Patrick W. Vriens

THE USE OF TECHNETIUM TC 99M ANNEXIN V FOR IN VIVO IMAGING OF APOPTOSIS DURING CARDIAC ALLOGRAFT REJECTION

OBJECTIVE Apoptosis, or programmed cell death, has been suggested as a mechanism of immunologic injury during cardiac allograft rejection. We tested the hypothesis that technetium Tc 99m annexin V, a novel radiopharmaceutical used to detect apoptosis, can be used to detect cardiac allograft rejection by nuclear imaging. METHODS Untreated ACI rats served as recipients of allogeneic PVG rat (n = 66) or syngeneic ACI rat (n = 30) cardiac grafts. Untreated recipient animals underwent 99mTc-annexin V imaging daily for 7 days. Region of interest analysis was used to quantify the uptake of 99mTc-annexin V. Immediately after imaging grafts were procured for histopathologic analysis and terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate-biotin nick-end labeling of apoptotic nuclei. One group was treated with 10 mg/kg/d cyclosporine (INN: ciclosporin) commencing on day 4 after transplantation (n = 6). RESULTS Untreated allografts showed histologic signs of rejection 4 days after transplantation. Apoptotic nuclei could be demonstrated in myocytes, endothelial cells, and graft-infiltrating cells of all rejecting allografts. Nuclear imaging revealed a significantly greater uptake of 99mTc-annexin V in rejecting allogeneic grafts than in syngeneic grafts on day 4 (P = .05), day 5 (P < .001), day 6 (P < .001), and day 7 (P = .013) after transplantation. A correlation between the histologic grade of acute rejection and uptake of 99mTc-annexin V was observed (r2 = 0.87). After treatment of rejection with cyclosporine, no apoptotic nuclei could be identified in allografts and uptake of 99mTc-annexin V decreased to baseline. CONCLUSIONS Apoptosis occurs during acute cardiac allograft rejection and disappears after treatment of rejection. 99mTc-annexin V can be used to detect and monitor cardiac allograft rejection.

Tissue-specific differences in the establishment of tolerance. Tolerogenic effects of lung allografts in rats.

With the increasing frequency of transplantation of two or more organs into a single recipient, it has become evident that different organs are rejected with different kinetics. In this study the kinetics of skin, lung, and heart allograft rejection were compared in a rodent model. To study the influence of different allografts on the recipients immune system, simultaneous or sequential skin, lung, or heart transplants were performed in various combinations, using DA rats as recipients for PVG allografts. Recipients receiving primary allografts were treated postoperatively with ten doses of cyclosporine (CsA) or preoperatively with 4 doses of rabbit antirat thymocyte globulin (ATG). Subsequent transplants were performed a minimum of 40 days later without additional immunosuppression. All primary skin allografts and 60% of primary lung allografts were rejected, while 100% of the heart allografts were accepted indefinitely. Recipients of primary skin allografts rejected subsequent skin, lung, or heart allografts with accelerated kinetics. Recipients of primary heart allografts accepted subsequent skin, lung, and heart allografts indefinitely without further immunosuppression. Surprisingly, animals that had rejected a primary lung allograft accepted subsequent skin or heart allografts indefinitely. Simultaneously transplanted skin and lung allografts were concordantly rejected. However, these animals accepted a subsequent heart allograft indefinitely, suggesting a strong tolerizing effect of lung allografts. Our results indicate that tissue-specific differences are critical, not only in determining acceptance or rejection of a primary allograft but also in determining the fate of subsequent allografts.

Optimization Of Ex Vivo Pressure Mediated Delivery Of Antisense Oligodeoxynucleotides To Icam-1 Reduces Reperfusion Injury In Rat Cardiac Allografts1

BACKGROUND Our purpose was to optimize hyperbaric pressure as a vector for ex vivo transfection of antisense oligodeoxynucleotides (AS-ODN) to intercellular adhesion molecule-1 to limit reperfusion injury (RI) in cardiac allografts. We investigated the effects of increased pressure, incubation time, and AS-ODN concentrations on transfection efficiency and toxicity. METHODS AND RESULTS PVG (RT1c) donor hearts were heterotopically transplanted to ACI (RT1a) recipients. Donor hearts were harvested and the various groups were treated at: (1) different pressure (1-9 atm) for 45 min with 80 micromol/liter AS-ODN; (2) different incubation times (15 min to 6 hr) at 5 atm with 80 micromol/liter AS-ODN; 3) different AS-ODN concentrations (80-240 micromol/liter) at 5 atm for 45 min. Hearts were procured 24 or 72 hr after transplantation. Transfection efficiency was determined with fluorescein-labeled AS-ODN. The degree of RI was determined with biochemical and histological analysis. Increasing pressure from ambient (1 atm) pressure to pressures as high as 9 atm leads to a increase in transfection efficiency from 1.7+/-.5 to 62+/-3.9% and a reduction in RI. Increased incubation time up to 45 min increased transfection efficiency and reduced RI, but longer incubation times induced significant toxicity to the allograft. Increased AS-ODN concentrations improved transfection and reduced RI. CONCLUSIONS Hyperbaric pressure is a safe and effective vector for the ex vivo delivery of AS-ICAM-1-ODN to rodent cardiac allografts and results in a reduction in reperfusion injury.

In vivo studies of the maintenance of peripheral transplant tolerance after cyclosporine : radiosensitive antigen-specific suppressor cells mediate lasting graft protection against primed effector cells

Cellular mechanisms responsible for maintenance of peripheral transplant tolerance in a rodent model were evaluated. Donor-specific tolerance was established in ACI rats given a vascularized heterotopic cardiac allograft followed by a 10-day course of cyclosporine. Tolerance was associated with a reduction in donor-specific cytotoxic T lymphocyte precursors and the presence within the spleen of cells capable of transferring suppression in adoptive transfer assays. Experiments using thymectomized animals revealed that the establishment and maintenance of tolerance occurred peripherally, independently of the thymus. Adoptive transfer experiments demonstrated that ongoing graft tolerance was mediated by suppressor cells that were antigen-restricted, radiosensitive, and capable of preventing allograft rejection by naive as well as sensitized cells in vivo. Studies designed to disrupt tolerance demonstrated a remarkable durability of graft protection once established, and give insight into the identity and mechanism of action of suppressor cells generated in this model.

TISSUE-SPECIFIC DIFFERENCES IN THE ESTABLISHMENT OF TOLERANCE

With the increasing frequency of transplantation of two or more organs into a single recipient, it has become evident that different organs are rejected with different kinetics. In this study the kinetics of skin, lung, and heart allograft rejection were compared in a rodent model. To study the influence of different allografts on the recipients immune system, simultaneous or sequential skin, lung, or heart transplants were performed in various combinations, using DA rats as recipients for PVG allografts. Recipients receiving primary allografts were treated postoperatively with ten doses of cyclosporine (CsA) or preoperatively with 4 doses of rabbit antirat thymocyte globulin (ATG). Subsequent transplants were performed a minimum of 40 days later without additional immunosuppression. All primary skin allografts and 60% of primary lung allografts were rejected, while 100% of the heart allografts were accepted indefinitely. Recipients of primary skin allografts rejected subsequent skin, lung, or heart allografts with accelerated kinetics. Recipients of primary heart allografts accepted subsequent skin, lung, and heart allografts indefinitely without further immunosuppression. Surprisingly, animals that had rejected a primary lung allograft accepted subsequent skin or heart allografts indefinitely. Simultaneously transplanted skin and lung allografts were concordantly rejected. However, these animals accepted a subsequent heart allograft indefinitely, suggesting a strong tolerizing effect of lung allografts. Our results indicate that tissue-specific differences are critical, not only in determining acceptance or rejection of a primary allograft but also in determining the fate of subsequent allografts.

Report of two in situ reconstructions with a saphenous spiral vein graft of Coxiella burnetii-infected aneurysms of the abdominal aorta

Coxiella burnetii is a rare cause of vascular infections. Yet, Q fever is endemic in the southern part of The Netherlands. This report describes two patients--from the southern part of The Netherlands--with infected aneurysms of the abdominal aorta caused by Coxiella burnetii. Both patients underwent surgical debridement, in situ reconstruction with a great saphenous vein spiral graft, and a transmesenteric omentumplasty. One patient fully recovered, while the other died due to ischemic complications. A multidisciplinary work-up approach to treat infected abdominal aneurysms is proposed, including adequate surgical treatment and long-term antibiotic administration.

Vascular access outcome in the elderly dialysis patient in combination with the quality of life.

Purpose: We performed a retrospective study on hemodialysis fistulae in patients aged 75 years and older. Methods: Dialysis records of 2 hospitals were searched for patients of 75 years and older who had primary autologous radiocephalic arteriovenous fistulae (RCAVFs) and brachiocephalic arteriovenous fistulae (BCAVFs). Outcome measures were primary, primary-assisted, and secondary patency rates. Also, quality of life (QOL) was measured. Results: A total of 107 fistulae were placed in 90 patients; 65 (61%) RCAVFs and 42 (39%) BCAVFs were created. The primary patency rate (P = .026) and the primary-assisted patency rate (P = .016) of BCAVFs were significantly higher than that of RCAVFs. Secondary patency rates at 1 year (P = .01) and 2 years (P = .035) were higher in BCAVFs than in RCAVFs. Conclusions: The BCAVFs give significantly higher primary and primary-assisted patency rates and also significantly higher secondary patency rates at 1 and 2 years. Therefore, we suggest the placement of elbow fistulae in the elderly patients. The QOL was surprisingly high in this population despite a high mortality rate.

Self-reported symptoms on questionnaires and anatomic lesions on duplex ultrasound examinations in patients with peripheral arterial disease

OBJECTIVE Whether a typical patient and symptom profile is associated with proximal or distal lesions in lower extremity peripheral arterial disease (PAD) is unknown. Knowing which patient characteristics, exertional leg symptoms, and cardiovascular risk profile accompany the anatomic lesion location may facilitate a more tailor-made management of PAD. METHODS This cross-sectional study comprised 701 patients from two vascular surgery outpatient clinics with new-onset symptoms of PAD (Fontaine 2) who underwent duplex ultrasound (DUS) examinations from March 2006 to March 2011. The main outcome measures were patient characteristics, self-reported leg symptoms, and cardiovascular risk factors as documented from questionnaires and medical records. Peripheral lesion information, categorized by proximal and distal lesions, was obtained from DUS examinations. Multivariable logistic regression analyses were performed of proximal vs nonproximal lesions, distal vs nondistal lesions, and proximal and distal vs absence of having both lesions to assess relationships between patient characteristics, leg symptom categories (typical vs atypical leg symptoms), cardiovascular risk factors, and anatomic lesion location. RESULTS Lesions were proximal in 270 (38.5%), distal in 441 (62.9%), and proximal and distal in 94 (13.4%). Patients with proximal lesions were younger (odds ratio [OR], 0.94; P < .0001) and less likely to be obese (OR, 0.34; P < .0001) than those without proximal lesions. Older age (OR, 1.07; P < .0001), male sex (OR, 1.96; P = .003), being without a partner (OR, 2.24; P = .004), and lower anxiety scores (OR, 0.42; P = .003) were associated with distal lesions. Patients with both lesions were more likely to be single (OR, 2.30; P = .010) and less likely to be obese (OR, 0.24; P = .009). No distinguishing leg symptom pattern was observed for patients with proximal lesions. Intermittent claudication was more frequently reported in those with distal lesions (P = .011). Although buttock and thigh pain seemed to be somewhat more present in proximal lesions (P < .01) and calf pain more in distal lesions (P < .001), patients still reported pain at a variety of levels throughout their legs, regardless of the anatomic lesion location. CONCLUSIONS Two distinctive PAD phenotypes-each with its own characteristics and risk factors-emerged by anatomic lesion location; however, PAD-specific leg symptoms did not always reflect the anatomic lesion location. These findings may open new opportunities to better tailor PAD management to these two PAD subgroups and may raise awareness about not relying on self-reported symptoms to guide further diagnostic imaging and peripheral lesion management.

Pre‐transplant blood transfusion and cyclosporin A induce long‐term hamster cardiac xenograft survival in immunocompetent rats

Abstract:  Background:  In previous studies we have shown that pre‐transplant hamster blood transfusion (HBT) can induce non‐responsiveness in the T cell independent immunecompartment and result in tolerance towards hamster cardiac xenografts (Xgs) in T cell deficient athymic nude rats. In this study we test the combination of pre‐transplant HBT with cyclosporin A (CSA) in immunocompetent Lewis rats.

Pre‐transplant blood transfusion induces tolerance to hamster cardiac xenografts in athymic nude rats

Abstract: The effects of pre‐transplant blood transfusion vary from induction of antibodies and accelerated graft rejection, to prolonged survival and even tolerance. The beneficial ‘transfusion effect’ in allotransplantation is believed to be merely T‐cell mediated. In xenotransplantation, T‐cell independent mechanisms form a major hurdle. In this study we investigated the effects of pre‐transplant hamster blood transfusion on the survival of hamster cardiac xenografts in T‐cell deficient athymic nude rats. Nude rats rejected xenografts after 3.8 ± 0.5 d (n = 8), and immunocompetent Lewis rats after 4.0 ± 0.5 d (n = 8), following a similar IgM response (P = NS). Hamster blood transfusion 3 d before transplantation in nude rats led to an IgM response and long‐term xenograft survival in 17/20. Timing was of importance: blood transfusion 7 d before transplantation resulted in 45% long‐term xenograft survival (n = 20). Injection of purified hamster erythrocytes, leukocytes or minced heart also led to survival of xenografts for > 100 d in nude rats, but not in all cases. Second xenografts transplanted to long‐term survivors did not provoke an IigM response, and were accepted for > 100 d (n = 4). Transfer of serum from long‐term survivors to untreated nude rats resulted in survival of xenografts for > 100 d (n = 4). In Lewis rats pre‐transplant blood transfusion induced hyperacute rejection of xenografts after 158 ± 128 min (n = 8, P < 0.01). We conclude that pre‐transplant hamster blood transfusion can induce long‐term survival of hamster cardiac xenografts in T‐cell deficient athymic nude rats. This blood transfusion effect is mediated by humoral factors and can be transferred by serum. Elucidation of underlying mechanisms might provide some insight into xenotransplantation nonresponsiveness of T‐cell independent immunefactors.