Historical and Molecular Perspectives on the Presence of Helicobacter pylori in Latin America: A Niche to Improve Gastric Cancer Risk Assessment.

Helicobacter pylori (H. pylori) is responsible for causing chronic gastritis, which can cause peptic ulcer and premalignant lesions such as atrophic gastritis, intestinal metaplasia, and dysplasia, with the risk of developing gastric cancer. Recent data describe that H. pylori colonizes the gastric mucosa of more than 50% of the world's population; however, this bacterium has been described as infecting the human population since its prehistory. This review focuses on the populations and subpopulations of H. pylori, differentiated by the polymorphisms present in their constitutive and virulence genes. These genes have spread and associated with different human populations, showing variability depending on their geographical distribution, and have evolved together with the human being. The predominant genotypes worldwide, Latin America and Chile, are described to understand the genetic diversity and pathogenicity of H. pylori in different populations and geographic regions. The high similarity in the sequence of virulence genes between H. pylori strains present in Peruvian and Spanish natives in Latin America suggests a European influence. The presence of cagA-positive strains and vacA s1 m1 allelic variants is observed with greater prevalence in Chilean patients with more severe gastrointestinal diseases and is associated with its geographical distribution. These findings highlight the importance of understanding the genetic diversity of H. pylori in different regions of the world for a more accurate assessment of the risk of associated diseases and their potential impact on health.

Intersections between innate immune response and gastric cancer development.

Worldwide, gastric cancer (GC) is the fifth most commonly diagnosed malignancy. It has a reduced prevalence but has maintained its poor prognosis being the fourth leading cause of deaths related to cancer. The highest mortality rates occur in Asian and Latin American countries, where cases are usually diagnosed at advanced stages. Overall, GC is viewed as the consequence of a multifactorial process, involving the virulence of the Helicobacter pylori (H. pylori) strains, as well as some environmental factors, dietary habits, and host intrinsic factors. The tumor microenvironment in GC appears to be chronically inflamed which promotes tumor progression and reduces the therapeutic opportunities. It has been suggested that inflammation assessment needs to be measured qualitatively and quantitatively, considering cell-infiltration types, availability of receptors to detect damage and pathogens, and presence or absence of aggressive H. pylori strains. Gastrointestinal epithelial cells express several Toll-like receptors and determine the first defensive line against pathogens, and have been also described as mediators of tumorigenesis. However, other molecules, such as cytokines related to inflammation and innate immunity, including immune checkpoint molecules, interferon-gamma pathway and NETosis have been associated with an increased risk of GC. Therefore, this review will explore innate immune activation in the context of premalignant lesions of the gastric epithelium and established gastric tumors.

Recombinant Domain of Flagellin Promotes In Vitro a Chemotactic Inflammatory Profile in Human Immune Cells Independently of a Dendritic Cell Phenotype.

Flagellin is the major component of the flagellum in gram-positive and -negative bacteria and is also the ligand for the Toll-like receptor 5 (TLR5). The activation of TLR5 promotes the expression of proinflammatory cytokines and chemokines and the subsequent activation of T cells. This study evaluated a recombinant domain from the amino-terminus D1 domain (rND1) of flagellin from Vibrio anguillarum, a fish pathogen, as an immunomodulator in human peripheral blood mononuclear cells (PBMCs) and monocyte-derived dendritic cells (MoDCs). We demonstrated that rND1 induced an upregulation of proinflammatory cytokines in PBMCs, characterized at the transcriptional level by an expression peak of 220-fold for IL-1ß, 20-fold for IL-8, and 65-fold for TNF-α. In addition, at the protein level, 29 cytokines and chemokines were evaluated in the supernatant and were correlated with a chemotactic signature. MoDCs treated with rND1 showed low levels of co-stimulatory and HLA-DR molecules and kept an immature phenotype with a decreased phagocytosis of dextran. We probed that rND1 from a non-human pathogen promotes modulation in human cells, and it may be considered for further studies in adjuvant therapies based on pathogen-associated patterns (PAMPs).

Simultaneous and Spatially-Resolved Analysis of T-Lymphocytes, Macrophages and PD-L1 Immune Checkpoint in Rare Cancers.

Penile, vulvar and anal neoplasms show an incidence lower than 0.5% of the population per year and therefore can be considered as rare cancers but with a dramatic impact on quality of life and survival. This work describes the experience of a Chilean cancer center using multiplexed immunofluorescence to study a case series of four penile cancers, two anal cancers and one vulvar cancer and simultaneous detection of CD8, CD68, PD-L1, Cytokeratin and Ki-67 in FFPE samples. Fluorescent image analyses were performed using open sources for automated tissue segmentation and cell phenotyping. Our results showed an objective and reliable counting of objects with a single or combined labeling or within a specific tissue compartment. The variability was below 10%, and the correlation between analytical events was 0.92-0.97. Critical cell phenotypes, such as TILs, PD-L1+ or proliferative tumor cells were detected in a supervised and unsupervised manner with a limit of detection of less than 1% of relative abundance. Finally, the observed diversity and abundance of the different cell phenotypes within the tumor microenvironment for the three studied tumor types confirmed that our methodology is useful and robust to be applicable for many other solid tumors.

Associations between bacterial communities and microplastics from surface seawater of the Northern Patagonian area of Chile.

The presence of microplastics in oceans and coastlines has increased during recent years due anthropogenic activities and represents a serious environmental problem. The establishment and assembly of microbial communities in these microplastics, specifically located near aquaculture activities, is not well understood. In this study, we analyzed unique and core members of bacterial communities attached to microplastics collected from three coastal environments of the South Pacific, which represent low, medium and high anthropogenic activity derived from the aquaculture industry. Microplastics were analyzed with Fourier-transform infrared spectroscopy, scanning electron microscopy, and next-generation sequencing to assess the prevailing microplastics types, and to characterize microbial communities attached to them. We identified four main types of microplastics (polypropylene, polyethylene, nylon and polystyrene) and 3102 Operational Taxonomic Units (OTUs) at the sampled sites, which were dominated by the phylum Cyanobacteria, Bacteroidetes and Proteobacteria (mainly Alpha and Gammaproteobacteria). Similarity index analysis showed that bacterial communities in microplastics differed from those found in the surrounding seawaters, and also that they varied among locations, suggesting a role of the environment and level of anthropogenic activities on the plastisphere taxa. Despite this difference, 222 bacterial OTUs were shared among the three sites representing between 34 and 51% of OTUs of each sampled site, and thus constituted a core microbiome of microplastics. Comparison of the core microbiome with bacterial communities of the surrounding seawater suggested that the plastisphere constituted a selective habitat for diverse microbial communities. Computational predictions also provided evidence of significantly enriched functions in the core microbiome. Co-occurrence networks revealed that putative ecological interactions among microplastics OTUs was dominated by positive correlations. To the best of our knowledge, this is the first study that evaluated the composition of microbial communities found in microplastics from the Patagonia region of the Southern Pacific Ocean.

Insights into neutralizing antibody responses in individuals exposed to SARS-CoV-2 in Chile.

Chile has one of the worst numbers worldwide in terms of SARS-CoV-2 positive cases and COVID-19-related deaths per million inhabitants; thus, characterization of neutralizing antibody (NAb) responses in the general population is critical to understanding of immunity at the local level. Given our inability to perform massive classical neutralization assays due to the scarce availability of BSL-3 facilities in the country, we developed and fully characterized an HIV-based SARS-CoV-2 pseudotype, which was used in a 96-well plate format to investigate NAb responses in samples from individuals exposed to SARS-CoV-2 or treated with convalescent plasma. We also identified samples with decreased or enhanced neutralization activity against the D614G spike variant compared with the wild type, indicating the relevance of this variant in host immunity. The data presented here represent the first insights into NAb responses in individuals from Chile, serving as a guide for future studies in the country.

Full recombinant flagellin B from Vibrio anguillarum (rFLA) and its recombinant D1 domain (rND1) promote a pro-inflammatory state and improve vaccination against P. salmonis in Atlantic salmon (S. salar).

Flagellin is the major component of the flagellum, and a ligand for Toll-like receptor 5. As reported, recombinant flagellin (rFLA) from Vibrio anguillarum and its D1 domain (rND1) are able to promote in vitro an upregulation of pro-inflammatory genes in gilthead seabream (Sparus aurata) and rainbow trout (Oncorhynchus mykiss) macrophages. This study evaluated the in vitro and in vivo stimulatory/adjuvant effect for rFLA and rND1 during P. salmonis vaccination in Atlantic salmon (Salmo salar). We demonstrated that rFLA and rND1 are molecules able to generate an acute upregulation of pro-inflammatory cytokines (IL-1ß, IL-8, IL-12ß), allowing the expression of genes associated with T-cell activation (IL-2, CD4, CD8ß), and differentiation (IFNγ, IL-4/13, T-bet, Eomes, GATA3), in a differential manner, tissue/time dependent way. Altogether, our results suggest that rFLA and rND1 are valid candidates to be used as an immuno-stimulant or adjuvants with existing vaccines in farmed salmon.

Biofilm Produced In Vitro by Piscirickettsia salmonis Generates Differential Cytotoxicity Levels and Expression Patterns of Immune Genes in the Atlantic Salmon Cell Line SHK-1.

Piscirickettsia salmonis is the causative agent of Piscirickettsiosis, an infectious disease with a high economic impact on the Chilean salmonid aquaculture industry. This bacterium produces biofilm as a potential resistance and persistence strategy against stressful environmental stimuli. However, the in vitro culture conditions that modulate biofilm formation as well as the effect of sessile bacteria on virulence and immune gene expression in host cells have not been described for P. salmonis. Therefore, this study aimed to analyze the biofilm formation by P. salmonis isolates under several NaCl and iron concentrations and to evaluate the virulence of planktonic and sessile bacteria, together with the immune gene expression induced by these bacterial conditions in an Atlantic salmon macrophage cell line. Our results showed that NaCl and Fe significantly increased biofilm production in the LF-89 type strain and EM-90-like isolates. Additionally, the planktonic EM-90 isolate and sessile LF-89 generated the highest virulence levels, associated with differential expression of il-1ß, il-8, nf-κb, and iκb-α genes in SHK-1 cells. These results suggest that there is no single virulence pattern or gene expression profile induced by the planktonic or sessile condition of P. salmonis, which are dependent on each strain and bacterial condition used.

Biochemical and Genomic Characterization of the Cypermethrin-Degrading and Biosurfactant-Producing Bacterial Strains Isolated from Marine Sediments of the Chilean Northern Patagonia.

Pesticides cause severe environmental damage to marine ecosystems. In the last ten years, cypermethrin has been extensively used as an antiparasitic pesticide in the salmon farming industry located in Northern Patagonia. The objective of this study was the biochemical and genomic characterization of cypermethrin-degrading and biosurfactant-producing bacterial strains isolated from cypermethrin-contaminated marine sediment samples collected in southern Chile (MS). Eleven strains were isolated by cypermethrin enrichment culture techniques and were identified by 16S rDNA gene sequencing analyses. The highest growth rate on cypermethrin was observed in four isolates (MS13, MS15a, MS16, and MS19) that also exhibited high levels of biosurfactant production. Genome sequence analyses of these isolates revealed the presence of genes encoding components of bacterial secondary metabolism, and the enzymes esterase, pyrethroid hydrolase, and laccase, which have been associated with different biodegradation pathways of cypermethrin. These novel cypermethrin-degrading and biosurfactant-producing bacterial isolates have a biotechnological potential for biodegradation of cypermethrin-contaminated marine sediments, and their genomes contribute to the understanding of microbial lifestyles in these extreme environments.

New immunomodulatory role of neuropeptide Y (NPY) in Salmo salar leucocytes.

Neuropeptide Y (NPY) plays different roles in mammals such as: regulate food intake, memory retention, cardiovascular functions, and anxiety. It has also been shown in the modulation of chemotaxis, T lymphocyte differentiation, and leukocyte migration. In fish, NPY expression and functions have been studied but its immunomodulatory role remains undescribed. This study confirmed the expression and synthesis of NPY in S. salar under inflammation, and validated a commercial antibody for NPY detection in teleost. Additionally, immunomodulatory effects of NPY were assayed in vitro and in vivo. Phagocytosis and superoxide anion production in leukocytes and SHK cells were induced under stimulation with a synthetic peptide. IL-8 mRNA was selectively and strongly induced in the spleen, head kidney, and isolated cells, after in vivo challenge with NPY. All together suggest that NPY is expressed in immune tissues and modulates the immune response in teleost fish.

Polyglucosan Molecules Induce Mitochondrial Impairment and Apoptosis in Germ Cells Without Affecting the Integrity and Functionality of Sertoli Cells.

Glycogen is the main storage form of glucose; however, the accumulation of glycogen-like glucose polymers can lead to degeneration and cellular death. Previously, we reported that the accumulation of glycogen in testis of transgenic animals overexpressing a constitutively active form of glycogen synthase enhances the apoptosis of pre-meiotic male germ cells and a complete disorganization of the seminiferous tubules. Here we sought to further identify the effects of glycogen storage in cells from the seminiferous tubules and the mechanism behind the pro-apoptotic activity induced by its accumulation. Using an in vitro culture of Sertoli cells (line 42GPA9) and spermatocyte-like cells (line GC-1) expressing a superactive form of glycogen synthase or the Protein Targeting to Glycogen (PTG), we found that glycogen synthesized in both cell lines is poorly branched. In addition, the immunodetection of key molecules of apoptotic events suggests that cellular death induced by polyglucosan molecules affects GC-1 cells, but not 42GPA9 cells by mitochondrial impairment and activation of an intrinsic apoptotic pathway. Furthermore, we analyzed the effects of glycogen deposition during the establishment of an in vitro blood-testis barrier. The results using a non-permeable fluorescent molecule showed that, in conditions of over-synthesis of glycogen, 42GPA9 cells do not lose their capacity to generate an impermeable barrier and the levels of connexin43, occludin, and ZO1 proteins were not affected. These results suggest that the accumulation of polyglucosan molecules has a selective effect-triggered by the intrinsic activation of the apoptotic pathway-in germ cells without directly affecting Sertoli cells. J. Cell. Physiol. 231: 2142-2152, 2016. © 2016 Wiley Periodicals, Inc.

A synthetic peptide derived from the D1 domain of flagellin induced the expression of proinflammatory cytokines in fish macrophages.

Flagellin is the main protein component of flagellum in Gram negative and positive bacteria, and it is also the ligand that activates the Toll-like receptor 5 (TLR5) in fish and mammals. In higher vertebrates, flagellin induces the activation of the membrane-bound TLR5 (TLR5M), which promotes the expression of proinflammatory cytokines and chemokines, and other immunological functions. We have previously reported that recombinant flagellin from Vibrio anguillarum and its ND1 domain are able to upregulate the expression of genes encoding major the proinflammatory mediators in gilthead seabream and rainbow trout macrophages. Considering the key role of D1 domain of flagellin for binding to TLR5M and its immunostimulatory activity, we designed and chemically synthesized a peptide derived of this region. The effects of the synthetic peptide were evaluated in vitro using head kidney macrophages from gilthead seabream (Sparus aurata L., Perciformes, Sparidae) and rainbow trout (Oncorhynchus mykiss W., Salmoniformes, Salmonidae). In both species the expression of genes encoding the proinflammatory cytokines interleukin-1ß (IL-1ß) and tumour necrosis factor-α (TNF-α), and the chemokine IL-8, was induced upon stimulation of macrophages with the D1 domain synthetic peptide. IL-1ß and IL-8 were the most upregulated genes and to a lesser extent TNF-α. Interestingly, however, the induction activity of the synthetic peptide was higher in gilthead seabream than in rainbow trout macrophages. The results were confirmed at the protein levels for IL-8. Collectively, these results suggest that synthetic peptide derived from flagelling could be a promising approach for the immunostimulation and vaccination of farmed fish.

Identification and genetic characterization of Piscirickettsia salmonis in native fish from southern Chile.

Piscirickettsia salmonis is the etiological agent of piscirickettsiosis, a severe disease causing high mortalities in salmonids. This bacterium has been previously identified and isolated in all cultivated salmonids in Chile and worldwide, including Salmo salar, Oncorhynchus kisutch, and O. mykiss, in addition to being found in non-salmonid species such as Dicentrarchus labrax and Atractoscion nobilis. In this study, the 16S rRNA gene and intergenic spacer ITS-1 of P. salmonis were amplified by PCR from DNA samples extracted from the native Chilean fish species Eleginops maclovinus, Odontesthes regia, Sebastes capensis, and Salilota australis. Analysis of the 16S rRNA sequences from O. regia demonstrated a close phylogenetic relationship with the 16S rRNA gene in the Chilean EM-90 strain. The 16S rRNA sequences from E. maclovinus, S. capensis, and S. australis were related to the Chilean LF-89 sequence and Scottish strains. To confirm these findings, analysis of P. salmonis ITS-1 sequences obtained from the 4 sampled native species demonstrated a high degree of identity and a close phylogenetic relationship with Chilean P. salmonis sequences, including LF-89 and EM-90. These results suggest a strong relationship between the nucleotide sequences from the 16S rRNA and ITS-1 genes amplified from native fish with those sequences described in the first P. salmonis strains to be identified and isolated in Chile.

Effects of recombinant flagellin B and its ND1 domain from Vibrio anguillarum on macrophages from gilthead seabream (Sparus aurata L.) and rainbow trout (Oncorhynchus mykiss, W.).

Flagellin is the principal component of flagellum in Gram negative and positive bacteria, and it is also the ligand that activates the Toll-like receptor 5 (TLR5) in mammals and fish. In higher vertebrates, flagellin induces the activation of the membrane-bound TLR5 (TLR5M), which promotes the expression of proinflammatory cytokines and chemokines and the co-stimulatory molecules present in antigen-presenting cells needed for the activation of T cells. In the present study, we report the production of two recombinant proteins of Vibrio anguillarum: i) a full length flagellin B (FlaB) (rFla) and ii) the amino-terminus of the D1 domain (rND1) of the same protein, the region mainly responsible for binding to TLR5 and for the immunostimulatory activity of flagellin. The effects of these recombinant proteins were assessed in vitro using head kidney macrophages of gilthead seabream (Sparus aurata L., Perciformes, Sparidae) and rainbow trout (Oncorhynchus mykiss W., Salmoniformes, Salmonidae). In both species, 3 h of stimulation with rFla and rND1 induced expression of the proinflammatory cytokines interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α), and of the chemokine IL-8. In gilthead seabream macrophages stimulated with rFla and rND1, a 900- and 6-fold increase were observed for IL-1ß transcription, while a 900- and 3-fold increase were recorded for IL-8 transcription, respectively, as compared to non-stimulated macrophages. In rainbow trout, rFla increased expression of IL-8 40-fold in macrophages, whereas rND1 increased expression of the chemokine 3-fold, as compared to non-stimulated cells. The results obtained for rFla and rND1 demonstrate their modulatory capabilities in vitro, suggesting that rFla and rND1 could be evaluated as immunostimulatory candidates for use in farmed fish. However, further in vivo studies are needed to confirm and expand on the present results.