Hepatocyte growth factor, hepatocyte growth factor activator and arginine in a rat fulminant colitis model

INTRODUCTION: Dextran sodium sulfate (DSS) is commonly used to induce a murine fulminant colitis model. Hepatocyte growth factor (HGF) has been shown to decrease the symptoms of inflammatory bowel disease (IBD) but the effect of its activator, HGFA, is not well characterized. Arginine reduces effects of oxidative stress but its effect on IBD is not well known. The primary aim is to determine whether HGF and HGFA, or arginine will decrease IBD symptoms such as pain and diarrhea in a DSS-induced fulminant colitis murine model. METHODS: A severe colitis was induced in young, male Fischer 344 rats with 4% (w/v) DSS oral solution for seven days; rats were sacrificed on day 10. Rats were divided into five groups of 8 animals: control, HGF (700 mcg/kg/dose), HGF and HGFA (10 mcg/dose), HGF and arginine, and high dose HGF (2800 mcg/kg/dose). Main clinical outcomes were pain, diarrhea and weight loss. Blinded pathologists scored the terminal ileum and distal colon. RESULTS: DSS reliably induced severe active colitis in 90% of animals (n = 36/40). There were no differences in injury scores between control and treatment animals. HGF led to 1.38 fewer days in pain (p = 0.036), while arginine led to 1.88 fewer days of diarrhea (P = 0.017) compared to controls. 88% of HGFA-treated rats started regaining weight (P \u3c 0.001). DISCUSSION/CONCLUSION: Although treatment was unable to reverse fulminant disease, HGF and arginine were associated with decreased days of pain and diarrhea. These clinical interventions may reduce associated symptoms for severe IBD patients, even when urgent surgical intervention remains the only viable option

Mixed Chimerism Achieved by a Nonlethal Conditioning Regimen Induces Donor-Specific Tolerance to Lung Allografts

Graft rejection and toxicity associated with chronic immunosuppressive therapy remain a major problem in lung transplantation (Tx). Mixed hematopoietic chimerism has been shown to produce long-lasting donor-specific transplant tolerance without immunosuppressive drugs in animal models; however, most conditioning regimens required to achieve mixed chimerism are too toxic for clinical use. The aim of this study was to develop a nonlethal conditioning regimen to induce tolerance to lung allografts. Four to 6-wk old ACI (RT1.A a) and Wistar Furth (RT1.A u) rats were used as organ donors and recipients, respectively. The recipient conditioning regimen included: 10 mg/animal antilymphocyte globulin (on day-5), 1 mg/kg/d tacrolimus (days 1 to 10), total body irradiation (500 cGy; day 0), and donor bone marrow (DBM) Tx (100 × 10 6 T-cell depleted cells on day 0 following irradiation). Six weeks after DBM Tx, chimeric animals received orthotopic left lung Tx. Graft survival was monitored by chest X-ray and histology. Long-term DBM engraftment was observed: hematopoietic chimerism in the peripheral blood was 12.4 ± 3.4%, 36.7 ± 14.1%, and 31.9 ± 14.1% at 30 d, 6 mo, and 16 mo following DBM Tx, respectively. There was no graft versus host disease. Chimeric recipients (RT1.A u) permanently accepted (>400 d) donor-specific lungs (RT1.A a; n = 8), yet rapidly rejected (<8 d) third party hearts (RT1.A l; n = 5). Graft (lung) tolerant (>150 d) chimeric recipients accepted secondary donor-specific heart grafts (>150 d; n = 4) but rejected third party heart grafts (<7 d; n = 3). Graft tolerant recipients demonstrated reduced ( P < 0.05) in vitro donor-specific lymphoproliferative response and cytotoxicity, and no evidence of acute or chronic graft rejection. Mixed chimerism achieved by a nonlethal conditioning regimen induced long-term donor-specific tolerance to lung allografts

CTLA4-Ig-Based Conditioning Regimen to Induce Tolerance to Cardiac Allografts

Transplant rejection and toxicity associated with chronic immunosuppressive therapy remain a major problem. Mixed hematopoietic chimerism has been shown to produce tolerance to solid organ transplants. However, currently available protocols to induce mixed hematopoietic chimerism invariably require toxic pre-conditioning. In this study, we investigated a non-toxic CTLA4-Ig-based protocol to induce donor-specific tolerance to cardiac allografts in rats. Fully mismatched, 4 to 6 week old ACI (RT1.A a) and Wistar Furth (RT1.A u) rats were used as cell/organ donors and recipients, respectively. Recipients were treated with CTLA4-Ig 2 mg/kg/day (on days 0, 2, 4, 6, 8), tacrolimus 1 mg/kg/day (daily, from days 0 to 9), and a single dose of anti-lymphocyte serum (10 mg) on day 10, soon after total body irradiation (300 cGy) and donor bone marrow (100 × 10 6 T-cell depleted cells) transplantation (BMT). Six weeks after BMT, chimeric animals received heterotopic heart transplants. Hematopoietic chimerism was 18.8 ± 10.6% at day 30, and was stable (24 ± 10%) at 1 year post-BMT; there was no graft versus host disease. Chimeric recipients (RT1.A u) permanently accepted (>360 days) donor-specific (RT1.A a; n = 6) hearts, yet rapidly rejected (<9 days) third-party hearts (RT1.A l; n = 5). Graft (heart) tolerant (>100 days) recipients accepted donor-specific secondary skin grafts (>200 days) while rejected the third-party skin grafts (<9 days). Lymphocytes of graft tolerant animals demonstrated hyporesponsiveness in mixed lymphocyte cultures in a donor-specific manner. Tolerant graft histology showed no obliterative arteriopathy or chronic rejection. The CTLA4-Ig based conditioning regimen with donor BMT produced mixed chimerism and induced donor- specific tolerance to cardiac allografts

Effects of Valproic Acid and Dexamethasone Administration on Early Bio-Markers and Gene Expression Profile in Acute Kidney Ischemia-Reperfusion Injury in the Rat

<div><p>Renal ischemia-reperfusion (IR) causes acute kidney injury (AKI) with high mortality and morbidity. The objective of this investigation was to ameliorate kidney IR injury and identify novel biomarkers for kidney injury and repair. Under general anesthesia, left renal ischemia was induced in Wister rats by occluding renal artery for 45 minutes, followed by reperfusion and right nephrectomy. Thirty minutes prior to ischemia, rats (n = 8/group) received Valproic Acid (150 mg/kg; VPA), Dexamethasone (3 mg/kg; Dex) or Vehicle (saline) intraperitoneally. Animals were sacrificed at 3, 24 or 120 h post-IR. Plasma creatinine (mg/dL) at 24 h was reduced (P<0.05) in VPA (2.7±1.8) and Dex (2.3±1.2) compared to Vehicle (3.8±0.5) group. At 3 h, urine albumin (mg/mL) was higher in Vehicle (1.47±0.10), VPA (0.84±0.62) and Dex (1.04±0.73) compared to naïve (uninjured/untreated control) (0.14±0.26) group. At 24 h post-IR urine lipocalin-2 (μg/mL) was higher (P<0.05) in VPA, Dex and Vehicle groups (9.61–11.36) compared to naïve group (0.67±0.29); also, kidney injury molecule-1 (KIM-1; ng/mL) was higher (P<0.05) in VPA, Dex and Vehicle groups (13.7–18.7) compared to naïve group (1.7±1.9). Histopathology demonstrated reduced (P<0.05) ischemic injury in the renal cortex in VPA (Grade 1.6±1.5) compared to Vehicle (Grade 2.9±1.1). Inflammatory cytokines IL1β and IL6 were downregulated and anti-apoptotic molecule BCL2 was upregulated in VPA group. Furthermore, kidney DNA microarray demonstrated reduced injury, stress, and apoptosis related gene expression in the VPA administered rats. VPA appears to ameliorate kidney IR injury via reduced inflammatory cytokine, apoptosis/stress related gene expression, and improved regeneration. KIM-1, lipocalin-2 and albumin appear to be promising early urine biomarkers for the diagnosis of AKI.</p></div

Histopathology of hematoxylin and eosin stained kidney sections.

<p>A, B, C = Renal cortex at 3 hours (h) post ischemia-reperfusion (IR); D, E, F = Renal outer medulla at 24 h post-IR; A, D = Vehicle (saline control); B, E = Valproic acid (VPA); C, F = Dexamethasone (Dex) treated animals. Three high power fields (400x) representing approximately 50 tubules from cortex and outer medulla of each kidney were evaluated for ischemic changes (injury), tubular necrosis and regenerative changes. Collectively kidney injury and regeneration were graded (0–4) based on the mean percentage of tubules affected: 0, None; 1, <25%; 2, ≥25 but <50%; 3, ≥50 but <75%; 4, >75–100%. Ischemic changes included nuclear condensation <b>(nc)</b>, cytoplasmic eosinophilia, individual cell necrosis and tubular dilation <b>(td)</b>; tubular necrosis <b>(tn)</b> included confluent cell necrosis or sloughing of the tubular epithelium; and regenerative changes included tubular dilation, cytoplasmic basophilia and contraction of the cytoplasm, as well as vesicular chromatin with nucleoli. Hemorrhage <b>(hg)</b> was predominant in the vehicle control group. G, H, I = represent Histopathology quantification: renal cortex (black bars ■) and renal outer medulla (white bars □). The histologic injury score was significantly (P<0.05) lower in the VPA treated group compared to the Vehicle control at 3 h post-IR (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126622#pone.0126622.t003" target="_blank">Table 3</a>).</p

Gene transcription analysis as determined by Affymetrix Gene Array Technology.

<p>The Venn diagrams presented show the number of transcriptomes expressed and overlaps in the kidneys of animals treated with valproic acid (VPA), dexamethasone (Dex) or untreated (None; Vehicle control) at 3, 24 and 120 hours (h) post ischemia-reperfusion (IR). All relative changes in gene expression are derived from comparison with naïve rats. Fewer genes were up-regulated in the VPA group compared to untreated Vehicle control group at 3 h post-IR. At 120 h post-IR only two genes were upregulated and none was downregulated in VPA treated animals. Specific gene expression information is presented in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126622#pone.0126622.s001" target="_blank">S1</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126622#pone.0126622.s006" target="_blank">S6</a> Tables</b>. The DNA microarray data have been deposited in NCBI’s Gene Expression Omnibus (GEO) repository and are accessible through Accession No. GSE58438 (<a href="http://www.ncbi.nlm.nih.gov/geo/" target="_blank">http://www.ncbi.nlm.nih.gov/geo/</a>).</p

Experimental design.

<p>Lewis rats were pre-medicated with Valproic acid (VPA; 150 mg/kg body weight) or Dexamethasone (Dex; 3 mg/kg/body weight) or saline (Vehicle control) intraperitoneally 30 minutes (min) prior to cross clamping left renal artery and inducing left renal ischemia. The cross clamp was removed after 45 min allowing kidney reperfusion, and at the same time a right nephrectomy was performed. Animals were sacrificed at 3, 24 and 120 hours post ischemia-reperfusion. Left kidney, blood and urine were collected for cellular and molecular analyses.</p