Carboxylesterase-1 Assisted Targeting of HDAC Inhibitors to Mononuclear Myeloid Cells in Inflammatory Bowel Disease
Ahmed M I ElfikyMohammed GhiboubAndrew Y. F. Li YimIshtu HagemanJan VerhoeffManon de KrijgerPatricia H. P. van HamersveldOlaf WeltingIris AdmiraalShafaque RahmanJuan J. García‐VallejoManon E. WildenbergLaura TomlinsonRichard GregoryInmaculada RiojaRab K. PrinjhaRebecca C. FurzeHuw D. LewisPalwinder K. ManderSigrid E.M. HeinsbroekMatthew BellWouter J. de Jonge
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Histone deacetylase inhibitors [HDACi] exert potent anti-inflammatory effects. Because of the ubiquitous expression of HDACs, clinical utility of HDACi is limited by off-target effects. Esterase-sensitive motif [ESM] technology aims to deliver ESM-conjugated compounds to human mononuclear myeloid cells, based on their expression of carboxylesterase 1 [CES1]. This study aims to investigate utility of an ESM-tagged HDACi in inflammatory bowel disease [IBD].CES1 expression was assessed in human blood, in vitro differentiated macrophage and dendritic cells, and Crohn's disease [CD] colon mucosa, by mass cytometry, quantitative polymerase chain reaction [PCR], and immunofluorescence staining, respectively. ESM-HDAC528 intracellular retention was evaluated by mass spectrometry. Clinical efficacy of ESM-HDAC528 was tested in dextran sulphate sodium [DSS]-induced colitis and T cell transfer colitis models using transgenic mice expressing human CES1 under the CD68 promoter.CES1 mRNA was highly expressed in human blood CD14+ monocytes, in vitro differentiated and lipopolysaccharide [LPS]-stimulated macrophages, and dendritic cells. Specific hydrolysis and intracellular retention of ESM-HDAC528 in CES1+ cells was demonstrated. ESM-HDAC528 inhibited LPS-stimulated IL-6 and TNF-α production 1000 times more potently than its control, HDAC800, in CES1high monocytes. In healthy donor peripheral blood, CES1 expression was significantly higher in CD14++CD16- monocytes compared with CD14+CD16++ monocytes. In CD-inflamed colon, a higher number of mucosal CD68+ macrophages expressed CES1 compared with non-inflamed mucosa. In vivo, ESM-HDAC528 reduced monocyte differentiation in the colon and significantly improved colitis in a T cell transfer model, while having limited potential in ameliorating DSS-induced colitis.We demonstrate that monocytes and inflammatory macrophages specifically express CES1, and can be preferentially targeted by ESM-HDAC528 to achieve therapeutic benefit in IBD.Cite
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Objective To study the effect of staphylococcal enterotoxin B(SEB) on human peripheral blood mononuclear cells and to explore whether apoptosis is one of its immunosuppressive mechanism.Methods Human peripheral blood mononuclear cells in vitro were stimulated by different concentrations of SEB(100 μg/ml,50 μg/ml,25 μg/ml,10 μg/ml,5 μg/ml,1 μg/ml,0.1 μg/ml,0.01 μg/ml,0.001 μg/ml and 0.0001 μg/ml).PBMCs in negative control group were cultivated in RPMI1640,and PHA was added in positive control group.After cultivation for 48 h,MTT colorimetric assay was used to analyze proliferation of PBMC.Same process was also carried out in negative control group in RPMI1640 and positive control group of PHA.After 48 h,the repeated stimulation with 10 μg/ml SEB was done on PBMC for two times.Results Different concentrations of SEB could initially stimulate human peripheral blood mononuclear cells in vitro to proliferate,but the proliferation of PBMC activated by 10 μg/ml SEB was most dramatic.There was no significant difference between group of 10 μg/ml SEB and group of PHA.Their rates of proliferation were 23.1% and 24.3% respectively.After repeated stimulation at 48 hr later,PHA could continue to stimulate the proliferation of PBMC,but 10 μg/ml SEB could inhibit the proliferation of PBMC.Conclusion SEB may initially stimulate the proliferation of human PBMC in vitro.The repeated stimulation with SEB may inhibit the proliferation of PBMC.
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Objective To observe the significance of expressions of CD14+CD16+ on peripheral monocytes in children with Kawasaki di-sease (KD).Methods The expression of CD14+ and CD14+CD16+ monocytes in 16 children with KD (1-11 years old) were analyzed by flow cytomety both pre-treatment and post-treatment.And the percentages of CD14+CD16+ monocytes among CD14+ monocytes were calculated.Sixteen healthy children (10 months -10 years old) were served as normal control group.Statistical analysis was performed using t test.Results The levels of CD14+ monocytes,percentage of CD14+CD16+ monocytes among CD14+ monocytes and CD14+CD16+ monocytes in children with KD during acute phase (n=16) were (1.03±0.58)×109 L-1,(12.53±5.31)% and(1.20±0.79)×108 L-1.They were significantly higher than those in the normal controls[(0.57±0.21)×109 L-1,(3.86±1.84)% and (0.21±0.10)×108 L-1](Pa0.05).Followed by the patients' condition improved(n=15), the increased expressive levels[(1.03±0.60) ×109 L-1,( 12.83±6.51)%,(1.22±0.82)×108 L-1] decreased [(0.49±0.21)×109 L-1,(5.07±4.77)%,(0.24±0.20)×108 L-1)(Pa0.01)].The expressive levels of CD14+CD16+ monocytes were no significant differences between the post-treatment and the normal controls(Pa0.05).And the expressive levels remained high when the patient recurred.Conclusions The expressive levels of CD14+CD16+ monocytes increase in children with KD.And they change when the patient's clinical condition change.
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CD14 has been reported to be the lipopolysaccharide (LPS)-LPS binding protein receptor. The effects of interferon-γ (IFN-γ) on CD14 expression have not been clearly established. The purpose of this investigation was to examine the effects of IFN-γ alone and IFN-γ followed by bacterial LPS on CD14 expression. Human peripheral blood monocytes were isolated by counterflow centrifugal elutriation (CCE). Monocytes were cultured for 48 h with IFN-γ alone or for 24 h with IFN-γ followed by LPS for a second 24 h. IFN-γ alone caused a down-regulation of CD14 expression, as assessed by flow cytometry, relative to CD14 expression in untreated monocytes. In addition, CD14 expression was even more significantly down-regulated after IFN-γ pretreatment followed by either Prevotella intermedia or Salmonella typhimurium LPS. Likewise, the percentage of CD14+ monocytes decreased after IFN-γ alone and even more dramatically after IFN-γ treatment followed by either LPS. This study clearly demonstrated that IFN-γ down-regulates CD14 expression and that LPS following IFN-γ pretreatment potentiates this effect.
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Objective To explore the killing efficacy of cord blood mononuclear cells in HO9810 and the activation effect of rhIFNα-2b on it.Methods Mononuclear cells were isolated from fetal blood and adult peripheral blood respectively,whose killing efficacy in HO9810 at the presence or absence of rhIFNα-2b were measured with MTT.Results The natural killing efficacy of cord blood mononuclear cells was lower than that of the peripheral blood.When activated,however,it was enhanced markedly and could be equal to that of the latter.Conclusions The mononuclear cells in the cord blood can be activated into killing cells by rhIFNα-2b,which shows increased killing efficacy to HO9810 and have a promised future in antitumor immunotherapy.
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Animal models of experimentally induced inflammatory bowel disease (IBD) are useful for understanding more about the mechanistic basis of disease, identifying new targets for therapeutic intervention, and testing novel therapeutic agents. This unit provides detailed protocols for four of the most commonly used mouse models of experimentally induced intestinal inflammation: chemical induction of colitis by dextran sodium sulfate (DSS), hapten-induced colitis via 2,4,6-trinitrobenzene sulfonic acid (TNBS), Helicobacter-induced colitis in mdr1a(-/-) mice, and the CD4(+) CD45RB(hi) SCID transfer colitis model.
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Inflammatory bowel disease (IBD) is a chronic inflammatory disease of the intestinal tract and is primarily comprised of Crohn's disease (CD) and ulcerative colitis (UC). Several murine models that include both chemical induced and genetic models have been developed that mimic some aspects of either CD or UC. These models have been instrumental in our understanding of IBD. Of the chemical induced colitis models, dextran sodium sulfate (DSS) induced colitis model is a relatively simple and very widely used model of experimental colitis.
Inflammatory Bowel Diseases
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Uncontrolled preanalytical variables can reduce the accuracy and reproducibility of downstream analytical results from peripheral blood mononuclear cells (PBMCs). PBMCs were isolated from EDTA and citrate-anticoagulated blood samples, obtained from healthy subjects and patients with inflammatory and infectious conditions. PBMC-derived RNA samples were examined for gene expression changes induced by extended blood pre-centrifugation delays at 4 °C and RT. We used Taqman RTqPCR to evaluate the combination of two target genes for their "diagnostic performance" in identifying EDTA and citrate-anticoagulated PBMC samples with extended pre-centrifugation times. We established the PBMC preanalytical score, a gene expression metric to asses the PBMC quality related to the pre-centrifugation delay at room temperature for different anticoagulants. The PBMC preanalytical score measurement can identify: EDTA PBMC samples or RNA extracted from these PBMCs with RT precentrifugation times >48 h with 98% sensitivity and 87% specificity at a cutoff of 57. citrate PBMC samples or RNA extracted from these PBMCs with RT precentrifugation times of >48 h with 92% sensitivity and 84% specificity at a cutoff of 348. The proposed PBMC preanalytical score may enable objective PBMC sample qualification for downstream applications, which may be influenced by blood precentrifugation delays.
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Animal models of experimentally induced inflammatory bowel disease (IBD) are useful for understanding more about the mechanistic basis of the disease, identifying new targets for therapeutic intervention, and testing novel therapeutics. This unit provides detailed protocols for five widely used mouse models of experimentally induced intestinal inflammation: chemical induction of colitis by dextran sodium sulfate (DSS), hapten-induced colitis via 2,4,6-trinitrobenzene sulfonic acid (TNBS), Helicobacter-induced colitis in mdr1a(-/-) mice, the CD4(+) CD45RB(hi) SCID transfer colitis model, and the IL-10(-/-) colitis model. © 2016 by John Wiley & Sons, Inc.
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