scholarly journals α-Difluoromethylornithine reduces gastric carcinogenesis by causing mutations in Helicobacter pylori cagY

2019 ◽  
Vol 116 (11) ◽  
pp. 5077-5085 ◽  
Author(s):  
Johanna C. Sierra ◽  
Giovanni Suarez ◽  
M. Blanca Piazuelo ◽  
Paula B. Luis ◽  
Dara R. Baker ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Epithelial Cells ◽  
Oxidative Dna Damage ◽  
Mongolian Gerbils ◽  
Gastric Epithelial Cells ◽  
Oncogenic Signaling ◽  
Cytotoxin Associated Gene A ◽  
H Pylori ◽  
Oncogenic Signaling Pathways

Infection by Helicobacter pylori is the primary cause of gastric adenocarcinoma. The most potent H. pylori virulence factor is cytotoxin-associated gene A (CagA), which is translocated by a type 4 secretion system (T4SS) into gastric epithelial cells and activates oncogenic signaling pathways. The gene cagY encodes for a key component of the T4SS and can undergo gene rearrangements. We have shown that the cancer chemopreventive agent α-difluoromethylornithine (DFMO), known to inhibit the enzyme ornithine decarboxylase, reduces H. pylori-mediated gastric cancer incidence in Mongolian gerbils. In the present study, we questioned whether DFMO might directly affect H. pylori pathogenicity. We show that H. pylori output strains isolated from gerbils treated with DFMO exhibit reduced ability to translocate CagA in gastric epithelial cells. Further, we frequently detected genomic modifications in the middle repeat region of the cagY gene of output strains from DFMO-treated animals, which were associated with alterations in the CagY protein. Gerbils did not develop carcinoma when infected with a DFMO output strain containing rearranged cagY or the parental strain in which the wild-type cagY was replaced by cagY with DFMO-induced rearrangements. Lastly, we demonstrate that in vitro treatment of H. pylori by DFMO induces oxidative DNA damage, expression of the DNA repair enzyme MutS2, and mutations in cagY, demonstrating that DFMO directly affects genomic stability. Deletion of mutS2 abrogated the ability of DFMO to induce cagY rearrangements directly. In conclusion, DFMO-induced oxidative stress in H. pylori leads to genomic alterations and attenuates virulence.

scholarly journals Helicobacter pylori Caga Protein Can Be Tyrosine Phosphorylated in Gastric Epithelial Cells

2000 ◽  
Vol 191 (4) ◽  
pp. 593-602 ◽  
Author(s):  
Momoyo Asahi ◽  
Takeshi Azuma ◽  
Shigeji Ito ◽  
Yoshiyuki Ito ◽  
Hiroyuki Suto ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Epithelial Cells ◽  
Gastric Epithelial Cells ◽  
Phosphorylated Proteins ◽  
Caga Protein ◽  
Host Cytoplasm ◽  
Cytotoxin Associated Gene A ◽  
Tyrosine Phosphorylated Protein ◽  
H Pylori

Attachment of Helicobacter pylori to gastric epithelial cells induces various cellular responses, including the tyrosine phosphorylation of an unknown 145-kD protein and interleukin 8 production. Here we show that this 145-kD protein is the cagA product of H. pylori, an immunodominant, cytotoxin-associated antigen. Epithelial cells infected with various H. pylori clinical isolates resulted in generation of tyrosine-phosphorylated proteins ranging from 130 to 145 kD in size that were also induced in vitro by mixing host cell lysate with bacterial lysate. When epithelial cells were infected with [35S]methionine-labeled H. pylori, a radioactive 145-kD protein was detected in the immunoprecipitates with antiphosphotyrosine antibody or anti-CagA (cytotoxin-associated gene A) antibody. Consistently, the 145-kD protein recognized by the anti-CagA and antiphosphotyrosine antibodies was induced in epithelial cells after infection of wild-type H. pylori but not the cagA::Km mutant. Furthermore, the amino acid sequence of the phosphorylated 145-kD protein induced by H. pylori infection was identical to the H. pylori CagA sequence. These results reveal that the tyrosine-phosphorylated 145-kD protein is H. pylori CagA protein, which may be delivered from attached bacteria into the host cytoplasm. The identification of the tyrosine-phosphorylated protein will thus provide further insights into understanding the precise roles of CagA protein in H. pylori pathogenesis.

scholarly journals Helicobacter pylori Induces Gastric Epithelial Cell Apoptosis in Association with Increased Fas Receptor Expression

1999 ◽  
Vol 67 (8) ◽  
pp. 4237-4242 ◽  
Author(s):  
Nicola L. Jones ◽  
Andrew S. Day ◽  
Hilary A. Jennings ◽  
Philip M. Sherman
Keyword(s):  
Cell Death ◽  
Helicobacter Pylori ◽  
Epithelial Cells ◽  
Receptor Expression ◽  
Gastric Epithelial Cells ◽  
Ags Cells ◽  
Fas Receptor ◽  
Epithelial Cell Apoptosis ◽  
H Pylori

ABSTRACT The mechanisms involved in mediating the enhanced gastric epithelial cell apoptosis observed during infection withHelicobacter pylori in vivo are unknown. To determine whether H. pylori directly induces apoptosis of gastric epithelial cells in vitro and to define the role of the Fas-Fas ligand signal transduction cascade, human gastric epithelial cells were infected with H. pylori for up to 72 h under microaerophilic conditions. As assessed by both transmission electron microscopy and fluorescence microscopy, incubation with acagA-positive, cagE-positive, VacA-positive clinical H. pylori isolate stimulated an increase in apoptosis compared to the apoptosis of untreated AGS cells (16.0% ± 2.8% versus 5.9% ± 1.4%, P < 0.05) after 72 h. In contrast, apoptosis was not detected following infection withcagA-negative, cagE-negative, VacA-negative clinical isolates or a Campylobacter jejuni strain. In addition to stimulating apoptosis, infection with H. pylorienhanced Fas receptor expression in AGS cells to a degree comparable to that of treatment with a positive control, gamma interferon (12.5 ng/ml) (148% ± 24% and 167% ± 24% of control, respectively). The enhanced Fas receptor expression was associated with increased sensitivity to Fas-mediated cell death. Ligation of the Fas receptor with an agonistic monoclonal antibody resulted in an increase in apoptosis compared to the apoptosis of cells infected with the bacterium alone (38.5% ± 7.1% versus 16.0% ± 2.8%,P < 0.05). Incubation with neutralizing anti-Fas antibody did not prevent apoptosis of H. pylori-infected cells. Taken together, these findings demonstrate that the gastric pathogen H. pylori stimulates apoptosis of gastric epithelial cells in vitro in association with the enhanced expression of the Fas receptor. These data indicate a role for Fas-mediated signaling in the programmed cell death that occurs in response toH. pylori infection.

scholarly journals Helicobacter pylori infection downregulates the DNA glycosylase NEIL2, resulting in increased genome damage and inflammation in gastric epithelial cells

2020 ◽  
Vol 295 (32) ◽  
pp. 11082-11098 ◽  
Author(s):  
Ibrahim M. Sayed ◽  
Ayse Z. Sahan ◽  
Tatiana Venkova ◽  
Anirban Chakraborty ◽  
Dibyabrata Mukhopadhyay ◽  
...  
Keyword(s):  
Dna Damage ◽  
Helicobacter Pylori ◽  
Epithelial Cells ◽  
Inflammatory Response ◽  
Oxidative Dna Damage ◽  
Expression Profiles ◽  
Dna Glycosylase ◽  
Gastric Epithelial Cells ◽  
Genome Damage ◽  
H Pylori

Infection with the Gram-negative, microaerophilic bacterium Helicobacter pylori induces an inflammatory response and oxidative DNA damage in gastric epithelial cells that can lead to gastric cancer (GC). However, the underlying pathogenic mechanism is largely unclear. Here, we report that the suppression of Nei-like DNA glycosylase 2 (NEIL2), a mammalian DNA glycosylase that specifically removes oxidized bases, is one mechanism through which H. pylori infection may fuel the accumulation of DNA damage leading to GC. Using cultured cell lines, gastric biopsy specimens, primary cells, and human enteroid-derived monolayers from healthy human stomach, we show that H. pylori infection greatly reduces NEIL2 expression. The H. pylori infection-induced downregulation of NEIL2 was specific, as Campylobacter jejuni had no such effect. Using gastric organoids isolated from the murine stomach in coculture experiments with live bacteria mimicking the infected stomach lining, we found that H. pylori infection is associated with the production of various inflammatory cytokines. This response was more pronounced in Neil2 knockout (KO) mouse cells than in WT cells, suggesting that NEIL2 suppresses inflammation under physiological conditions. Notably, the H. pylori-infected Neil2-KO murine stomach exhibited more DNA damage than the WT. Furthermore, H. pylori-infected Neil2-KO mice had greater inflammation and more epithelial cell damage. Computational analysis of gene expression profiles of DNA glycosylases in gastric specimens linked the reduced Neil2 level to GC progression. Our results suggest that NEIL2 downregulation is a plausible mechanism by which H. pylori infection impairs DNA damage repair, amplifies the inflammatory response, and initiates GC.

scholarly journals Helicobacter pylori Infection Stimulates Plasminogen Activator Inhibitor 1 Production by Gastric Epithelial Cells

2008 ◽  
Vol 76 (9) ◽  
pp. 3992-3999 ◽  
Author(s):  
A. C. Keates ◽  
S. Tummala ◽  
R. M. Peek ◽  
E. Csizmadia ◽  
B. Kunzli ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Epithelial Cells ◽  
Plasminogen Activator Inhibitor ◽  
Gastric Epithelial Cells ◽  
Plasminogen Activator Inhibitor 1 ◽  
Ags Cells ◽  
H Pylori ◽  
Activator Inhibitor ◽  
Pai 1

ABSTRACT Chronic infection with the gastric pathogen Helicobacter pylori significantly increases the risk of developing atrophic gastritis, peptic ulcer disease, and gastric adenocarcinoma. H. pylori strains that possess the cag pathogenicity island, which translocates CagA into the host cells, augment these risks. The aim of this study was to determine the molecular mechanisms through which H. pylori upregulates the expression of plasminogen activator inhibitor 1 (PAI-1), a member of the urokinase activator system that is involved in tumor metastasis and angiogenesis. Levels of PAI-1 mRNA and protein were examined in tissues from H. pylori-infected patients and in vitro using AGS gastric epithelial cells. In vitro, cells were infected with toxigenic cag-positive or nontoxigenic cag-negative strains of H. pylori or isogenic mutants. The amount of PAI-1 secretion was measured by enzyme-linked immunosorbent assay, and mRNA levels were determined using real-time PCR. The regulation of PAI-1 was examined using the extracellular signal-regulated kinase 1/2 (ERK1/2) inhibitor and small interfering RNA. Analysis of human biopsy samples revealed an increase in both PAI-1 mRNA and protein levels in patients with H. pylori gastritis compared to those of uninfected controls. Infection of AGS cells with H. pylori significantly increased PAI-1 mRNA expression and the secretion of PAI-1 protein. Moreover, PAI-1 mRNA and protein production was more pronounced when AGS cells were infected by H. pylori strains carrying a functional cag secretion system than when cells were infected by strains lacking this system. PAI-1 secretion was also reduced when cells were infected with either cagE-negative or cagA-negative mutants. The ectopic overexpression of CagA significantly increased the levels of PAI-1 mRNA and protein, whereas blockade of the ERK1/2 pathway inhibited H. pylori-mediated PAI-1 upregulation. These findings suggest that the upregulation of PAI-1 in H. pylori-infected gastric epithelial cells may contribute to the carcinogenic process.

scholarly journals Regulation of Human β-Defensins by Gastric Epithelial Cells in Response to Infection with Helicobacter pylori or Stimulation with Interleukin-1

2000 ◽  
Vol 68 (9) ◽  
pp. 5412-5415 ◽  
Author(s):  
Deborah A. O'Neil ◽  
Sheri P. Cole ◽  
Edith Martin-Porter ◽  
Michael P. Housley ◽  
Lide Liu ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Epithelial Cells ◽  
Interleukin 1 ◽  
Peptide Antibiotic ◽  
Gastric Epithelial Cells ◽  
Proinflammatory Mediators ◽  
H Pylori

ABSTRACT Gastric epithelial cells in vitro and in vivo are shown to constitutively express the peptide antibiotic human β-defensin type 1 (hBD-1). In contrast, hBD-2 expression is regulated in gastric epithelial cells and increases in response to infection withHelicobacter pylori or stimulation with the proinflammatory cytokine interleukin-1. These data suggest that hBD-2 is a component of the regulated host gastric epithelial cell response to H. pylori infection and proinflammatory mediators.

scholarly journals N-Acetylcysteine Reduces ROS-Mediated Oxidative DNA Damage and PI3K/Akt Pathway Activation Induced by Helicobacter pylori Infection

2018 ◽  
Vol 2018 ◽  
pp. 1-9 ◽  
Author(s):  
Chuan Xie ◽  
Jian Yi ◽  
Jing Lu ◽  
Muwen Nie ◽  
Meifang Huang ◽  
...  
Keyword(s):  
Dna Damage ◽  
Gastric Mucosa ◽  
Epithelial Cells ◽  
Oxidative Dna Damage ◽  
Akt Pathway ◽  
Gastric Epithelial Cells ◽  
H Pylori

Background. H. pylori infection induces reactive oxygen species- (ROS-) related DNA damage and activates the PI3K/Akt pathway in gastric epithelial cells. N-Acetylcysteine (NAC) is known as an inhibitor of ROS; the role of NAC in H. pylori-related diseases is unclear. Aim. The aim of this study was to evaluate the role of ROS and the protective role of NAC in the pathogenesis of H. pylori-related diseases. Method. An in vitro coculture system and an in vivo Balb/c mouse model of H. pylori-infected gastric epithelial cells were established. The effects of H. pylori infection on DNA damage and ROS were assessed by the comet assay and fluorescent dichlorofluorescein assay. The level of PI3K/Akt pathway-related proteins was evaluated by Western blotting. The protective role of N-acetylcysteine (NAC) was also evaluated with in vitro and in vivo H. pylori infection models. Results. The results revealed that, in vitro and in vivo, H. pylori infection increased the ROS level and induced DNA damage in gastric epithelial cells. NAC treatment effectively reduced the ROS level and inhibited DNA damage in GES-1 cells and the gastric mucosa of Balb/c mice. H. pylori infection induced ROS-mediated PI3K/Akt pathway activation, and NAC treatment inhibited this effect. However, the gastric mucosa pathological score of the NAC-treated group was not significantly different from that of the untreated group. Furthermore, chronic H. pylori infection decreased APE-1 expression in the gastric mucosa of Balb/c mice. Conclusions. An increased ROS level is a critical mechanism in H. pylori pathogenesis, and NAC may be beneficial for the treatment of H. pylori-related gastric diseases linked to oxidative DNA damage.

scholarly journals Rabeprazole inhibits inflammatory reaction by inhibition of cell pyroptosis in gastric epithelial cells

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Jing Xie ◽  
Long Fan ◽  
Liya Xiong ◽  
Peiyu Chen ◽  
Hongli Wang ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Clinical Sample ◽  
Critical Role ◽  
Enzyme Linked Immunosorbent Assay ◽  
Inflammasome Activation ◽  
Peptic Ulcers ◽  
Gastric Epithelial Cells ◽  
Caspase 1 ◽  
H Pylori

Abstract Background Helicobacter pylori (H. pylori) is a common pathogen in development of peptic ulcers with pyroptosis. Rabeprazole, a critical component of standard triple therapy, has been widely used as the first-line regimen for H. pylori infectious treatment. The aim of this study to explore the function of Rabeprazole on cell pyroptosis in vitro. Methods The clinical sample from patients diagnosed with or without H. pylori-infection were collected to analyze by Immunohistochemistry (IHC). Real-time quantitative PCR (qPCR), western blot (WB) and enzyme linked immunosorbent assay (Elisa) were performed to analyze the effect of Rabeprazole on cell pyroptosis, including LDH, IL-1β and IL-18. Results In this study, we showed that Rabeprazole regulated a phenomenon of cell pyroptosis as confirmed by lactate dehydrogenase (LDH) assay. Further results showed that Rabeprazole inhibited cell pyroptosis in gastric epithelial cells by alleviating GSDMD-executed pyroptosis, leading to decrease IL-1β and IL-18 mature and secretion, which is attributed to NLRP3 inflammasome activation inhibition. Further analysis showed that ASC, NLRP3 and Caspase-1, was significantly repressed in response to Rabeprazole stimulation, resulting in decreasing cleaved-caspase-1 expression. Most important, NLRP3 and GSDMD is significantly increased in gastric tissue of patients with H. pylori infection. Conclusion These findings revealed a critical role of Rabeprazole in cell pyroptosis in patients with H. pylori infection, suggesting that targeting cell pyroptosis is an alternative strategy in improving H. pylori treatment.

scholarly journals Effects ofHelicobacter pyloriand Heat Shock Protein 70 on the Proliferation of Human Gastric Epithelial Cells

2014 ◽  
Vol 2014 ◽  
pp. 1-5 ◽  
Author(s):  
Liping Tao ◽  
Hai Zou ◽  
Zhimin Huang
Keyword(s):  
Helicobacter Pylori ◽  
Heat Shock ◽  
Epithelial Cells ◽  
Heat Shock Protein ◽  
Mtt Assay ◽  
Heat Shock Protein 70 ◽  
Hsp70 Expression ◽  
Gastric Epithelial Cells ◽  
Ags Cells ◽  
H Pylori

Infection ofHelicobacter pylori (H. pylori)changed the proliferation of gastric epithelial cells and decreased the expression of heat shock protein 70 (HSP70). However, the effects ofH. pylorion the proliferation of gastric epithelial cells and the roles of HSP70 during the progress need further investigation.Objective.To investigate the effects ofHelicobacter pylori (H. pylori)and heat shock protein 70 (HSP70) on the proliferation of human gastric epithelial cells.Methods. H. pyloriand a human gastric epithelial cell line (AGS) were cocultured. The proliferation of AGS cells was quantitated by an MTT assay, and the expression of HSP70 in AGS cells was detected by Western blotting. HSP70 expression in AGS cells was silenced by small interfering RNA (siRNA) to investigate the role of HSP70. ThesiRNA-treated AGS cells were cocultured withH. pyloriand cell proliferation was measured by an MTT assay.Results.The proliferation of AGS cells was accelerated by coculturing withH. pylorifor 4 and 8 h, but was suppressed at 24 and 48 h. HSP70 expression was decreased in AGS cells infected byH. pylorifor 48 h. The proliferation in HSP70-silenced AGS cells was inhibited after coculturing withH. pylorifor 24 and 48 h compared with the control group.Conclusions.Coculture ofH. pylorialtered the proliferation of gastric epithelial cells and decreased HSP70 expression. HSP70 knockdown supplemented the inhibitory effect ofH. pylorion proliferation of epithelial cells. These results indicate that the effects ofH. pylorion the proliferation of gastric epithelial cells at least partially depend on the decreased expression of HSP70 induced by the bacterium.

scholarly journals TIFA Signaling in Gastric Epithelial Cells Initiates the cag Type 4 Secretion System-Dependent Innate Immune Response to Helicobacter pylori Infection

mBio ◽  
2017 ◽  
Vol 8 (4) ◽  
Author(s):  
Alevtina Gall ◽  
Ryan G. Gaudet ◽  
Scott D. Gray-Owen ◽  
Nina R. Salama
Keyword(s):  
Immune Response ◽  
Helicobacter Pylori ◽  
Epithelial Cells ◽  
Inflammatory Response ◽  
Secretion System ◽  
Innate Immune ◽  
Gastric Ulcers ◽  
Bacterial Clearance ◽  
Gastric Epithelial Cells ◽  
H Pylori

ABSTRACT Helicobacter pylori is a bacterial pathogen that colonizes the human stomach, causing inflammation which, in some cases, leads to gastric ulcers and cancer. The clinical outcome of infection depends on a complex interplay of bacterial, host genetic, and environmental factors. Although H. pylori is recognized by both the innate and adaptive immune systems, this rarely results in bacterial clearance. Gastric epithelial cells are the first line of defense against H. pylori and alert the immune system to bacterial presence. Cytosolic delivery of proinflammatory bacterial factors through the cag type 4 secretion system ( cag -T4SS) has long been appreciated as the major mechanism by which gastric epithelial cells detect H. pylori . Classically attributed to the peptidoglycan sensor NOD1, recent work has highlighted the role of NOD1-independent pathways in detecting H. pylori ; however, the bacterial and host factors involved have remained unknown. Here, we show that bacterially derived heptose-1,7-bisphosphate (HBP), a metabolic precursor in lipopolysaccharide (LPS) biosynthesis, is delivered to the host cytosol through the cag -T4SS, where it activates the host tumor necrosis factor receptor-associated factor (TRAF)-interacting protein with forkhead-associated domain (TIFA)-dependent cytosolic surveillance pathway. This response, which is independent of NOD1, drives robust NF-κB-dependent inflammation within hours of infection and precedes NOD1 activation. We also found that the CagA toxin contributes to the NF-κB-driven response subsequent to TIFA and NOD1 activation. Taken together, our results indicate that the sequential activation of TIFA, NOD1, and CagA delivery drives the initial inflammatory response in gastric epithelial cells, orchestrating the subsequent recruitment of immune cells and leading to chronic gastritis. IMPORTANCE H. pylori is a globally prevalent cause of gastric and duodenal ulcers and cancer. H. pylori antibiotic resistance is rapidly increasing, and a vaccine remains elusive. The earliest immune response to H. pylori is initiated by gastric epithelial cells and sets the stage for the subsequent immunopathogenesis. This study revealed that host TIFA and H. pylori -derived HBP are critical effectors of innate immune signaling that account for much of the inflammatory response to H. pylori in gastric epithelial cells. HBP is delivered to the host cell via the cag -T4SS at a time point that precedes activation of the previously described NOD1 and CagA inflammatory pathways. Manipulation of the TIFA-driven immune response in the host and/or targeting of ADP-heptose biosynthesis enzymes in H. pylori may therefore provide novel strategies that may be therapeutically harnessed to achieve bacterial clearance.

Lactobacillus fermentum UCO-979C beneficially modulates the innate immune response triggered by Helicobacter pylori infection in vitro

2018 ◽  
Vol 9 (5) ◽  
pp. 829-841 ◽  
Author(s):  
V. Garcia-Castillo ◽  
H. Zelaya ◽  
A. Ilabaca ◽  
M. Espinoza-Monje ◽  
R. Komatsu ◽  
...  
Keyword(s):  
Immune Response ◽  
Helicobacter Pylori ◽  
Epithelial Cells ◽  
Morphological Changes ◽  
Helicobacter Pylori Infection ◽  
Lactobacillus Fermentum ◽  
Gastric Tissue ◽  
Gastric Epithelial Cells ◽  
Ags Cells ◽  
H Pylori

Helicobacter pylori infection is associated with important gastric pathologies. An aggressive proinflammatory immune response is generated in the gastric tissue infected with H. pylori, resulting in gastritis and a series of morphological changes that increase the susceptibility to cancer development. Probiotics could present an alternative solution to prevent or decrease H. pylori infection. Among them, the use of immunomodulatory lactic acid bacteria represents a promising option to reduce the severity of chronic inflammatory-mediated tissue damage and to improve protective immunity against H. pylori. We previously isolated Lactobacillus fermentum UCO-979C from human gastric tissue and demonstrated its capacity to reduce adhesion of H. pylori to human gastric epithelial cells (AGS cells). In this work, the ability of L. fermentum UCO-979C to modulate immune response in AGS cells and PMA phorbol 12-myristate 13-acetate (PMA)-differentiated THP-1 (human monocytic leukaemia) macrophages in response to H. pylori infection was evaluated. We demonstrated that the UCO-979C strain is able to differentially modulate the cytokine response of gastric epithelial cells and macrophages after H. pylori infection. Of note, L. fermentum UCO-979C was able to significantly reduce the production of inflammatory cytokines and chemokines in AGS and THP-1 cells as well as increase the levels of immunoregulatory cytokines, indicating a remarkable anti-inflammatory effect. These findings strongly support the probiotic potential of L. fermentum UCO-979C and provide evidence of its beneficial effects against the inflammatory damage induced by H. pylori infection. Although our findings should be proven in appropriate experiments in vivo, in both H. pylori infection animal models and human trials, the results of the present work provide a scientific rationale for the use of L. fermentum UCO-979C to prevent or reduce H. pylori-induced gastric inflammation in humans.

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