The Helicobacter pylori CagY Protein Drives Gastric Th1 and Th17 Inflammation and B Cell Proliferation in Gastric MALT Lymphoma

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.     

Morphological and Cellular Features of Innate Immune Reaction in Helicobacter pylori Gastritis: A Brief Review

Innate and adaptive immunity are both involved in acute and chronic inflammatory processes. The main cellular players in the innate immune system are macrophages, mast cells, dendritic cells, neutrophils, eosinophils, and natural killer (NK), which offer antigen-independent defense against infection. Helicobacter pylori (H. pylori) infection presents peculiar characteristics in gastric mucosa infrequently occurring in other organs; its gastric colonization determines a causal role in both gastric carcinomas and mucosa-associated lymphoid tissue lymphoma. In contrast, an active role for Epstein-Barr virus (EBV) has been identified only in 9% of gastric carcinomas. The aim of the present review is to discuss the role of cellular morphological effectors in innate immunity during H. pylori infection and gastric carcinogenesis.     

Different Role of Tumor Necrosis Factor-α Polymorphism in Non-Hodgkin Lymphomas among Caucasian and Asian Populations: A Meta-Analysis

Tumor necrosis factor-α (TNF-α) is an immunoregulatory cytokine involved in B- and T-cell function, and also plays an important role in inflammation and cancer. TNF-α-308G>A has been associated with constitutively elevated TNF-α expression. Several studies have reported the association between the TNF-α-308G>A polymorphism and non-Hodgkin lymphomas (NHL) risk, however, results are still inconsistent. To solve these conflicts, we conducted the first meta-analysis to assess the effect of TNF-α-308G>A polymorphism on the risk of NHL and various subtypes (additive model) including 10,619 cases and 12,977 controls in Caucasian and Asian populations. Our meta-analysis indicated that TNF-α-308G>A polymorphism is not associated with NHL risk when pooling all studies together (OR = 1.06, 95% CI: 0.92–1.23, p = 0.413). In stratified analyses, we found TNF-α-308A allele was significantly associated with higher risk of NHL, B-cell lymphomas (BCL), T-cell lymphomas (TCL) and diffuse large B-cell lymphomas (DLBCL) in Caucasians (OR = 1.22, 95% CI: 1.06–1.40, p = 0.007; OR = 1.18, 95% CI: 1.03–1.34, p = 0.014; OR = 1.20, 95% CI: 1.01–1.42, p = 0.040; OR = 1.21, 95% CI: 1.11–1.32, p < 0.001, respectively). Interestingly, it was associated with decreased risk of NHL, BCL and DLBCL in Asians (OR = 0.75, 95% CI: 0.66–0.86, p < 0.001; OR = 0.70, 95% CI: 0.52–0.94, p = 0.018; OR = 0.70, 95% CI: 0.57–0.86, p = 0.001). These findings also suggest TNF-α might play a distinct role in pathogenesis of NHL in different populations.     

Tracking the Genetic Susceptibility Background of B-Cell Non-Hodgkin’s Lymphomas from Genome-Wide Association Studies

B-cell non-Hodgkin’s lymphoma (NHL) risk associations had been mainly attributed to family history of the disease, inflammation, and immune components including human leukocyte antigen (HLA) genetic variations. Nevertheless, a broad range of genome-wide association studies (GWAS) have shed light into the identification of several genetic variants presumptively associated with B-cell NHL etiologies, survival or shared genetic risk with other diseases. The present review aims to overview HLA structure and diversity and summarize the evidence of genetic variations, by GWAS, on five NHL subtypes (diffuse large B-cell lymphoma DLBCL, follicular lymphoma FL, chronic lymphocytic leukemia CLL, marginal zone lymphoma MZL, and primary central nervous system lymphoma PCNSL). Evidence indicates that the HLA zygosity status in B-cell NHL might promote immune escape and that genome-wide significance variants can give biological insight but also potential therapeutic markers such as WEE1 in DLBCL. However, additional studies are needed, especially for non-DLBCL, to replicate the associations found to date.     

BH3 Mimetics in Hematologic Malignancies

Hematologic malignancies (HM) comprise diverse cancers of lymphoid and myeloid origin, including lymphomas (approx. 40%), chronic lymphocytic leukemia (CLL, approx. 15%), multiple myeloma (MM, approx. 15%), acute myeloid leukemia (AML, approx. 10%), and many other diseases. Despite considerable improvement in treatment options and survival parameters in the new millennium, many patients with HM still develop chemotherapy-refractory diseases and require re-treatment. Because frontline therapies for the majority of HM (except for CLL) are still largely based on classical cytostatics, the relapses are often associated with defects in DNA damage response (DDR) pathways and anti-apoptotic blocks exemplified, respectively, by mutations or deletion of the TP53 tumor suppressor, and overexpression of anti-apoptotic proteins of the B-cell lymphoma 2 (BCL2) family. BCL2 homology 3 (BH3) mimetics represent a novel class of pro-apoptotic anti-cancer agents with a unique mode of action—direct targeting of mitochondria independently of TP53 gene aberrations. Consequently, BH3 mimetics can effectively eliminate even non-dividing malignant cells with adverse molecular cytogenetic alterations. Venetoclax, the nanomolar inhibitor of BCL2 anti-apoptotic protein has been approved for the therapy of CLL and AML. Numerous venetoclax-based combinatorial treatment regimens, next-generation BCL2 inhibitors, and myeloid cell leukemia 1 (MCL1) protein inhibitors, which are another class of BH3 mimetics with promising preclinical results, are currently being tested in several clinical trials in patients with diverse HM. These pivotal trials will soon answer critical questions and concerns about these innovative agents regarding not only their anti-tumor efficacy but also potential side effects, recommended dosages, and the optimal length of therapy as well as identification of reliable biomarkers of sensitivity or resistance. Effective harnessing of the full therapeutic potential of BH3 mimetics is a critical mission as it may directly translate into better management of the aggressive forms of HM and could lead to significantly improved survival parameters and quality of life in patients with urgent medical needs.     

Evaluation of Orbital Lymphoproliferative and Inflammatory Disorders by Gene Expression Analysis

Non-specific orbital inflammation (NSOI) and IgG4-related orbital disease (IgG4-ROD) are often challenging to differentiate. Furthermore, it is still uncertain how chronic inflammation, such as IgG4-ROD, can lead to mucosa-associated lymphoid tissue (MALT) lymphoma. Therefore, we aimed to evaluate the diagnostic value of gene expression analysis to differentiate orbital autoimmune diseases and elucidate genetic overlaps. First, we established a database of NSOI, relapsing NSOI, IgG4-ROD and MALT lymphoma patients of our orbital center (2000–2019). In a consensus process, three typical patients of the above mentioned three groups (mean age 56.4 ± 17 years) at similar locations were selected. Afterwards, RNA was isolated using the RNeasy FFPE kit (Qiagen) from archived paraffin-embedded tissues. The RNA of these 12 patients were then subjected to gene expression analysis (NanoString nCounter^®), including a total of 1364 target genes. The most significantly upregulated and downregulated genes were used for a machine learning algorithm to distinguish entities. This was possible with a high probability (p < 0.0001). Interestingly, gene expression patterns showed a characteristic overlap of lymphoma with IgG4-ROD and NSOI. In contrast, IgG4-ROD shared only altered expression of one gene regarding NSOI. To validate our potential biomarker genes, we isolated the RNA of a further 48 patients (24 NSOI, 11 IgG4-ROD, 13 lymphoma patients). Then, gene expression pattern analysis of the 35 identified target genes was performed using a custom-designed CodeSet to assess the prediction accuracy of the multi-parameter scoring algorithms. They showed high accuracy and good performance (AUC ROC: IgG4-ROD 0.81, MALT 0.82, NSOI 0.67). To conclude, genetic expression analysis has the potential for faster and more secure differentiation between NSOI and IgG4-ROD. MALT-lymphoma and IgG4-ROD showed more genetic similarities, which points towards progression to lymphoma.     

Distinct Chemokine Receptor Expression Profiles in De Novo DLBCL,Transformed Follicular Lymphoma,Richter’s Trans-Formed DLBCL and Germinal Center B-Cells

Chemokine receptors and their ligands have been identified as playing an important role in the development of diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, and Richter syndrome (RS). Our aim was to investigate the different expression profiles in de novo DLBCL, transformed follicular lymphoma (tFL), and RS. Here, we profiled the mRNA expression levels of 18 chemokine receptors (CCR1–CCR9, CXCR1–CXCR7, CX3CR1 and XCR1) using RQ-PCR, as well as immunohistochemistry of seven chemokine receptors (CCR1, CCR4–CCR8 and CXCR2) in RS, de novo DLBCL, and tFL biopsy-derived tissues. Tonsil-derived germinal center B-cells (GC-B) served as non-neoplastic controls. The chemokine receptor expression profiles of de novo DLBCL and tFL substantially differed from those of GC-B, with at least 5-fold higher expression of 15 out of the 18 investigated chemokine receptors (CCR1–CCR9, CXCR1, CXCR2, CXCR6, CXCR7, CX3CR1 and XCR1) in these lymphoma subtypes. Interestingly, the de novo DLBCL and tFL exhibited at least 22-fold higher expression of CCR1, CCR5, CCR8, and CXCR6 compared with RS, whereas no significant difference in chemokine receptor expression profile was detected when comparing de novo DLBCL with tFL. Furthermore, in de novo DLBCL and tFLs, a high expression of CCR7 was associated with a poor overall survival in our study cohort, as well as in an independent patient cohort. Our data indicate that the chemokine receptor expression profile of RS differs substantially from that of de novo DLBCL and tFL. Thus, these multiple dysregulated chemokine receptors could represent novel clinical markers as diagnostic and prognostic tools. Moreover, this study highlights the relevance of chemokine signaling crosstalk in the tumor microenvironment of aggressive lymphomas.     

Innate Immunity Crosstalk with Helicobacter pylori: Pattern Recognition Receptors and Cellular Responses

Helicobacter pylori is one of the most successful gastric pathogens that has co-existed with human for centuries. H. pylori is recognized by the host immune system through human pattern recognition receptors (PRRs), such as toll-like receptors (TLRs), C-type lectin like receptors (CLRs), NOD-like receptors (NLRs), and RIG-I-like receptors (RLRs), which activate downstream signaling pathways. Following bacterial recognition, the first responders of the innate immune system, including neutrophils, macrophages, and dendritic cells, eradicate the bacteria through phagocytic and inflammatory reaction. This review provides current understanding of the interaction between the innate arm of host immunity and H. pylori, by summarizing H. pylori recognition by PRRs, and the subsequent signaling pathway activation in host innate immune cells.     

Polymorphisms in DNA Repair Genes and MDR1 and the Risk for Non-Hodgkin Lymphoma

The damage caused by oxidative stress and exposure to cigarette smoke and alcohol necessitate DNA damage repair and transport by multidrug resistance-1 (MDR1). To explore the association between polymorphisms in these genes and non-Hodgkin lymphoma risk, we analyzed 15 polymorphisms of 12 genes in a population-based study in Korea (694 cases and 1700 controls). Four genotypes of DNA repair pathway genes (XRCC1 399 GA, OGG1 326 GG, BRCA1 871 TT, and WRN 787 TT) were associated with a decreased risk for NHL [odds ratio (OR)_{XRCC1 GA} = 0.80, p = 0.02; OR_{OGG1 GG} = 0.70, p = 0.008; OR_{BRCA1 TT} = 0.71, p = 0.048; OR_{WRN TT} = 0.68, p = 0.01]. Conversely, the MGMT 115 CT genotype was associated with an increased risk for NHL (OR = 1.25, p = 0.04). In the MDR1 gene, the 1236 CC genotype was associated with a decreased risk for NHL (OR = 0.74, p = 0.04), and the 3435 CT and TT genotypes were associated with an increased risk (OR_3435CT = 1.50, p < 0.0001; OR_3435TT = 1.43, p = 0.02). These results suggest that polymorphisms in the DNA repair genes XRCC1, OGG1, BRCA1, WRN1, and MGMT and in the MDR1 gene may affect the risk for NHL in Korean patients.     

Selective Inhibition of Helicobacter pylori Carbonic Anhydrases by Carvacrol and Thymol Could Impair Biofilm Production and the Release of Outer Membrane Vesicles

Helicobacter pylori, a Gram-negative neutrophilic pathogen, is the cause of chronic gastritis, peptic ulcers, and gastric cancer in humans. Current therapeutic regimens suffer from an emerging bacterial resistance rate and poor patience compliance. To improve the discovery of compounds targeting bacterial alternative enzymes or essential pathways such as carbonic anhydrases (CAs), we assessed the anti-H. pylori activity of thymol and carvacrol in terms of CA inhibition, isoform selectivity, growth impairment, biofilm production, and release of associated outer membrane vesicles-eDNA. The microbiological results were correlated by the evaluation in vitro of H. pylori CA inhibition, in silico analysis of the structural requirements to display such isoform selectivity, and the assessment of their limited toxicity against three probiotic species with respect to amoxicillin. Carvacrol and thymol could thus be considered as new lead compounds as alternative H. pylori CA inhibitors or to be used in association with current drugs for the management of H. pylori infection and limiting the spread of antibiotic resistance.     

Characterization and Genomic Analysis of a New Phage Infecting Helicobacter pylori

Helicobacter pylori, a significant human gastric pathogen, has been demonstrating increased antibiotic resistance, causing difficulties in infection treatment. It is therefore important to develop alternatives or complementary approaches to antibiotics to tackle H. pylori infections, and (bacterio)phages have proven to be effective antibacterial agents. In this work, prophage isolation was attempted using H. pylori strains and UV radiation. One phage was isolated and further characterized to assess potential phage-inspired therapeutic alternatives to H. pylori infections. HPy1R is a new podovirus prophage with a genome length of 31,162 bp, 37.1% GC, encoding 36 predicted proteins, of which 17 were identified as structural. Phage particles remained stable at 37 °C, from pH 3 to 11, for 24 h in standard assays. Moreover, when submitted to an in vitro gastric digestion model, only a small decrease was observed in the gastric phase, suggesting that it is adapted to the gastric tract environment. Together with its other characteristics, its capability to suppress H. pylori population levels for up to 24 h post-infection at multiplicities of infection of 0.01, 0.1, and 1 suggests that this newly isolated phage is a potential candidate for phage therapy in the absence of strictly lytic phages.     

Selective Targeting of Human and Animal Pathogens of the Helicobacter Genus by Flavodoxin Inhibitors: Efficacy,Synergy, Resistance and Mechanistic Studies

Antimicrobial resistant (AMR) bacteria constitute a global health concern. Helicobacter pylori is a Gram-negative bacterium that infects about half of the human population and is a major cause of peptic ulcer disease and gastric cancer. Increasing resistance to triple and quadruple H. pylori eradication therapies poses great challenges and urges the development of novel, ideally narrow spectrum, antimicrobials targeting H. pylori. Here, we describe the antimicrobial spectrum of a family of nitrobenzoxadiazol-based antimicrobials initially discovered as inhibitors of flavodoxin: an essential H. pylori protein. Two groups of inhibitors are described. One group is formed by narrow-spectrum compounds, highly specific for H. pylori, but ineffective against enterohepatic Helicobacter species and other Gram-negative or Gram-positive bacteria. The second group includes extended-spectrum antimicrobials additionally targeting Gram-positive bacteria, the Gram-negative Campylobacter jejuni, and most Helicobacter species, but not affecting other Gram-negative pathogens. To identify the binding site of the inhibitors in the flavodoxin structure, several H. pylori-flavodoxin variants have been engineered and tested using isothermal titration calorimetry. An initial study of the inhibitors capacity to generate resistances and of their synergism with antimicrobials commonly used in H. pylori eradication therapies is described. The narrow-spectrum inhibitors, which are expected to affect the microbiota less dramatically than current antimicrobial drugs, offer an opportunity to develop new and specific H. pylori eradication combinations to deal with AMR in H. pylori. On the other hand, the extended-spectrum inhibitors constitute a new family of promising antimicrobials, with a potential use against AMR Gram-positive bacterial pathogens.     

Myricetin as an Antivirulence Compound Interfering with a Morphological Transformation into Coccoid Forms and Potentiating Activity of Antibiotics against Helicobacter pylori

Helicobacter pylori, a gastric pathogen associated with a broad range of stomach diseases, has a high tendency to become resistant to antibiotics. One of the most important factors related to therapeutic failures is its ability to change from a spiral to a coccoid form. Therefore, the main aim of our original article was to determine the influence of myricetin, a natural compound with an antivirulence action, on the morphological transformation of H. pylori and check the potential of myricetin to increase the activity of antibiotics against this pathogen. We observed that sub-minimal inhibitory concentrations (sub-MICs) of this compound have the ability to slow down the process of transformation into coccoid forms and reduce biofilm formation of this bacterium. Using checkerboard assays, we noticed that the exposure of H. pylori to sub-MICs of myricetin enabled a 4–16-fold reduction in MICs of all classically used antibiotics (amoxicillin, clarithromycin, tetracycline, metronidazole, and levofloxacin). Additionally, RT-qPCR studies of genes related to the H. pylori morphogenesis showed a decrease in their expression during exposure to myricetin. This inhibitory effect was more strongly seen for genes involved in the muropeptide monomers shortening (csd3, csd6, csd4, and amiA), suggesting their significant participation in the spiral-to-coccoid transition. To our knowledge, this is the first research showing the ability of any compound to synergistically interact with all five antibiotics against H. pylori and the first one showing the capacity of a natural substance to interfere with the morphological transition of H. pylori from spiral to coccoid forms.     

Outer Membrane Vesicle Production by Helicobacter pylori Represents an Approach for the Delivery of Virulence Factors CagA,VacA and UreA into Human Gastric Adenocarcinoma (AGS) Cells

Helicobacter pylori infection is the etiology of several gastric-related diseases including gastric cancer. Cytotoxin associated gene A (CagA), vacuolating cytotoxin A (VacA) and α-subunit of urease (UreA) are three major virulence factors of H. pylori, and each of them has a distinct entry pathway and pathogenic mechanism during bacterial infection. H. pylori can shed outer membrane vesicles (OMVs). Therefore, it would be interesting to explore the production kinetics of H. pylori OMVs and its connection with the entry of key virulence factors into host cells. Here, we isolated OMVs from H. pylori 26,695 strain and characterized their properties and interaction kinetics with human gastric adenocarcinoma (AGS) cells. We found that the generation of OMVs and the presence of CagA, VacA and UreA in OMVs were a lasting event throughout different phases of bacterial growth. H. pylori OMVs entered AGS cells mainly through macropinocytosis/phagocytosis. Furthermore, CagA, VacA and UreA could enter AGS cells via OMVs and the treatment with H. pylori OMVs would cause cell death. Comparison of H. pylori 26,695 and clinical strains suggested that the production and characteristics of OMVs are not only limited to laboratory strains commonly in use, but a general phenomenon to most H. pylori strains.     

Helicobacter pylori Infection Acts Synergistically with a High-Fat Diet in the Development of a Proinflammatory and Potentially Proatherogenic Endothelial Cell Environment in an Experimental Model

Classic atherosclerosis risk factors do not explain all cases of chronic heart disease. There is significant evidence that gut microbiota may influence the development of atherosclerosis. The widespread prevalence of chronic Helicobacter pylori (H. pylori, HP) infections suggests that HP can be the source of components that stimulate local and systemic inflammatory responses. Elevated production of reactive oxygen species during HP infection leads to cholesterol oxidation, which drives atherogenesis. The aim of this study is to explore the link between persistent HP infection and a high-fat diet in the development of proinflammatory conditions that are potentially proatherogenic. An in vivo model of Caviae porcellus infected with HP and exposed to an experimental diet was investigated for the occurrence of a proinflammatory and proatherogenic endothelial environment. Vascular endothelial primary cells exposed to HP components were tested in vitro for oxidative stress, cell activation and apoptosis. The infiltration of inflammatory cells into the vascular endothelium of animals infected with HP and exposed to a high-fat diet was observed in conjunction with an increased level of inflammatory markers systemically. The arteries of such animals were the least elastic, suggesting the role of HP in arterial stiffness. Soluble HP components induced transformation of macrophages to foam cells in vitro and influenced the endothelial life span, which was correlated with Collagen I upregulation. These preliminary results support the hypothesis that HP antigens act synergistically with a high-fat diet in the development of proatherogenic conditions.     

Use of Fourier-Transform Infrared Spectroscopy (FT-IR) for Monitoring Experimental Helicobacter pylori Infection and Related Inflammatory Response in Guinea Pig Model

Infections due to Gram-negative bacteria Helicobacter pylori may result in humans having gastritis, gastric or duodenal ulcer, and even gastric cancer. Investigation of quantitative changes of soluble biomarkers, correlating with H. pylori infection, is a promising tool for monitoring the course of infection and inflammatory response. The aim of this study was to determine, using an experimental model of H. pylori infection in guinea pigs, the specific characteristics of infrared spectra (IR) of sera from H. pylori infected (40) vs. uninfected (20) guinea pigs. The H. pylori status was confirmed by histological, molecular, and serological examination. The IR spectra were measured using a Fourier-transform (FT)-IR spectrometer Spectrum 400 (PerkinElmer) within the range of wavenumbers 3000–750 cm⁻¹ and converted to first derivative spectra. Ten wavenumbers correlated with H. pylori infection, based on the chi-square test, were selected for a K-nearest neighbors (k-NN) algorithm. The wavenumbers correlating with infection were identified in the W2 and W3 windows associated mainly with proteins and in the W4 window related to nucleic acids and hydrocarbons. The k-NN for detection of H. pylori infection has been developed based on chemometric data. Using this model, animals were classified as infected with H. pylori with 100% specificity and 97% sensitivity. To summarize, the IR spectroscopy and k-NN algorithm are useful for monitoring experimental H. pylori infection and related inflammatory response in guinea pig model and may be considered for application in humans.