Stem cell, Granulocyte-Colony Stimulating Factor and/or Dihexa to promote limb function recovery in a rat sciatic nerve damage-repair model: Experimental animal studies.

BACKGROUND: Optimizing nerve regeneration and re-innervation of target muscle/s is the key for improved functional recovery following peripheral nerve damage. We investigated whether administration of mesenchymal stem cell (MSC), Granulocyte-Colony Stimulating Factor (G-CSF) and/or Dihexa can improve recovery of limb function following peripheral nerve damage in rat sciatic nerve transection-repair model. MATERIALS AND METHODS: There were 10 experimental groups (n = 6-8 rats/group). Bone marrow derived syngeneic MSCs (2 × 106; passage≤6), G-CSF (200-400 µg/kg b.wt.), Dihexa (2-4 mg/kg b.wt.) and/or Vehicle were administered to male Lewis rats locally via hydrogel at the site of nerve repair, systemically (i.v./i.p), and/or to gastrocnemius muscle. The limb sensory and motor functions were assessed at 1-2 week intervals post nerve repair until the study endpoint (16 weeks). RESULTS: The sensory function in all nerve boundaries (peroneal, tibial, sural) returned to nearly normal by 8 weeks (Grade 2.7 on a scale of Grade 0-3 [0 = No function; 3 = Normal function]) in all groups combined. The peroneal nerve function recovered quickly with return of function at one week (∼2.0) while sural nerve function recovered rather slowly at four weeks (∼1.0). Motor function at 8-16 weeks post-nerve repair as determined by walking foot print grades significantly (P < 0.05) improved with MSC + G-CSF or MSC + Dihexa administrations into gastrocnemius muscle and mitigated foot flexion contractures. CONCLUSIONS: These findings demonstrate MSC, G-CSF and Dihexa are promising candidates for adjunct therapies to promote limb functional recovery after surgical nerve repair, and have implications in peripheral nerve injury and limb transplantation. IACUC No.215064.

"Stem cell therapy to promote limb function recovery in peripheral nerve damage in a rat model" - Experimental research.

BACKGROUND: Optimizing nerve regeneration and mitigating muscle atrophy are the keys to successful outcomes in peripheral nerve damage. We investigated whether mesenchymal stem cell (MSC) therapy can improve limb function recovery in peripheral nerve damage. MATERIALS AND METHODS: We used sciatic nerve transection/repair (SNR) and individual nerve transection/repair (INR; branches of sciatic nerve - tibial, peroneal, sural) models to study the effect of MSCs on proximal and distal peripheral nerve damages, respectively, in male Lewis rats. Syngeneic MSCs (5 × 106; passage≤6) or saline were administered locally and intravenously. Sensory/motor functions (SF/MF) of the limb were assessed. RESULTS: Rat MSCs (>90%) were CD29+, CD90+, CD34-, CD31- and multipotent. Total SF at two weeks post-SNR & INR with or without MSC therapy was ∼1.2 on a 0-3 grading scale (0 = No function; 3 = Normal); by 12 weeks it was 2.6-2.8 in all groups (n ≥ 9/group). MSCs accelerated SF onset. At eight weeks post-INR, sciatic function index (SFI), a measure of MF (0 = Normal; -100 = Nonfunctional) was -34 and -77 in MSC and vehicle groups, respectively (n ≥ 9); post-SNR it was -72 and -92 in MSC and vehicle groups, respectively. Long-term MF (24 weeks) was apparent in MSC treated INR (SFI -63) but not in SNR (SFI -100). Gastrocnemius muscle atrophy was significantly reduced (P < 0.05) in INR. Nerve histomorphometry revealed reduced axonal area (P < 0.01) but no difference in myelination (P > 0.05) in MSC treated INR compared to the naive contralateral nerve. CONCLUSION: MSC therapy in peripheral nerve damage appears to improve nerve regeneration, mitigate flexion-contractures, and promote limb functional recovery.

Mesenchymal stem cell therapy to promote limb transplant functional recovery.

BACKGROUND: Limb transplantation is a viable option for reconstruction after traumatic limb loss; however, functional recovery can be suboptimal. The aim of this study was to determine whether mesenchymal stem cell (MSC) administration can improve limb transplant functional recovery. METHODS: Orthotopic syngeneic hindlimb transplants were performed in Lewis rats, followed by topical and intravenous injections of syngeneic MSCs (5 × 106 ) or vehicle. Transplanted limb sensory and motor functions were tested by cutaneous pain reaction and walking track analysis, respectively. RESULTS: MSCs expanded ex vivo were CD29+ , CD31- , CD34- , CD44+ , CD45low , CD90+ , MHC Class-I+ , Class-II- , and pluripotent. Greater than 90% of limb transplants survived. At 4 weeks post-transplantation, the mean sensory nerve (tibial, peroneal, or sural) function in MSC (n = 9) and vehicle (n = 9) groups was <0.3 on a scale of Grades 0-3 (0 = No function; 3 = Normal). By 8 weeks, the sensory scores for tibial, peroneal, and sural nerves were 2.2 ± 0.7, 1.2 ± 0.5, and 1.7 ± 0.9 in the vehicle, and 2.6 ± 0.4, 1.0 ± 0.9, and 1.7 ± 0.9 in the MSC group, respectively (n = 9/group). At 4, 8, 16, and 24 weeks, the overall sensory function was higher in MSC group (≥7/group). Sciatic Function Index (SFI), a measure of motor function, could not be calculated because of poor foot prints; therefore, a novel grading system was developed. Bone fusion/vascularization as determined by X-ray films/laser Doppler (≥2 week post-transplantation) were normal (n = 3/group). Gastrocnemius muscle was atrophied (P < 0.05), and flexion contractures were evident by 24 weeks. CONCLUSIONS: Bone marrow-derived MSC therapy appears to improve sensory function recovery in a rat limb transplant model. Published 2016. This article is a U.S. Government work and is in the public domain in the USA Microsurgery 37:222-234, 2017.

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 < 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.

Effects of valproic acid and dexamethasone administration on early bio-markers and gene expression profile in acute kidney ischemia-reperfusion injury in the rat.

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 IL1ß 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.

Effects of histone deacetylase inhibition on 24-hour survival and end-organ injury in a porcine trauma model: a prospective, randomized trial.

BACKGROUND: Valproic acid (VPA) is a histone deacetylase inhibitor that has been shown to improve early resuscitation from hemorrhagic shock. We sought to examine whether there is a sustained benefit of VPA in a survival model of severe injury. METHODS: Yorkshire swine (n = 36) were randomized to three groups as follows: (a) control, (b) VPA (single dose), and (c) VPA (two doses at 12 hours apart). Animals underwent a 35% volume-controlled hemorrhage, followed by aortic cross-clamping for 50-minute duration, at which time VPA (400 mg/kg) was administered intravenously. Animals then underwent protocol guided resuscitation with crystalloid and vasopressor infusions for up to 24 hours. The primary end point was animal survival; secondary end points included hemodynamics, physiology, and histologic evidence of end-organ injury. RESULTS: Mean duration of survival was significantly longer in the control group (15.8 hours, n = 11) compared with single-dose VPA (12.6 hours, n = 9, p < 0.02). Redosing VPA at 12 hours provided no survival benefit. During cross-clamp, animals that received VPA required significantly less lidocaine compared with the control animals (32.8 mg vs. 159.4 mg, p = 0.03). Animals that received VPA also required significantly greater quantities of intravenous fluids per hour (p < 0.01) and higher epinephrine doses (p = 0.01). VPA administration was associated with earlier evidence of cardiac suppression (decreased cardiac output, increased pulmonary wedge pressures, and systemic vascular resistance; p < 0.05). VPA was associated with renal end-organ histologic protection and improved levels of blood urea nitrogen and creatinine at all time points (p < 0.05). CONCLUSION: Despite previous reports citing improved early outcomes with VPA administration, VPA did not improve resuscitation or mortality in a survival model with severe injury. VPA did show some evidence of prolonged renal protection. No benefit of redosing VPA was identified. VPA had a cardiac depressant effect that may be dose dependent and should be studied further.

Interleukin-10 delivery via mesenchymal stem cells: a novel gene therapy approach to prevent lung ischemia-reperfusion injury.

Ischemia-reperfusion (IR) injury is an important cause of primary graft failure in lung transplantation. In this study, viral interleukin-10 (vIL-10)-engineered mesenchymal stem cells (MSCs) were tested for their ability to prevent lung IR injury. Bone marrow-derived MSCs were transduced with rvIL-10-retrovirus. After 120 min of warm left lung ischemia, rats received approximately 15 x 10(6) vIL-10-engineered MSCs (MSC-vIL-10), empty vector-engineered MSCs (MSC-vec), or saline intravenously. Mean blood oxygenation (PaO(2)/FiO(2) ratio, mmHg) was measured at 4 hr, 24 hr, 72 hr, and 7 days. As early as 4 hr post-IR injury with MSC-vIL-10 treatment, blood oxygenation was significantly (p < 0.05) improved (319 +/- 94; n = 7) compared with untreated (saline) controls (63 +/- 19; n = 6). At 24 hr post-IR injury, in the MSC-vIL-10-treated group there was a further increase in blood oxygenation (353 +/- 105; n = 10) compared with the MSC-vec group (138 +/- 86; n = 9) and saline group (87 +/- 39; n = 10). By 72 hr, oxygenation reached normal (475 +/- 55; n = 9) in the MSC-vIL-10-treated group but not in the saline-treated and MSC-vec-treated groups. At 4 hr after IR injury, lungs with MSC-vIL10 treatment had a lower (p < 0.05) injury score (0.9 +/- 0.4) compared with lungs of the untreated (saline) group (2.5 +/- 1.4) or MSC-vec-treated group (2 +/- 0.4). Lung microvascular permeability and wet-to-dry weight ratios were markedly lower in the MSC-vIL10 group compared with untreated (saline) controls. ISOL (in situ oligonucleotide ligation for DNA fragmentation detection) and caspase-3 staining demonstrated significantly (p < 0.05) fewer apoptotic cells in MSC-vIL10-treated lungs. Animals that received MSC-vIL10 therapy had fewer (p < 0.05) CD4(+) and CD8(+) T cells in bronchoalveolar lavage fluid compared with untreated control animals. A therapeutic strategy using vIL-10-engineered MSCs to prevent IR injury in lung transplantation seems promising.

Mixed chimerism achieved by a nonlethal conditioning regimen induces donor-specific tolerance to lung allografts.

BACKGROUND: 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. METHODS: 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 x 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. RESULTS: 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. CONCLUSION: Mixed chimerism achieved by a nonlethal conditioning regimen induced long-term donor-specific tolerance to lung allografts.

Heme oxygenase-1 upregulation protects against intestinal ischemia/reperfusion injury: a laboratory based study.

OBJECTIVES: Tissue damage caused by ischemia/reperfusion injury (IRI) of the intestine may lead to organ dysfunction in several clinical conditions, and is associated with increased incidence of chronic rejection after transplantation. Heme oxygenase-1 (HO-1) is a stress-inducible protein capable of modulating inflammation, oxidative stress, and cell death. The aim of the present study was to assess the effects of HO-1 upregulation on intestinal IRI. METHODS: Lewis rats (seven groups, n=6 each) underwent intestinal warm ischemia induced by clamping the superior mesenteric artery and by ligating the inferior mesenteric artery for 60 min. After 120 or 240 min of reperfusion, tissue samples were collected for analysis. Cobalt protoporphyrin (CoPP) was administered IP at 10 or 20mg/kg 24h before IRI, to induce HO-1 upregulation. Control animals received vehicle alone. Tissue injury measurements included the following: histological changes, tissue myeloperoxidase (MPO) activity, nitrate/nitrite levels, and IL-6 levels. RESULTS: A significant HO-1 upregulation was demonstrated in pre-treated animals (p<0.05, 95% CI: -0.84 to -0.05). Intestinal IL-6 mRNA expression levels were significantly reduced in animals treated with CoPP 20mg/kg after 240 min of IRI (p<0.05, 95% CI: 0.09-2.25). Significant reduction in MPO activity and NO products was observed in treated animals when compared to controls (p<0.01, 95% CI: 0.07-0.24 and p<0.01, 95% CI: 5.58-12.75, respectively). CONCLUSIONS: Induction of HO-1 by CoPP administration before IRI was resulted in a significant reduction of intestinal tissue injury. Developing strategies to induce HO-1 upregulation before surgery will be important to reduce IRI in the clinical setting.

CTLA4-Ig-based conditioning regimen to induce tolerance to cardiac allografts.

BACKGROUND: 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. METHODS: 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 x 10(6) T-cell depleted cells) transplantation (BMT). Six weeks after BMT, chimeric animals received heterotopic heart transplants. RESULTS: 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. CONCLUSIONS: The CTLA4-Ig based conditioning regimen with donor BMT produced mixed chimerism and induced donor- specific tolerance to cardiac allografts.

Viral interleukin-10-engineered autologous hematopoietic stem cell therapy: a novel gene therapy approach to prevent graft rejection.

The Epstein-Barr virus-encoded protein BCRF1 (viral interleukin [vIL]-10) is a biologically active homologue of cellular interleukin (IL)-10. In this study, a novel gene therapy approach to prolong allograft survival was designed. Autologous (syngeneic) hematopoietic progenitor/stem cell-enriched (HSC; lineage(-ve)) population derived from CBA/J mouse bone marrow were transduced with retrovirus encoding vIL-10 gene (vIL-10-HSC), ex vivo; vIL-10-HSC were injected (4-6 x 10(6) cells intravenously) into lethally (9.5 Gy) or sublethally (4 Gy) irradiated CBA/J mice. Six weeks after vIL-10-HSC administration, vascular heterotopic heart (C57BL/6) transplantation was performed. Ex vivo, the vIL-10-HSC produced 5.4 +/- 0.5 ng of vIL-10 protein/2 x 10(5) cells per 24 hr. In vivo, serum vIL-10 production was 187 +/- 205 pg/ml during 3-10 weeks after vIL-10-HSC administration. Cardiac allograft survival was prolonged (p < 0.004) in lethally (71 +/- 40 days) and sublethally (114 +/- 15 days) irradiated mice that received vIL-10-HSC compared to controls that received unengineered (UE) HSC or vector DNA-engineered HSC (12-16 days). However, secondary skin graft (C57BL/6) survival was not prolonged in cardiac allograft-tolerant animals. In the vIL-10-HSC-administered group, graft histopathology demonstrated mild arteritis/venulitis (grade 0.7) and rejection (grade 1.0). Intragraft expression of costimulatory molecules (B7.1, B7.2), cytokines (IL-2, IL-4, mIL-10, interferon [IFN]-gamma), and inducible nitric oxide synthase (iNOS) molecules was markedly lower in vIL-10-HSC-treated tolerant grafts that survived more than 100 days compared to vector DNA-HSC- or UE-HSC-treated controls. Furthermore, T lymphocytes derived from vIL-10-HSC-treated tolerant recipients demonstrated hyporeactivity to donor antigens in mixed lymphocyte cultures. Administration of autologous vIL-10-engineered HSC prior to organ transplantation prolonged cardiac allograft survival significantly.

Anastomotic healing in a small bowel transplantation model in the rat.

Anastomotic healing is impaired after intestinal surgery because of ischemia and reperfusion injury (IRI), which can result in intestinal leaks leading to increased mortality. The objective of this study was to determine the effects of transplant IRI and immune mechanisms on intestinal graft anastomotic healing. Orthotopic intestinal transplantations (OIT) were performed in rats. The experimental design consisted of six groups A-F (n=5/group): A, allogeneic OIT treated with tacrolimus (1 mg/kg/day); B, syngeneic OIT treated with tacrolimus; C, syngeneic OIT; D, allogeneic OIT; E, proximal and distal anastomoses performed in nontransplanted animals; F, same as in group E but treated with tacrolimus. Anastomotic bursting pressure (ABP), hydroxyproline content (HPC), and mucosal inflammatory infiltrate (MII) were determined at the anastomotic sites (proximal and distal) and compared between groups. ABP was significantly (p<0.001) reduced in OIT groups A, B, C, and D compared to control groups E and F at both the proximal and distal anastomotic sites. HPC was approximately 1 microg/mg of tissue in groups A, B, C, and D, and approximately 5 microg/mg of tissue in groups E and F. This demonstrates a significant (p<0.001) reduction in HPC after OIT. MII was significantly (p<0.001) increased in OIT groups when compared to nontransplanted control groups. MII was also significantly (p<0.05) increased in allogeneic OIT groups A and D compared to syngeneic OIT groups B and C. Generally, ABP and HPC were inversely proportional to MII in both nontransplanted control and OIT groups. Reduced anastomotic strength was demonstrated in both syngeneic and allogeneic OIT anastomotic sites irrespective of immunosuppressive therapy, and is probably related to IRI.

Expression of MUC2 and MUC4 proteins and cytokines: early markers of intestinal graft rejection.

BACKGROUND: Histopathologic examination (HP) is the primary method of monitoring intestinal graft rejection. Alterations in mucin levels have been demonstrated in bowel diseases. The aim of this study was to detect early markers of intestinal graft rejection based on mucin and cytokine levels. METHODS: Allogeneic and syngeneic orthotopic intestinal transplantations were performed in untreated Lewis strain recipient rats from Dark Agouti and Lewis strain donors, respectively (unmodified rejection and nonrejection groups). Similarly, allogeneic and syngeneic orthotopic intestinal transplantations were performed in tacrolimus (immunosuppression)-treated groups. HP was performed on hematoxylin-eosin and periodic acid Schiff-stained sections. Expression of MUC2 and MUC4 proteins and of mRNA was detected by immunohistochemistry and Northern analysis, respectively. Interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, and transforming growth factor-beta(1) were measured by reverse transcription-polymerase chain reaction. RESULTS: HP revealed early or mild rejection on day 3, moderate rejection on day 5, and severe rejection on day 7 posttransplantation (posttx) in the unmodified rejection group. A significant (P<0.01) increase in MUC2 and MUC4 expression was observed on day 3 posttx in the allogeneic rejection group compared with syngeneic controls; the levels decreased by day 7. Goblet cells were significantly more frequent on day 3 compared with days 5 and 7 posttx (P<0.01). IFN-gamma and TNF-alpha expression were also higher in the rejection group. CONCLUSIONS: Early transplant rejection is associated with increased MUC2, MUC4, IFN-gamma, and TNF-alpha expression. These markers combined with HP may assist in the diagnosis of early intestinal graft rejection.

Gene delivery in renal tubular epithelial cells using recombinant adeno-associated viral vectors.

Gene therapy has the potential to provide a therapeutic strategy for numerous renal diseases such as diabetic nephropathy, chronic rejection, Alport syndrome, polycystic kidney disease, and inherited tubular disorders. In previous studies using cationic liposomes or adenoviral or retroviral vectors to deliver genes into the kidney, transgene expression has been transient and often associated with adverse host immune responses, particularly with the use of adenoviral vectors. The unique properties of recombinant adeno-associated viral (rAAV) vectors permit long-term stable transgene expression with a relatively low host immune response. The purpose of the present study was to evaluate gene expression in the rat kidney after intrarenal arterial infusion of a rAAV (serotype 2) vector encoding green fluorescence protein (GFP) induced by a cytomegalovirus-chicken beta-actin hybrid promoter. The left kidney of experimental animals was treated with either saline or transduced with rAAV2-GFP (0.125 ml/100 g body wt, 1 x 10(10)/ml infectious units) through the renal artery. A time-dependent expression of GFP was observed in all kidneys injected with rAAV2-GFP, with maximal expression observed at 6 wk posttransduction. The expression of GFP was restricted to cells in the S(3) segment of the proximal tubule and intercalated cells in the collecting duct, the latter identified by co-localization with H(+)-ATPase. No transduction was observed in the glomeruli or the intrarenal vasculature. These studies demonstrate successful transgene expression in tubular epithelial cells, specifically in the S(3) segment of the proximal tubule and intercalated cells, after intrarenal administration of a rAAV vector and provide the impetus for further studies to exploit its use as a tool for gene therapy in the kidney.