Journal: Frontiers in Immunology

Article Title: Sphingosine 1-Phosphate- and C-C Chemokine Receptor 2-Dependent Activation of CD4 + Plasmacytoid Dendritic Cells in the Bone Marrow Contributes to Signs of Sepsis-Induced Immunosuppression

doi: 10.3389/fimmu.2017.01622

Figure Lengend Snippet: The expansion of the CD11c hi MHCII + CD4 + DC population in the bone marrow is mediated by S1P receptors and does not rely on splenic dendritic cells (DCs). (A) Mice were treated with the functional sphingosine 1-phosphate (S1P) receptor antagonist FTY720 or with the solvent as control before sham or cecal ligation and puncture (CLP) operation. After 36 h, the number of CD11c hi MHCII + CD4 + DC in the bone marrow was determined. The scatter plot shows the values of individual mice ( n = 5–9 mice per group) and the median/interquartile range as horizontal lines. Statistical differences were tested using the Kruskal–Wallis test followed by Dunn’s posttest. (B,C) CD11c hi MHCII + CD4 + DCs in the spleen. The gating strategy (B) was equivalent to the gating of CD4 + DCs in the bone marrow. Numbers indicate the percentage of gated cells or cells within the respective quadrant. Representative dot plots of one sham and one CLP mouse (36 h after surgery) are shown. (C) At different time points after sham or CLP operation total spleen cells were isolated and the number of CD11c hi MHCII + CD4 + DCs was quantified ( n = 4–21 per group). (D) Mice underwent splenectomy or laparotomy as control surgery 4 weeks before sham or CLP operation. After 36 h, the number of CD4 + DC in the bone marrow was quantified. The scatter plot shows the values of individual mice. Horizontal lines depict the median/interquartile range ( n = 7–11 mice per group). Statistical differences between sham and CLP were tested using the Mann–Whitney U -test. * p < 0.05; *** p < 0.001. DCs, dendritic cells.

Article Snippet: Where indicated, the functional S1P receptor antagonist FTY720 (2 mg/kg body weight; Fingolimod, LC Labs, Woburn, MA, USA) dissolved in 20% 2-hydroxypropyl-β-cyclodextrin (2-HPCD) was intravenously (i.v.) applied immediately before sham and CLP operation and 12 h thereafter.

Techniques: Functional Assay, Solvent, Control, Ligation, Isolation, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: Sphingosine 1-Phosphate- and C-C Chemokine Receptor 2-Dependent Activation of CD4 + Plasmacytoid Dendritic Cells in the Bone Marrow Contributes to Signs of Sepsis-Induced Immunosuppression

doi: 10.3389/fimmu.2017.01622

Figure Lengend Snippet: CD4 + T cells accumulate in parallel to CD4 + DCs in the bone marrow during sepsis. (A) Representative dot plots of bone marrow cells (BMC) from one sham and one cecal ligation and puncture (CLP) mouse 36 h after surgery. CD11c − CD4 + T lymphocytes were gated. (B,C) Mice were treated with the functional sphingosine 1-phosphate receptor antagonist FTY720 or with the solvent before sham or CLP operation. After 36 h, the number and frequency of CD4 + T cells in the bone marrow (B) and blood (C) , respectively, were determined. Data show the values of individual mice ( n = 5–9 mice per group) with median/interquartile range. (D) Analysis of the bone marrow of one representative naive wildtype (wt) and one Rag2 −/− mouse for the presence of CD4 + T cells. Numbers indicate the percentage of gated cells. (E) Wildtype and Rag2 −/− mice underwent sham or CLP operation. After 36 h, BMC were isolated and the frequency of CD11c hi MHCII + CD4 + DCs was determined according to the gating strategy shown in Figure B. Data indicate the values of individual mice ( n = 4–8 per group). Horizontal lines depict the median/interquartile range. Significant differences between sham and CLP were tested using the Mann–Whitney U -test. * p < 0.05; ** p < 0.01; *** p < 0.001. DCs, dendritic cells.

Article Snippet: Where indicated, the functional S1P receptor antagonist FTY720 (2 mg/kg body weight; Fingolimod, LC Labs, Woburn, MA, USA) dissolved in 20% 2-hydroxypropyl-β-cyclodextrin (2-HPCD) was intravenously (i.v.) applied immediately before sham and CLP operation and 12 h thereafter.

Techniques: Ligation, Functional Assay, Solvent, Isolation, MANN-WHITNEY

Journal: Expert opinion on therapeutic patents

Article Title: Deuterated drugs; where are we now?

doi: 10.1517/13543776.2014.943184

Figure Lengend Snippet: Status of Some Recent Drug Deuteration US Patent Applications

Article Snippet: It is also interesting to note that the Public Relations (PR) materials were used in the rejection of Rao and Zhang [ 14 ], and although this appears isolated, it is clear that companies need to balance the desirability of disclosures to drive investment, with the potential for these disclosures to later be used as prior art. table ft1 table-wrap mode="anchored" t5 caption a7 Application Assignee and Status CONCERT Tetrahydronaphthalene derivatives [ 15 ] Not Yet Examined Substituted triazolophthalazine derivatives [ 16 ] Not Yet Examined Substituted xanthine derivatives [ 17 ]] Not Yet Examined Pyrimidine derivatives [ 18 ] Not Yet Examined Derivatives of pyrazole- [ 19 ] Not Yet Examined Substituted isoindoline-1,3-dione [ 20 ] Non-Final Rejection, No §103 Substituted triazolo-pyridazine [ 21 ] Non-Final Rejection, No §103 Morphinan compounds [ 22 ] Allowed Substituted triazolo-pyridazine [ 23 ] Non-Final Rejection, No §103 Substituted dioxopiperidinyl phthalimide [ 24 ] Non-Final Rejection §103 and §103 based upon Tung [ 25 ], not ‘Dyck’ Analogues of cilostazol [ 26 ] Allowed N-phenyl-2-pyrimidineamine derivatives [ 27 ] Non-Final Rejection Abbreviated ‘Dyck’ §103 citing Foster [ 28 ], Ito [ 29 ], Kushner [ 30 ], Fluorouracil derivatives [ 31 ] Non-Final Rejection ‘Dyck’ §103 Novel pyrimidinecarboxamide derivatives [ 32 ] Not Yet Examined Heterocyclic kinase inhibitors [ 33 ] Non-Final Rejection §103 citing Czarnik [ 34 ] Pyrazinoisoquinoline compounds [ 35 ] Non-Final Rejection, ‘Dyck’ §103 4-hydroxybutyric acid analogs [ 36 ] Allowed after Non-Final Rejection §103 citing Kushner [ 30 ] Substituted derivatives of [ 37 ] Not Yet Examined Prostacyclin derivatives [ 38 ] Restriction Required Analogues of cilostazol [ 39 ] Allowed Substituted isoindoline-1,3-dione [ 40 ] Allowed Fluorinated diaryl urea [ 41 ] Allowed Substituted xanthine derivatives [ 42 ] Non-Final Rejection, Abbreviated ‘Dyck’ §103 citing Ando [ 43 ], Buteau [ 3 ], Foster [ 28 ] and Armstrong [ 44 ] Pyrazinoisoquinoline compounds [ 45 ] Allowed Deuterated fingolimod [ 46 ] Restriction Required AUSPEX Cyclopropyl modulators of [ 14 ] Allowed after Final Rejection §103 Citing non-’Dyck’ deuteration references, and Auspex and Concert PR Announcements.

Techniques: Analogues

Journal: Rheumatology (Oxford, England)

Article Title: FTY720 ameliorates experimental MPO-ANCA-associated vasculitis by regulating fatty acid oxidation via the neutrophil PPARα–CPT1a pathway

doi: 10.1093/rheumatology/keae320

Figure Lengend Snippet: FTY720 alleviated renal injury in EAV rats. ( A , B ) Representative images of glomeruli ( A ) and tubulointerstitium ( B ) of PAS staining for kidneys of rats (scale bar: 100 µm). ( C , D ) Representative images of glomerular ( C ) and tubulointerstitial ( D ) neutrophil infiltration in EAV rats (scale bar: 50 µm). ( E , F ) Amelioration of haematuria ( E ) and proteinuria ( F ) of EAV rats following FTY720 treatment. ( G , H ) Assessment of crescent formation ( G ) and TIN score ( H ) in EAV rats following FTY720 treatment. ( I , J ) Quantification of infiltrating neutrophils in glomeruli ( I ) and tubulointerstitium ( J ). Data are presented as means ( s . d .) from five rats per group. * P < 0.05, ** P < 0.01, *** P < 0.001; ns: not significant. DAPI: 4′,6-diamidino-2-phenylindole; EAV: experimental autoimmune vasculitis; HSA: human serum albumin; MPO: myeloperoxidase; PAS: periodic acid–Schiff; TIN: tubulointerstitial nephritis

Article Snippet: According to the product instruction, FTY720 (A8548, APExBIO, Houston, TX, USA) was dissolved in a mixture of 10% dimethyl sulfoxide, 40% PEG300 and 50% phosphate-buffered saline (PBS).

Techniques: Staining

Journal: Rheumatology (Oxford, England)

Article Title: FTY720 ameliorates experimental MPO-ANCA-associated vasculitis by regulating fatty acid oxidation via the neutrophil PPARα–CPT1a pathway

doi: 10.1093/rheumatology/keae320

Figure Lengend Snippet: RNA sequencing analysis and gene enrichment analysis of renal cortex from rats. ( A , B ) Heat map ( A ) and scatter plot ( B ) presenting the distribution of DEGs between vehicle-treated and FTY720-treated groups. Fold change >2.0 or <0.5, P < 0.05. ( C ) A cluster profiler of the 10 most prominently enriched pathways identified in RNA sequencing and gene enrichment analysis between vehicle-treated and FTY720-treated groups. ( D , F ) Gene set enrichment analysis of FAO ( D ), PPAR signalling pathway ( E ) and respiratory burst pathway ( F ). FTY represents EAV rats treated with FTY720; AAV represents EAV rats treated with vehicle. AAV: ANCA-associated vasculitis; DEG: differentially expressed gene; FAO: fatty acid oxidation; FC: fold change; FDR: false discovery rate; NES: normalized enrichment score; PPAR: peroxisome proliferator activated receptor

Article Snippet: According to the product instruction, FTY720 (A8548, APExBIO, Houston, TX, USA) was dissolved in a mixture of 10% dimethyl sulfoxide, 40% PEG300 and 50% phosphate-buffered saline (PBS).

Techniques: RNA Sequencing

Journal: Rheumatology (Oxford, England)

Article Title: FTY720 ameliorates experimental MPO-ANCA-associated vasculitis by regulating fatty acid oxidation via the neutrophil PPARα–CPT1a pathway

doi: 10.1093/rheumatology/keae320

Figure Lengend Snippet: FTY720 inhibited ANCA-induced neutrophil activation while upregulating FAO. ( A ) Identification of increased neutrophil marker CD11b of differentiated HL-60 cells. ( B ) Inhibitory effect of FTY720 on respiratory burst of differentiated HL-60 cells stimulated with serum from AAV patients. ( C ) Inhibitory effect of FTY720 on respiratory burst of isolated human neutrophils activated by MPO-ANCA positive IgG. ( D ) Gene expression of the top six upregulated genes in FAO in RNA sequencing was measured in differentiated HL-60 cells treated with the 10% serum from AAV patients and FTY720 or vehicle. ( E ) mRNA level of PPARα and protein expression of CPT1a and PPARα in differentiated HL-60 cells treated with the 10% serum from AAV patients and FTY720 or vehicle. Data are shown as means ( s . d .). * P < 0.05, ** P < 0.01, *** P < 0.001; ns: not significant. AAV: ANCA-associated vasculitis; ABCD3: ATP binding cassette subfamily D member 3; CPT1a: carnitine palmitoyltransferase 1A; ECHDC2: enoyl-CoA hydratase domain containing 2; FAO: fatty acid oxidation; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; HSD17b4: 17β-hydroxysteroid dehydrogenase 4; MFI: mean fluorescence intensity; PPARα: peroxisome proliferator activated receptor α; ROS: reactive oxygen species; SESN2: sestrin 2; SLC27A2: solute carrier family 27 member 2

Article Snippet: According to the product instruction, FTY720 (A8548, APExBIO, Houston, TX, USA) was dissolved in a mixture of 10% dimethyl sulfoxide, 40% PEG300 and 50% phosphate-buffered saline (PBS).

Techniques: Activation Assay, Marker, Isolation, Gene Expression, RNA Sequencing, Expressing, Binding Assay, Fluorescence

Journal: Rheumatology (Oxford, England)

Article Title: FTY720 ameliorates experimental MPO-ANCA-associated vasculitis by regulating fatty acid oxidation via the neutrophil PPARα–CPT1a pathway

doi: 10.1093/rheumatology/keae320

Figure Lengend Snippet: FTY720 exerted an inhibitory effect on ANCA-induced neutrophil activation via the PPARα–CPT1a pathway. ( A ) Determination of CPT1a-knockdown efficiency by qPCR and western blot. ( B , C ) The inhibitory effect of FTY720 on respiratory burst in differentiated HL-60 cells upon stimulation with 10% serum from AAV patients was reversed by CPT1 antagonist etomoxir ( B ) and CPT1a knockdown ( C ). ( D , E ) The effect of GW6471 (a PPARα antagonist) on the inhibition of FTY720 on respiratory burst ( D ) and NET formation ( E ). ( F , G ) The effect of combination of GW7647 (a PPARα agonist) and etomoxir (a CPT1 inhibitor) on the inhibition of FTY720 on respiratory burst ( F ) and NET formation ( G ). ( H ) Representative images of NET formation with or without FTY720, etomoxir, GW6471 (scale bar: 50 µm). Data are shown as means ( s . d .). * P < 0.05, ** P < 0.01, *** P < 0.001; ns: not significant. AAV: ANCA-associated vasculitis; CPT1a: carnitine palmitoyltransferase 1A; Eto: etomoxir; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; MFI: mean fluorescence intensity; PPARα: peroxisome proliferator activated receptor α; ROS: reactive oxygen species

Article Snippet: According to the product instruction, FTY720 (A8548, APExBIO, Houston, TX, USA) was dissolved in a mixture of 10% dimethyl sulfoxide, 40% PEG300 and 50% phosphate-buffered saline (PBS).

Techniques: Activation Assay, Knockdown, Western Blot, Inhibition, Fluorescence