Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Interleukin-37 monomer is the active form for reducing innate immunity

doi: 10.1073/pnas.1819672116

Figure Lengend Snippet: IL-37 elicits a unique (parabolic) dose dependence observed under multiple conditions. (A) Recombinant IL-37 suppression of phorbol 12-myristate 13-acetate (PMA)-induced IL-1β secretion in human peripheral blood mononuclear cells (PBMCs) pretreated with indicated doses of IL-37 2 h before treatment with 25 ng/mL of PMA for 24 h in the presence of 10% FCS. IL-1Ra was also used at 10 μg/mL (B) Recombinant IL-37 (IL-3746–218) suppression of LPS-induced IL-6 secretion in bone marrow cells of mice pretreated with indicated doses of IL-37 2 h before treatment with 100 ng/mL LPS. Assays were performed in quadruplicate in 96 well plates with 500,000 cells/mL. J1 murine macrophage cells were stimulated with LPS, and the indicated doses of IL-3746–218 and supernatants were probed for both (C) IL-1β and (D) IL-6 as described in Fig. 5.

Article Snippet: The murine macrophage cell line J1 was purchased from American Type Culture Collection and cultured according to the distributor’s instructions in DMEM (Corning) supplemented with 10% FBS and 1% penicillin/streptomycin at 37 °C and 5% CO 2 .

Techniques: Recombinant

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Interleukin-37 monomer is the active form for reducing innate immunity

doi: 10.1073/pnas.1819672116

Figure Lengend Snippet: IL-37-mediated antiinflammatory activity is not increased by terminal truncations but only mutations that disrupt dimer formation. (A) J1 murine macrophage cells were pretreated 1 h with the indicated doses of WT IL-3746–218 and engineered terminal mutants of IL-37, stimulated with 1 µg/mL LPS for 4 h and, subsequently, treated with 20 µM of nigericin for 1 h before monitoring IL-1β secretion. (B) WT IL-3746–218 along with both point mutations that specifically disrupt the IL-37 dimer, Y85A, and D73K, was assayed identically to A. Assays were performed in triplicate in a 96 well plate with 750,000 cells/mL Cytokine production was quantified by ELISA. All assays are representative of, at least, five independent experiments. Error bars represent the SEM; *P < 0.05, **P < 0.001, and ***P < 0.0001, statistical significance was assessed using the unpaired Student’s t test.

Article Snippet: The murine macrophage cell line J1 was purchased from American Type Culture Collection and cultured according to the distributor’s instructions in DMEM (Corning) supplemented with 10% FBS and 1% penicillin/streptomycin at 37 °C and 5% CO 2 .

Techniques: Activity Assay, Enzyme-linked Immunosorbent Assay

Journal: mBio

Article Title: Lactobacillus johnsonii is a dominant Lactobacillus in the murine oral mucosa and has chitinase activity that compromises fungal cell wall integrity

doi: 10.1128/mbio.02416-24

Figure Lengend Snippet: Chitin content influences C. albicans susceptibility to MT4. ( A ) C. albicans strains SC5314, DAY286 and its isogenic cht2/cht2 mutant strain FJS5 were stained with CW. Chitin content as assessed by FACS analysis of CW binding. Strains DAY286 and FJS5 have higher CW binding than strain SC5314, indicating higher cell wall chitin content. Each dot represents the mean MFI of 20,000 cells from three replicates. ( B ) C. albicans strains SC5314, DAY286, and FJS5 were cocultured with MT4 in BHI broth, aerobically with 5% CO 2 at 37°C for 4 h, and fungal viability was assessed by the XTT assay. Strains with higher chitin cell wall content (DAY286 and FJS5) were less susceptible to MT4. Data from 2 to 3 independent experiments with technical replicates. One-way ANOVA with the uncorrected Dunn’s test.

Article Snippet: Primers were synthesized by Integrated DNA Technologies, Inc., and their specificity was confirmed experimentally against murine (C57BL/6 mice), fungal ( C. albicans SC5314), and bacterial DNA from ATCC reference strains and murine isolates.

Techniques: Mutagenesis, Staining, Binding Assay, XTT Assay

Journal: Nature clinical practice. Cardiovascular medicine

Article Title: Mitochondrial oxidative metabolism is required for the cardiac differentiation of stem cells

doi: 10.1038/ncpcardio0766

Figure Lengend Snippet: Embryonic stem cell cardiac differentiation is coordinated with metabolic transcriptome reprogramming and mitochondrial oxidative metabolism. (A) ES have no contractile activity on linescans and MEF2C, a cardiac transcription factor, and cardiac α-actinin, a tissue-specific protein of the contractile apparatus, are absent. (B) Cardiomyocytes derived from ES have distinct structures on light microscopy (upper panel) and electron microscopy (inset), contractile activity on linescans, and MEF2C and cardiac α-actinin are abundant in the nucleus and sarcomeres, respectively, on immunofluorescent microscopy. (Bars 10 μm [A,B left], 2 μm [A,B insets], 2 s [B middle], and 5 μm [A,B right]). (C) The basal respiratory rate and maximum respiratory capacity of cardiomyocytes were markedly higher, while the lactate production from anaerobic glycolysis was lower than in ES, underscoring distinct metabolic identities of the progeny compared with the embryonic source. A higher ADP:ATP ratio reflected an increased rate of energy use in cardiomyocytes than ES. (D) High-membrane-potential mitochondria (red) in ES on confocal microscopy. (E) Mitochondria have lower membrane potential (green) in cardiomyocytes, associated with increased energy use. (F) Intercellular cardiomyocyte connections with mitochondrial traffic (mitotrail) indicate cell–cell metabolic cross-talk in cardiogenesis. (D–F, bars 20 μm, sample >3.) (G) Microarray analysis of total mRNA in ES and cardiomyocytes revealed specific changes in genetic programming of the cellular energetic system. Genes were hierarchically clustered as mRNA copy numbers of cardiomyocytes versus ES transcripts (n = 3 in each group). Redundant probe sets were included to illustrate the individual dynamics of expression profiles. aP<0.05; n=3–12. Abbreviations: CM, cardiomyocytes; ES, embryonic stem cells; mRNA, messenger RNA.

Article Snippet: Murine embryonic stem cells, maintained in Glasgow’s Minimum Essential Medium (BioWhittaker-Cambrex, Walkersville, MD) with sodium pyruvate, nonessential amino acids, 2-mercaptoethanol, 7.5% fetal bovine serum (Invitrogen Corporation, Carlsbad, CA), and leukemia inhibitory factor (ESGRO; Chemicon International, Inc, Temecula, CA), were differentiated in media containing 20% fetal bovine serum via an inverted hanging-drop method.

Techniques: Activity Assay, Derivative Assay, Light Microscopy, Electron Microscopy, Microscopy, Membrane, Confocal Microscopy, Microarray, Expressing

Journal: Nature clinical practice. Cardiovascular medicine

Article Title: Mitochondrial oxidative metabolism is required for the cardiac differentiation of stem cells

doi: 10.1038/ncpcardio0766

Figure Lengend Snippet: Development and maturation of mitochondrial network in stem cell cardiac differentiation. (A) Gene array analysis of selected genes related to mitochondrial fission, fusion, and/or cristae maturation. Compared with the embryonic stem cell source, genes involved in mitochondrial fission and membrane structure remodeling in cardiomyocytes (Dnm1l, Mtp18, Opa1, and DAP3) were downregulated, whereas those involved in mitochondrial fusion and cristae maturation were typically upregulated (Mfn2 and IMMT). (B) Transmission electron microscopy revealed spherical mitochondria with underdeveloped cristae in embryonic stem cells versus elongated, cristae-rich mitochondria in cardiomyocytes. (C) Live-cell imaging showed discrete organelles with no apparent pattern of mitochondrial arrangement in ES versus an expanded network of aligned mitochondria in cardiomyoctyes. (D) Tracking cardiomyocyte development revealed an organization of the mitochondrial network (upper panels) ranging from random (left), to perinuclear (center), to transcellular (right), along with concomitant maturation of myofibrillar structure (lower panels). Mitochondria were visualized with MitoTracker Red, myofibrils with α-actinin staining (green), and nuclei with DAPI (blue). (E) Development of mechanoenergetic coupling through intercalation of mitochondria with myofibrils in cardiomyocytes observed by confocal microscopy (upper panel). Profile of fluorescence intensity (lower panel corresponds to arrow in upper panel) indicates an alternating distribution of mitochondria (red) and myofibrils (green). (F) Integration of mitochondrial (green) and electrical (red) activities in a beating area of an embryoid body (EB). Cardiac beating area delineated by the tight correlation of electrical activity staining with RH237, a probe for plasma membrane potential, and mitochondrial imaging with JC-1 (upper panel). Profiles of overlapping fluorescence intensity for both signals within the cardiac beating area are depicted in the lower panel (lower panel corresponds to line in upper panel). aP <0.05. Abbreviations: CM, cardiomyocytes; ES, embryonic stem cells.

Article Snippet: Murine embryonic stem cells, maintained in Glasgow’s Minimum Essential Medium (BioWhittaker-Cambrex, Walkersville, MD) with sodium pyruvate, nonessential amino acids, 2-mercaptoethanol, 7.5% fetal bovine serum (Invitrogen Corporation, Carlsbad, CA), and leukemia inhibitory factor (ESGRO; Chemicon International, Inc, Temecula, CA), were differentiated in media containing 20% fetal bovine serum via an inverted hanging-drop method.

Techniques: Membrane, Transmission Assay, Electron Microscopy, Live Cell Imaging, Staining, Confocal Microscopy, Fluorescence, Activity Assay, Clinical Proteomics, Imaging

Journal: PLoS ONE

Article Title: Vaccination with Embryonic Stem Cells Protects against Lung Cancer: Is a Broad-Spectrum Prophylactic Vaccine against Cancer Possible?

doi: 10.1371/journal.pone.0042289

Figure Lengend Snippet: ( A ) Bar graph showing GM-CSF expression in non-transduced and retrovirally transduced STO fibroblasts. Error bars represent mean ± SD. *, p<0.05; relative to non-transduced STO cells; t test. ( B ) Scheme of immunization. Male C57BL/6 mice were immunized twice (days 0 and 14) with HBSS (control), or irradiated 1×10 6 ESC+irradiated 1×10 6 GM-CSF-expressing STO murine embryonic fibroblasts (STO-GM) s.c. in the right flank. Seven days after boost, mice were challenged with 1×10 5 Lewis lung carcinoma cells (LLC) s.c. in the left flank. ( C ) C57BL/6 mice (10/group) were immunized twice (days 0 and 14) with HBSS (control), or irradiated 1×10 6 ESC+irradiated 1×10 6 STO-GM, or irradiated 1×10 6 STO-GM cells alone s.c. in the right flank prior to s.c. challenge with LLC on day 21. Tumor growth was monitored daily in all animals until sacrifice due to tumors exceeding 5% of body weight. The vaccinated tumor free mice remained so for up to 4 months later with no overt signs of distress or autoimmunity. are representative of three independent experiments. **, p <0.001; relative to control group; log-rank test. ( D ). Tumor growth was measured by calipers every 2nd or 3rd day and tumor volumes were plotted as indicated. The data represent the average tumor volumes of 10 mice/control group and 3 mice/ESC/STO-GM group and are representative of three independent experiments. Error bars represent mean ± SEM.

Article Snippet: As a vaccine, we employed the murine embryonic stem cell (ESC) line, ES-D3 (ATCC CRL-11632), derived from 129/Sv mice (expressing MHC class II I-E).

Techniques: Expressing, Control, Irradiation