scholarly journals Metagenomic and single-cell RNA-seq survey of the H. pylori-infected stomach in asymptomatic individuals

2021 ◽  
Author(s):  
Chiara Sorini ◽  
Kumar P. Tripathi ◽  
Shengru Wu ◽  
Shawn M. Higdon ◽  
Jing Wang ◽  
...  
Keyword(s):  
Gastric Mucosa ◽  
Single Cell ◽  
Immune Cell ◽  
Human Stomach ◽  
List Type ◽  
Whole Genome ◽  
Tertiary Lymphoid Structures ◽  
Lymphoid Structures ◽  
H Pylori ◽  
Asymptomatic Individuals

ABSTRACTObjectiveHelicobacter pylori colonization of the gastric niche can persist for years in asymptomatic individuals. Although latent H. pylori infection can progress to cancer, a detailed survey of the microbiome and immune composition in the chronically infected stomach is still lacking.DesignWe collected human gastric tissues and performed metagenomic sequencing, single-cell RNA sequencing (scRNA-seq), flow cytometry and fluorescent microscopy to deeply characterize the host-microbiota environment in H. pylori-infected (HPI) stomachs.ResultsHPI asymptomatic individuals showed dramatic changes in the composition of gastric microbiome and immune cells compared to non-infected individuals. With metagenomic data, we also demonstrated antibiotic resistant genes, enzymes and pathway alterations related to metabolism and immune response. scRNA-seq and flow cytometry data revealed that in contrast to murine stomachs, ILC2 are virtually absent in the human gastric mucosa, whereas ILC3 are the dominant population in asymptomatic HPI individuals. Specifically, NKp44+ ILC3s were highly increased in the gastric mucosa of asymptomatic HPI individuals, and their proportions correlated with the abundance of selected microbial taxa found to be enriched in the infected mucosa. In addition, CD11c+ myeloid cells, activated CD4 T cells and B cells were expanded in HPI individuals. In HPI individuals, B cells acquired an activated phenotype and progressed into a highly proliferating germinal center stage and plasmablast maturation, which correlated with the presence of tertiary lymphoid structures within the gastric lamina propria.ConclusionOur study provides a comprehensive atlas of the gastric mucosa-associated microbiome and immune cell landscape when comparing asymptomatic HPI and uninfected individuals.SIGNIFICANCE OF THE STUDYWhat is already known on this subject? Previous studies on the gastric microbiome were performed via 16S rRNA gene sequencing.Acute Helicobacter spp. infection in murine models and symptomatic H. pylori-driven pathology in humans result in remodeling of the stomach immune cell compartment.ILC2 is the dominant ILC population in the murine stomach.What are the new findings? We described the effect of chronic asymptomatic H. pylori infection on the gastric microbiome via whole-genome sequencing.Single cell census of the gastric mucosa reveals ILC3 to be the dominant ILC population in the human stomach, whereas ILC2 were virtually absent.scRNA-seq reveals the gastric immune cell programs in asymptomatic H. pylori-infected individuals, which is characterized by the formation of tertiary lymphoid structures.How might it impact on clinical practice in the foreseeable future? Whole genome sequencing of uninfected and H. pylori-infected gastric mucosa bolsters collective knowledge of stomach physiology with respect to the gastric microbiome and microbiota function.We present a comprehensive immune cellular landscape of the human stomach, which will be a valuable resource to interrogate pathology of gastric diseases.

scholarly journals Helicobacter pylori–binding nonacid glycosphingolipids in the human stomach

2018 ◽  
Vol 293 (44) ◽  
pp. 17248-17266 ◽  
Author(s):  
Chunsheng Jin ◽  
Angela Barone ◽  
Thomas Borén ◽  
Susann Teneberg
Keyword(s):  
Helicobacter Pylori ◽  
Gastric Mucosa ◽  
Structural Complexity ◽  
Human Stomach ◽  
Carbohydrate Binding ◽  
Human Gastric Mucosa ◽  
H Pylori ◽  
Contrast Information

Helicobacter pylori has a number of well-characterized carbohydrate-binding adhesins (BabA, SabA, and LabA) that promote adhesion to the gastric mucosa. In contrast, information on the glycoconjugates present in the human stomach remains unavailable. Here, we used MS and binding of carbohydrate-recognizing ligands to characterize the glycosphingolipids of three human stomachs from individuals with different blood group phenotypes (O(Rh−)P, A(Rh+)P, and A(Rh+)p), focusing on compounds recognized by H. pylori. We observed a high degree of structural complexity, and the composition of glycosphingolipids differed among individuals with different blood groups. The type 2 chain was the dominating core chain of the complex glycosphingolipids in the human stomach, in contrast to the complex glycosphingolipids in the human small intestine, which have mainly a type 1 core. H. pylori did not bind to the O(Rh−)P stomach glycosphingolipids, whose major complex glycosphingolipids were neolactotetraosylceramide, the Lex, Lea, and H type 2 pentaosylceramides, and the Ley hexaosylceramide. Several H. pylori-binding compounds were present among the A(Rh+)P and A(Rh+)p stomach glycosphingolipids. Ligands for BabA-mediated binding of H. pylori were the Leb hexaosylceramide, the H type 1 pentaosylceramide, and the A type 1/ALeb heptaosylceramide. Additional H. pylori-binding glycosphingolipids recognized by BabA-deficient strains were lactosylceramide, lactotetraosylceramide, the x2 pentaosylceramide, and neolactohexaosylceramide. Our characterization of human gastric receptors required for H. pylori adhesion provides a basis for the development of specific compounds that inhibit the binding of this bacterium to the human gastric mucosa.

scholarly journals The Immune System of Mesothelioma Patients: A Window of Opportunity for Novel Immunotherapies

2021 ◽  
Author(s):  
Fabio Nicolini ◽  
Massimiliano Mazza
Keyword(s):  
Immune System ◽  
Immune Cells ◽  
Immune Cell ◽  
Current Knowledge ◽  
Cell Types ◽  
Screening Strategies ◽  
Tertiary Lymphoid Structures ◽  
Lymphoid Structures ◽  
Review Current

The interplay between the immune system and the pleural mesothelium is crucial both for the development of malignant pleural mesothelioma (MPM) and for the response of MPM patients to therapy. MPM is heavily infiltrated by several immune cell types which affect the progression of the disease. The presence of organized tertiary lymphoid structures (TLSs) witness the attempt to fight the disease in situ by adaptive immunity which is often suppressed by tumor expressed factors. In rare patients physiological, pharmacological or vaccine-induced immune response is efficient, rendering their plasma a valuable resource of anti-tumor immune cells and molecules. Of particular interest are human antibodies targeting antigens at the tumor cell surface. Here we review current knowledge regarding MPM immune infiltration, MPM immunotherapy and the harnessing of this response to identify novel biologics as biomarkers and therapeutics through innovative screening strategies.

scholarly journals Nerve fibers in the Tumor Microenvironment are co-localized with Tertiary Lymphoid Structures

2020 ◽  
Author(s):  
Lara R. Heij ◽  
Xiuxiang Tan ◽  
Jakob N. Kather ◽  
Jan M. Niehues ◽  
Shivan Sivakumar ◽  
...  
Keyword(s):  
Pancreatic Ductal Adenocarcinoma ◽  
Immune Cells ◽  
Immune Cell ◽  
Nerve Fibers ◽  
Ductal Adenocarcinoma ◽  
Fiber Density ◽  
Tertiary Lymphoid Structures ◽  
Lymphoid Structures ◽  
Nerve Fiber Density ◽  
Small Nerve

ABSTRACTBackgroundB cells and tertiary lymphoid structures (TLS) are reported to be important in the improvement of survival of cancer patients. These secondary lymphoid organs have been associated with the generation of an anti-tumor response. Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancer types and the stromal architecture shapes the intratumoral heterogeneity. The stroma of PDAC is a complex system in which crosstalk takes place between cancer-associated fibroblasts, immune cells, endothelial cells and the cancer cells. Besides immune cells and fibroblasts, there is some limited data about the influence of nerve fibers on cancer progression.Patients and methodsNerve Fiber Density (NFD) was analysed in our cohort of 188 patients with Pancreatic Ductal Adenocarcinoma who underwent pancreatic surgery. We used immunohistochemistry and multiplex imaging to phenotype the immune cell infiltrate. The cell detection classifier measured distance from immune cell to cancer gland and with a heat map we could count TLS. By using Machine learning we were able to define the spatial distribution and counting Tertiary Lymphoid Structures.ResultsHigh NFD is significantly associated with prolonged overall survival (HR 1.676 (95%CI 1.126,2.495) for low vs. high NFD, p-value 0.0109). The immune cells surrounding the nerve fibers were phenotyped in B cells, T cells and dendritic follicular cells, matching a TLS. Here we show that small nerve fibers are located at the TLS in Pancreatic Cancer and a high Nerve Fiber Density combined with more than 5 TLS is associated with a better survival (HR 0.388 (95%CI 0.218, 0.689).ConclusionThe co-localization of small nerve fibers with TLS is a new finding which has not been described before. However the precise roles of these TLS and nerve fibers remains unknown. These findings unravel future pathways and has the potential to reach new directions into already existing targeted therapy.

scholarly journals Prognostic Significance of Gene Signature of Tertiary Lymphoid Structures in Patients With Lung Adenocarcinoma

2021 ◽  
Vol 11 ◽  
Author(s):  
Hong Feng ◽  
Fujun Yang ◽  
Lihong Qiao ◽  
Kai Zhou ◽  
Junfei Wang ◽  
...  
Keyword(s):  
Overall Survival ◽  
Lung Adenocarcinoma ◽  
Immune Cell ◽  
Prognostic Significance ◽  
Gene Signature ◽  
Driver Gene ◽  
Immune Microenvironment ◽  
High Group ◽  
Tertiary Lymphoid Structures ◽  
Lymphoid Structures

BackgroundLung adenocarcinoma (LUAD) is a highly mortal cancer. Tertiary lymphoid structures (TLS) are ectopic lymphoid organs with similar morphological and molecular characters to secondary lymphoid organ. The aim of this study is to investigate the prognostic effect of a gene signature associated with TLSs, including B-cell-specific genes.MethodsClinical data of 515 LUAD patients in the TGCA cohort were used to examine the relationship of TLS signature with immune microenvironment, tumor mutational burden (TMB), and driver gene mutations. Patients were divided into the TLS signature high group and TLS signature low group, and comparative analysis of survival and its influencing factors between the two groups was performed. The resulting data were then validated in the GSE37745 cohort.ResultsTLS signature high group had significantly better overall survival (OS) and progression-free interval (PFI) as well as significantly higher infiltration of immune cell subsets, cancer immune cycle (CIC) signature except for immunogram score2 (IGS2), and expression of major checkpoint genes than the TLS signature low group. Notably, while TLS signature was not markedly associated with TMB and mutation frequencies of driver genes, there were significant differences in overall survival of patients with given mutation status of EGFR, KRAS, BRAF and TP53 genes between the TLS signature high and low groups.ConclusionThis study provided evidence that LUAD patients with high TLS signature had a favorable immune microenvironment and better prognosis, suggesting that TLS signature is an independent positive prognostic factor for LUAD patients.

scholarly journals Comparison of induction of B45 Helicobacter pylori prophage by acid and UV radiation

2013 ◽  
Vol 19 (S4) ◽  
pp. 27-28 ◽  
Author(s):  
A.P. Alves de Matos ◽  
P. Lehours ◽  
A. Timóteo ◽  
M. Roxo-Rosa ◽  
F.F. Vale
Keyword(s):  
Electron Microscopy ◽  
Gastric Mucosa ◽  
Uv Radiation ◽  
Human Stomach ◽  
Lytic Cycle ◽  
Acidic Environment ◽  
H Pylori ◽  
Citrate Phosphate ◽  
Microaerophilic Conditions ◽  
Citrate Phosphate Buffer

Helicobacter pylori is a Gram-negative microorganism that grows on microaerophilic conditions and has only one known natural reservoir: the gastric mucosa. The infection by H. pylori is very common worldwide and this bacterium is associated with the development of gastritis, peptic ulcer gastric cancer or gastric Mucosa Associated Lymphoid Tissue (MALT) lymphoma. Although its natural habitat is the acidic gastric mucosa, H. pylori is considered to be a neutralophile. The bacterium survives brief exposure to pHs of <4, but growth occurs only at the relatively narrow pH range of 5.5 to 8.0, with optimal growth at neutral pH. Recently we have identified a prophage sequence (prophage phiHP33) in the strain B45, isolated from a patient diagnosed with gastric MALT lymphoma. This prophage revealed to be very difficult to induce. In fact, only few phage particles were observed on electron microscopy micrographs after exposure to UV radiation.In the present work we have compared the exposure to UV and to acidic environment in the induction of the prophage into a lytic cycle. We have tested two strains, the strain B45 carrying the prophage phiHP33 and a clinical strain 1152, isolated from a patient with peptic ulcer, that was revealed to be negative for the presence of integrase gene (a prophage gene essential for genome integration of prophage) by PCR, as negative control. Since the H. pylori reservoir is the human stomach the exposition to acid is very common, and with this experiment we intended to test if acid can trigger a phage lytic cycle.The induction using UV radiation has been previously described. For acid induction we have used a protocol adapted from Karita and Blaser. A 48 hours culture of H. pylori was grown in Brucella broth (Oxoid) supplemented with 10% of fetal bovine serum (Gibco) and 1% of Polivitex (BioMérieaux) in microaerophilic conditions at 37ºC. The liquid culture was centrifuged and the cell pellet ressuspended in citrate-phosphate buffer pH 6 and incubated 15 minutes, centrifuged again and ressuspended in citrate-phosphate buffer pH 3 and incubated for 30 minutes. After centrifugation the supernatant was recovered and incubated for 3 hours in phage precipitant (33% polyethylene glycol [PEG], 3M NaCl). After centrifugation at 10000 rpm for 10 minutes at 4ºC the pellet was ressuspended in phage buffer. These samples were analysed by transmission electron microscopy (TEM) after negative staining with 1% aqueous uranyl acetate, using a JEOL 100SX electron microscope.For B45 strain the induction using UV radiation (previously reported in Lehours, 2011) and acid exposure produced similar results (Figure 1 and Figure 2) showing numerous phage-like particles of about 100 nm diameter, apparently lacking a tail, after UV or acid exposition, respectively. These particles were not observed in the control strain 1152. Currently we are analysing the samples using molecular biology techniques and fixation embedding followed by ultrathin sectioning for TEM analysis, to detect the presence of phages.These preliminary results suggest that acid also appears to induce the H. pylori prophage phiHP33. However, since the number of phage particles observed is small, we can not rule out that the observed particles were released spontaneously. The exposition to the natural acidic environment of the human stomach may induce H. pylori prophage into a lytic cycle and to the propagation of phages among different H. pylori strains colonizing the same individual. Although highly speculative, transduction may be another form of horizontal gene transfer, which has not been described for this bacterium yet.Financial support received from the Portuguese Science and Technology foundation under the contract PTDC/EBB-EBI/119860/2010.

584 Therapeutic vascular normalization to promote tumor-associated tertiary lymphoid structures

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A619-A619
Author(s):  
Jessica Filderman ◽  
Manoj Chelvanambi ◽  
Walter Storkus
Keyword(s):  
Immune Cell ◽  
Tumor Vasculature ◽  
Cell Infiltration ◽  
Global Changes ◽  
Antiangiogenic Agents ◽  
Immune Cell Infiltration ◽  
Low Doses ◽  
Vascular Normalization ◽  
Tertiary Lymphoid Structures ◽  
Lymphoid Structures

BackgroundTertiary lymphoid structures (TLS) are non-encapsulated immune cell aggregates that form at sites of chronic inflammation, including in and around tumors. Recent studies have shown that the presence of TLS in human tumors is an indicator of positive clinical outcome. However, due to dysregulated angiogenesis, many tumors have a poorly-organized and leaky vasculature that impedes entry of immune effector cells into tumors and consequently, TLS formation. It has been shown in pre-clinical studies that low doses of antiangiogenic agents normalize the tumor vasculature, leading us to hypothesize that treating tumors with low-doses (well below drug MTD) of vascular normalizing (VN) therapies will improve immune cell infiltration and TLS formation within the tumor microenvironment (TME).MethodsTo test this hypothesis, melanoma-bearing mice were treated intratumorally with VN agents. Five days post-treatment, tumors were digested into single cell suspensions and RNA was isolated and used for RT-PCR. Transcript levels of TLS-promoting factors (CCL19, CCL21, CXCL13) and markers of vascular normalization (HIF1A, GLUT1) and inflammation/immune cell infiltration (CXCL10, VCAM1, CD8A) were measured and compared to PBS treated controls. Changes in tumor vasculature were evaluated using immunofluorescence microscopy (IFM) of tumor sections stained with CD31, PNAd, and PDGFRβ. Fluorescently-labeled lectin was injected into the mice to observe tumor perfusion. TLS formation was evaluated in tumor sections using IFM, with TLS being defined as PNAd+ vessels surrounded by clusters of CD45+ cells.ResultsWe observed that the VN agents dasatinib, STING agonist, bevacizumab, and agonist anti-TNFR1 antibody each induced global changes in the TME that are consistent with enhanced immune cell infiltration and TLS formation. These changes include increases in expression of CCL19, CCL21, and VCAM1. Dasatinib and STING agonists were shown to promote VN, as demonstrated by improved lectin perfusion into the tumor and increased pericyte coverage of blood vessels on-treatment.ConclusionsVN agents induce global changes in immune cell infiltration and TLS-promoting factors in the TME. In vivo, these agents induce VN in the TME and promote TLS formation. This knowledge can be used to develop optimal combination immunotherapy designs in the cancer setting.

scholarly journals Single-cell atlas of a non-human primate reveals new pathogenic mechanisms of COVID-19

2020 ◽  
Author(s):  
Lei Han ◽  
Xiaoyu Wei ◽  
Chuanyu Liu ◽  
Giacomo Volpe ◽  
Zhifeng Wang ◽  
...  
Keyword(s):  
Single Cell ◽  
Viral Entry ◽  
Immune Cell ◽  
Cell Types ◽  
Chromatin Accessibility ◽  
List Type ◽  
Innate Immune Responses ◽  
Cell Transcriptome ◽  
Single Cell Transcriptome ◽  
Substantial Heterogeneity

ABSTRACTStopping COVID-19 is a priority worldwide. Understanding which cell types are targeted by SARS-CoV-2 virus, whether interspecies differences exist, and how variations in cell state influence viral entry is fundamental for accelerating therapeutic and preventative approaches. In this endeavor, we profiled the transcriptome of nine tissues from a Macaca fascicularis monkey at single-cell resolution. The distribution of SARS-CoV-2 facilitators, ACE2 and TMRPSS2, in different cell subtypes showed substantial heterogeneity across lung, kidney, and liver. Through co-expression analysis, we identified immunomodulatory proteins such as IDO2 and ANPEP as potential SARS-CoV-2 targets responsible for immune cell exhaustion. Furthermore, single-cell chromatin accessibility analysis of the kidney unveiled a plausible link between IL6-mediated innate immune responses aiming to protect tissue and enhanced ACE2 expression that could promote viral entry. Our work constitutes a unique resource for understanding the physiology and pathophysiology of two phylogenetically close species, which might guide in the development of therapeutic approaches in humans.Bullet pointsWe generated a single-cell transcriptome atlas of 9 monkey tissues to study COVID-19.ACE2+TMPRSS2+ epithelial cells of lung, kidney and liver are targets for SARS-CoV-2.ACE2 correlation analysis shows IDO2 and ANPEP as potential therapeutic opportunities.We unveil a link between IL6, STAT transcription factors and boosted SARS-CoV-2 entry.

scholarly journals Developing ovine mammary terminal duct lobular units have a dynamic mucosal and stromal immune microenvironment

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Dorottya Nagy ◽  
Clare M. C. Gillis ◽  
Katie Davies ◽  
Abigail L. Fowden ◽  
Paul Rees ◽  
...  
Keyword(s):  
Mammary Gland ◽  
Immune Cell ◽  
Leading Edge ◽  
Breast Development ◽  
Cell Aggregates ◽  
Human Breast ◽  
Immune Microenvironment ◽  
Ki67 Expression ◽  
Tertiary Lymphoid Structures ◽  
Lymphoid Structures

AbstractThe human breast and ovine mammary gland undergo striking levels of postnatal development, leading to formation of terminal duct lobular units (TDLUs). Here we interrogate aspects of sheep TDLU growth as a model of breast development and to increase understanding of ovine mammogenesis. The distributions of epithelial nuclear Ki67 positivity differ significantly between younger and older lambs. Ki67 expression is polarised to the leading edge of the developing TDLUs. Intraepithelial ductal macrophages exhibit periodicity and considerably increased density in lambs approaching puberty. Stromal macrophages are more abundant centrally than peripherally. Intraepithelial T lymphocytes are more numerous in older lambs. Stromal hotspots of Ki67 expression colocalize with immune cell aggregates that exhibit distinct organisation consistent with tertiary lymphoid structures. The lamb mammary gland thus exhibits a dynamic mucosal and stromal immune microenvironment and constitutes a valuable model system that provides new insights into postnatal breast development.

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