Project description:In the present study, we aimed to determine the genes involved in inflammatory process of diabetic nephropathy. ICAM-1+/+ and ICAM-1-/- mice aged 8 weeks were divided into four groups: 1) nondiabetic ICAM-1+/+ mice (ND-WT), 2) nondiabetic ICAM-1-/- mice (ND-KO), 3) streptozotocin (STZ)-induced diabetic ICAM-1+/+ mice (DM-WT), and 4) STZ-induced diabetic ICAM-1-/- mice (DM-KO). Three months after the induction of diabetes, total RNA was extracted from each specimen of renal cortex. We examined gene expression profiles of four groups. We identified 193 genes; the ratio of expression level of DM-WT was >2 or <0.5 of that of DM-KO. Of 193 genes, hierarchical clustering identified 33 genes that were significantly upregulated only in DM-WT but not remarkable in ND-WT and ND-KO. Functional annotation of these 33 genes revealed that the significant functions of them were related to the immune or inflammatory process: immune response, response to stimulus, defense response, immune effector process and antigen processing and presentation. These genes contained several inflammatory related genes, such as chemokine (C-X-C motif) ligand 10, chemokine (c-c motif) ligand 12, and chemokine (c-c motif) ligand 8. In this cluster, we focused on cholecystokinin (CCK) because CCK is one of the most up-regulated genes. Real-time RT-PCR revealed that CCK mRNA expression was significantly up-regulated in DM-WT compared with DM-KO. These results suggest that CCK may play a critical role in the progression of diabetic nephropathy by controlling inflammation in diabetic kidney. Male ICAM-1-/- mice (C57BL/6J background) were purchased from The Jackson Laboratory (Bar Harbor, ME). Male C57BL/6J mice (ICAM-1+/+) mice were used as controls. ICAM-1+/+ and ICAM-1-/- mice aged 8 weeks were divided into four groups (n = 5 each): 1) nondiabetic ICAM-1+/+ mice (ND-WT), 2) nondiabetic ICAM-1-/- mice (ND-KO), 3) streptozotocin (STZ)-induced diabetic ICAM-1+/+ mice (DM-WT), and 4) STZ-induced diabetic ICAM-1-/- mice (DM-KO). Mice in the diabetic groups received an intraperitoneal injection of STZ (Sigma Chemical, St.Louis, MO) at 200 mg/kg in citrate buffer (pH 4.5). Blood glucose levels were determined 7 days after STZ injection and only mice with blood glucose concentrations >16 mmol/L were used in the study. Nondiabetic ICAM-1+/+ and ICAM-1-/- mice received citrate buffer injections only. All mice had free access to standard diet and tap water. All animal procedures were performed according to the Guidelines for Animal Experiments at Okayama University Medical School, Japanese Government Animal Protection and Management Law (no. 105), and Japanese Government Notification on Feeding and Safekeeping of Animals (no. 6). Three months after the induction of diabetes, all mice were killed, and the kidneys were harvested. Total RNA was extracted from each specimen of renal cortex using standard protocol from RNeasy midi kit (Qiagen, Valencia, CA) at 3 months. The concentration and the quality of the total RNA sample were assessed by Agilent Bioanalyzer. Biotin-labeled target cRNA was prepared using First and Second strand cDNA Synthesis Kit (Amersham Biosciences, No. 320000) and In Vitro Transcription Kit (Amersham Biosciences, No.320001). Incubate total RNA (5 Î¼g), diluted bacterial mRNA spike controls and T7 oligo (dT) primer for 10 min at 70 C. Add first-strand reaction components and incubate 1 hour at 42C. Add the first-strand cDNA product to the second-strand reaction components and incubate for 2 hours at 16 C. Double-strand cDNA was purified using QIAquick purification kit (Qiagen), eluted twice, each time with 30Î¼l of nuclease-free water then dried in a SpeedVac concentrator. Prepare IVT mix of biotinylated UTP, ribonucleotides, and the 10x T7 enzyme mix and add to the resuspended cDNA. Incubate at 37C for 14 hours. Purify cRNA using RNeasy Mini Kit (Qiagen) and elute the cRNA twice, each time with 50 Î¼l nuclease-free water. Measure the absorbance at 260 nm and 280 nm to determine the ratio (A260:A280=2). In 25 Î¼l total volume, add 10 Î¼g of cRNA to 5 Î¼l of 5x fragmentation buffer and incubate at 94 C for 20 min. Bring 10 Î¼g of fragmented cRNA, 78 Î¼l of hybridization buffer component A, and 130 Î¼l of hybridization buffer component B to a final volume of 260 Î¼l with water. Incubate at 90C for 5 min and immediately chill on ice for 5 min. Slowly inject 250 Î¼l of hybridization reaction mixture into array input port and seal ports with sealing strips. Set the shaker speed to 300 rpm and incubate slides for 18 hours at 37C in an Innove 4080 shaker. Remove the Flex Chamber using the hybridization removal tool. Then, place the bioarrays into the bioarray rack while it sits inside the medium reagent reservoir containing 0.75xTNT. Transfer the bioarray rack to the large reagent reservoir containing preheated 0.75xTNT, and incubate at 46 C for exactly 1 hour. Fill each slot of the small reagent reservoir with 3.4 ml of Cy5-Streptavidin working solution. Transfer the bioarray rack from the large reagent reservoir in to the small reagent reservoir and incubate bioarrays at RT for 30 min. Wash the bioarrays four times with 1xTNT at RT for 5 min. Rinse the bioarrays in 0.1xSSC/0.05%Tween by moving rack up and down 5 times in 5 seconds. Immediately follow the rinse with centrifugation to dry bioarrays, and store dried bioarrays in the dark. We scanned bioarrays with Axon GenePix 4000B and analyzed with CodeLink Expression Analysis software version 2.3.2. The 10,000 spot intensities on the scanned microarray image were normalized to a median value of 1 and data were exported for analysis with CodeLink Expression Analysis software version 2.3.2

Project description:Pemphigus foliaceus (PF) is a complex autoimmune disease characterized by bullous skin lesions and the presence of antibodies against desmoglein 1. In this study we sought to contribute to a better understanding of the molecular processes in endemic PF, since the identification of factors that participate in the pathogenesis is a prerequisite for understanding its biological basis and may lead to novel therapeutic interventions. CD4+ T lymphocytes are central to the development of the disease. Therefore, we compared genome-wide gene expression profiles of peripheral CD4+ T cells of various PF patient subgroups with each other and with that of healthy individuals. The patients sample was subdivided in three groups: untreated patients with the generalized form of the disease, patients submitted to immunosuppressive treatment, and patients with the localized form of the disease. Comparisons between different subgroups resulted in 135, 54 and 64 genes differentially expressed. These genes are mainly related with lymphocyte adhesion and migration, apoptosis, cellular proliferation, cytotoxicity and antigen presentation. Several of these genes were differentially expressed when comparing lesional and uninvolved skin from the same patient. The chromosomal regions 19q31 and 12p13 concentrate differentially expressed genes and are candidate regions for PF susceptibility genes and disease markers. Our results reveal genes involved in severity, potential therapeutic targets and previously unsuspected processes involved in the pathogenesis. Besides, this study adds original information that will contribute to the understanding of PFM-bM-^@M-^Ys pathogenesis and of the still poorly defined in vivo functions of most of these genes. 24 samples were analyzed, no replicates. Three groups of patients were analyzed: (1) patients with the generalized form of the disease and without treatment; (2) patients with the generalized form undergoing immunosuppressive therapy (prednisone); and (3) patients with the localized form of the disease and without treatment. A fourth group consisted of healthy control subjects.

Project description:BACKGROUND: Patients encountering severe trauma are at risk of developing sepsis syndrome and subsequent multiple organ failure. This is often associated with fatal outcome despite survival of the initial injury. We undertook a prospective cohort study of trauma patients to examine the role of tumor necrosis factor alpha (TNF-alpha) in the pathogenesis of sepsis syndrome and mortality. METHODS: 159 severely traumatized patients from a single centre were included. Serial blood samples were analyzed for serum concentrations of TNF-alpha and lymphotoxin alpha (LT-alpha). We genotyped nine polymorphisms in the TNF gene and tested for an association with sepsis syndrome and outcome. Genetic associations were validated in an external replication sample (n=76). We examined the peripheral blood transcriptome in n=28 patients by whole genome-based profiling and validated the results. RESULTS: Carriage of the TNF rs1800629 A allele was associated with higher TNF-alpha serum concentrations on the first day after trauma and during follow up (two-sided p=5.0x10-5), with development of sepsis syndrome (OR 7.14, two-sided p=1.2x10-6; external validation sample (n=76): OR 3.3, one-sided p=0.03), and with fatal outcome (OR 7.65, two-sided p=1.9x10-6). Carriage of the TNF rs1800629 A allele was associated with differential expression of genes representing stronger pro-inflammatory and apoptotic responses as compared to carriage of the wild type allele. CONCLUSIONS: Common TNF gene variants are associated with sepsis syndrome and death after severe injury. These findings are strongly supported by functional data and may be important for developing preemptive anti-inflammatory interventions in carriers of the risk-associated allele. Keywords: Disease state analysis 159 consecutive traumatized patients were included in the study upon admission to the intensive care unit (ICU), fulfilling all of the following inclusion criteria: severe injuries of at least two body regions or three major fractures, between 18 and 65 years of age, an estimated Injury Severity Score (ISS)21 of ≥12 points after thorough assessment of injuries, <12 hours between the occurrence of the accident and time of admission to the ICU, and at least more than 3 days of survival. None of the patients underwent neuro- or cardiac surgery. We excluded patients with severe intracranial head injuries, coagulation abnormalities known at the day of admission to the ICU, acute renal failure, liver failure, malignant disease, or hemofiltration in the patient`s history. All patients were genotyped for nine polymorphisms in the TNF gene and tested for an association with sepsis syndrome and outcome. The genotyping results revealed significant association for the carriage of a TNF rs1800629 A allele with higher TNF-alpha serum concentrations on the first day after trauma and during follow up, with development of sepsis syndrome (OR 7.14), and with fatal outcome (OR 7.65). To obtain more insight into the biological mechanisms underlying the findings in the genotyping study, we searched for differences in the peripheral blood transcriptome from patients with and without the TNF rs1800629 A variant. From 28 patients, we examined the transcpriptome from peripheral blood using the CodeLink UniSet Human 10 K Bioarrays (GE Healthcare, Freiburg, Germany). Only whole blood samples drawn upon admission to the ICU were used for this investigation. Microarray results were validated by quantification of mRNA by TaqMan® technology. The patients were dichotomized into two groups: group A) 16 patients without the TNF rs1800629 A variant (WT1-16) and group B) 12 patients carrying the TNF rs1800629 A variant (MUT1-12). Each patient sample was hybridized in duplicate on microarrays (label-extract technical replicate). A total of 75 arrays (42 group A, 33 group B) were subjected to microarray quality analysis. Of these, 17 arrays (12 group A, 5 group B) were excluded from the data set due to quality reasons and a final set of 58 arrays (30 group A and 28 group B) was subjected to microarray analysis. The arrays were designated WTx.y or MUTx.y with WT for patients with and MUT for patients without the TNF rs1800629 A variant, with x for the patient-ID (1,2,3,4..) and y for the technical replicate number (1,2 or 3).

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