Immunotherapy for Infarcts: In Vivo Postinfarction Macrophage Modulation Using Intramyocardial Microparticle Delivery of Map4k4 Small Interfering RNA.

The myeloid cells infiltrating the heart early after acute myocardial infarction elaborate a secretome that largely orchestrates subsequent ventricular wall repair. Regulating this innate immune response could be a means to improve infarct healing. To pilot this concept, we utilized (ß1,3-d-) glucan-encapsulated small interfering RNA (siRNA)-containing particles (GeRPs), targeting mononuclear phagocytes, delivered to mice as a one-time intramyocardial injection immediately after acute infarction. Findings demonstrated that cardiac macrophages phagocytosed GeRPs in vivo and had little systemic dissemination, thus providing a means to deliver local therapeutics. Acute infarcts were then injected in vivo with phosphate-buffered saline (PBS; vehicle) or GeRPs loaded with siRNA to Map4k4, and excised hearts were examined at 3 and 7 days by quantitative polymerase chain reaction, flow cytometry, and histology. Compared with infarcted PBS-treated hearts, hearts with intrainfarct injections of siRNA-loaded GeRPs exhibited 69-89% reductions in transcripts for Map4k4 (mitogen-activated protein kinase kinase kinase kinase 4), interleukin (IL)-1ß, and tumor necrosis factor α at 3 days. Expression of other factors relevant to matrix remodeling-monocyte chemoattractant protein-1 (MCP-1), matrix metalloproteinases, hyaluronan synthases, matricellular proteins, and profibrotic factors transforming growth factor beta (TGF-ß), and connective tissue growth factor (CTGF)-were also decreased. Most effects peaked at 3 days, but, in some instances (Map4k4, IL-1ß, TGF-ß, CTGF, versican, and periostin), suppression persisted to 7 days. Thus, direct intramyocardial GeRP injection could serve as a novel and clinically translatable platform for in vivo RNA delivery to intracardiac macrophages for local and selective immunomodulation of the infarct microenvironment.

Comparing web-based video interventions to enhance university student willingness to donate organs: A randomized controlled trial.

BACKGROUND: The efficacy of video interventions to increase organ donation willingness remains unclear. METHODS: Three-arm web-based randomized controlled trial involving 2261 students at 3 northeastern Ohio universities. Intervention students watched a live-action (n = 755) or animated (n = 753) donation video. Control students (n = 753) viewed wellness information from the Centers for Disease Control and Prevention (CDC). The primary outcome was proportion of students who visited their state electronic donor registry to consent. The secondary outcome was intervention quality. Logistic regression assessed the effects of interventions on visiting the state registry to provide donation consent while controlling for baseline variables. RESULTS: Students in the live-action video arm visited their state registry more frequently than students in the CDC arm (OR = 1.86, 95% CI = 1.20-2.88). There was no difference between students in the animated video and CDC arms (OR = 1.10, 95% CI = 0.69-1.76). The quality of the live-action video was rated lower than the animated video and the CDC text (75% ± 18, 84% ± 16, 80% ± 16, respectively; P < 0.001). CONCLUSION: Students who watched the live-action video were more willing to visit their electronic donor registry to register as organ donors, but rated it lower in satisfaction. Future work should identify the most potent components of organ donation interventions.

Effects of a Video on Organ Donation Consent Among Primary Care Patients: A Randomized Controlled Trial.

BACKGROUND: Low organ donation rates remain a major barrier to organ transplantation. OBJECTIVE: We aimed to determine the effect of a video and patient cueing on organ donation consent among patients meeting with their primary care provider. DESIGN: This was a randomized controlled trial between February 2013 and May 2014. SETTING: The waiting rooms of 18 primary care clinics of a medical system in Cuyahoga County, Ohio. PATIENTS: The study included 915 patients over 15.5 years of age who had not previously consented to organ donation. INTERVENTIONS: Just prior to their clinical encounter, intervention patients (n = 456) watched a 5-minute organ donation video on iPads and then choose a question regarding organ donation to ask their provider. Control patients (n = 459) visited their provider per usual routine. MAIN MEASURES: The primary outcome was the proportion of patients who consented for organ donation. Secondary outcomes included the proportion of patients who discussed organ donation with their provider and the proportion who were satisfied with the time spent with their provider during the clinical encounter. KEY RESULTS: Intervention patients were more likely than control patients to consent to donate organs (22 % vs. 15 %, OR 1.50, 95%CI 1.10-2.13). Intervention patients were also more likely to have donation discussions with their provider (77 % vs. 18 %, OR 15.1, 95%CI 11.1-20.6). Intervention and control patients were similarly satisfied with the time they spent with their provider (83 % vs. 86 %, OR 0.87, 95%CI 0.61-1.25). LIMITATION: How the observed increases in organ donation consent might translate into a greater organ supply is unclear. CONCLUSION: Watching a brief video regarding organ donation and being cued to ask a primary care provider a question about donation resulted in more organ donation discussions and an increase in organ donation consent. Satisfaction with the time spent during the clinical encounter was not affected. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT01697137.

Can Cytoprotective Cobalt Protoporphyrin Protect Skeletal Muscle and Muscle-derived Stem Cells From Ischemic Injury?

BACKGROUND: Extremity trauma is the most common injury seen in combat hospitals as well as in civilian trauma centers. Major skeletal muscle injuries that are complicated by ischemia often result in substantial muscle loss, residual disability, or even amputation, yet few treatment options are available. A therapy that would increase skeletal muscle tolerance to hypoxic damage could reduce acute myocyte loss and enhance preservation of muscle mass in these situations. QUESTIONS/PURPOSES: In these experiments, we investigated (1) whether cobalt protoporphyrin (CoPP), a pharmacologic inducer of cytoprotective heme oxygenase-1 (HO-1), would upregulate HO-1 expression and activity in skeletal muscle, tested in muscle-derived stem cells (MDSCs); and (2) whether CoPP exposure would protect MDSCs from cell death during in vitro hypoxia/reoxygenation. Then, using an in vivo mouse model of hindlimb ischemia/reperfusion injury, we examined (3) whether CoPP pharmacotherapy would reduce skeletal muscle damage when delivered after injury; and (4) whether it would alter the host inflammatory response to injury. METHODS: MDSCs were exposed in vitro to a single dose of 25 µΜ CoPP and harvested over 24 to 96 hours, assessing HO-1 protein expression by Western blot densitometry and HO-1 enzyme activity by cGMP levels. To generate hypoxia/reoxygenation stress, MDSCs were treated in vitro with phosphate-buffered saline (vehicle), CoPP, or CoPP plus an HO-1 inhibitor, tin protoporphyrin (SnPP), and then subjected to 5 hours of hypoxia (< 0.5% O2) followed by 24 hours of reoxygenation and evaluated for apoptosis. In vivo, hindlimb ischemia/reperfusion injury was produced in mice by unilateral 2-hour tourniquet application followed by 24 hours of reperfusion. In three postinjury treatment groups (n = 7 mice/group), CoPP was administered intraperitoneally during ischemia, at the onset of reperfusion, or 1 hour later. Two control groups of mice with the same injury received phosphate-buffered saline (vehicle) or the HO-1 inhibitor, SnPP. Myocyte damage in the gastrocnemius and tibialis anterior muscles was determined by uptake of intraperitoneally delivered Evans blue dye (EBD), quantified by image analysis. On serial sections, inflammation was gauged by the mean myeloperoxidase staining intensity per unit area over the entirety of each muscle. RESULTS: In MDSCs, a single exposure to CoPP increased HO-1 protein expression and enzyme activity, both of which were sustained for 96 hours. CoPP treatment of MDSCs reduced apoptotic cell populations by 55% after in vitro hypoxia/reoxygenation injury (from a mean of 57.3% apoptotic cells in vehicle-treated controls to 25.7% in CoPP-treated cells, mean difference 31.6%; confidence interval [CI], 28.1-35.0; p < 0.001). In the hindlimb ischemia/reperfusion model, CoPP delivered during ischemia produced a 38% reduction in myocyte damage in the gastrocnemius muscle (from 86.4% ± 7% EBD(+) myofibers in vehicle-treated, injured controls to 53.2% EBD(+) in CoPP-treated muscle, mean difference 33.2%; 95% CI, 18.3, 48.4; p < 0.001). A 30% reduction in injury to the gastrocnemius was seen with drug delivery at the onset of reperfusion (to 60.6% ± 13% EBD(+) with CoPP treatment, mean difference 25.8%; CI, 12.2-39.4; p < 0.001). In the tibialis anterior, however, myocyte damage was decreased only when CoPP was given at the onset of reperfusion, resulting in a 27% reduction in injury (from 78.8% ± 8% EBD(+) myofibers in injured controls to 58.3% ± 14% with CoPP treatment, mean difference 20.5%; CI, 6.1-35.0; p = 0.004). Delaying CoPP delivery until 1 hour after tourniquet release obviated the protective effect in both muscles. Mean MPO staining intensity per unit area, indicating the host inflammatory response, decreased by 27-34% across both the gastrocnemius and tibialis anterior muscles when CoPP was given either during ischemia or at the time of reperfusion. Delaying drug delivery until 1 hour after the start of reperfusion abrogated this antiinflammatory effect. CONCLUSIONS: CoPP can decrease skeletal muscle damage when given early in the course of ischemia/reperfusion injury and also provide protection for regenerative stem cell populations. CLINICAL RELEVANCE: Pharmacotherapy with HO-1 inducers, delivered in the field, on hospital arrival, or during trauma surgery, may improve preservation of muscle mass and muscle-inherent stem cells after severe ischemic limb injury.

Targeting survival pathways to create infarct-spanning bridges of human embryonic stem cell-derived cardiomyocytes.

OBJECTIVE: Generating myocyte grafts that bridge across infarcts could maximize their functional impact and best utilize small numbers of stem cells. To date, however, graft survival within acute infarcts has not been feasible. To enhance intrainfarct graft viability, human embryonic stem cell-derived cardiomyocytes (hESC-CMs) were pretreated before implantation with cobalt protoporphyrin (CoPP), a pharmacologic inducer of cytoprotective heme oxygenase-1. METHODS: After preculturing with CoPP (vs phosphate-buffered saline), hESC-CMs were injected intramyocardially into acutely infarcted rat hearts, using directed injections to span the infarct. A further group received CoPP-pretreated hESC-CMs plus 4 weekly doses of systemic CoPP to prolong exposure to cytoprotectants. Two control groups with infarcts received vehicle-only intramyocardial injections or weekly systemic CoPP without cell therapy. Postinfarct ventricular function was gauged by echocardiography and graft size quantified at 8 weeks by histomorphometry. RESULTS: CoPP-preconditioned hESC-CMs formed stable grafts deep within infarcted myocardium, while grafts without CoPP exposure survived mainly at the infarct periphery. Fractional shortening was improved at 4 and 8 weeks in all hearts receiving cell therapies (P < .01 vs vehicle-only injections). CoPP treatment of both graft hESC-CMs and recipient animals resulted in the largest grafts, highest fractional shortening, preserved wall thickness, and reduced infarct dimensions. CONCLUSIONS: Cellular therapy delivered acutely after infarction significantly improved postinfarct ventricular function at 1 and 2 months. CoPP pretreatment of cells resulted in stable hESC-CM grafts within infarcted myocardium. This design enables construction of directionally oriented, infarct-spanning bands of new cardiomyocytes that might further improve functional restoration as engrafted myocytes proliferate and mature.

Cobalt protoporphyrin pretreatment protects human embryonic stem cell-derived cardiomyocytes from hypoxia/reoxygenation injury in vitro and increases graft size and vascularization in vivo.

Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) can regenerate infarcted myocardium. However, when implanted into acutely infarcted hearts, few cells survive the first week postimplant. To improve early graft survival, hESC-CMs were pretreated with cobalt protoporphyrin (CoPP), a transcriptional activator of cytoprotective heme oxygenase-1 (HO-1). When hESC-CMs were challenged with an in vitro hypoxia/reoxygenation injury, mimicking cell transplantation into an ischemic site, survival was significantly greater among cells pretreated with CoPP versus phosphate-buffered saline (PBS)-pretreated controls. Compared with PBS-pretreated cells, CoPP-pretreated hESC-CM preparations exhibited higher levels of HO-1 expression, Akt phosphorylation, and vascular endothelial growth factor production, with reduced apoptosis, and a 30% decrease in intracellular reactive oxygen species. For in vivo translation, 1 × 10(7) hESC-CMs were pretreated ex vivo with CoPP or PBS and then injected intramyocardially into rat hearts immediately following acute infarction (permanent coronary ligation). At 1 week, hESC-CM content, assessed by quantitative polymerase chain reaction for human Alu sequences, was 17-fold higher in hearts receiving CoPP- than PBS-pretreated cells. On histomorphometry, cardiomyocyte graft size was 2.6-fold larger in hearts receiving CoPP- than PBS-pretreated cells, occupying up to 12% of the ventricular area. Vascular density of host-perfused human-derived capillaries was significantly greater in grafts composed of CoPP- than PBS-pretreated cells. Taken together, these experiments demonstrate that ex vivo pretreatment of hESC-CMs with a single dose of CoPP before intramyocardial implantation more than doubled resulting graft size and improved early graft vascularization in acutely infarcted hearts. These findings open the door for delivery of these, or other, stem cells during acute interventional therapy following myocardial infarction or ischemia.

Effect of an iPod video intervention on consent to donate organs: a randomized trial.

BACKGROUND: The gap between the supply of organs available for transplantation and demand is growing, especially among ethnic groups. OBJECTIVE: To evaluate the effect of a video designed to address concerns of ethnic groups about organ donation. DESIGN: Cluster randomized, controlled trial. Randomization was performed by using a random-number table with centralized allocation concealment. Participants and investigators assessing outcomes were not blinded to group assignment. (ClinicalTrials.gov registration number: NCT00870506) SETTING: Twelve branches of the Ohio Bureau of Motor Vehicles in northeastern Ohio. PARTICIPANTS: 952 participants aged 15 to 66 years. INTERVENTION: Video (intervention; n = 443) or usual Bureau of Motor Vehicles license practices (control; n = 509). MEASUREMENTS: The primary outcome was the proportion of participants who provided consent for organ donation on a newly acquired driver's license, learner's permit, or state identification card. Secondary outcomes included willingness to make a living kidney donation to a family member in need and personal beliefs about donation. RESULTS: More participants who viewed the video consented to donate organs than control participants (84% vs. 72%; difference, 12 percentage points [95% CI, 6 to 17 percentage points]). The video was effective among black participants (76% vs. 54%; difference, 22 percentage points [CI, 9 to 35 percentage points]) and white participants (88% vs. 77%; difference, 11 percentage points [CI, 5 to 15 percentage points]). At the end of the trial, fewer intervention than control participants reported having insufficient information about organ donation (34% vs. 44%; difference, -10 percentage points [CI, -16 to -4 percentage points]), wanting to be buried with all of their organs (14% vs. 25%; difference, -11 percentage points [CI, -16 to -6 percentage points]), and having conflicts with organ donation (7% vs. 11%; difference, -4 percentage points [CI, -8 to -2 percentage points]). LIMITATION: How the observed increases in consent to donate organs might translate into a greater organ supply in the region is unclear. CONCLUSION: Exposure to a brief video addressing concerns that ethnic groups have about organ donation just before obtaining a license, permit, or identification card increased consent to donate organs among white and black participants. PRIMARY FUNDING SOURCE: National Institutes of Health and the Robert Wood Johnson Foundation.

Heme oxygenase-1 induction enhances cell survival and restores contractility to unvascularized three-dimensional adult cardiomyocyte grafts implanted in vivo.

Autologous adult cardiomyocytes are not utilized for heart repair strategies because of their rapid apoptosis after implantation. We examined whether induction of heme oxygenase-1 (HO-1), a mediator of preconditioning, could enhance early postimplant myocyte survival. Three-dimensional 5×5 mm patches of full-thickness adult murine atrial wall, including cardiomyocytes, capillary networks, and extracellular matrix, were cultured with or without HO-1 inducer cobalt protoporphyrin (CoPP), or the HO-1 inhibitor, tin protoporphyrin (SnPP), or both. Patches were then implanted subcutaneously. Freshly procured atrial wall patches implanted without preculturing served as additional controls. By 14 days postimplant, graft cardiomyocyte content was significantly greater in CoPP-treated patches than in either control group (p<0.02). Adult cardiomyocytes did not contract in culture or immediately after implantation. However, by 14 days postimplant, spontaneous contraction had recovered in 47% of CoPP-treated patches, but in only 6% of precultured patches without CoPP, 0% of SnPP-treated patches, and 0% of uncultured patches (p<0.03). CoPP-treated adult cardiomyocyte patches were also observed to remodel spontaneously into endothelial-lined chambers that pumped nonclotting blood. These findings demonstrate that adult cardiomyocytes have more plasticity and capacity for functional recovery than previously recognized and could have application as an autologous cardiomyocyte source for tissue engineering.

Primary care physicians' attitudes and practices regarding discussing organ donation with their patients.

PURPOSE: Among the general population, discussing organ donation with a primary care provider may be associated with increased willingness to donate. However, the frequency with which primary care providers hold these discussions with their patients has not been reported. SETTING: Cross-sectional mail and an Internet survey of validated questions regarding organ donation were done. SUBJECTS: A national sample of 831 primary care physicians. black, and Hispanic physicians were oversampled. RESULTS: Few physicians reported receiving formal training in donation (17%). Only 5% of physicians have donor cards available in their practice, and only 11% have donation information available in their practice. While 30% of physicians reported discussing end-of-life care with their patients, fewer than 4% reported discussing donation with their patients. However, only 36% felt that discussing donation was outside of their scope of practice. In a multivariate regression model, predictors of discussing donation with patients included having received formal education about organ donation (odds ratio [OR], 2.6; p < .05) and discussing end-of-life care with patients (OR, 12.8; p < .001). CONCLUSIONS: Very few primary care physicians reported discussing organ donation with their patients despite the majority agreeing that it was within their scope of practice. Primary care physicians who had received education on the subject or who regularly discuss end-of-life care with their patients were more likely to discuss donation. Efforts to improve donation in the general population should include a focus on understanding and improving communication about organ donation between providers and their patients.

Effects of classroom education on knowledge and attitudes regarding organ donation in ethnically diverse urban high schools.

School-based health education is a promising approach for improving organ donation rates, but little is known about its efficacy among ethnically diverse youth. The impact of a classroom intervention was examined in a multicultural high school population where students' ethnicities were 45% African American, 30% Asian American, and 33% Caucasian (allowing for multiracial choices). A baseline survey was administered to all health classes within two wk prior to intervention. On the intervention day, classes randomly assigned to the intervention group received an educational session, followed by a second survey; in control classes, the second survey was taken before the educational session. At baseline, non-Caucasian ethnicity and male gender were each associated with lower levels of willingness to donate. Following the intervention, students in the intervention group demonstrated a significant increase in knowledge scores (p < 0.001), as well as positive movement of opinion regarding willingness to donate (p < 0.0001). Most importantly, the positive changes in opinion occurred independently of ethnicity and gender, in spite of these both being negative predictors of opinion at baseline. These results demonstrate that even a single classroom exposure can impact knowledge levels, correct misinformation, and effect opinion change on organ donation among an ethnically diverse adolescent population.

How do communities want their information? Designing educational outreach on organ donation for Asian Americans.

CONTEXT: Support of organ donation among Asian Americans has been limited, but lack of access to information and prevalence of misinformation are 2 barriers that might be counteracted by public education. OBJECTIVE: To solicit advice from 4 Asian American communities on the design of a culturally appropriate educational campaign on organ donation and transplantation. DESIGN AND SETTING: Cross-sectional, multilingual survey administered at community festivals and supermarkets. PARTICIPANTS: 201 Asian American respondents. MAIN OUTCOME MEASURES: The components of an effective public education outreach campaign on organ donation were defined for 4 Asian American communities. RESULTS: Media venues ranked highest for information dissemination on organ donation/transplantation were, in descending order, mainstream television, ethnic newspapers, mainstream newspapers, and ethnic television. Most respondents preferred a spokesperson of Asian American descent, but opinions differed by ethnicity as to whether an effective spokesperson needed to be of the same Asian ethnicity as the respondents. Respondents were further divided by ethnicity on their preference for a locally or nationally well-known spokesperson. The most compelling scenario to promote organ donation was an Asian American waiting for a transplant, followed by an organ donor family or individual, and, last, a transplant recipient. Different advertisements for organ donation appealed to different Asian ethnic groups. CONCLUSIONS: Community-based research gives communities the opportunity to collaborate with health professionals in designing health education programs that target their own populations. Because key aspects influencing campaign efficacy can vary by ethnicity, these important differences need to be taken into account in outreach planning.

Ethnic and gender differences in willingness among high school students to donate organs.

PURPOSE: To explore ethnic and gender differences in willingness to donate organs among teenagers and determine factors associated with those differences. METHODS: A cross-sectional survey was administered to 883 students attending health science class at nine inner-city high schools in Seattle, Washington. Knowledge and personal experience regarding donation and transplantation, willingness to donate on an ordinal scale, and student demographics were measured. RESULTS: Although only 40% of the cohort had a driver's license, 24% of those with driver's licenses had signed a donor card. Girls were more willing to donate than boys (p < .001) and whites more willing to donate than minorities (p < .001). In a multivariate ordinal logistic regression model, after controlling for school, age, religious preference, home zip code, knowledge regarding donation, willingness to receive a transplant, conversations with others regarding donation, and knowing someone who had donated or received an organ, girls remained more willing to donate compared to boys (odds ratio [OR] 2.10), and white students remained more willing to donate than black (OR 2.38), Chinese (OR 3.03), Hispanic (OR 2.5), Southeast Asian (OR 2.86) and other ethnic students (OR 3.33) (all p < .05). CONCLUSIONS: Gender and ethnic differences in willingness to donate organs exist among high school students. Efforts to increase teenage donation should focus on increasing knowledge and promoting communication about donation with others while remaining cognizant of gender and ethnic differences regarding motivators for donation.

Completion of advanced care directives is associated with willingness to donate.

OBJECTIVES: A useful framework for initiating organ donation discussions in the primary care setting may help increase willingness to donate and thereby increase the frequency of organ transplantation. Given the lower willingness to donate among African Americans and that a higher proportion of African Americans die while waiting for an organ transplant, this is an important group to consider in such an approach. We examined the association among completion of a living will and willingness to donate and the influence of race in this relationship. METHODS: A nationwide telephone interview survey using random digit dialing of households in high- and low-density African-American census blocks. RESULTS: One hundred-eighty-eight adults participated (41% cooperation rate). In a multivariate model, factors associated with willingness to donate included having signed a living will (OR=2.43, 95% CI=1.13-5.23), talking with a physician about organ donation (OR=3.04, 95% CI=1.07-8.67) and white race (OR=2.5, 95% CI=1.23-5). CONCLUSION: The public is generally supportive of organ donation although African Americans remain less willing to donate after controlling for confounding variables. Physicians interested in increasing donation rates should consider incorporating organ donation into discussions of advance care planning and end-of-life care.

Widespread and early myocardial gene expression by adeno-associated virus vector type 6 with a beta-actin hybrid promoter.

Gene therapy for acute cardiac events such as myocardial infarction requires early gene expression over an entire region of myocardium, which has not been possible using adeno-associated virus (AAV) vectors to date. Here we demonstrate marked improvement in the distribution and rapidity of gene expression in myocardium using the AAV pseudotype 6 (AAV6) vector, compared to the standard serotype 2 (AAV2) vector. An alkaline phosphatase (AP) reporter construct driven by the chicken beta-actin promoter was packaged in either AAV6 or AAV2 capsids and delivered to rat hearts in vivo by direct injection. AP expression was evident in both AAV6 and AAV2 vector-treated hearts as early as 1 day after injection, but increased rapidly in AAV6 vector-treated hearts during the first 7 days. The amplitude of AP activity produced by the AAV6 vector was 5-fold greater than that produced by the equivalent AAV2 vector at both 3 and 7 days postinjection. Additionally, the AAV6 vector transduced a myocardial volume that was 10-fold larger than the AAV2 vector. These results indicate the significant potential of AAV6 serotype vectors for early gene expression and widespread regional transduction of myocardium, both auspicious results for in vivo applications in acute cardiac disease.

A CMV-actin-globin hybrid promoter improves adeno-associated viral vector gene expression in the arterial wall in vivo.

BACKGROUND: Adeno-associated virus (AAV) vectors are attractive tools for direct intralumenal arterial gene transfer in interventional cardiology or cardiovascular surgery, but clinical application has been constrained by poor gene expression in this setting. METHODS: To improve arterial wall gene expression, a hybrid promoter consisting of a cytomegalovirus (CMV) immediate-early enhancer, a chicken beta-actin transcription start site, and a rabbit beta-globin intron (CAG promoter) was substituted for the Rous sarcoma virus (RSV) promoter in an AAV type 2 vector with an alkaline phosphatase (AP) reporter gene. RESULTS: Intralumenal transduction of rabbit carotid arteries by an AAV2 vector containing a CAG promoter resulted in gene expression in a mean of > or = 80% of the lumenal area at 14 days following exposure, compared to < or = 25% gene-expressing area with the RSV promoter-based control vector. The high prevalence of gene expression was maintained at 3, 7, 14, and 28 days. Importantly, in carotid arteries transduced with the CAG promoter, gene product expression was readily visible by the third day following transduction whereas gene expression was rarely seen before day 10 using the RSV promoter in the same animal model. On histology, AP gene expression was predominantly in vascular smooth muscle cells although some endothelial cell expression was also present. CONCLUSIONS: Substituting the CAG for the RSV promoter results in widespread gene expression, demonstrating efficient arterial wall transduction by AAV2 vectors. This finding plus the early time to gene expression hold promise for AAV vectors as agents for direct intralumenal arterial wall gene delivery during cardiovascular interventions.

Tolerance to vascularized kidney grafts in canine mixed hematopoietic chimeras.

BACKGROUND: Recent progress in allogeneic hematopoietic stem cell transplantation provides new methods for reliable engraftment with nonlethal conditioning regimens. These techniques have been successfully applied in the treatment of both malignant and nonmalignant diseases, but have not been fully exploited for their potential to tolerize recipients for organ transplantation. These studies were undertaken to test whether the tolerance of host immune cells toward donor hematopoietic cells in mixed hematopoietic chimeras extends to include a vascularized organ, the kidney. METHODS: Using nonmyeloablative doses of total body irradiation, a short course of immunosuppression, and hematopoietic stem cells from marrow or peripheral blood sources, five dog lymphocyte antigen-identical canines were made to become stable mixed hematopoietic chimeras with no development of graft-versus-host disease or posttransplant lymphoproliferative disorder. Subsequently, renal transplantations were performed between stem cell donor and recipient littermates, and no additional immunosuppressive therapy was given after stem cell transplantation. RESULTS: All mixed chimeric dogs demonstrate different, but stable, levels of donor peripheral blood lymphocyte and granulocyte chimerism. With follow-up of longer than 1 year, all of the mixed chimeric dogs (five/five) have excellent renal function with normal serum creatinines (<1.5 mg/dl) and no pathological evidence of rejection on biopsies. CONCLUSIONS: In a major histocompatibility-matched model, minor antigen differences between donor and recipient are not sufficient to induce a host immune response to a vascularized kidney transplant in mixed hematopoietic chimeras.

Intracoronary E-/L-selectin blockade reduces neutrophil infiltration in heart transplantation.

BACKGROUND: This study examined the effect of local intracoronary delivery of a unique monoclonal antibody (mAb) to both E- and L-selectin (EL-246) on neutrophil infiltration after global ischemia during cardiac transplantation. METHODS: In 12 ovine heart transplants, allograft coronary arteries were locally perfused with EL-246 (n = 6), or isotype-matched control antibodies (n = 2) or saline (n = 4). At 24 hours posttransplant, myocardium was analyzed for neutrophil infiltration and myocardial water content. RESULTS: The mean number of intramyocardial neutrophils per area (PMN/hpf) was greatly reduced in the allografts perfused with EL-246 (3.45 +/- 0.4 PMN/hpf), compared with an average 6.5 +/- 0.97 PMN/hpf in control hearts (p = 0.004). Peripheral leukocyte counts were unaffected; myocardial water content was not significantly reduced. CONCLUSIONS: Local perfusion of cardiac allografts with blocking antibody EL-246 before reperfusion significantly reduced the neutrophilic infiltration that occurs early after transplantation. Prohibiting neutrophil-endothelial adhesion and transmigration may be useful in decreasing neutrophil-dependent post-reperfusion injury in transplantation and routine cardiac surgery.

Organ donation and transplantation: ethnic differences in knowledge and opinions among urban high school students.

PURPOSE: To assess knowledge and opinions about the process of human organ donation and transplantation among American teenagers. METHODS: A culturally sensitive 35-item self-administered survey assessing knowledge, opinions, and family discussion about organ donation and transplantation was conducted with 247 students in 13 separate classrooms encompassing three urban high schools in the same city. RESULTS: More than 50% of the students did not know the correct answers to 13 of the 16 questions on factual knowledge. The sources of information about organ donation and transplantation among students were primarily television and school. African-Americans and Asian-Americans were significantly less likely to want to become organ donors when compared to non-African-Americans and non-Asian-Americans, respectively. Asian-Americans were significantly less likely to have discussed the matter with family members. CONCLUSIONS: Accurate, up-to-date, culturally sensitive youth-oriented health education that emphasizes family discussions about organ donation and transplantation is needed.