Romain Guillemain

Human Leukocyte Antigen-G Expression After Heart Transplantation Is Associated With a Reduced Incidence of Rejection

Background—Human leukocyte antigen (HLA)-G, a nonclassic major histocompatibility complex class I molecule expressed in the extravillous cytotrophoblast at the feto-maternal interface, is known to protect the fetus from maternal cellular immunity. In a preliminary study, we showed that HLA-G is expressed in the hearts of some patients after heart transplantation. Methods and Results—In the present study, a larger number of patients was investigated to confirm this finding and to look for possible correlations between HLA-G expression and the number and types of rejection. Expression of HLA-G in endomyocardial biopsy specimens was investigated by immunohistochemical analysis, and detection of the soluble HLA-G in the serum was performed by immunoprecipitation followed by Western blot analysis. HLA-G was detected in the biopsy specimens and serum of 9 of 51 patients (18%). The number of episodes of acute rejection was significantly lower in HLA-G-positive patients (1.2±1.1) as compared with HLA-G-negative patients (4.5±2.8) (P <0.001). No chronic rejection was observed in HLA-G-positive patients, whereas 15 HLA-G-negative patients had chronic rejection (P <0.032). A longitudinal study of these patients reveals that the status of HLA-G expression was maintained after 6 months both in serum and in biopsy specimens. During this period, HLA-G-positive patients did not have chronic rejection. Conclusions—There is a significant correlation between rejection and HLA-G expression in the heart after transplantation. HLA-G expression and its effect in reducing the incidence and severity of rejection seem to be stable throughout the evolution.

Human embryonic stem cell-derived cardiac progenitors for severe heart failure treatment: first clinical case report

AIMS Comparative studies suggest that stem cells committed to a cardiac lineage are more effective for improving heart function than those featuring an extra-cardiac phenotype. We have therefore developed a population of human embryonic stem cell (ESC)-derived cardiac progenitor cells. METHODS AND RESULTS Undifferentiated human ESCs (I6 line) were amplified and cardiac-committed by exposure to bone morphogenetic protein-2 and a fibroblast growth factor receptor inhibitor. Cells responding to these cardio-instructive cues express the cardiac transcription factor Isl-1 and the stage-specific embryonic antigen SSEA-1 which was then used to purify them by immunomagnetic sorting. The Isl-1(+) SSEA-1(+) cells were then embedded into a fibrin scaffold which was surgically delivered onto the infarct area in a 68-year-old patient suffering from severe heart failure [New York Heart Association [NYHA] functional Class III; left ventricular ejection fraction (LVEF): 26%]. A coronary artery bypass was performed concomitantly in a non-infarcted area. The implanted cells featured a high degree of purity (99% were SSEA-1(+)), had lost the expression of Sox-2 and Nanog, taken as markers for pluripotency, and strongly expressed Isl-1. The intraoperative delivery of the patch was expeditious. The post-operative course was uncomplicated either. After 3 months, the patient is symptomatically improved (NYHA functional Class I; LVEF: 36%) and a new-onset contractility is echocardiographically evident in the previously akinetic cell/patch-treated, non-revascularized area. There have been no complications such as arrhythmias, tumour formation, or immunosuppression-related adverse events. CONCLUSION This observation demonstrates the feasibility of generating a clinical-grade population of human ESC-derived cardiac progenitors and combining it within a tissue-engineered construct. While any conclusion pertaining to efficacy would be meaningless, the patients functional outcome yet provides an encouraging hint. Beyond this case, the platform that has been set could be useful for generating different ESC-derived lineage-specific progenies.

Cardiac stem cells in the real world

OBJECTIVE Cardiac stem cell transplantation as a potential means of regenerating infarcted myocardium is currently receiving a great deal of interest. However, data on these endogenous cardiac precursors are primarily derived from animal studies, and their clinical relevance still remains elusive. METHODS We prospectively screened 32 endomyocardial biopsies harvested from heart transplant recipients (off rejection episodes) and 18 right appendage biopsies collected during coronary artery bypass surgery, and processed the tissue specimens for the immunohistochemical detection of markers of stemness (c-kit, MDR-1, Isl-1), hematopoietic origin (CD45), mast cells (tryptase), endothelial cells (CD105), and cardiac lineage (Nkx2.5). Confocal microscopy was used for colocalization experiments. Three right appendage biopsies were also cultured for 2 to 3 weeks, at the completion of which c-kit-positive cells were sorted by flow cytometry. RESULTS In endomyocardial biopsies, a median number of 2.7 (1.8-4) c-kit-positive cells/mm(2) were found, and this number was even significantly smaller in right appendage biopsies (1 [0.5-1.8] c-kit-positive cell/mm(2), P = .01). All of these c-kit-positive cells co-stained for CD45 and were more specifically identified as mast cells by their positive staining for the specific tryptase marker. However, none of the c-kit-positive cells expressed the markers of stemness MDR-1 and Isl-1 or colocalized with CD105. Flow cytometry confirmed the small number of c-kit-positive cells in cultured right atrial appendages. CONCLUSION These data raise a cautionary note on the therapeutic exploitation of cardiac stem cells in patients with ischemic cardiomyopathy, who may be the elective candidates for regenerative therapy.

Combined lung and liver transplantation in patients with cystic fibrosis: A 4 1/2-year experience ☆ ☆☆ ★ ★★ ♢

Patients with cystic fibrosis who have end-stage respiratory failure and associated liver cirrhosis have been considered poor candidates for lung transplantation because of high morbidity and mortality resulting from hepatic insufficiency after the operation. Since April 1989, our policy has been to combine heart-lung or lung and liver transplantation in this group of patients. Between June 1990 and March 1995, among 25 patients accepted in the program for combined transplantation, nine died awaiting transplantation and 10 underwent one of the following procedures: heart-lung-liver transplantation (n = 5), en bloc double lung-liver transplantation (n = 1), sequential double lung-liver transplantation (n = 3), and bilateral lobar lung transplantation from a split left lung and reduced liver transplantation (n = 1). There were 5 male and 5 female patients. The ages of the recipients ranged from 10 to 24 years. Mean forced expiratory volume in 1 second was 29% and mean forced vital capacity was 35% of predicted values. All patients were infected with resistant Pseudomonas, three with Pseudomonas cepaceia, and two patients had Aspergillus species in addition. All patients had severe cirrhosis with portal hypertension. Four patients had a history of esophageal variceal bleeding and two had had previous portosystemic shunts. The operation was performed as a two-stage procedure, the intrathoracic operation being completed before the abdominal stage was begun. Cardiopulmonary bypass was used in all patients because of poor clinical condition. Immunosuppression consisted of azathioprine, cyclosporine, and prednisone, as for isolated lung transplantation. There were two perioperative deaths, one caused by primary liver failure and the second by early lung dysfunction. For the first 3 months after transplantation pulmonary infection was the most common cause of morbidity. Other complications included tracheal stenosis (n = 1), bronchial stenosis (n = 1), biliary stricture (n = 2), and severe ascites (n = 3). All were successfully treated. Obliterative bronchiolitis developed in three patients. This was stabilized with FK 506 in two patients; the other patient underwent retransplantation at 38 months but eventually died of bleeding. Actuarial survival was 70% at 1 year and remained unchanged at 3 years. Significant functional improvement was observed in all survivors. For patients who have chronic respiratory failure with advanced cirrhosis, lung transplantation combined with liver transplantation can be performed with a satisfactory outcome.

MicroRNAs as non-invasive biomarkers of heart transplant rejection

AIM Rejection is one of the major causes of late cardiac allograft failure and at present can only be diagnosed by invasive endomyocardial biopsies. We sought to determine whether microRNA profiling could serve as a non-invasive biomarker of cardiac allograft rejection. METHODS We included 113 heart transplant recipients from four referral French institutions (test cohort, n = 60, validation cohort, n = 53). In the test cohort, we compared patients with acute biopsy-proven allograft rejection (n = 30) to matched control patients without rejection (n = 30), by assessing microRNAs expression in the heart allograft tissue and patients concomitant serum using RNA extraction and qPCR analysis. Fourteen miRNAs were selected on the basis of their implication in allograft rejection, endothelial activation, and inflammation and tissue specificity. RESULTS We identified seven miRNAs that were differentially expressed between normal and rejecting heart allografts: miR-10a, miR-21, miR-31, miR-92a, miR-142-3p miR-155, and miR-451 (P < 0.0001 for all comparisons). Four out of seven miRNAs also showed differential serological expression (miR-10a, miR-31, miR-92a, and miR-155) with strong correlation with their tissular expression. The receiver-operating characteristic analysis showed that these four circulating miRNAs strongly discriminated patients with allograft rejection from patients without rejection: miR-10a (AUC = 0.975), miR-31 (AUC = 0.932), miR-92a (AUC = 0.989), and miR-155 (AUC = 0.998, P < 0.0001 for all comparisons). We confirmed in the external validation set that these four miRNAs highly discriminated patients with rejection from those without. The discrimination capability of the four miRNAs remained significant when stratified by rejection diagnosis (T-cell-mediated rejection or antibody-mediated rejection) and time post-transplant. CONCLUSION This study demonstrates that a differential expression of miRNA occurs in rejecting allograft patients, not only at the tissue level but also in the serum, suggesting their potential relevance as non-invasive biomarkers in heart transplant rejection.

Clinical outcome following lung transplantation in patients with cystic fibrosis colonised with Burkholderia cepacia complex: results from two French centres

Background: Infection with Burkholderia cepacia complex (BCC) is a life threatening complication of cystic fibrosis (CF), often seen as a contraindication for lung transplantation. Methods: A long term retrospective study was conducted of all patients with CF undergoing lung transplants from January 1990 to October 2006 in two French centres allowing transplantation in patients colonised with BCC. Results: 22 of the 247 lung transplant patients with CF were infected with BCC (B cenocepacia genomovar III (n = 8), B multivorans genomovar II (n = 11), B vietnamiensis genomovar V (n = 2) and B stabilis genomovar IV (n = 1)). BCC colonisation was not associated with any significant excess mortality (HR 1.5, 95% CI 0.7 to 3.2; p = 0.58). However, early mortality rates tended to be higher in the BCC group than in the non-BCC group (3 month survival: 85% vs 95%, respectively; log rank p = 0.05). Univariate analysis showed that the risk of death was significantly higher for the eight patients infected with B cenocepacia than for the other 14 colonised patients (HR 3.2, 95% CI 1.1 to 5.9; p = 0.04). None of the other risk factors tested—primary graft failure, late extubation, septicaemia—had a significant effect. The 5 year cumulative incidence rate of bronchiolitis obliterans syndrome was not significantly higher in the BCC group than in the non-BCC group (38% vs 24%, respectively; p = 0.35). Conclusion: Our results suggest that BCC infection with a non-genomovar III organism may not be associated with excess mortality after lung transplantation in patients with CF and should not be seen as sufficient reason to exclude lung transplantation. However, colonisation with B cenocepacia remains potentially detrimental.

Very Late Heart Transplant Rejection Is Associated with Microvascular Injury, Complement Deposition and Progression to Cardiac Allograft Vasculopathy

In heart transplants, the significance of very late rejection (after 7 years post‐transplant, VLR) detected by routine endomyocardial biopsies (EMB) remains uncertain. Here, we assessed the prevalence, histopathological and immunological phenotype, and outcome of VLR in clinically stable patients. Between 1985 and 2009, 10 662 protocol EMB were performed at our institution in 398 consecutive heart transplants recipients. Among the 196 patients with >7‐year follow‐up, 20 (10.2%) presented subclinical ≥3A/2R‐ISHLT rejection. The VLR group was compared to a matched control group of patients without rejection. All biopsies were stained for C4d/C3d/CD68 with sera screened for the presence of donor‐specific antibodies (DSAs). In addition to cellular infiltrates with myocyte damage, 60% of VLR patients had evidence of intravascular macrophages. C4d and/or C3d‐capillary deposition was found in 55% VLR EMB. All cases of VLR associated with microcirculation injury had DSAs (mean DSAmax−MFI = 1751 ± 583). This entity was absent from the control group (p < 0.0001). Finally, after a similar follow‐up postreference EMB of 6.4 ± 1 years, the mean of CAV grade was 0.76 ± 0.18 in the control group compared to 2.06 ± 0.26 in the VLR group respectively, p = 0.001). There was no difference in patient survival between study and control groups. In conclusion, VLR is frequently associated with complement‐cascade activation, microvascular injury and DSA, suggesting an antibody‐mediated process. VLR is associated with a dramatic progression to severe CAV in long‐term follow‐up.

Voriconazole pharmacokinetic variability in cystic fibrosis lung transplant patients.

Background. Aspergillosis is a high‐risk complication in cystic fibrosis (CF) lung transplant patients. Azole antifungal drugs inhibit CYP3A4, resulting in significant metabolic drug–drug interactions. Voriconazole (VRZ) was marketed without therapeutic drug monitoring (TDM) recommendations, consistent with favorable pharmacokinetics, but regular determinations of plasma VRZ concentration were introduced in our center to manage interactions with calcineurin inhibitors and to document the achievement of therapeutic levels.

Lung transplantation for lymphangioleiomyomatosis: the French experience.

Background. Lymphangioleiomyomatosis (LAM) is a rare disease, leading in some cases to end-stage respiratory failure. Lung transplantation (LT) represents a therapeutic option in advanced pulmonary LAM. Methods. We conducted a retrospective multicenter study of 44 patients who underwent LT for LAM at 9 centers in France between 1988 and 2006. Results. All patients were women with a mean age of 41±10 years at LT. There were 34 single-lung transplants and 11 bilateral transplants (one retransplantation). Prior clinical events related to LAM were present in 75% of the patients and previous thoracic surgical procedures were noted in 86.6% of cases. At the latest preoperative evaluation, 30 patients had an obstructive pattern (mean forced expiratory volume in 1 second: 26%±14% of predicted) and 15 had a combined restrictive and obstructive pattern, with a mean KCO=27%±8.8% of predicted, PaO2=52.8±10.4 and PaCO2=42.6±9.8 mm Hg. Intraoperative cardiopulmonary bypass was required in 13 cases. The length of mechanical ventilation was 7.5±12.8 days. The median duration of follow-up was 37 months. The 1, 2, 5, and 10 years survival rates were 79.6%, 74.4%, 64.7%, and 52.4%, respectively. Extensive pleural adhesions were found in 21 patients leading to severe intraoperative hemorrhage. Postoperative LAM-related complications were pneumothorax in the native lung in five patients, chylothorax in six, bronchial dehiscence or stenosis in seven. There were two cases of recurrence of LAM. Conclusion. Despite a high morbidity mainly caused by previous surgical interventions and disease-related complications, LT is a satisfactory therapeutic option for end-stage respiratory failure in LAM.

Renal histopathological lesions after lung transplantation in patients with cystic fibrosis.

We have analyzed the evolution of renal status beyond the perioperative period in patients with cystic fibrosis (CF) undergoing lung transplantation and presented histological analysis of 15 patients biopsied for an episode of accelerated renal function loss (RFL).