The nontoxic B-subunit of the intestinal pathogen-produced Shiga toxin (STxB) binds specifically to the glycosphingolipid Gb3, overexpressed Double click the 'setup' file.Delivery of drugs to the appropriate target cells would improve efficacy and reduce potential side effects. If the file is in a 'zip' format double click the zip file and choose 'unzip' and select a location on your computer to save the files. Find the files on your system. Right click on the link to the file and choose 'Save Target As' and save the zip file to your desktop.GraphPad Software.Intra-arterial PBS perfusion was done in order to wash out the circulating drugs in blood vessels. Perkin Elmer Health Sciences Inc. 2Centre de recherche en Cancérologie de Marseille, INSERM U1068, CNRS U7258, Aix Marseille Université, Institut Paoli – Calmettes, Marseille, FranceIVIS Spectrum Xenogen.
Xenogen Living Image Software Download A WidePrevious studies using a mouse model of GVHD have shown that targeting the T-cell Inducible COStimulator (ICOS, CD278) molecule is beneficial, but it is unclear whether the same applies to human cells.Browse and download a wide variety of award-winning video, audio, business, utility, or graphics software programs for both PC and Mac. Targeting costimulatory molecules with antagonist antibodies could dampen the excessive immune response that occurs, while preserving the beneficial graft vs leukemia (GVL) of the allogeneic response. The fluorescent signal intensities in the tumor and different organs were analyzed by Xenogen Living Image software, Version 2.50 (WaveMetrix, USA).Background: Graft-vs-host disease (GVHD) is a major complication of allogenic bone marrow transplantation (BMT).Moreover, a significant improvement in survival was obtained in a curative xeno-GVHD setting. In contrast, 65% of the mice receiving a single injection of the anti-hICOS mAb survived beyond 100 days. To test this hypothesis, we used a xenogeneic model of GVHD where human peripheral blood mononuclear cells were adoptively transferred in immunocompromised NOD.SCID.gc-null mice (NSG).Results: In this model, control mice invariably lost weight and died by day 50.However, the allogeneic T-cell response is often necessary to eliminate residual malignant cells, an effect referred to as graft vs leukemia (GVL). Thus, ICOS represents a promising target in the management of BMT, preventing GVHD while preserving GVL.Graft-vs-host disease (GVHD) is a major complication of allogeneic bone marrow transplantation (BMT), used to treat hematological malignancies. Importantly, GVHD-protected mice retained the ability to control the P815 mastocytoma cell line, mimicking GVL in humans.Conclusion: A mAb-targeting human ICOS alleviated GVHD without impairing GVL in a xenograft murine model. In addition, the mAb prevented T-cell expansion in the blood during xeno-GVHD. ICOS/ICOS-L interaction has been described to play an important role in T-cell activation and function such as cytokine production ( 8) and antibody class switching by B cells ( 9, 10). ICOS-ligand (ICOS-L, CD275, B7-H1, or B7RP-1) is the only known ICOS partner, constitutively expressed by B cells, and can be induced on macrophages and dendritic cells ( 4, 7). Among other costimulatory molecules of the CD28 immunoglobulin superfamily, the Inducible T-cell COStimulator (ICOS, CD278) has been shown to be an important mediator of T-cell proliferation and survival ( 3– 6). Targeting costimulatory molecules such as CD28 with monoclonal antibodies (mAbs) has the potential to inhibit T-cell activation and, thus, be favorable for GVHD control ( 2). Other studies have evaluated the efficacy of ICOS blockade in mice models of GVHD. In transplantation, inhibition of the ICOS/ICOS-L interaction also allows better allograft survival ( 13, 14). For instance, ICOS/ICOS-L interaction is critical for T helper cell-mediated lung mucosal inflammatory responses ( 5) and for collagen-induced arthritis ( 11, 12). Materials and Methods AntibodiesThe 314.8 mouse anti-human ICOS mAb was produced as ascites and purified by protein A binding and elution with the Affi-gel Protein A MAPS II Kit (Bio-rad). Using a model of xeno-GVHD in NSG mice that we previously established ( 20), we assessed here whether a murine mAb to human ICOS, previously reported as an antagonist of ICOS/ICOS-L interaction in vitro ( 21), would prevent GVHD while preserving GVL. Activation of human cells in this xenogeneic and lymphopenic environment then leads to GVHD symptoms, such as weight loss, infiltration of human cells in various tissues, and ultimately death. Furthermore, the impact of ICOS blockade on GVL is currently unknown.To address this question in a human setting, we used the transfer of human peripheral blood mononuclear cells (PBMC) in irradiated immunocompromised NOD.SCID.γc −/− (NSG) mice that lacks T, B, and NK cells, as described earlier ( 19). Thus, the precise role of ICOS in murine GVHD is still controversial. However, others have found more severe GVHD when ICOS −/− CD8 + T cells were transferred ( 17, 18). Mice and Xeno-GVHD ModelAll animals used were NSG (NOD.SCID gamma-c −/− H2-K d, H2-D b) mice (stock ≠005557) purchased from the Jackson laboratory (USA). Cells were washed in PBS 3% FCS and diluted at the appropriate cell concentration in 1× PBS before injection into mice. Preparation of Human Peripheral Blood Mononuclear Cells (huPBMC)Human peripheral blood mononuclear cells were collected by Etablissement Français du Sang from healthy adult volunteers after informed consent in accordance with the Declaration of Helsinki and isolated on a Ficoll gradient (Biocoll). Anti-hICOS antibody or control isotype was injected intraperitoneally at various doses (see figure legends). For xeno-GVHD induction, 7–12-weeks-old female mice were irradiated (2 Gy) and engrafted the same day with 2.10 6 huPBMC by retro orbital injection. All protocols were approved by the Ethics Committee for Animal Experimentation Charles Darwin (Ce5/2012/025). Cells were then fixed and permeabilized using the Cytofix/Cytoperm kit according to manufacturer’s instructions (eBioscience) and stained with CD45, CD4, CD8, and anti-human IFNγ-FITC, antihuman TNFα-PE-Cy7, and anti-human IL-2-PE (all from eBioscience). Data were analyzed using the FlowJo software (TreeStar, Ashland, OR, USA).For cytokine production analysis, freshly harvested splenocytes were stimulated 4 h with PMA (50 ng/ml) and ionomycin (500 ng/ml), both from Sigma (Saint-Louis, MO, USA) prior to extracellular staining. Cells were stained during 20 min at room temperature and were washed with 1× PBS 3% FCS before acquisition on LSRII cytometer (Becton Dickinson, San Jose, CA, USA). Splenocytes were washed with 1× PBS 3% FCS and stained with human-specific antibodies used for flow cytometry: phycoerythrin (PE)-CF594-labeled CD45 (HI30 clone), allophycocyanin (APC)-H7-labeled CD8 (SK1 clone), and Alexa-fluor 700-labeled CD45RA (HI100) purchased from Becton Dickinson (San Jose, CA, USA) phycoerythrin-cyanin7-labeled CD3 (SK7 clone), peridinin chlorophyll-labeled CD4 (clone RPA-T4 clone), and APC-labeled ICOS (C398.4A clone) purchased from Biolegend (San Diego, CA, USA) and eFluor 450-labeled Ki67 (clone 20Raj1) and PE-labeled ICOS-L (clone MIH12) purchased from eBioscience (San Diego, CA, USA). Phenotypic Analysis by Flow CytometrySpleen cells were isolated by mechanical dissociation. Mice were euthanized when they reached 80% of their initial weight or exhibited signs of GVHD, such as hunched back, ruffled fur, and reduced mobility. Vbox addition for mac clientThe absolute count of different populations was determined using the following formula. Then, 100 µl of fluorescent beads (Stem kit Beckman Coulter) were added and samples were run on LSRII cytometer. Red blood cells’ lysis was performed using the Beckman Coulter T q-prep automaton. Extracellular staining for CD45, CD3, CD4, and CD8 was performed on total blood. For cell counting, 100 µl of blood was collected on 20 µl of heparin (Panpharma, Luitré, France). Proportion of CD8 + cells producing one, two, or three cytokines was determined using boolean gating in FlowJo. For liver, localized infiltrates were numbered and normalized with vessel number. Slides were coded without reference to prior treatment and examined in a blinded fashion for quantification. Organs were further sectioned (6 µm thick) for hematoxylin–eosin staining.
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