Korea Research Institute of Bioscience and Biotechnology
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Maturation of human intestinal organoids in vitro facilitates colonization by commensal lactobacilli by reinforcing the mucus layer
No full textLactobacilli, which are probiotic commensal bacteria that mainly reside in the human small intestine, have attracted attention for their ability to exert health-promoting effects and beneficially modulate host immunity. However, host epithelial-commensal bacterial interactions are still largely unexplored because of limited access to human small intestinal tissues. Recently, we described an in vitro maturation technique for generating adult-like, mature human intestinal organoids (hIOs) from human pluripotent stem cells (hPSCs) that closely resemble the in vivo tissue structure and cellular diversity. Here, we established an in vitro human model to study the response to colonization by commensal bacteria using luminal microinjection into mature hIOs, allowing for the direct examination of epithelial-bacterial interactions. Lactobacillus reuteri and Lactobacillus plantarum were more likely to survive and colonize when microinjected into the lumen of mature hIOs than when injected into immature hIOs, as determined by scanning electron microscopy, colony formation assay, immunofluorescence, and real-time imaging with L plantarum expressing red fluorescent protein. The improved mature hIO-based host epithelium system resulted from enhanced intestinal epithelial integrity via upregulation of mucus secretion and tight junction proteins. Our study indicates that mature hIOs are a physiologically relevant in vitro model system for studying commensal microorganisms.
3,4,5-Trihydroxycinnamic acid exerts anti-asthmatic effects in vitro and in vivo
No full text3,4,5-Trihydroxycinnamic acid (THCA) has been reported to possess anti-inflammatory activity. However, the effect of THCA for treating allergic asthma was unknown. Therefore, in the present study, the anti-asthmatic effects of THCA were studied in both in vitro and in vivo studies. In phorbol 12-myristate 13-acetate (PMA)-stimulated A549 airway epithelial cells, THCA pretreatment decreased the mRNA expression and secretion of interleukin (IL)-8, monocyte chemoattractant protein-1 (MCP-1), and intercellular adhesion molecules 1 (ICAM-1), and reduced the mRNA expression of matrix metalloproteinase 9 (MMP-9). THCA also inhibited PMA-induced protein kinase B (AKT), mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-κB) activation in A549 cells. In lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages, THCA pretreatment suppressed the mRNA expression of ICAM-1 and MMP-9. In addition, THCA suppressed the adhesion of EOL and A549 cells. In ovalbumin (OVA)-administered asthmatic mice, THCA exerted inhibitory activity on IL-5, IL-13, and MCP-1 in bronchoalveolar lavage fluid (BALF) and on OVA-specific immunoglobulin E (IgE) in serum. THCA attenuated the numbers of inflammatory cells in BALF and the influx of inflammatory cell in lung tissues. Furthermore, THCA downregulated the levels of inducible nitric oxide (iNOS), cyclooxygenase-2 (COX-2), and leukotriene B4 (LTB4) expression, mucus production and CREB phosphorylation as well as Penh value. These effects were accompanied by suppression of AKT, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and NF-κB activation. Therefore, the results of the current study suggest that THCA may be a valuable adjuvant or therapeutic in the prevention or treatment of allergic asthma
Human cytomegalovirus-Induced Interleukin-10 production promotes the proliferation of Mycobacterium massiliense in macrophages
No full textHuman cytomegalovirus (HCMV) exploits the interleukin-10 (IL-10) pathway as a part of its infection cycle through the manipulation of the host IL-10 signaling cascade. Based on its immunomodulatory nature, HCMV attenuates the host immune response and facilitates the progression of co-infection with other pathogens in an immune-competent host. To investigate the impact of HCMV infection on the burden of non-tuberculous mycobacteria (NTM), whose prevalence is growing rapidly worldwide, macrophages were infected with HCMV and further challenged with Mycobacterium massiliense in vitro. The results showed that HCMV infection significantly increased host IL-10 synthesis and promoted the proliferation of M. massiliense in an IL-10-dependent manner. Transcriptomic analysis revealed that HCMV infection dampened the regulatory pathways of interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), and interleukin-1 (IL-1), consequently abrogating the immune responses to M. massiliense coinfection in macrophages. These findings provide a mechanistic basis of how HCMV infection may facilitate the development of pathogenic NTM co-infection by upregulating IL-10 expression.
Methylation of the CDX2 promoter in H. pylori-infected gastric mucosa increases with age and its rapid demethylation in gastric tumors is associated with upregulated gene expression
No full textPathological changes in the epigenetic landscape of chromatin are hallmarks of cancer. The caudal-type homeobox gene CDX2 is not expressed in normal gastric epithelia but rather in adult intestinal epithelia, and it is overexpressed in intestinal metaplasia (IM). However, it remains unclear how CDX2 transcription is suppressed in normal gastric epithelial cells and overexpressed in IM. Here, we demonstrate that methylation of the CDX2 promoter increases with age in Helicobacter pylori-positive, noncancerous gastric tissue, whereas the promoter is demethylated in paired gastric tumors in which CDX2 is upregulated. Moreover, we also found that the CDX2 promoter is demethylated in IM as well as gastric tumor. Immunohistochemistry revealed that CDX2 is present in foci of parts of the gastric mucosae but highly expressed in IM as well as in gastric tumors, suggesting that the elevated level of CDX2 in IM and gastric tumors may be attributable to promoter demethylation. Our data suggest that CDX2 repression may be associated with promoter methylation in noncancerous H. pylori-positive mucosa but its upregulation might be attributable to increased promoter activity mediated by chromatin remodeling during gastric carcinogenesis.
Chemosensitivity to HM90822, a novel synthetic IAP antagonist, is determined by p-AKT-inducible XIAP phosphorylation in human pancreatic cancer cells
No full textInhibitor of apoptosis proteins (IAPs) are overexpressed in the majority of cancers and prevent apoptosis by inhibiting caspases. IAPs have therefore attracted considerable attention as potential targets for anticancer therapy. Here, we demonstrated that HM90822 (abbreviated HM822; a new synthetic IAP antagonist) induced apoptotic cell death via proteasome-dependent degradation of BIR2/3 domain-containing IAPs in human pancreatic cancer cells. HM822 inhibited the expression of XIAP and cIAP1/2 proteins in Panc-1 and BxPC-3 cells, which are sensitive to HM822. HM822 also induced IAP ubiquitination and promoted proteasome-dependent IAP degradation. However, cells expressing phospho-XIAP (Ser87) and AKT exhibited resistance to HM822. In other words, the overexpression of AKT-CA (constitutive active form for AKT) or AKT-WT induced resistance to HM822. In addition, in Panc-1 xenograft and orthotopic mouse models, we revealed that tumor growth was suppressed by the administration of HM822. Taken together, these results suggest that HM822 induces apoptosis through ubiquitin/proteasome-dependent degradation of BIR3 domain-containing IAPs. These findings suggest that phospho-XIAP and phospho-AKT may be used as biomarkers for predicting the efficacy of HM822 in pancreatic cancer patients.
Aglycosylated antibody-producing mice for aglycosylated antibody-lectin coupled immunoassay for the quantification of tumor markers (ALIQUAT)
No full textTargeting aberrant glycoforms has been validated for in vitro cancer diagnostic development, and several assays are currently in routine clinical use. Because N-glycans in Fc region of antibodies show cross-reactivity with various lectins, high-quality aglycosylated antibodies are exceptionally important for immunoassay platform-based quantitative measurements. Previously, aglycosylated antibody acquisition relied on incomplete, uneconomical and onerous enzymatic and chemical methods. Here, we edited four murine immunoglobulin G genes using adenine base-editing and homology-directed recombination (HDR)-mediated gene editing methods to generate aglycosylated antibody-producing mice. Resulting aglycosylated antibodies showed required analytical performances without compromised protein stability. Thus, this aglycosylated monoclonal antibody-lectin coupled immunoassay for the quantification of tumour markers (ALIQUAT) method can provide a robust, versatile and accessible immunoassay platform to quantify specific glycoforms in precision cancer diagnostics. Moreover, the engineered mice can be used as a host to produce various aglycosylated antibodies in a convenient and robust fashion, thereby expanding in vitro diagnostic development opportunities that utilize glycoforms as a disease-specific biomarkers.
Taxonomy and molecular phylogenetics of Ensiculiferaceae, fam. nov. (Peridiniales, Dinophyceae), with consideration of their life-history
No full textIn the current circumscription, the Thoracosphaeraceae comprise all dinophytes exhibiting calcified coccoid cells produced during their life-history. Species hitherto assigned to Ensiculifera and Pentapharsodinium are mostly based on the monadoid stage of life-history, while the link to the coccoid stage (occasionally treated taxonomically distinct) is not always resolved. We investigated the different life-history stages and DNA sequence data of Ensiculifera mexicana and other species occurring in samples collected from all over the world. Based on concatenated ribosomal RNA gene sequences Ensiculiferaceae represented a distinct peridinalean branch, which showed a distant relationship to other calcareous dinophytes. Both molecular and morphological data (particularly of the coccoid stage) revealed the presence of three distinct clades within Ensiculiferaceae, which may include other dinophytes exhibiting a parasitic life-history stage. At a higher taxonomic level, Ensiculiferaceae showed relationships to parasites and endosymbionts (i.e., Blastodinium and Zooxanthella) as well as to dinophytes harbouring diatoms instead of chloroplasts. These unexpected phylogenetic relationships are corroborated by the presence of five cingular plates in all such taxa, which differs from the six cingular plates of most other Thoracosphaeraceae. We herein describe Ensiculiferaceae, emend the descriptions of Ensiculifera and Pentapharsodinium, erect Matsuokaea and provide several new combinations at the species level.
Immunotherapy in hematologic malignancies: emerging therapies and novel approaches
No full textImmunotherapy is extensively investigated for almost all types of hematologic tumors, from preleukemic to relapse/refractory malignancies. Due to the emergence of technologies for target cell characterization, antibody design and manufacturing, as well as genome editing, immunotherapies including gene and cell therapies are becoming increasingly elaborate and diversified. Understanding the tumor immune microenvironment of the target disease is critical, as is reducing toxicity. Although there have been many successes and newly FDA-approved immunotherapies for hematologic malignancies, we have learned that insufficient efficacy due to disease relapse following treatment is one of the key obstacles for developing successful therapeutic regimens. Thus, combination therapies are also being explored. In this review, immunotherapies for each type of hematologic malignancy will be introduced, and novel targets that are under investigation will be described.
MAM domain containing 2 is a potential breast cancer biomarker that exhibits tumour-suppressive activity
No full textObjectives
The aim of this study was to discover new potential biomarkers of breast cancer and investigate their cellular functions.
Materials and methods
We analysed the gene expression profiles of matched pairs of breast tumour and normal tissues from 24 breast cancer patients. Tetracycline？inducible MAMDC2 expression system was established and used to evaluate cell proliferation in vitro and in vivo. MAMDC2？mediated signalling was determined using immunoblot analysis.
Results
We identified MAMDC2 as a down？regulated gene showing significant prognostic capability. Overexpression of MAMDC2 or treatment with MAMDC2？containing culture medium significantly inhibited the cell proliferation of T？47D cells. Furthermore, MAMDC2 expression reduced in vivo growth of T？47D xenograft tumours. MAMDC2 may exert its growth？inhibitory functions by attenuating the MAPK signalling pathway.
Conclusion
We report that MAMDC2 has a tumour？suppressive role and, as a secretory protein, it might be useful as a biomarker for breast cancer treatment.
Probing the neuraminidase activity of influenza virus using a cytolysin A protein nanopore
No full textNeuraminidase (NA), one of the major surface glycoproteins of influenza A virus (IAV), is an important diagnostic biomarker and antiviral therapeutic target. Cytolysin A (ClyA) is a nanopore sensor with an internal constriction of 3.3 nm, enabling the detection of protein conformations at the single-molecule level. In this study, a nanopore-based approach is developed for analysis of the enzymatic activity of NA, which facilitates rapid and highly sensitive diagnosis of IAV. Current blockade analysis of the d-glucose/d-galactose-binding protein (GBP) trapped within a type I ClyA-AS (ClyA mutant) nanopore reveals that galactose cleaved from sialyl-galactose by NA of the influenza virus can be detected in real time and at the single-molecule level. Our results show that this nanopore sensor can quantitatively measure the activity of NA with 40？80-fold higher sensitivity than those previously reported. Furthermore, the inhibition of NA is monitored using small-molecule antiviral drugs, such as zanamivir. Taken together, our results reveal that the ClyA protein nanopore can be a valuable platform for the rapid and sensitive point-of-care diagnosis of influenza and for drug screening against the NA target.