scholarly journals Regulatory Mechanism for Exfoliative Toxin Production inStaphylococcus aureus

2011 ◽  
Vol 79 (4) ◽  
pp. 1660-1670 ◽  
Author(s):  
Fuminori Kato ◽  
Noriko Kadomoto ◽  
Yuko Iwamoto ◽  
Katsuaki Bunai ◽  
Hitoshi Komatsuzawa ◽  
...  
Keyword(s):  
Mouse Model ◽  
Regulatory Mechanism ◽  
Toxin Production ◽  
Neonatal Mouse ◽  
Epidermal Keratinocytes ◽  
Global Regulators ◽  
Exfoliative Toxin ◽  
Blistering Disease

ABSTRACTThe exfoliative toxin (ET) is a major virulence factor ofStaphylococcus aureusthat causes bullous impetigo and its disseminated form, staphylococcal scalded-skin syndrome (SSSS). ET selectively digests one of the intracellular adhesion molecules, desmoglein 1, of epidermal keratinocytes and causes blisters due to intraepidermal cell-cell dissociation. MostS. aureusstrains that cause blistering disease produce either ETA or ETB. They are serologically distinct molecules, where ETA is encoded on a phage genome and ETB is enocded on a large plasmid. ETA-producingS. aureusstrains are frequently isolated from impetigo patients, and ETB-producingS. aureusstrains are isolated from SSSS. ET-induced blister formation can be reproduced with the neonatal mouse. To determine the regulatory mechanism of ET production, we investigated the role of the two-component systems and global regulators foretaoretbexpressionin vitroandin vivowith the mouse model. Western blot and transcription analyses using a series of mutants demonstrate ETA production was downregulated bysigB,sarS, andsarA, while ETB production was downregulated bysigBandsarAbut not bysarS. Production of both toxins is upregulated bysaeRS,arlRS, andagrCA. Furthermore, by thein vivoneonatal mouse model,sigBandsarSbut notsarAnegatively regulate the exfoliation activity of the ETA-producing strain, whilesarAnegatively regulates the ETB-producing strain. In both strains,saeRS,arlRS, andagrCApositively regulate the exfoliation activityin vivo. The data illustrate similar but distinct regulatory mechanisms for ETA and ETB productionin S. aureus in vitroas well asin vivo.

scholarly journals LAP+ Cells Modulate Protection Induced by Oral Vaccination with Rhesus Rotavirus in a Neonatal Mouse Model

2019 ◽  
Vol 93 (19) ◽  
Author(s):  
Laura María Rey ◽  
José Ángel Gil ◽  
José Mateus ◽  
Luz-Stella Rodríguez ◽  
Martín Alonso Rondón ◽  
...  
Keyword(s):  
T Cells ◽  
Mouse Model ◽  
Cd4 T Cells ◽  
Transforming Growth Factor ◽  
T Cell Response ◽  
Neonatal Mouse ◽  
Oral Vaccination ◽  
Rotavirus Vaccines

ABSTRACT Transforming growth factor β (TGF-β) has been shown to play a role in immunity against different pathogens in vitro and against parasites in vivo. However, its role in viral infections in vivo is incompletely understood. Using a neonatal mouse model of heterologous rhesus rotavirus (RV) vaccination, we show that the vaccine induced rotavirus-specific CD4 T cells, the majority of which lacked expression of KLRG1 or CD127, and a few regulatory rotavirus-specific CD4 T cells that expressed surface latency-associated peptide (LAP)–TGF-β. In these mice, inhibiting TGF-β, with both a neutralizing antibody and an inhibitor of TGF-β receptor signaling (activin receptor-like kinase 5 inhibitor [ALK5i]), did not change the development or intensity of the mild diarrhea induced by the vaccine, the rotavirus-specific T cell response, or protection against a subsequent challenge with a murine EC-rotavirus. However, mice treated with anti-LAP antibodies had improved protection after a homologous EC-rotavirus challenge, compared with control rhesus rotavirus-immunized mice. Thus, oral vaccination with a heterologous rotavirus stimulates regulatory RV-specific CD4 LAP-positive (LAP+) T cells, and depletion of LAP+ cells increases vaccine-induced protection. IMPORTANCE Despite the introduction of several live attenuated animal and human rotaviruses as efficient oral vaccines, rotaviruses continue to be the leading etiological agent for diarrhea mortality among children under 5 years of age worldwide. Improvement of these vaccines has been partially delayed because immunity to rotaviruses is incompletely understood. In the intestine (where rotavirus replicates), regulatory T cells that express latency-associated peptide (LAP) play a prominent role, which has been explored for many diseases but not specifically for infectious agents. In this paper, we show that neonatal mice given a live oral rotavirus vaccine develop rotavirus-specific LAP+ T cells and that depletion of these cells improves the efficiency of the vaccine. These findings may prove useful for the design of strategies to improve rotavirus vaccines.

scholarly journals Flagellum and toxin phase variation impacts intestinal colonization and disease development in a mouse model of Clostridioides difficile infection

2021 ◽  
Author(s):  
Dominika Trzilova ◽  
Mercedes A. H. Warren ◽  
Nicole C. Gadda ◽  
Caitlin L. Williams ◽  
Rita Tamayo
Keyword(s):  
Mouse Model ◽  
Dna Recombination ◽  
Phase Variation ◽  
Toxin Production ◽  
Disease Development ◽  
Hamster Model ◽  
Clostridioides Difficile ◽  
The Right

AbstractClostridioides difficile is a major nosocomial pathogen that can cause severe, toxin-mediated diarrhea and pseudomembranous colitis. Recent work has shown that C. difficile exhibits heterogeneity in swimming motility and toxin production in vitro through phase variation by site-specific DNA recombination. The recombinase RecV reversibly inverts the flagellar switch sequence upstream of the flgB operon, leading to the ON/OFF expression of flagellum and toxin genes. How this phenomenon impacts C. difficile virulence in vivo remains unknown. We identified mutations in the right inverted repeat that reduced or prevented flagellar switch inversion by RecV. We introduced these mutations into C. difficile R20291 to create strains with the flagellar switch “locked” in either the ON or OFF orientation. These mutants exhibited a loss of flagellum and toxin phase variation during growth in vitro, yielding precisely modified mutants suitable for assessing virulence in vivo. In a hamster model of acute C. difficile infection, the phase-locked ON mutant caused greater toxin accumulation than the phase locked OFF mutant but did not differ significantly in the ability to cause acute disease symptoms. In contrast, in a mouse model, preventing flagellum and toxin phase variation affected the ability of C. difficile to colonize the intestinal tract and to elicit weight loss, which is attributable to differences in toxin production during infection. These results show that the ability of C. difficile to phase vary flagella and toxins influences colonization and disease development and suggest that the phenotypic variants generated by flagellar switch inversion have distinct capacities for causing disease.

scholarly journals A novel phosphoramide compound, DCZ0805, shows potent anti-myeloma activity via the NF-κB pathway

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Xuejie Gao ◽  
Bo Li ◽  
Anqi Ye ◽  
Houcai Wang ◽  
Yongsheng Xie ◽  
...  
Keyword(s):  
Mouse Model ◽  
Tumor Burden ◽  
High Rate ◽  
Cell Counting ◽  
Tumor Xenograft ◽  
Luciferase Reporter ◽  
Therapeutic Effects ◽  
Xenograft Mouse Model

Abstract Background Multiple myeloma (MM) is a highly aggressive and incurable clonal plasma cell disease with a high rate of recurrence. Thus, the development of new therapies is urgently needed. DCZ0805, a novel compound synthesized from osalmide and pterostilbene, has few observed side effects. In the current study, we intend to investigate the therapeutic effects of DCZ0805 in MM cells and elucidate the molecular mechanism underlying its anti-myeloma activity. Methods We used the Cell Counting Kit-8 assay, immunofluorescence staining, cell cycle assessment, apoptosis assay, western blot analysis, dual-luciferase reporter assay and a tumor xenograft mouse model to investigate the effect of DCZ0805 treatment both in vivo and in vitro. Results The results showed that DCZ0805 treatment arrested the cell at the G0/G1 phase and suppressed MM cells survival by inducing apoptosis via extrinsic and intrinsic pathways. DCZ0805 suppressed the NF-κB signaling pathway activation, which may have contributed to the inhibition of cell proliferation. DCZ0805 treatment remarkably reduced the tumor burden in the immunocompromised xenograft mouse model, with no obvious toxicity observed. Conclusion The findings of this study indicate that DCZ0805 can serve as a novel therapeutic agent for the treatment of MM.

scholarly journals MODL-12. DEVELOPMENT OF A NOVEL IMMUNOCOMPETENT MOUSE MODEL FOR DIFFUSE INTRINSIC PONTINE GLIOMA

2020 ◽  
Vol 22 (Supplement_3) ◽  
pp. iii413-iii413
Author(s):  
Maggie Seblani ◽  
Markella Zannikou ◽  
Katarzyna Pituch ◽  
Liliana Ilut ◽  
Oren Becher ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Mouse Model ◽  
Growth Factor Receptor ◽  
Diffuse Intrinsic Pontine Glioma ◽  
Pontine Glioma ◽  
Time Of Application ◽  
Tumor Associated Antigen ◽  
Sarcoma Virus

Abstract Diffuse intrinsic pontine glioma (DIPG) is a devastating brain tumor affecting young children. Immunotherapies hold promise however the lack of immunocompetent models recreating a faithful tumor microenvironment (TME) remains a challenge for development of targeted immunotherapeutics. We propose to generate an immunocompetent DIPG mouse model through induced overexpression of interleukin 13 receptor alpha 2 (IL13Rα2), a tumor-associated antigen overexpressed by glioma cells. A model with an intact TME permits comprehensive preclinical assessment of IL13Rα2-targeted immunotherapeutics. Our novel model uses the retroviral avian leucosis and sarcoma virus (RCAS) for in vivo gene delivery leading to IL13Rα2 expression in proliferating progenitor cells. Transfected cells expressing IL13Rα2 and PDGFB, a ligand for platelet derived growth factor receptor, alongside induced p53 loss via the Cre-Lox system are injected in the fourth ventricle in postnatal pups. We validated the expression of PDGFB and IL13Rα2 transgenes in vitro and in vivo and will characterize the TME through evaluation of the peripheral and tumor immunologic compartments using immunohistochemistry and flow cytometry. We confirmed expression of transgenes via flow cytometry and western blotting. Comparison of survival dynamics in mice inoculated with PDGFB alone with PDGFB+IL13Rα2 demonstrated that co-expression of IL13Rα2 did not significantly affect mice survival compared to the PDGFB model. At time of application, we initiated experiments to characterize the TME. Preliminary data demonstrate establishment of tumors within and adjacent to the brainstem and expression of target transgenes. Preclinical findings in a model recapitulating the TME may provide better insight into outcomes upon translation to clinical application.

scholarly journals TMOD-24. GENERATION AND CHARACTERIZATION OF A NOVEL TRANSGENIC IDO REPORTER MOUSE FOR IDO POSTTRANSLATIONAL MODIFICATION ANALYSIS IN SITU

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii233-ii233
Author(s):  
April Bell ◽  
Lijie Zhai ◽  
Erik Ladomersky ◽  
Kristen Lauing ◽  
Lakshmi Bollu ◽  
...  
Keyword(s):  
Mouse Model ◽  
Tumor Cell ◽  
Central Nervous System Tumor ◽  
Post Translational Modifications ◽  
Reporter Mouse ◽  
C Terminus ◽  
Human Gbm

Abstract Glioblastoma (GBM) is the most common and aggressive primary central nervous system tumor in adults with a median survival of 14.6 months. GBM is a potently immunosuppressive cancer due in-part to the prolific expression of immunosuppressive indoleamine 2,3 dioxygenase 1 (IDO). Tumor cell IDO facilitates the intratumoral accumulation of regulatory T cells (Tregs; CD4+CD25+FoxP3+). Although immunosuppressive IDO activity is canonically characterized by the conversion of tryptophan into kynurenine, we have utilized transgenic and syngeneic mouse models and mutant glioma lines to demonstrate that tumor cell IDO increases Treg accumulation independent of tryptophan metabolism. Here, we address the gap in our understanding of IDO signaling activity in vivo. Subcutaneously-engrafted human GBM expressing human IDO-GFP cDNA was isolated from immunodeficient humanized NSG-SGM3 mice. The tumor was immunoprecipitated for the GFP tag using GFP-TRAP followed by mass spectrometry which revealed a novel methylation site on a lysine residue at amino acid 373 in the IDO C-terminus region. Western blot analysis of IDO protein also revealed the presence of tyrosine phosphorylation. Additionally, we recently created a new transgenic IDO reporter mouse model whereby endogenous IDO is fused to GFP via a T2A linker (IDO→GFP). This model allows for the isolation of IDO+ cells in real-time and without causing cell death, thereby creating the opportunity for downstream molecular analysis of in situ-isolated GFP+ cells. Collectively, our work suggests that IDO non-enzyme activity may involve the post-translational modifications we recently identified. As IDO activity may differ between in vitro and in vivo modeling systems, we will use the new IDO→GFP reporter mouse model for an improved mechanistic understanding of how immunosuppressive IDO facilitates Treg accumulation in vivo.

scholarly journals Exosome-transported circRNA_0001236 enhances chondrogenesis and suppress cartilage degradation via the miR-3677-3p/Sox9 axis

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Guping Mao ◽  
Yiyang Xu ◽  
Dianbo Long ◽  
Hong Sun ◽  
Hongyi Li ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mouse Model ◽  
Chondrogenic Differentiation ◽  
Cartilage Degradation ◽  
Human Bone ◽  
Human Bone Marrow ◽  
Luciferase Reporter ◽  
Gene And Protein Expression

Abstract Objectives Aberrations in exosomal circular RNA (circRNA) expression have been identified in various human diseases. In this study, we investigated whether exosomal circRNAs could act as competing endogenous RNAs (ceRNAs) to regulate the pathological process of osteoarthritis (OA). This study aimed to elucidate the specific MSC-derived exosomal circRNAs responsible for MSC-mediated chondrogenic differentiation using human bone marrow-derived MSCs (hMSCs) and a destabilization of the medial meniscus (DMM) mouse model of OA. Methods Exosomal circRNA deep sequencing was performed to evaluate the expression of circRNAs in human bone marrow-derived MSCs (hMSCs) induced to undergo chondrogenesis from day 0 to day 21. The regulatory and functional roles of exosomal circRNA_0001236 were examined on day 21 after inducing chondrogenesis in hMSCs and were validated in vitro and in vivo. The downstream target of circRNA_0001236 was also explored in vitro and in vivo using bioinformatics analyses. A luciferase reporter assay was used to evaluate the interaction between circRNA_0001236 and miR-3677-3p as well as the target gene sex-determining region Y-box 9 (Sox9). The function and mechanism of exosomal circRNA_0001236 in OA were explored in the DMM mouse model. Results Upregulation of exosomal circRNA_0001236 enhanced the expression of Col2a1 and Sox9 but inhibited that of MMP13 in hMSCs induced to undergo chondrogenesis. Moreover, circRNA_0001236 acted as an miR-3677-3p sponge and functioned in human chondrocytes via targeting miR-3677-3p and Sox9. Intra-articular injection of exosomal circRNA_0001236 attenuated OA in the DMM mouse model. Conclusions Our results reveal an important role for a novel exosomal circRNA_0001236 in chondrogenic differentiation. Overexpression of exosomal circRNA_0001236 promoted cartilage-specific gene and protein expression through the miR-3677-3p/Sox9 axis. Thus, circRNA_0001236-overexpressing exosomes may alleviate cartilage degradation, suppressing OA progression and enhancing cartilage repair. Our findings provide a potentially effective therapeutic strategy for treating OA.

scholarly journals Indirect cholinergic activation slows down pancreatic cancer growth and tumor-associated inflammation

2020 ◽  
Vol 39 (1) ◽  
Author(s):  
Paulo L. Pfitzinger ◽  
Laura Fangmann ◽  
Kun Wang ◽  
Elke Demir ◽  
Engin Gürlevik ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Mouse Model ◽  
Tumor Stage ◽  
Ache Inhibitors ◽  
Impact On Survival ◽  
The Impact ◽  
Ache Expression ◽  
Cholinergic Activation

Abstract Background Nerve-cancer interactions are increasingly recognized to be of paramount importance for the emergence and progression of pancreatic cancer (PCa). Here, we investigated the role of indirect cholinergic activation on PCa progression through inhibition of acetylcholinesterase (AChE) via clinically available AChE-inhibitors, i.e. physostigmine and pyridostigmine. Methods We applied immunohistochemistry, immunoblotting, MTT-viability, invasion, flow-cytometric-cell-cycle-assays, phospho-kinase arrays, multiplex ELISA and xenografted mice to assess the impact of AChE inhibition on PCa cell growth and invasiveness, and tumor-associated inflammation. Survival analyses were performed in a novel genetically-induced, surgically-resectable mouse model of PCa under adjuvant treatment with gemcitabine+/−physostigmine/pyridostigmine (n = 30 mice). Human PCa specimens (n = 39) were analyzed for the impact of cancer AChE expression on tumor stage and survival. Results We discovered a strong expression of AChE in cancer cells of human PCa specimens. Inhibition of this cancer-cell-intrinsic AChE via pyridostigmine and physostigmine, or administration of acetylcholine (ACh), diminished PCa cell viability and invasion in vitro and in vivo via suppression of pERK signaling, and reduced tumor-associated macrophage (TAM) infiltration and serum pro-inflammatory cytokine levels. In the novel genetically-induced, surgically-resectable PCa mouse model, adjuvant co-therapy with AChE blockers had no impact on survival. Accordingly, survival of resected PCa patients did not differ based on tumor AChE expression levels. Patients with higher-stage PCa also exhibited loss of the ACh-synthesizing enzyme, choline-acetyltransferase (ChAT), in their nerves. Conclusion For future clinical trials of PCa, direct cholinergic stimulation of the muscarinic signaling, rather than indirect activation via AChE blockade, may be a more effective strategy.

scholarly journals Inhibition of Kirsten-Ras reduces fibrosis and protects against renal dysfunction in a mouse model of chronic folic acid nephropathy

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Lucy J. Newbury ◽  
Jui-Hui Wang ◽  
Gene Hung ◽  
Bruce M. Hendry ◽  
Claire C. Sharpe
Keyword(s):  
Folic Acid ◽  
Kidney Disease ◽  
Mouse Model ◽  
Renal Dysfunction ◽  
Antisense Oligonucleotides ◽  
Underlying Mechanism ◽  
Therapeutic Benefit ◽  
End Stage

Abstract Chronic Kidney Disease is a growing problem across the world and can lead to end-stage kidney disease and cardiovascular disease. Fibrosis is the underlying mechanism that leads to organ dysfunction, but as yet we have no therapeutics that can influence this process. Ras monomeric GTPases are master regulators that direct many of the cytokines known to drive fibrosis to downstream effector cascades. We have previously shown that K-Ras is a key isoform that drives fibrosis in the kidney. Here we demonstrate that K-Ras expression and activation are increased in rodent models of CKD. By knocking down expression of K-Ras using antisense oligonucleotides in a mouse model of chronic folic acid nephropathy we can reduce fibrosis by 50% and prevent the loss of renal function over 3 months. In addition, we have demonstrated in vitro and in vivo that reduction of K-Ras expression is associated with a reduction in Jag1 expression; we hypothesise this is the mechanism by which targeting K-Ras has therapeutic benefit. In conclusion, targeting K-Ras expression with antisense oligonucleotides in a mouse model of CKD prevents fibrosis and protects against renal dysfunction.

Anticancer effect of arsenic trioxide on cholangiocarcinoma: in vitro experiments and in vivo xenograft mouse model

2013 ◽  
Vol 14 (2) ◽  
pp. 215-224 ◽  
Author(s):  
Eun-Young Kim ◽  
Sang Soo Lee ◽  
Ji Hoon Shin ◽  
Soo Hyun Kim ◽  
Dong-Ho Shin ◽  
...  
Keyword(s):  
Mouse Model ◽  
Arsenic Trioxide ◽  
In Vitro Experiments ◽  
Anticancer Effect ◽  
Xenograft Mouse Model
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