He rejection group, 4 cases showed abnormalities which were graded as BL rejection according to Banff [15]. Demographics of these patients can be found here [16].immunosuppression, Cyclosporine (Cys). Animal activity, body weight and graft function were assessed daily. The latter was measured by direct abdominal palpation and expressed as graft beating score (BS) using the Stanford cardiac surgery laboratory graft scoring system (0: no contraction; 1: contraction barely palpable; 2: obvious decrease in contraction strength, but still contracting in a coordinated manner, rhythm disturbance; 3: strong, coordinated beat but noticeable decrease in strength or rate, distention/stiffness; or 4: strong contraction of both ventricles, regular rate, no enlargement or stiffness).Concise Methods Human Tissue Microarray ExperimentsTotal RNA extraction, quality control, complementary (c)-DNA 23727046 amplification and microarray hybridization for human renal allograft biopsies was essentially performed as published [19] and described in SM. In brief, total RNA was extracted from biopsies stored in RNAlater (Ambion, Texas, TX) using TRIzol Reagent (Invitrogen, Carlsbad, CA). After quality control, RNA was amplified to cDNA, biotin labeled and hybridized onto Affymetrix GeneChip Human Genome U133 plus 2.0 Arrays.Human PBMC StimulationHuman PBMC for in-vitro drug efficacy assays were Apocynin isolated from whole blood of 5 healthy individuals (2 female, 3 males, mean age 31+/214 years) using Ficoll gradient centrifugation (FicollPaqueTM PLUS, Amersham Biosciences, Uppsala, Sweden). Isolated PBMCs were pretreated with 100 mMol Fenofibrate (Sigma Aldrich, St. Luis, MO) for 2 hours and then stimulated with anti-human CD3/CD28 antibodies for 65 hours. Thereafter cells were harvested and snap frozen at 280uC until downstream analysis of gene expression. In vitro experiments are described in detail in SM.Mouse TreatmentFenofibrate (F6020; Sigma Aldrich, St. Louis, MO) was dosed at 100 mg/kg body weight/day and administered daily either by i.p. injection in the 7-day inflammatory model, or by oral gavage in the 30-day graft survival study. Treatment started the day prior to transplantation and lasted until the day before sacrifice (SM). Cyclosporine (Cys) was dosed at 20 mg/kg/day and was administered daily by i.p. injection. The dosage of Cyclosporine was based on published literature in experimental cardiac allograft rejection [20], the dosage of Fenofibrate was based on literature of an experimental mouse model of atherosclerosis that showed efficacy but no toxicity during the treatment [21]. A 1:1 translation of either Cyclosporine or of Fenofibrate dosages used to treat humans were not possible, as the ADME Met-Enkephalin site profiles between mice and humans are not comparable.Mice for Experimental Transplant RejectionFor in vivo experimental heart transplantation, recipient C57BL/6J (H2b) and donor FVB (H2q) mice were used (SM and [8]). All animal experiments were approved by Stanford University Institutional Animal Care and performed in accordance with the Guide for the Care and Use of Laboratory Animals (Ref; National Research Council 1996; Guide for the Care and Use of Laboratory Animals, Washington D.C., National Academy Press). All surgery was performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering. In brief, FVB donor hearts were implanted into the abdomen of C57BL/6 WT mice representing a complete MHC-class 1 and -2 mismatch [17,18]. Animals.He rejection group, 4 cases showed abnormalities which were graded as BL rejection according to Banff [15]. Demographics of these patients can be found here [16].immunosuppression, Cyclosporine (Cys). Animal activity, body weight and graft function were assessed daily. The latter was measured by direct abdominal palpation and expressed as graft beating score (BS) using the Stanford cardiac surgery laboratory graft scoring system (0: no contraction; 1: contraction barely palpable; 2: obvious decrease in contraction strength, but still contracting in a coordinated manner, rhythm disturbance; 3: strong, coordinated beat but noticeable decrease in strength or rate, distention/stiffness; or 4: strong contraction of both ventricles, regular rate, no enlargement or stiffness).Concise Methods Human Tissue Microarray ExperimentsTotal RNA extraction, quality control, complementary (c)-DNA 23727046 amplification and microarray hybridization for human renal allograft biopsies was essentially performed as published [19] and described in SM. In brief, total RNA was extracted from biopsies stored in RNAlater (Ambion, Texas, TX) using TRIzol Reagent (Invitrogen, Carlsbad, CA). After quality control, RNA was amplified to cDNA, biotin labeled and hybridized onto Affymetrix GeneChip Human Genome U133 plus 2.0 Arrays.Human PBMC StimulationHuman PBMC for in-vitro drug efficacy assays were isolated from whole blood of 5 healthy individuals (2 female, 3 males, mean age 31+/214 years) using Ficoll gradient centrifugation (FicollPaqueTM PLUS, Amersham Biosciences, Uppsala, Sweden). Isolated PBMCs were pretreated with 100 mMol Fenofibrate (Sigma Aldrich, St. Luis, MO) for 2 hours and then stimulated with anti-human CD3/CD28 antibodies for 65 hours. Thereafter cells were harvested and snap frozen at 280uC until downstream analysis of gene expression. In vitro experiments are described in detail in SM.Mouse TreatmentFenofibrate (F6020; Sigma Aldrich, St. Louis, MO) was dosed at 100 mg/kg body weight/day and administered daily either by i.p. injection in the 7-day inflammatory model, or by oral gavage in the 30-day graft survival study. Treatment started the day prior to transplantation and lasted until the day before sacrifice (SM). Cyclosporine (Cys) was dosed at 20 mg/kg/day and was administered daily by i.p. injection. The dosage of Cyclosporine was based on published literature in experimental cardiac allograft rejection [20], the dosage of Fenofibrate was based on literature of an experimental mouse model of atherosclerosis that showed efficacy but no toxicity during the treatment [21]. A 1:1 translation of either Cyclosporine or of Fenofibrate dosages used to treat humans were not possible, as the ADME profiles between mice and humans are not comparable.Mice for Experimental Transplant RejectionFor in vivo experimental heart transplantation, recipient C57BL/6J (H2b) and donor FVB (H2q) mice were used (SM and [8]). All animal experiments were approved by Stanford University Institutional Animal Care and performed in accordance with the Guide for the Care and Use of Laboratory Animals (Ref; National Research Council 1996; Guide for the Care and Use of Laboratory Animals, Washington D.C., National Academy Press). All surgery was performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering. In brief, FVB donor hearts were implanted into the abdomen of C57BL/6 WT mice representing a complete MHC-class 1 and -2 mismatch [17,18]. Animals.
