Carlo Pellegrini

Desmin accumulation restrictive cardiomyopathy and atrioventricular block associated with desmin gene defects

Primary desminopathies are caused by desmin gene [DES (MIM*125660)] mutations. The clinical spectrum includes pure myopathies, cardiomuscular diseases and cardiomyopathies. Patients with restrictive cardiomyopathy (RCM) plus atrioventricular block (AVB) due to DES defects are frequently unrecognized unless desmin accumulation is specifically investigated in endomyocardial biopsy (EMB) by ultrastructural study.

Human cytomegalovirus pp67 mRNAemia versus pp65 antigenemia for guiding preemptive therapy in heart and lung transplant recipients: A prospective, randomized, controlled, open-label trial

Background. Preemptive therapy of human cytomegalovirus (HCMV) infections has gained popularity in transplantation centers. However, standardized protocols are not available. In particular, whether a qualitative molecular assay for detection of a late (pp67) HCMV mRNA represents a valuable alternative to quantitative antigenemia remains to be defined. Methods. Overall, 82 heart (HTR) and lung (LTR) transplant recipients were randomized into two arms, where therapy was guided by qualitative pp67 mRNA NASBA (40 patients) or quantitative antigenemia (42 patients). In the NASBA arm, both primary and recurrent infections were treated upon first confirmed positive NASBA result. In the antigenemia arm, primary infections were treated upon first confirmed positive result, while recurrent infections were treated upon cutoff of 100 pp65-positive leukocytes. In both arms, therapy was stopped upon virus disappearance. Primary endpoint was duration of therapy. Results. The number of treated/infected patients was significantly higher in the NASBA arm (25/30 vs. 15/39;P =0.015), as was the number of treated/relapsing patients (5/8 vs. 1/11;P =0.040), whereas the number of HCMV-infected/total number of patients was significantly higher in the antigenemia arm (39/42 vs. 30/40;P =0.026). Thus, in the NASBA arm, although the median duration of therapy was shorter compared to antigenemia (17 vs. 21 days, P >0.05), the overall number of days of therapy was significantly higher. No patient developed HCMV disease. Conclusion. pp67 mRNA NASBA can safely replace antigenemia, with some apparent advantages (semiautomation and objectivity of test results) and disadvantages (overtreatment of patients and greater duration of overall treatment).

Venoarterial extracorporeal membrane oxygenation for acute fulminant myocarditis in adult patients: A 5-year multi-institutional experience

BACKGROUND Acute fulminant myocarditis (AFM) may represent a life-threatening event, characterized by rapidly progressive cardiac compromise that ultimately leads to refractory cardiogenic shock or cardiac arrest. Venoarterial extracorporeal membrane oxygenation (VA-ECMO) provides effective cardiocirculatory support in this circumstance, but few clinical series are available about early and long-term results. Data from a multicenter study group are reported which analyzed subjects affected by AFM and treated with VA-ECMO during a 5-year period. METHOD From hospital databases, 57 patients with diagnoses of AFM treated with VA-ECMO in the past 5 years were found and analyzed. Mean age was 37.6 ± 11.8 years; 37 patients were women. At VA-ECMO implantation, cardiogenic shock was present in 38 patients, cardiac arrest in 12, and severe hemodynamic instability in 7. A peripheral approach was used with 47 patients, whereas 10 patients had a central implantation or other access. RESULTS Mean VA-ECMO support was 9.9 ± 19 days (range, 2 to 24 days). Cardiac recovery with ECMO weaning was achieved in 43 patients (75.5%), major complications were observed in 40 patients (70.1%), and survival to hospital discharge occurred in 41 patients (71.9%). After hospital discharge (median follow-up, 15 months) there were 2 late deaths. The 5-year actual survival was 65.2% ± 7.9%, with recurrent self-recovering myocarditis observed in 2 patients (at 6 and 12 months from the first AFM event), and 1 heart transplantation. CONCLUSIONS Cardiopulmonary support with VA-ECMO provides an invaluable tool in the treatment of AFM, although major complications may characterize the hospital course. Long-term outcome appears favorable with rare episodes of recurrent myocarditis or cardiac-related events.

Port-access minimally invasive surgery for atrial septal defects: A 10-year single-center experience in 166 patients

OBJECTIVE We assessed the surgical results and the benefits to the patient of a minimally invasive surgical approach for atrial septal defects. METHODS Between May 1998 and May 2008, 166 patients (median age, 44 years) had surgery for atrial septal defects in our institution. Of these patients, 118 (71%) had a patent foramen ovale (associated with atrial septal aneurysm in 48 cases), 33 (20%) had a wide ostium secundum defect, 6 (3.6%) had an ostium primum defect, 6 (3.6%) had a sinus venosus defect with abnormal pulmonary vein connection, and 1 (0.6%) had a coronary sinus defect. In 2 cases (1.2%) patients were referred to our department for surgical correction after failure of interventional occluder placement. All patients were operated on via a right minithoracotomy (mean incision, 5.5 + or - 1 cm) in the fourth intercostal space and under cardiopulmonary bypass. RESULTS The HeartPort access system was used in 106 patients (64%), with an endoaortic clamp (central kit in 50 cases and peripheral kit in 56). In the remaining patients (36%), we preferred the Portaclamp system (37 cases) or the Chitwood clamp (23 cases). Average crossclamp time was 38.4 + or - 22.2 minutes with a mean cardiopulmonary bypass time of 64.9 + or - 34.5 minutes. There was no conversion in classic sternotomy. There were no early or late hospital deaths. Surgical revision was performed in 6 patients for bleeding from the thoracic wall. The mean hospital stay was 5.8 days. At 51 months mean follow-up, 4 patients died of non-cardiac-related causes. CONCLUSIONS Port-access minimally invasive surgery for atrial septal defects is a safe, less-invasive, reproducible, and cosmetic operation, providing an excellent outcome and an effective correction, and could be now considered the standard approach for this type of patient.

Port-access surgery as elective approach for mitral valve operation in re-do procedures

BACKGROUND Re-do mitral valve procedures performed through median sternotomy carry substantial mortality and morbidity. To avoid complications of sternal re-entry and to provide adequate mitral valve exposure, antero-lateral thoracotomy has been suggested by some authors. METHODS From October 1997 to January 2007, 677 mitral valve operations have been performed in our centre using port-access video-assisted right mini-thoracotomy. Among these, 241 (35.6%) were performed on patients who had undergone one or more previous cardiac surgery procedures. RESULTS Mean cardio-pulmonary bypass time and endo-clamp time were 117+/-46 min and 71+/-31 min, respectively. Arterial cannulation was performed either on the ascending aorta, with the endo-direct cannula (112 patients, 46.5%), or peripherally with a femoral artery approach (129 patients, 53.5%). Conversion to median sternotomy was necessary in only two patients (0.8%) due to aortic dissection (one case) and left ventricle free wall rupture (one case). Median intensive care unit stay was 24h, median mechanical ventilation time was 12h; median hospital stay was 8 days. Bleeding requiring surgical revision occurred in 12 patients (4.9%). Hospital mortality was 4.9% (12/241 patients). CONCLUSIONS Port-access video-assisted right mini-thoracotomy allows good results in a difficult subset of patients; it allows minimal adhesion dissection, short ICU and hospital stay. In our practice, this technique has become the treatment of choice for mitral valve re-do surgery.

Clodronate treatment of established bone loss in cardiac recipients: a randomized study.

Background. Bone loss has been reported as a complication after heart transplantation (HTx), and the increase in bone fractures is an effective problem. Treatment of osteoporosis has obtained mixed results. In this study we evaluate the effect of treatment with an oral bisphosphonate. Methods. Sixty-four patients with low mineral density 6 months after HTx were randomized as follows: Group A received oral clodronate (1600 mg/day in two divided doses), and Group B received placebo. Every patient was also treated with 2000 mg/day of oral calcium carbonate. Bone mineral density (BMD) was measured by dual energy x-ray absorptiometry at the lumbar spine, 1/3 and 1/10 of the distal nondominant forearm before and after 12 months of treatment. Laboratory tests were performed at 3, 6, and 12 months of treatment. Results. All patients demonstrated manifest bone loss 6 months after HTx compared with normal non-HTx controls (P =0.0001). After 1 year of clodronate therapy, BMD at the lumbar spine increased from 0.77±1.4 g/cm2 to 0.86 g/cm2 (P =0.02). Laboratory tests did not show any significant variation, except for the bone isoenzyme of alkaline phosphatase, which showed a significant decrease after 1 year of treatment. The incidence of new fractures was 9.3% in the placebo group and 0% in the clodronate group. Therapy was well tolerated without impact on graft function. Conclusions. One year of clodronate therapy induced a significant increase in BMD at the lumbar spine in our HTx patients. Treatment was well tolerated without onset of new bone fractures.

Influence of temperature on adenovirus-mediated gene transfer.

OBJECTIVE The transfer of recombinant genes to donor organs may allow for novel therapeutic approaches to the challenges of acute and chronic rejection. Adenoviral vectors are capable of efficient gene transfer, but use of these vectors during donor organ preservation may be less efficient due to the low temperature. This study was designed to examine the effect of temperature on the efficiency of adenovirus-mediated gene transfer. METHODS Gene transfer to human endothelial cells, porcine vascular smooth muscle cells and cultured rat thoracic aortas was examined. Incubation with an adenoviral vector encoding for E. coli beta-galactosidase was performed for 1 h at three different temperatures: 4 degrees C, 10 degrees C and 37 degrees C. Transgene expression was assessed by histochemical staining for beta-galactosidase in transduced cells and by evaluation of beta-galactosidase activity in organ cultures. RESULTS Both in human endothelial cells and vascular smooth muscle cells the percentage of positively staining cells following transduction at 37 degrees C was significantly greater than at 4 degrees C and at 10 degrees C (30.55 +/- 7.26% vs. 14.29 +/- 3.79% and 12.43 +/- 2.47%, respectively for endothelial cells, P < 0.01 vs. 4 degrees C and 10 degrees C; and 28.25 +/- 4.52% vs. 17.91 +/- 3.76% and 16.63 +/- 3.92%, respectively for smooth muscle cells, P < 0.05 vs. 4 degrees C, P < 0.01 vs. 10 degrees C). Beta-galactosidase activity was significantly greater in aortas transduced at 37 degrees C than in vessels transduced at 4 degrees C and 10 degrees C (289,700 +/- 113,300 vs. 149,600 +/- 54,390 and 108,800 +/- 23,140 relative chemiluminesce units/mg of total protein, respectively; P < 0.05 vs. 4 degrees C, P < 0.001 vs. 10 degrees C). CONCLUSIONS The present study demonstrates that the efficiency of adenovirus-mediated gene transfer is significantly reduced at lower temperatures. The need for cold preservation of donor organs may render efficient adenovirus-mediated gene transfer more difficult in the transplantation setting.

Gene Therapy In Lung Transplantation: Effective Gene Transfer Via The Airways

OBJECTIVES Gene therapy may provide a means of modifying factors that contribute to the development of pathologic processes in transplanted lungs. Experiments were designed to study the feasibility of adenovirus-mediated gene transfer by way of the airways to the transplanted lung. METHODS Orthotopic left lung transplantation (Lewis to Lewis rats) was performed on four groups of animals. 300 microl of adenovirus solution encoding for beta-galactosidase was infused into the left bronchus of donor rats at viral concentrations of 10(8) pfu/ml (n = 5), 10(9) pfu/ml (n = 6), and 10(10) pfu/ml (n = 6), and the lung was ventilated for 5 minutes. Controls (n = 6) received medium only. Seven days after transplantation, native and transduced, transplanted lungs were harvested. Sections of lung were fixed and stained with a solution of X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside) and staining was evaluated for distribution by cell type and intensity. RESULTS Beta-galactosidase expression was absent in the control group and in the native lungs. Two of five lungs in the 10(8) group expressed beta-galactosidase, but in a limited distribution and intensity. All six lungs in the 10(9) group and five of six lungs in the 10(10) group expressed beta-galactosidase. The distribution and intensity of beta-galactosidase expression ranged from only a few cells staining per slide to up to 75%. Pneumocytes were the most frequently stained cell type followed by alveolar macrophages. CONCLUSIONS Gene transfer to the transplanted lung via the bronchial route is feasible and offers a novel technique to modify pathologic processes in the transplanted lung.

Highly efficient ex vivo gene transfer to the transplanted heart by means of hypothermic perfusion with a low dose of adenoviral vector

BACKGROUND Hypothermic conditions required for donor heart preservation may reduce gene-transfer efficiency. Experiments were designed to determine whether a perfusion technique could improve the efficiency of gene transfer to donor hearts. METHODS An adenoviral vector encoding beta-galactosidase (3.5 x 10(8) plaque-forming units) was infused into explanted rat hearts under 4 conditions (each n = 6): (1) the virus was diluted in 350 microL of University of Wisconsin solution and infused as a high-pressure bolus into the coronary arteries of donor hearts through the aortic root; (2) the virus was diluted in 5 mL of University of Wisconsin solution and circulated by means of a peristaltic pump (flow, 0.75 mL/min) through the vasculature of the donor heart for 30 minutes; (3) 5 mL of viral solution was circulated as for group 2 for 15 minutes; and (4) 5 mL of viral solution was circulated for 5 minutes at a flow rate of 2.4 mL/min. Transduced hearts were transplanted into the abdomen of syngeneic rats, and transgene expression was assessed by means of immunoassay 4 days later. RESULTS The median beta-galactosidase content was (1) 45.0 ng/mg protein (25th-75th percentile, 33-73 ng/mg), (2) 640 ng/mg protein (25th-75th percentile, 614-878 ng/mg), (3) 493.8 ng/mg protein (25th-75th percentile, 456-527 ng/mg), and (4) 503.3 ng/mg protein (25th-75th percentile, 475-562 ng/mg; P <.01 for group 2 vs group 1, and P <.05 for groups 3 and 4 vs group 1). Transgene expression was predominantly in myocytes and favored the subepicardial region of the right ventricle. CONCLUSION Hypothermic perfusion of the donor heart with an adenoviral vector resulted in efficient transgene expression compared with that induced by a single bolus injection.

Transbronchial gene transfer of endothelial nitric oxide synthase to transplanted lungs

BACKGROUND Experiments were designed to study the efficiency, distribution, and toxicity of transbronchial adenoviral-mediated transfer of endothelial constitutive nitric oxide synthase (ecNOS) gene to transplanted lungs. METHODS Syngeneic orthotopic single-lung transplantation in the rat was performed after airway administration (300 microL, 1 x 10(9) pfu/mL) of either the ecNOS gene or the marker gene beta-Gal (control group) to donor lungs (n=4 each). After 4 days, transgene expression, inflammation, and the presence of apoptosis in the transplanted lungs were assessed by molecular, immunohistochemical, and histologic techniques. RESULTS Gene transfer was confirmed by a positive polymerase chain reaction signal for the recombinant ecNOS gene, and recombinant messenger RNA by reverse transcription polymerase chain reaction. Positive immunohistochemical staining for ecNOS was present in more than 75% of pneumocytes only in ecNOS transduced lungs. Calcium-dependent nitric oxide synthase activity was increased in ecNOS- compared with betaGal-transduced lungs (2,139+/-756 versus 47+/-28 pmol x mg protein(-1) x h(-1); p < 0.05). Minimal to mild inflammation was observed in all transplanted lungs; fewer than 0.5% of cells in both groups were apoptotic. CONCLUSIONS Transbronchial transfer of ecNOS gene to the transplanted lung results in transduction of pneumocytes with expression of a functionally active transgene product.