) pathway induced by H. pylori infection. It consequently might contribute to reduction of interleukin (IL)-8 production in AGS cells. Collectively, these results suggest anti-bacterial and anti-inflammatory effects of evodiamine against H. pylori. Full article
(This article belongs to the Special Issue Helicobacter: Infection, Diagnosis and Treatment)
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<p>Anti-bacterial activity of evodiamine against <span class="html-italic">H. pylori</span> and downregulation of replication and transcription genes of <span class="html-italic">H. pylori</span>. (<b>A</b>) Chemical structure of evodiamine. (<b>B</b>) The MIC of evodiamine against five <span class="html-italic">H. pylori</span> reference strains (ATCC 49503, ATCC 43504, ATCC 51932, ATCC 700392 and SS1) was determined by agar dilution method. (<b>C</b>) The MIC of evodiamine against <span class="html-italic">H. pylori</span> ATCC 49503 strain was confirmed by broth dilution method. Optical density of bacterial broth was measured at 600 nm wavelength by spectrophotometry. <span class="html-italic">H. pylori</span> was treated with indicated concentrations of evodiamine (0.5 and 1 μM) for 72 h. RNA was subjected to RT-PCR to detect the mRNA expression levels of (<b>D</b>) replication machineries (<span class="html-italic">dna</span>A, <span class="html-italic">dna</span>B, <span class="html-italic">dna</span>E, <span class="html-italic">dna</span>N, <span class="html-italic">dna</span>Q and <span class="html-italic">gyr</span>A) and (<b>E</b>) transcription machineries (<span class="html-italic">rpo</span>A, <span class="html-italic">rpo</span>B, <span class="html-italic">rpo</span>D and <span class="html-italic">rpo</span>N). The expression of <span class="html-italic">efp</span> was used as an internal control. (<b>F</b>) Each band intensity was normalized to <span class="html-italic">efp</span>. Data are presented as mean ± SEM of three independent experiments and analyzed by Student’s <span class="html-italic">t</span>-test (** <span class="html-italic">p</span> &lt; 0.01 and *** <span class="html-italic">p</span> &lt; 0.001).</p> Full article ">Figure 2
<p>The inhibitory effects of evodiamine on the expression and activity of urease in <span class="html-italic">H. pylori</span>. (<b>A</b>) <span class="html-italic">H. pylori</span> was treated with indicated concentrations of evodiamine (0.5 and 1 μM) for 72 h. RNA was subjected to RT-PCR to detect the mRNA expression levels of urease subunits (<span class="html-italic">ure</span>A and <span class="html-italic">ure</span>B). The expression of <span class="html-italic">efp</span> was used as an internal control. (<b>B</b>) Each band intensity was normalized using to <span class="html-italic">efp</span>. (<b>C</b>) The cell lysates were subjected to Western blot to detect UreA and UreB. The rabbit anti-<span class="html-italic">H. pylori</span> polyclonal antibody was used as an internal control. (<b>D</b>) After 72 h, the supernatant and cell lysate were collected and then urease activity was determined by measuring ammonia levels. Each band intensity was normalized by protein quantification. Data were presented as mean ± SEM of three independent experiments and analyzed by Student’s <span class="html-italic">t</span>-test (* <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01 and *** <span class="html-italic">p</span> &lt; 0.001).</p> Full article ">Figure 3
<p>The inhibitory effects of evodiamine on CagA and VacA translocation to the gastric cell line. (<b>A</b>) AGS gastric cancer cells were treated with indicated dose of evodiamine (0.5, 1, 2.5 and 5 μM) for 24 h and cell viability was measured by the WST assay. AGS cells were infected with <span class="html-italic">H. pylori</span> (200 MOI) and treated with indicated concentrations of evodiamine (0.5 and 1 μM) for 24 h. (<b>B</b>) After incubation, the cell lysates were collected and Western blot was then performed to detect CagA and VacA proteins. β-actin was used as an internal control. (<b>C</b>) Each band intensity was normalized to β-actin. <span class="html-italic">H. pylori</span> was treated with indicated concentrations of evodiamine (0.5 and 1 μM) for 72 h. (<b>D</b>) The cell lysates were subjected to Western blot to detect CagA, VacA and SecA. The rabbit anti-<span class="html-italic">H. pylori</span> polyclonal antibody was used as an internal control. (<b>E</b>) RNA was subjected to RT-PCR to detect the mRNA expression level of <span class="html-italic">cag</span>A, T4SS components, <span class="html-italic">vac</span>A and <span class="html-italic">sec</span>A. The expression of <span class="html-italic">efp</span> was used as an internal control. (<b>F</b>) Each band intensity was normalized to <span class="html-italic">efp</span>. Data were presented as mean ± SEM of three independent experiments and analyzed by Student’s <span class="html-italic">t</span>-test (<sup>###</sup> <span class="html-italic">p</span> &lt; 0.001, * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01 and *** <span class="html-italic">p</span> &lt; 0.001).</p> Full article ">Figure 4
<p>The inhibitory effects of evodiamine on <span class="html-italic">H. pylori</span>-induced activation of MAPK and NF-κB and degradation of IκBα in AGS cells infected with <span class="html-italic">H. pylori</span>. AGS cells were infected with <span class="html-italic">H. pylori</span> (200 MOI) and treated with indicated concentrations of evodiamine (0.5 and 1 μM) for 12 h. (<b>A</b>) After incubation, cell lysates were collected and Western blot analysis of phosphorylated and total ERK 1/2, JNK and p38 was performed. β-actin was used as an internal control. (<b>B</b>) Each band intensity was normalized to β-actin. Data were presented as mean ± SEM. ** <span class="html-italic">p</span> &lt; 0.01 and *** <span class="html-italic">p</span> &lt; 0.001 vs. uninfected control. <sup>#</sup> <span class="html-italic">p</span> &lt; 0.05, <sup>##</sup> <span class="html-italic">p</span> &lt; 0.01 and <sup>###</sup> <span class="html-italic">p</span> &lt; 0.001 vs. <span class="html-italic">H. pylori</span>-infected control without evodiamine treatment. (<b>C</b>) Western blot analysis of IκBα was performed. β-actin was used as an internal control. (<b>D</b>) Cell lysates were separated into nuclear and cytosolic fractions, then performed to Western blot for NF-κB. Lamin B1 was used as an internal control for nuclear fraction and α-tubulin was used as an internal control for cytosolic fraction. EVD, evodiamine.</p> Full article ">Figure 5
<p>Inhibitory effect of evodiamine on IL-8 secretion induced by <span class="html-italic">H. pylori</span> in AGS cells. AGS cells were uninfected or infected with <span class="html-italic">H. pylori</span> (200 MOI) with indicated concentrations of evodiamine (0.5 and 1 μM). Culture medium were collected after 24 h and ELISA was performed to determine the IL-8 concentration. Data were presented as mean ± SEM of three independent experiments and analyzed by Student’s <span class="html-italic">t</span>-test (** <span class="html-italic">p</span> &lt; 0.01 and *** <span class="html-italic">p</span> &lt; 0.001). EVD, evodiamine.</p> Full article ">
16 pages, 1807 KiB  
Article
Fusion of Dendritic Cells Activating Rv2299c Protein Enhances the Protective Immunity of Ag85B-ESAT6 Vaccine Candidate against Tuberculosis
by Yong Woo Back, Hyun Shik Bae, Han-Gyu Choi, Dang Thi Binh, Yeo-Jin Son, Seunga Choi and Hwa-Jung Kim
Pathogens 2020, 9(11), 865; https://doi.org/10.3390/pathogens9110865 - 22 Oct 2020
Cited by 6 | Viewed by 2826
Abstract
In Mycobacterium tuberculosis infection, naïve T cells that encounter mycobacterial antigens through dendritic cells (DCs) induce various CD4+ T-cell responses; therefore, appropriate DC activation is the key for protective immunity against tuberculosis. We previously found that Rv2299c-matured DCs induce Th1 differentiation with [...] Read more.
In Mycobacterium tuberculosis infection, naïve T cells that encounter mycobacterial antigens through dendritic cells (DCs) induce various CD4+ T-cell responses; therefore, appropriate DC activation is the key for protective immunity against tuberculosis. We previously found that Rv2299c-matured DCs induce Th1 differentiation with bactericidal activity. In this study, to prove that Rv2299c could enhance the protective immunity of other vaccine candidates comprising T-cell-stimulating antigens, Ag85B-ESAT6, a well-known vaccine candidate, was selected as a fusion partner of Rv2299c. Recombinant Rv2299c-Ag85B-ESAT6 protein induced DC maturation and activation. Furthermore, fusion of Rv2299c enhanced the protective efficacy of the Ag85B-ESAT6 vaccine in a mouse model and significantly higher production of TNF-α and IL-2 was detected in the lungs, spleen, and lymph nodes of the group immunized with the Rv2299c-fused protein than with Ag85B-ESAT6. In addition, fusion of Rv2299c enhanced the Ag85B-ESAT6-mediated expansion of multifunctional CD4+ T cells. These data suggested that the DC-activating protein Rv2299c may potentiate the protective immunity of the vaccine candidate comprising T cell antigens. Full article
(This article belongs to the Special Issue Feature Papers of Vaccines and Therapeutic Developments)
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<p>Rv2299c-Ag85B-ESAT6 induces maturation of dendritic cells (DCs) and Th1 responses. (<b>a</b>) Schematic diagram of Rv2299c-Ag85B-ESAT6 fusion protein (RAE6). (<b>b</b>) Recombinant Rv2299c-Ag85B-ESAT6 was produced in BL21 cells and purified using Ni-NTA resin. The purified protein was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot (WB) analysis using a mouse anti-His antibody (Ab). (<b>c</b>) To ensure that Rv2299c-Ag85B-ESAT6-induced RAW264.7 cells (5 × 10<sup>4</sup> cells) activation was not owing to endotoxin contamination in the protein preparation, Rv2299c-Ag85B-ESAT6 (10 μg/mL) or lipopolysaccharide (LPS, 100 ng/mL) was pretreated with polymyxin B (20 μg/mL) for 1 h prior to stimulation of RAW264.7 cell cultures. After 24 h, the quantities of MCP-1 in the culture medium were measured by enzyme-linked immunosorbent (ELISA) assay. All data are expressed as the mean values ± standard deviation (SD; <span class="html-italic">n</span> = 3); ** <span class="html-italic">p</span> &lt; 0.01 = a significant difference compared to the Rv2299c-Ag85B-ESAT6-treated RAW264.7 cells, as determined by unpaired Student’s t-test. Treatments with no significant effect are indicated as n.s. (<b>d</b>) Analysis of functional activation of DCs by Rv2299c-Ag85B-ESAT6. Immature DCs (5 × 10<sup>5</sup> cells) were cultured in the presence of 10 μg/mL Rv2299c-Ag85B-ESAT6 or 100 ng/mL LPS for 24 h. The quantities of TNF-α, IL-1β, IL-6, and IL-12p70 in the culture supernatant were determined by ELISA. All data are expressed as mean ± SD (<span class="html-italic">n</span> = 3). The levels of significance (* <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, or *** <span class="html-italic">p</span> &lt; 0.001 determined by one-way analysis of variance [<a href="#B17-pathogens-09-00865" class="html-bibr">17</a>]) of the differences between the treatment data and the control data are indicated. (<b>e</b>) The cells were gated on CD11c. The DCs were stained with anti-CD80, anti-CD86, anti-MHC class I, or anti-MHC class II monoclonal Abs (mAbs). The percentage of positive cells is shown in each panel. The bar graphs depict data as the mean ± SD (<span class="html-italic">n</span> = 3). The levels of significance (** <span class="html-italic">p</span> &lt; 0.01 or *** <span class="html-italic">p</span> &lt; 0.001, determined by one-way ANOVA) of the differences between the treatment data and the control data are indicated. (<b>f</b>) To examine Th1 differentiation by Rv2299c-Ag85B-ESAT6-treated DCs, naïve splenocytes activated by unstimulated DCs, LPS-stimulated DCs, or Rv2299c-Ag85B-ESAT6-stimulated DCs were co-cultured for 3 days with splenocytes of naïve mice, at a DC to T cell ratio of 1:10. The quantities of IFN-γ in the culture supernatant were determined by ELISA. The data shown are the mean values ± SD (<span class="html-italic">n</span> = 3); * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, and *** <span class="html-italic">p</span> &lt; 0.001 compared to each group.</p> Full article ">Figure 2
<p>Rv2299c-Ag85B-ESAT6 induces Ag85B-specific Th1 responses and protects the lungs of mice. (<b>a</b>) Schematic diagram of the experimental design. (<b>b</b>) Lungs after 4 weeks of the last vaccination or 3 weeks post-challenge. (<b>c</b>) Three weeks post-challenge, the mice were sacrificed and the bacterial burden (CFU) was measured in the lung. (<b>d</b>) lungs and (<b>e</b>) spleen were harvested from mice in each group. Each group of cells (10<sup>6</sup> cells) was treated with Ag85B (2 μg/well) for 24 h. IFN-γ, TNF-α, IL-2, and IL-17 levels were analyzed by enzyme-linked immunosorbent assay (ELISA). The data are shown as the mean ± standard deviation (SD) of 3 samples. One representative plot from three independent experiments is shown. * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, and *** <span class="html-italic">p</span> &lt; 0.001, compared to the non-vaccinated control or Ag85B-ESAT6 group. Treatments with no significant effect are indicated as n.s. Group G1: non-vaccinated control; G2: adjuvant alone; G3: Ag85B-ESAT6 (AE6); and G4: Rv2299c-Ag85B-ESAT6 (RAE6).</p> Full article ">Figure 3
<p>Rv2299c-Ag85B-ESAT6 induces Ag85B-specific Th1 responses and expansion of the effector/memory T-cell population in the lungs of mice. (<b>a</b>) Three weeks post-challenge, lung cells (10<sup>6</sup> cells) were stimulated with Ag85B (2 μg/well) for 12 h at 37 °C in the presence of GolgiStop. The percentage of antigen-specific CD4<sup>+</sup> T cells producing IFN-γ, TNF-α, and/or IL-2 among the cells isolated from the lungs of each group of mice were analyzed by multicolor flow cytometry by gating for CD4<sup>+</sup> T cells. (<b>b</b>) Lung cells (10<sup>6</sup> cells) were treated with Ag85B (2 μg/well) for 24 h. The lung cells were stained with anti-CD4, anti-CD62L, and anti-CD44 monoclonal antibodies (mAbs). Bar graphs show antigen-specific CD4<sup>+</sup>CD62L<sup>–</sup>CD44<sup>+</sup> T-cell or CD4<sup>+</sup>CD62L<sup>+</sup>CD44<sup>+</sup> T-cell populations among the lung cells. The data are shown as the mean ± SD of three samples. One representative plot from three independent experiments is shown. *** <span class="html-italic">p</span> &lt; 0.001, compared to the non-vaccinated control or Ag85B-ESAT6 group. Group G1: non-vaccinated control; G2: adjuvant alone; G3: Ag85B-ESAT6 (AE6); and G4: Rv2299c-Ag85B-ESAT6 (RAE6).</p> Full article ">Figure 4
<p>Rv2299c-Ag85B-ESAT6 induces Ag85B-specific Th1 responses in lymph nodes. (<b>a</b>) Three weeks post-challenge, cervical lymph nodes were harvested from mice in each group. The lymphocytes (10<sup>6</sup> cells) were treated with Ag85B (2 μg/well) for 24 h. IFN-γ, TNF-α, IL-2, and IL-17 cytokine levels were analyzed by ELISA. The data are shown as the mean ± standard deviation (SD) of 3 samples. One representative plot from three independent experiments is shown. ** <span class="html-italic">p</span> &lt; 0.01 and *** <span class="html-italic">p</span> &lt; 0.001, compared to the non-vaccinated control or Ag85B-ESAT6 group. Treatments with no significant effect are indicated as n.s. (<b>b</b>) Three weeks post-challenge, lymphocytes (10<sup>6</sup> cells) were stimulated with Ag85B (2 μg/well) for 12 h at 37 °C in the presence of GolgiStop. The percentage of antigen-specific CD4<sup>+</sup> T cells producing IFN-γ, TNF-α, and/or IL-2 among the cells isolated from the lymph nodes of each group of mice were analyzed by multicolor flow cytometry by gating for CD4<sup>+</sup> T cells.</p> Full article ">
13 pages, 1152 KiB  
Article
Helicobacter pylori Infection and Autoimmune Thyroid Diseases: The Role of Virulent Strains
by Natale Figura, Giovanni Di Cairano, Elena Moretti, Francesca Iacoponi, Annalisa Santucci, Giulia Bernardini, Stefano Gonnelli, Nicola Giordano and Antonio Ponzetto
Antibiotics 2020, 9(1), 12; https://doi.org/10.3390/antibiotics9010012 - 30 Dec 2019
Cited by 30 | Viewed by 4932
Abstract
Aim: To verify a possible association between overall H. pylori and CagA+ H. pylori infection and autoimmune thyroid diseases (AITDs). Methods: Consecutive patients with AITDs admitted to one single centre of Endocrinology during one solar year were examined. The diagnoses were Hashimoto thyroiditis [...] Read more.
Aim: To verify a possible association between overall H. pylori and CagA+ H. pylori infection and autoimmune thyroid diseases (AITDs). Methods: Consecutive patients with AITDs admitted to one single centre of Endocrinology during one solar year were examined. The diagnoses were Hashimoto thyroiditis (HT) in 76, Graves’ Disease (GD) in 39, and aspecific thyroiditis (AT) in 44 patients. Controls were 136 individuals without AITDs. Median values of fT3, fT4, anti-thyreoglobulin (Tg) antibodies, IL-1β, IL-6, and TNF-α in patients were compared with those in controls. H. pylori infection and CagA status were determined serologically. Structural homology of some thyroid proteins with H. pylori antigens was investigated. Results: H. pylori infection prevalence was significantly increased in GD (66.6%) and HT (64.4%) patients, vs. 29.4% of controls and 34.0% of AT. CagA seropositivity was significantly more frequent in GD (46.1%) and HT (46.9%) infected patients, vs. infected controls (20%). fT3 and fT4 median values were significantly decreased in infected CagA+ GD patients vs. uninfected GD patients. IL-1β median values were increased in patients respect to controls, independently of the clinical form of AITD. Median values of IL-6, TNF-α and anti-Tg autoantibodies in CagA infected patients were significantly higher than those measured in infected CagA− and uninfected patients and in infected CagA+ controls. The examined thyroid proteins shared putative conserved domains with numerous bacterial antigens. Conclusions: Overall H. pylori and CagA+ H. pylori infection were associated with GD and HT, putatively through an increased inflammatory status and molecular mimicry. Full article
(This article belongs to the Special Issue Helicobacter pylori Virulence Factors and Antibiotic Susceptibility)
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<p>Median (IQR) values of fT3 (<b>left</b> and fT4 (<b>right</b>) in patients with different clinical forms of AITD and controls according to the overall <span class="html-italic">H. pylori</span> and CagA infectious status. ° <span class="html-italic">p</span> &lt; 0.05: HP+/CagA+ vs. HP+/CagA−, §§ <span class="html-italic">p</span> &lt; 0.01 AITDs vs. no-AITDs.</p> Full article ">Figure 2
<p>Median values (IQR) of anti-Tg serum antibodies in patients and controls according the overall <span class="html-italic">H. pylori</span> infectious and CagA status. * <span class="html-italic">p</span> &lt; 0.05: HP+ vs. HP− patients, ° <span class="html-italic">p</span> &lt; 0.05: HP+/CagA+ vs. HP+/CagA− patients, §§ <span class="html-italic">p</span> &lt; 0.01 AITDs vs. no-AITD.</p> Full article ">Figure 3
<p>Median values (IQR) of IL-6 in patients and controls according to the overall <span class="html-italic">H. pylori</span> and CagA infectious status. ° <span class="html-italic">p</span> &lt; 0.05: HP+/CagA+ vs. HP+/CagA− patients, §§ <span class="html-italic">p</span> &lt; 0.01 AITDs vs. no-AITD.</p> Full article ">Figure 4
<p>Median values (IQR) of TNF-α in patients and controls according to the overall <span class="html-italic">H. pylori</span> and the CagA infectious status. ° <span class="html-italic">p</span> &lt; 0.05: HP+/CagA+ vs. HP+/CagA− patients, § <span class="html-italic">p</span> &lt; 0.05 AITDs vs. no-AITD.</p> Full article ">Figure 5
<p>Main putative conserved domains detected by aligning thyroglobulin, thyroid peroxidase, and thyroid stimulating hormone receptor with <span class="html-italic">H. pylori</span> proteins. “Query”: Amino acid sequence of the human proteins. “Subject”: Amino acid sequence of the bacterial protein present in the data base. “+” indicates that the amino acid in the sequence “query” and that one in the sequence “subject” are similar from a chemical-physic point of view. The numbers in the sequence represent the initial and final positions of the amino acid sequence.</p> Full article ">
20 pages, 1091 KiB  
Review
Novel Insights of Lymphomagenesis of Helicobacter pylori-Dependent Gastric Mucosa-Associated Lymphoid Tissue Lymphoma
by Sung-Hsin Kuo, Ming-Shiang Wu, Kun-Huei Yeh, Chung-Wu Lin, Ping-Ning Hsu, Li-Tzong Chen and Ann-Lii Cheng
Cancers 2019, 11(4), 547; https://doi.org/10.3390/cancers11040547 - 17 Apr 2019
Cited by 30 | Viewed by 5873
Abstract
Gastric mucosa-associated lymphoid tissue (MALT) lymphoma is the most common subtype of gastric lymphoma. Most gastric MALT lymphomas are characterized by their association with the Helicobacter pylori (HP) infection and are cured by first-line HP eradication therapy (HPE). Several studies have been conducted [...] Read more.
Gastric mucosa-associated lymphoid tissue (MALT) lymphoma is the most common subtype of gastric lymphoma. Most gastric MALT lymphomas are characterized by their association with the Helicobacter pylori (HP) infection and are cured by first-line HP eradication therapy (HPE). Several studies have been conducted to investigate why most gastric MALT lymphomas remain localized, are dependent on HP infection, and show HP-specific intratumoral T-cells (e.g., CD40-mediated signaling, T-helper-2 (Th2)-type cytokines, chemokines, costimulatory molecules, and FOXP3+ regulatory T-cells) and their communication with B-cells. Furthermore, the reason why the antigen stimuli of these intratumoral T-cells with tonic B-cell receptor signaling promote lymphomagenesis of gastric MALT lymphoma has also been investigated. In addition to the aforementioned mechanisms, it has been demonstrated that the translocated HP cytotoxin-associated gene A (CagA) can promote B-cell proliferation through the activation of Src homology-2 domain-containing phosphatase (SHP-2) phosphorylation-dependent signaling, extracellular-signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (MAPK), B-cell lymphoma (Bcl)-2, and Bcl-xL. Furthermore, the expression of CagA and these CagA-signaling molecules is closely associated with the HP-dependence of gastric MALT lymphomas (completely respond to first-line HPE). In this article, we summarize evidence of the classical theory of HP-reactive T-cells and the new paradigm of direct interaction between HP and B-cells that contributes to the HP-dependent lymphomagenesis of gastric MALT lymphomas. Although the role of first-line HPE in the treatment of HP-negative gastric MALT lymphoma remains uncertain, several case series suggest that a proportion of HP-negative gastric MALT lymphomas remains antibiotic-responsive and is cured by HPE. Considering the complicated interaction between microbiomes and the genome/epigenome, further studies on the precise mechanisms of HP- and other bacteria-directed lymphomagenesis in antibiotic-responsive gastric MALT lymphomas are warranted. Full article
(This article belongs to the Special Issue Helicobacter pylori Associated Cancer)
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Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>The cytotoxin-associated gene A (CagA) deregulates intracellular signaling pathways in B-lymphocytes in tyrosine phosphorylation-dependent and -independent manners to initiate the lymphomagenesis of <span class="html-italic">Helicobacter pylori</span> (HP)-dependent gastric mucosa-associated lymphoid tissue (MALT) lymphoma. The HP protein CagA was translocated into B lymphocytes and co-immunoprecipitated with phospho-Src homology-2 domain-containing phosphatase (SHP-2), and further activated extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (MAPK), B cell lymphoma (Bcl)-2-associated death promoter (BAD), Bcl-2, and Bcl-xL. The tyrosine phosphorylation-dependent signaling pathway promotes the proliferation and inhibits the apoptosis of B lymphocytes, allowing these B-lymphocytes to acquire an oncogenic survival ability and develop hematological malignancies. In contrast, the signaling pathways from the CagA tyrosine phosphorylation-independent pathway, including the impairment of p53 and inhibition of Janus kinase (JAK)/STAT, resulted in the suppression of apoptosis and the inhibition of proliferation of B-lymphocytes, respectively. The imbalance between apoptosis and proliferation leads to the pathogenesis and development of HP-dependent gastric MALT lymphoma.</p> Full article ">Figure 2
<p>Involvement of ctotoxin-associated gene (CagA)-derived signals, T-cell-derived signals, and tumor microenvironment-related mediators in the <span class="html-italic">Helicobacter pylori</span> (HP)-induced lymphomagenesis of gastric mucosa-associated lymphoid tissue (MALT) lymphoma. HP infection stimulates T lymphocytes and cytokines in the gastric mucosa and indirectly induces the development of MALT, from which B lymphocytes migrate and infiltrate the site of MALT in the stomach. (<b>A</b>) The CagA protein translocates into B lymphocytes as it is secreted by HP on gastric epithelial surfaces through the T4SS system, thereby resulting in a cascade of survival signaling in the B lymphocytes. (<b>B</b>) Simultaneously, CagA restricts the proliferation of these lymphoma B cells by upregulating the G1 cell-cycle phase regulator, p21, by triggering nuclear factor of activated T cell (NFAT) c1 signaling. (<b>C</b>) HP infection also indirectly promotes lymphomagenesis through T-cell-stimulatory pathways, such as CD40-mediated signaling, Th-2-type cytokine-mediated signaling, and forkhead box P3 (FOXP3)+ regulatory T cells (Tregs) and B-cell receptor (BCR)-signaling that mediate the interaction between co-stimulatory molecules such as CD86 and CTLA4 in T-lymphocytes. (<b>D</b>) Molecular cross-talk between lymphoma B cells and immune-associated molecules in the tumor microenvironment (T cells, FOXP3+ Tregs, and Th17 helper cell-regulated cytokines including interleukin (IL)-22, chemokines, and chemokine receptors, and the interaction between macrophage and a proliferation-inducing ligand (APRIL)) stimulates the survival signaling in B-lymphocytes.</p> Full article ">
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