Granulocyte colony-stimulating factor therapy is associated with a reduced incidence of acute rejection episodes or allograft vasculopathy in heart transplant recipients.

BACKGROUND: We evaluated the potential effects of granulocyte colony-simulating factor (G- CSF) on the incidence of rejection and allograft vasculopathy in heart transplant recipients. METHODS: Of 247 patients undergoing heart transplantation from 2000 to 2007, 52 (21%) developed leukopenia (white blood cell [WBC] <2.5 × 10(9) cells/L) in the absence of active infection, rejection, or malignancy. In 24 (46%) patients a clinical decision was made to treat the leukopenia with G-CSF (G-CSF group), and 28 (54%) Patients received no G-CSF (non-GCSF group). Patients followed up for 1 year after the period of leukopenia were assessed for allograft vasculopathy and acute rejection incidence. RESULTS: At baseline, the G-CSF group and the non-GCSF group did not differ in age, gender, race, heart failure etiology, creatinine, left ventricular ejection fraction (LVEF) or immunosupressive regimen. During 1-year follow-up there were no deaths in the G-CSF group, and 1 death in the non-GCSF group (P = .34). The incidence of rejection or progressive allograft vasculopathy was lower in the G-CSF group when compared with the non-GCSF group (2 [8%] vs 15 [53%]; P < .01). Multivariate analysis identified both prior rejection episodes and G-CSF therapy as factors associated with the combined end-point of rejection or progressive allograft vasculopathy (odds ratio [OR] = 7.89 [1.67-37.2] and OR = 0.09 [0.02-0.52], respectively). CONCLUSIONS: G-CSF therapy appears to be associated with a decreased incidence of acute rejection episodes or allograft vasculopathy in heart transplant recipients, suggesting a potential immunomodulatory effect of G-CSF.

Angiogenic effects despite limited cell survival of bone marrow-derived mesenchymal stem cells under ischemia.

Bone marrow-derived mesenchymal stem cells (MSCs) are multipotent and secrete angiogenic factors, which could help patients with occlusive arterial diseases. We hypothesize that MSCs, in comparison to fibroblasts, survive better under hypoxic conditions in vitro and in vivo. MSCs and fibroblasts from L2G mice expressing firefly luciferase and GFP were cultured in normoxic and hypoxic conditions for 24 hours. In vitro cell viability was tested by detecting apoptosis and necrosis. MSCs released higher amounts of VEGF (281.1 +/- 62.6 pg/ml) under hypoxic conditions compared to normoxia (154.9 +/- 52.3 pg/ml, p = NS), but were less tolerant to hypoxia (45 +/- 7.9%) than fibroblasts (28.1 +/- 3.6%, p = NS). A hindlimb ischemia model was created by ligating the femoral artery of 18 FVB mice. After one week, 1 x 106 cells (MSCs, fibroblasts or saline) were injected into the limb muscles of each animal (n = 6 per group). Bioluminescence measurement to assess the viability of luciferase positive cells showed significant proliferation of MSCs on day four compared to fibroblasts (p = 0.001). Three weeks after cell delivery, the capillary to muscle fiber ratio of ischemic areas was analyzed. In the MSC group, vessel density was significantly higher than in the fibroblast or control group (0.5 +/- 0.08 and 0.3 +/- 0.03). Under hypoxia, MSCs produced more VEGF compared to normal conditions and MSC transplantation into murine ischemic limbs led to an increase in vessel density, although MSC survival was limited. This study suggests that MSC transplantation may be an effective and clinically relevant tool in the therapy of occlusive arterial diseases.

Effect of inhaled tacrolimus on cellular and humoral rejection to prevent posttransplant obliterative airway disease.

This study aimed to investigate the pharmacokinetics after tacrolimus aerosol inhalation and to assess its efficacy to suppress acute and chronic airway allograft rejection. Orthotopic tracheal transplantations were performed and tacrolimus (4 mg/kg) was administered orally (PO) or via aerosol (AER). Tracheal tissue level AUCs(0-12) were similar in both treatment groups, but blood AUCs(0-12) were approximately 5.5-fold lower with AER (p < 0.001). Interestingly, only PO animals showed elevated BUN, cholesterol and triglycerides on POD 60 (p < 0.05). Histology of grafts harvested after 6 and 60 days revealed that both treatment groups were similarly effective in suppressing graft mononuclear infiltration (p < 0.001). Cellular immune activation (assessed by IFN-gamma- and IL-4-ELISPOTS), however, was far more effectively suppressed by tacrolimus PO (p < 0.001). In both treatment groups, the vigorous alloreactive IgM-antibody surge was effectively inhibited (p < 0.001). Due to the insufficient systemic cellular immunosuppression, discontinuation of tacrolimus AER resulted in a far stronger (3.5-fold) graft infiltration on POD 8 compared to PO (p < 0.001). Tacrolimus aerosol reduces systemic side effects and effectively protects the airway graft from early cellular rejection and chronic obliterative airway disease.

Is the malononitrilamide FK778 better for the prevention of acute or chronic rejection?

OBJECTIVE: The aim of this study was to assess the efficacy of FK778 to prevent acute and chronic allograft rejection compared with other immunosuppressive agents. MATERIALS AND METHODS: Heterotopic Brown-Norway (BN)-to-Lewis rat cardiac transplantations and heterotopic BN-to-Lewis tracheal transplantations were performed to study acute heart rejection and the development of chronic obliterative airway disease (OAD), respectively. Recipients were treated with FK778, tacrolimus, MMF, or sirolimus for 10 days (acute rejection study) or 28 days (chronic OAD study) at varying doses. RESULTS: In untreated recipients, cardiac allograft survival was 6.2 +/- 0.4 days. FK778 (20 mg/kg), tacrolimus (2 or 8 mg/kg), mycophenolate mofetil (MMF; 40 mg/kg), or sirolimus (0.5 or 2 mg/kg) significantly prolonged graft survival to 17.0 +/- 2.8, 18.5 +/- 2.7, 25.0 +/- 2.5, 20.7 +/- 3.8, 14.5 +/- 2.2, and 23.2 +/- 1.5 days, respectively (P < .05). Tracheal grafts in untreated recipients showed intense infiltration and complete luminal obliteration by day 28. FK778 (20 mg/kg), tacrolimus (1 or 4 mg/kg), MMF (10 or 40 mg/kg), or sirolimus (0.5 or 2 mg/kg) significantly inhibited tracheal luminal obliteration (19.5% +/- 16.4%, 44.2% +/- 33.6%, 12.3% +/- 3.3%, 61.7% +/- 18.6%, 18.3% +/- 11.3%, 55.0% +/- 30.9%, and 8.5% +/- 3.5% (P < .05). All 4 high-dose groups showed similar efficacy. CONCLUSIONS: When used in therapeutic doses, tacrolimus and sirolimus were more effective than FK778 to prolong cardiac allograft survival. However, with its antiproliferative effects on smooth muscle cells, its good tolerability, and its blockade of cytomegalovirus replication, FK778 proved effective to prevent chronic OAD development. Thus, FK778 may acquire an important role in maintenance therapy for the prevention of long-term fibroproliferative complications.

Stem cell transplantation: the lung barrier.

BACKGROUND: Mesenchymal stem cells (MSCs) show differentiation capacity along mesenchymal lineages and have the potential to aid tissue regeneration. MSC transplantation strategies are therefore currently being assessed following injury to various organs. However, potential MSC migration to these organs after intravenous (IV) MSC injection continues to be impeded by cell trapping within the lung. METHODS: Mouse MSCs were isolated, purified, transfected with firefly luciferase, and labeled with CSFE. Their size was assessed in vitro. To estimate the diameter of mouse pulmonary capillaries, fluorescence-labeled microspheres of different sizes were injected with or without sodium nitroprusside (SN) pretreatment. The lungs were harvested after 30 seconds and mean numbers of trapped microspheres per high-power field (HPF) were calculated. After IV injection of MSC suspensions (with or without SN), their dynamic distribution was monitored by in vivo luciferine imaging as well as by histopathology. RESULTS: The diameter of suspended MSCs in vitro was 15 to 19 microm. Whereas nearly no 4-microm microspheres could be detected in lung sections, the numbers of trapped 10- and 15-microm microspheres could be significantly decreased by prior SN injection from 19.3 +/- 11.1 to 6.0 +/- 1.6 cells/HPF (P = .004) and from 34.9 +/- 11.9 to 25.6 +/- 8.1 cells/HPF (P = .028), respectively. Within seconds after MSC IV injection, the vast majority of cells was found in the lungs. However, cell trapping in the pulmonary microvasculature was significantly reduced by pre-treatment with SN. CONCLUSIONS: We demonstrate that cell trapping in lungs can be reduced with IV SN pretreatment, increasing MSC passage through the lung capillaries, and potentially facilitating cell access to injured organs.

Tracheal allograft transplantation in rats: the role of different immunosuppressants on preservation of respiratory epithelium.

BACKGROUND: Bronchiolitis obliterans is the most significant complication adversely affecting the survival of lung allograft recipients. Injury and loss of epithelium are associated with obliteration of the airway lumen. The aim of this study was to examine the effects of various immunosuppressants on airway epithelium. METHODS: Tracheae from Brown Norway donors were heterotopically transplanted into the greater omentum of Lewis (allografts) or Brown Norway (isografts) animals. Recipients were treated for 28 days with FK778 (20 mg/kg), tacrolimus (4 mg/kg), or sirolimus (2 mg/kg). Tracheal segments were evaluated for the degree of luminal occlusion as well as the type and percent of luminal epithelial cell coverage. RESULTS: All agents inhibited peritracheal infiltration and luminal obliteration. Tacrolimus- more than sirolimus-treated recipients showed partial preservation of the luminal epithelial coverage, whereas animals that received FK778 showed no respiratory epithelium. The epithelial loss was accompanied by the appearance of fibrous tissue, which replaced the mucosa. CONCLUSIONS: Tacrolimus as well as sirolimus effectively prevented the development of obliterative airway disease whereas tacrolimus and, to a lesser degree, sirolimus preserved epithelial cells as a source of protective cytokines. With FK778 significant airway obliteration was suppressed despite complete epithelial loss. Thus, FK778-treated animals displayed an epithelial-independent inhibitory effect on myofibroblast proliferation.

FK778: new cellular and molecular mechanisms of action.

PURPOSE: The new malononitrilamide FK778 is currently being evaluated as an immunosuppressant for organ transplantation. Its main mechanism is inhibition of a pivotal enzyme of pyrimidine biosynthesis. This report revealed new mechanisms of action on different cell types involved in acute and chronic allograft rejection. METHODS: Purified Brown-Norway rat aortic endothelial cell (EC) cultures were pretreated with several concentrations of FK778. Endothelial adhesion molecule expression (ICAM-1/VCAM-1) stimulated with TNF-alpha was quantified by immunofluorescence. Purified Lewis rat lymphocytes (LC) incubated with FK778 were stimulated via TCR/CD28 signals, and CD25 expression was quantified using FACS analysis. Uridine addition was used in all assays to reverse the pyrimidine synthesis blockade. Lymphocyte-EC interaction was assessed by micromanipulator-assisted single-cell adhesion assays. Finally, smooth muscle cell (SMC) proliferation and migration was analyzed. Uridine addition was used in all assays to reverse the pyrimidine synthesis blockade. RESULTS: TNF-alpha stimulation and TCR/CD28 co-stimulation significantly increased EC ICAM-1/VCAM-1-expression and LC CD25 surface expression, respectively. These effects were dose-dependently inhibited by FK778 and were not reversed by the addition of uridine. FK778 dose-dependently attenuated LC adhesion to allogeneic EC. The dose-dependent inhibition of SMC proliferation by FK778 was abolished by uridine addition, whereas the inhibitory effect on SMC migration was not affected by uridine supplementation. CONCLUSIONS: FK778 directly reduced endothelial adhesion molecule up-regulation, inhibited lymphocyte activation, and attenuated lymphocyte-endothelium interactions, critical early steps in graft rejection. These effects were separate from the blockade of pyrimidine synthesis. The antiproliferative potency of FK778 on SMC may be an important mechanism to inhibit the fibroproliferative lesions of chronic organ rejection.

Correlation between mononuclear infiltration and changes in VASP phosphorylation patterns after heterotopic cardiac transplantation in the rat.

Chronic cardiac transplant vasculopathy still remains the major cause of late graft failure after the 1st postoperative year, with iNOS playing a central role in the progression of this disease. Since VASP, a recently identified microfilament-associated protein in smooth muscle cells, endothelial cells, and platelets, is phosphorylated by cyclic nucleotide dependent protein kinases, changing amounts of NO-producing mononuclear infiltration cells during cardiac rejection are supposed to change platelet VASP phosphorylation patterns. We investigated whether platelet VASP Ser(157) phosphorylation (VASP shift) after coronary passage of rat cardiac allografts correlates with graft infiltration. The Lew-F344 heterotopic rat cardiac transplantation model was used. Native hearts and grafts were harvested 3-150 days after transplantation and were used for Langendorff perfusion. The platelet VASP shift after native heart and graft perfusion was identified. Additional iNOS stimulation and iNOS inhibition were achieved pharmacologically. Immunohistology revealed graft mononuclear infiltration. Platelet VASP Ser(157) and Ser(239) phosphorylation significantly increased after coronary passage of native hearts and grafts (p < 0.01). Though platelet VASP Ser(157) phosphorylation failed to directly express graft infiltration, we showed a significant correlation between changes of platelet VASP shift and extent of grafts' mononuclear infiltration after competitive iNOS inhibition (p < 0.01). The platelet VASP shift is modified during coronary perfusion, and this modification correlates with mononuclear infiltration in the graft. This emphasizes the influence of mononuclear infiltration cells on microfilamental structures of the cytoskeleton in adjacent cells.

Five years of experience with mitral valve homografts.

INTRODUCTION: The main advantages of mitral homografts are preservation of the subvalvular apparatus and avoidance of life-long anticoagulation. In this communication, we will present our five-year experience using mitral homografts in mitral valve surgery. PATIENTS AND METHODS: Since 1996, 14 patients (mean age 46 +/- 8 years, range 27 - 65 years have had mitral homografts implanted. Thirteen patients had mitral valve replacement; the septal leaflet of the tricuspid valve was replaced in one case. The indications were mitral (n = 6) or tricuspid endocarditis (n = 1), mitral valve stenosis (n = 3), and combined mitral valve disease (n = 4). Complete mitral homografts were implanted in eight patients; partial homografts were used in six cases. Preoperatively, the dimensions of the left ventricle and the mitral valve were measured by transoesophageal echocardiography (TOE). The mean left ventricular ejection fraction was 56 +/- 9%, the mean end-diastolic diameter 58 +/- 6 mm. The technique described by Acar/Carpentier was adapted for implantation; a Carpentier ring was implanted in all cases for annular stabilization. The patients had anticoagulative therapy which was discontinued when stable sinus rhythm was present after three months postoperatively. Follow-up included clinical examination, ECG, and echocardiography, and was initiated six months postoperatively and continued on a yearly basis. The following parameters were determined by echocardiography--left atrial size, left ventricular end-diastolic and end-systolic diameter, pressure gradient across the mitral valve (c/w Doppler, Bernoulli's equation), and mitral regurgitation. RESULTS: All patients survived surgery; the mean operation-time was 281 +/- 37 minutes. Intraoperative TOE revealed a first degree insufficiency in 7 patients. Follow-up was completed in all patients, with a mean period of 30 months (6 - 60 months). Two patients had an acute endocarditis two years postoperatively, requiring repeat valve replacement with a mechanical prosthesis. An additional patient had to be reoperated due to chordal rupture three years postoperatively. All three patients had mitral valve stenosis as the initial indication for surgery and had received a complete homograft. In the remaining eleven patients, the morphological and functional state of the implanted grafts remained unchanged during follow-up. The freedom from valve-related events was 93% after one year, 86% after two years, and 79% after three years. At six-month follow-up, ECG and echocardiography revealed sinus rhythm and sufficient atrial contractions in 13 cases. At the last follow-up, the pressure gradients were 3.4 +/- 0.6 mmHg for complete homografts and 2.8 +/- 0.6 mmHg for partial homografts. In five cases, a mild insufficiency was documented, while six patients presented with competent grafts. CONCLUSIONS: Mitral homografts can be used with acceptable mid-term results in selected cases with good left ventricular function and only slightly dilated left ventricles. Partial mitral homografts represent an additional technique, especially for mitral valve repair in patients with acute endocarditis. The susceptibility to bacterial infections of a homograft makes strict prophylaxis against endocarditis mandatory.

ERP and behavioral changes during the wake/sleep transition.

Event-related potentials (ERPs) following infrequent and frequent stimuli were studied as subjects moved from wakefulness to sleep. Subjects were instructed to respond to the infrequent "target" stimuli (attend condition) or to ignore the stimuli (ignore condition). Parietal P300, prominent following target ERPs in wakefulness under the attend condition, disappeared in association with reduced behavioral responsiveness and emergence of a central negativity (N350). The N350 and preceding and following positivities (P220 and P450) became the dominant feature of both target and nontarget ERPs under both attend and ignore conditions. The P220-N350-P450 complex was larger and peak latencies were shorter under the attend condition. Peak amplitudes tended to be larger following targets, especially under the attend condition. The findings suggest that, although the processes underlying P300 are less likely to be engaged, processing of stimulus deviance and task relevance continues in sleepiness and sleep, and is reflected by variance in N350 and related activity.