Multiple breath washout in pediatric patients after lung transplantation.

Forced expiratory volume in 1 second (FEV1 ) from spirometry is the most commonly used parameter to detect early allograft dysfunction after lung transplantation (LTx). There are concerns regarding its sensitivity. Nitrogen-multiple breath washout (N2 -MBW) is sensitive at detecting early global (lung clearance index [LCI]) and acinar (Sacin ) airway inhomogeneity. We investigated whether N2 -MBW indices indicate small airways pathology after LTx in children with stable spirometry. Thirty-seven children without bronchiolitis obliterans syndrome [BOS] at a median of 1.6 (0.6-3.0) years after LTx underwent N2 -MBW and spirometry, 28 of those on 2 occasions (≤6 months apart) during clinically stable periods. Additional longitudinal data (11 and 8 measurements, respectively) are provided from 2 patients with BOS. In patients without BOS, LCI and Sacin were significantly elevated compared with healthy controls. LCI was abnormal at the 2 test occasions in 81% and 71% of patients, respectively, compared with 30% and 39% of patients with abnormal FEV1 /forced vital capacity (FVC). Correlations of LCI with FEV1 /FVC (r = 0.1, P = .4) and FEV1 (r = -0.1, P = .6) were poor. N2 -MBW represents a sensitive and reproducible tool for the early detection of airways pathology in stable transplant recipients. Moreover, indices were highly elevated in both patients with BOS. Spirometry and LCI showed poor correlation, indicating distinct and complementary physiologic measures.

Single lung transplantation from a donor 8 months after double lung transplantation.

Scarcity of donors leads transplant surgeons to consider extended-criteria lungs and occasionally to accept the unlikely. Here we report a case of successful single lung transplantation from a donor 8 months after double lung transplantation.

Association of Higher CD4+ CD25high CD127low , FoxP3+ , and IL-2+ T Cell Frequencies Early After Lung Transplantation With Less Chronic Lung Allograft Dysfunction at Two Years.

Regulatory T cells (Treg) can regulate alloantigens and may counteract chronic lung allograft dysfunction (CLAD) in lung transplantation. We analyzed Treg in peripheral blood prospectively and correlated percentages of subpopulations with the incidence of CLAD at 2 years. Among lung-transplanted patients between January 2009 and July 2011, only patients with sufficient Treg measurements were included into the study. Tregs were measured immediately before lung transplantation, at 3 weeks and 3, 6, 12, and 24 months after transplantation and were defined as CD4+ CD25high T cells and further analyzed for CTLA4, CD127, FoxP3, and IL-2 expressions. Between January 2009 and July 2011, 264 patients were transplanted at our institution. Among the 138 (52%) patients included into the study, 31 (22%) developed CLAD within 2 years after transplantation. As soon as 3 weeks after lung transplantation, a statistically significant positive association was detected between Treg frequencies and later absence of CLAD. At the multivariate analysis, increasing frequencies of CD4+ CD25high CD127low , CD4+ CD25high FoxP3+ and CD4+ CD25high IL-2+ T cells at 3 weeks after lung transplantation emerged as protective factors against development of CLAD at 2 years. In conclusion, higher frequencies of specific Treg subpopulations early after lung transplantation are protective against CLAD development.

Everolimus Versus Mycophenolate Mofetil De Novo After Lung Transplantation: A Prospective, Randomized, Open-Label Trial.

The role of mammalian target of rapamycin (mTOR) inhibitors in de novo immunosuppression after lung transplantation is not well defined. We compared Everolimus versus mycophenolate mofetil in an investigator-initiated single-center trial in Hannover, Germany. A total of 190 patients were randomly assigned 1:1 on day 28 posttransplantation to mycophenolate mofetil (MMF) or Everolimus combined with cyclosporine A (CsA) and steroids. Patients were followed up for 2 years. The primary endpoint was freedom from bronchiolitis obliterans syndrome (BOS). The secondary endpoints were incidence of acute rejections, infections, treatment failure and kidney function. BOS-free survival in intention-to-treat (ITT) analysis was similar in both groups (p = 0.174). The study protocol was completed by 51% of enrolled patients. The per-protocol analysis shows incidence of bronchiolitis obliterans syndrome (BOS): 1/43 in the Everolimus group and 8/54 in the MMF group (p = 0.041). Less biopsy-proven acute rejection (AR) (p = 0.005), cytomegalovirus (CMV) antigenemia (p = 0.005) and lower respiratory tract infection (p = 0.003) and no leucopenia were seen in the Everolimus group. The glomerular filtration rate (GFR) decreased in both groups about 50% within 6 months. Due to a high withdrawal rate, the study was underpowered to prove a difference in BOS-free survival. The dropout rate was more pronounced in the Everolimus group. Secondary endpoints indicate potential advantages of Everolimus-based protocols but also a potentially higher rate of drug-related serious adverse events.

In Vivo Development of Transplant Arteriosclerosis in Humanized Mice Reflects Alloantigen Recognition and Peripheral Treg Phenotype of Lung Transplant Recipients.

Experimentally, regulatory T cells inhibit rejection. In clinical transplantations, however, it is not known whether T cell regulation is the cause for, or an epiphenomenon of, long-term allograft survival. Here, we study naïve and alloantigen-primed T cell responses of clinical lung transplant recipients in humanized mice. The pericardiophrenic artery procured from human lung grafts was implanted into the aorta of NODrag-/- /IL-2rγc-/- mice reconstituted with peripheral blood mononuclear cells (PBMCs) from the respective lung recipient. Naïve or primed allogeneic PBMCs procured 21 days post-lung transplantation with or without enriching for CD4+ CD25high T cells were used. Transplant arteriosclerosis was assessed 28 days later by histology. Mice reconstituted with alloantigen-primed PBMCs showed significantly more severe transplant arteriosclerosis than did mice with naïve PBMCs (p = 0.005). Transplant arteriosclerosis was equally suppressed by enriching for autologous naïve (p = 0.012) or alloantigen-primed regulatory T cells (Tregs) (p = 0.009). Alloantigen priming in clinical lung recipients can be adoptively transferred into a humanized mouse model. Transplant arteriosclerosis elicited by naïve or alloantigen-primed PBMCs can be similarly controlled by potent autologous Tregs. Cellular therapy with expanded autologous Tregs in lung transplantation might be a promising future strategy.

Prolonged Mechanical Ventilation After Lung Transplantation-A Single-Center Study.

This single-center study examines the incidence, etiology, and outcomes associated with prolonged mechanical ventilation (PMV), defined as time to definite spontaneous ventilation >21 days after double lung transplantation (LTx). A total of 690 LTx recipients between January 2005 and December 2012 were analyzed. PMV was necessary in 95 (13.8%) patients with decreasing incidence during the observation period (p < 0.001). Independent predictors of PMV were renal replacement therapy (odds ratio [OR] 11.13 [95% CI, 5.82-21.29], p < 0.001), anastomotic dehiscence (OR 8.74 [95% CI 2.42-31.58], p = 0.001), autoimmune comorbidity (OR 5.52 [95% CI 1.86-16.41], p = 0.002), and postoperative neurologic complications (OR 5.03 [95% CI 1.98-12.81], p = 0.001), among others. Overall 1-year survival was 86.0% (90.4% for LTx between 2010 and 2012); it was 60.7% after PMV and 90.0% in controls (p < 0.001). Conditional long-term outcome among hospital survivors, however, did not differ between the groups (p = 0.78). Multivariate analysis identified renal replacement therapy (hazard ratio [HR] 3.55 [95% CI 2.40-5.25], p < 0.001), post-LTx extracorporeal membrane oxygenation (HR 3.47 [95% CI 2.06-5.83], p < 0.001), and prolonged inotropic support (HR 1.95 [95% CI 1.39-2.75], p < 0.001), among others, as independent predictors of mortality. In conclusion, PMV complicated 14% of LTx procedures and, although associated with increased in-hospital mortality, outcomes among patients surviving to hospital discharge were unaffected.

Augmentation of Transient Donor Cell Chimerism and Alloantigen-Specific Regulation of Lung Transplants in Miniature Swine.

Donor alloantigen infusion induces T cell regulation and transplant tolerance in small animals. Here, we study donor splenocyte infusion in a large animal model of pulmonary transplantation. Major histocompatibility complex-mismatched single lung transplantation was performed in 28 minipigs followed by a 28-day course of methylprednisolone and tacrolimus. Some animals received a perioperative donor or third party splenocyte infusion, with or without low-dose irradiation (IRR) before surgery. Graft survival was significantly prolonged in animals receiving both donor splenocytes and IRR compared with controls with either donor splenocytes or IRR only. In animals with donor splenocytes and IRR, increased donor cell chimerism and CD4(+) CD25(high+) T cell frequencies were detected in peripheral blood associated with decreased interferon-γ production of leukocytes. Secondary third-party kidney transplants more than 2 years after pulmonary transplantation were acutely rejected despite maintained tolerance of the lung allografts. As a cellular control, additional animals received third-party splenocytes or donor splenocyte protein extracts. While animals treated with third-party splenocytes showed significant graft survival prolongation, the subcellular antigen infusion showed no such effect. In conclusion, minipigs conditioned with preoperative IRR and donor, or third-party, splenocyte infusions may develop long-term donor-specific pulmonary allograft survival in the presence of high levels of circulating regulatory T cells.

Impact of CLAD Phenotype on Survival After Lung Retransplantation: A Multicenter Study.

Chronic lung allograft dysfunction (CLAD) remains a major problem after lung transplantation with no definitive treatment except redo lung transplantation (re-LTx) in selected candidates. However, CLAD is not a homogeneous entity and different phenotypes exist. Therefore, we aimed to evaluate the effect of CLAD phenotypes on survival after re-LTx for CLAD. Patients who underwent re-LTx for respiratory failure secondary to CLAD in four LTx centers between 2003 and 2013 were included in this retrospective analysis. Bronchiolitis obliterans syndrome (BOS) and restrictive CLAD (rCLAD) were distinguished using pulmonary function, radiology and explant lung histopathology. Patient variables pre- and post-re-LTx were collected and analyzed. A total of 143 patients underwent re-LTx for CLAD resulting in 94 BOS (66%) and 49 rCLAD (34%) patients. Unadjusted and adjusted survival after re-LTx for rCLAD was worse compared to BOS (HR = 2.60, 1.59-4.24; p < 0.0001 and HR = 2.61, 1.51-4.51; p = 0.0006, respectively). Patients waiting at home prior to re-LTx experienced better survival compared to hospitalized patients (HR 0.40; 0.23-0.72; p = 0.0022). Patients with rCLAD redeveloped CLAD earlier and were more likely to redevelop rCLAD. Survival after re-LTx for rCLAD is worse compared to BOS. Consequently, re-LTx for rCLAD should be critically discussed, particularly when additional peri-operative risk factors are present.

Paclitaxel-coated balloons in refractory nonanastomostic airway stenosis following lung transplantation.

Airway stenosis represents the commonest airway complication following lung transplantation, affecting between 7% and 18% of patients. Existing treatment options offer limited efficacy and can cause additional patient morbidity. Paclitaxel-coated balloons (PCB) have proved effective in managing postinterventional coronary artery re-stenosis. In a first-in-man study, we evaluated similar PCBs in refractory nonanastomotic airway stenosis in 12 patients. Following a single application, luminal patency was maintained in 50% at 270 days. No significant peri-interventional or early postinterventional complications occurred. Given these encouraging initial findings, further studies appear warranted.

Significance of new lung infiltrates in outpatients after lung and heart-lung transplantation.

BACKGROUND: Infection and rejection represent major complications following lung transplantation and are often associated with pulmonary infiltrates. The differential diagnosis of these infiltrates depends on their timing after transplantation. The aim of this study was to characterize lung transplant recipients (LTR) presenting with new pulmonary infiltrates. METHODS: A retrospective analysis of all LTR and heart-lung transplant recipients attending outpatient follow-up at our institution between September 1, 2006 and October 14, 2011 was performed. All patients presenting with new pulmonary infiltrates on chest x-ray who underwent bronchoscopy were included. RESULTS: A total of 913 patients accounted for 13,156 attendances, with 3,912 bronchoscopies being performed. Seventy-eight patients (9%) exhibited new pulmonary infiltrates and proceeded to bronchoscopy. Infiltrates occurred at a median 15 (interquartile range [IQR] 5-39) months after transplantation. Forty-eight patients (62%) were male, and median patient age was 47 (IQR 29-57) years. Subsequent investigation revealed pneumonia to be the underlying cause in 63 patients (81%). In the remaining patients, chronic lung allograft dysfunction (CLAD) was responsible in 6 (8%), acute rejection in 5 (6%), and toxic pneumonitis in 4 (5%) patients. Overall 1-year survival in LTR presenting with new infiltrates was 97%, compared with 96% for all LTR attending our Outpatient Department. CONCLUSIONS: New pulmonary infiltrates occurring after the first month in LTR are most likely due to infection. Through prompt diagnosis and treatment, early mortality appears unaffected. Late mortality remains attributable to CLAD.

The impact of abdominal complications on the outcome after thoracic transplantation--a single center experience.

BACKGROUND: Abdominal complications after thoracic transplantation (Tx) are potentially associated with an increased risk of mortality. We recently reported about the severe outcome after bowel perforation in patients following lung transplantation (LuTx). The aim of the present study was to likewise identify the risk factors with an impact on patient survival following heart transplantation (HTx). METHODS: A retrospective analysis for the frequency and outcome of abdominal interventions following HTx was performed in 342 patients, and these data thereafter compared to a re-evaluated pool of 1,074 patients following LuTx. All patients were transplanted at Hanover Medical School, Germany, between January 2000 and October 2011. RESULTS: The incidence for abdominal surgery was comparable between patients following HTx (n = 33; 9.6 %) and LuTx (n = 90; 8.4 %). Elective operations were more frequently performed in patients after HTx (8.5 vs. 5.1 %). In contrast, the incidence of emergency interventions was higher after LuTx (5.3 %) than that following HTx (2.3 %). Herewith associated was the mortality observed in these transplant recipients (15.3 and 9.9 % for LuTx and HTx, respectively). Leading diagnosis for emergency surgery was bowel perforation (n = 18, regarding all cases). In 11 of these patients, perforation occurred within the first 6 months after Tx and eight of them died in the course of this complication (one patient after HTx and seven patients after LuTx). CONCLUSIONS: Abdominal complications after HTx are less frequently than after LuTx but equally correlate with a high mortality rate. In finding or even reasonable suspicion of an acute abdomen after thoracic Tx, a broad practice for extended diagnostics and a low barrier for an early explorative laparotomy thus are recommended.

Phenotyping established chronic lung allograft dysfunction predicts extracorporeal photopheresis response in lung transplant patients.

Chronic lung allograft dysfunction (CLAD) remains the leading cause of mortality in lung transplant recipients after the first year. Treatment remains limited and unpredictable. Existing data suggests extracorporeal photopheresis (ECP) may be beneficial. This study aimed to identify factors predicting treatment response and the prognostic implications. A single center retrospective analysis of all patients commencing ECP for CLAD between November 1, 2007 and September 1, 2011 was performed. In total 65 patients were included, 64 of whom had deteriorated under azithromycin. Median follow-up after commencing ECP was 503 days. Upon commencing ECP, all patients were classified using proposed criteria for emerging clinical phenotypes, including "restrictive allograft syndrome (RAS)", "neutrophilic CLAD (nCLAD)" and "rapid decliners". At follow-up, 8 patients demonstrated ≥10% improvement in FEV1 , 27 patients had stabilized and 30 patients exhibited ≥10% decline in FEV1 . Patients fulfilling criteria for "rapid decliners" (n=21, p=0.005), RAS (n=22, p=0.002) and those not exhibiting neutrophilia in bronchoalveolar lavage (n=44, p=0.01) exhibited poorer outcomes. ECP appears an effective second line treatment in CLAD patients progressing under azithromycin. ECP responders demonstrated improved progression-free survival (median 401 vs. 133 days). Proposed CLAD phenotypes require refinement, but appear to predict the likelihood of ECP response.

Clinical significance of quantitative cytomegalovirus detection in bronchoalveolar lavage fluid in lung transplant recipients.

OBJECTIVES: Cytomegalovirus (CMV) is an important pathogen after lung transplant (LTx) and may be associated with bronchiolitis obliterans syndrome (BOS). We assessed the outcome of LTx patients with CMV DNA-positive bronchoalveolar lavage (BAL) during surveillance. METHODS: A single-center retrospective study was performed. Outpatients transplanted between September 2007 and February 2010, who had undergone at least 3 BALs, were included. CMV DNA load in BAL was measured by polymerase chain reaction (PCR). Monitoring of peripheral blood antigenemia was performed simultaneously. RESULTS: In total, 103 LTx patients underwent 1118 BALs. During median follow-up of 639 days (interquartile range, 495-780), 53 (51%) patients exhibited at least 1 positive CMV PCR in BAL. The incidence of positive CMV PCR varied post transplantation, with 6%, 30%, and 25% of BALs testing positive at <6 months, 6-24 months, and >24 months, respectively. Neither CMV BAL positivity, positive CMV antigenemia, nor dual positivity were significantly associated with BOS-free survival during follow-up. Patients with CMV-positive BAL more frequently developed CMV antigenemia in the first year (44% vs. 5%, respectively, log-rank P < 0.001). CONCLUSIONS: Detection of CMV-positive BAL after the sixth month appears common, but did not correlate with BOS-free survival after LTx in this study. An increased risk of developing blood antigenemia was observed in patients with positive CMV PCR in BAL, warranting close follow-up.

Concept of "awake venovenous extracorporeal membrane oxygenation" in pediatric patients awaiting lung transplantation.

In patients awaiting LuTx, MV and ECMO are often the last ways to create a bridge to LuTx. Both interventions are associated with a poor posttransplant outcome and survival rate. To improve the results of these patients, new "bridging-strategies" are necessary. Recent reports demonstrate promising results for the concept of "awake ECMO" in adult patients. To date, no data on this approach in pediatric patients have been available. We therefore describe the use of VV-ECMO as a treatment strategy for RF in awake pediatric patients. It presents our experiences with the first three children treated using this new concept. Mean amount of time on ECMO was 44 days (range, 11.5-109 days). Two patients were successfully bridged to their LuTx. Both are still alive without any recurrences (24 and three months following LuTx). One patient died before a further LuTx after 109 days on ECMO due to adenoviral infection. Although reintubation was necessary in two patients, and total time being awake while on ECMO was <50%, we conclude that the concept of "awake VV-ECMO" is feasible for the treatment of RF and can be used as a "bridging therapy" to LuTx.

Calcium domains around single and clustered IP3 receptors and their modulation by buffers.

We study Ca(2+) release through single and clustered IP(3) receptor channels on the ER membrane under presence of buffer proteins. Our computational scheme couples reaction-diffusion equations and a Markovian channel model and allows our investigating the effects of buffer proteins on local calcium concentrations and channel gating. We find transient and stationary elevations of calcium concentrations around active channels and show how they determine release amplitude. Transient calcium domains occur after closing of isolated channels and constitute an important part of the channel's feedback. They cause repeated openings (bursts) and mediate increased release due to Ca(2+) buffering by immobile proteins. Stationary domains occur during prolonged activity of clustered channels, where the spatial proximity of IP(3)Rs produces a distinct [Ca(2+)] scale (0.5-10 microM), which is smaller than channel pore concentrations (>100 microM) but larger than transient levels. While immobile buffer affects transient levels only, mobile buffers in general reduce both transient and stationary domains, giving rise to Ca(2+) evacuation and biphasic modulation of release amplitude. Our findings explain recent experiments in oocytes and provide a general framework for the understanding of calcium signals.

Extracorporeal membrane oxygenation in nonintubated patients as bridge to lung transplantation.

We report on the use of veno-arterial extracorporeal membrane oxygenation (ECMO) as a bridging strategy to lung transplantation in awake and spontaneously breathing patients. All five patients described in this series presented with cardiopulmonary failure due to pulmonary hypertension with or without concomitant lung disease. ECMO insertion was performed under local anesthesia without sedation and resulted in immediate stabilization of hemodynamics and gas exchange as well as recovery from secondary organ dysfunction. Two patients later required endotracheal intubation because of bleeding complications and both of them eventually died. The other three patients remained awake on ECMO support for 18-35 days until the time of transplantation. These patients were able to breathe spontaneously, to eat and drink, and they received passive and active physiotherapy as well as psychological support. All of them made a full recovery after transplantation, which demonstrates the feasibility of using ECMO support in nonintubated patients with cardiopulmonary failure as a bridging strategy to lung transplantation.

Modeling of the modulation by buffers of Ca2+ release through clusters of IP3 receptors.

Intracellular Ca(2+) release is a versatile second messenger system. It is modeled here by reaction-diffusion equations for the free Ca(2+) and Ca(2+) buffers, with spatially discrete clusters of stochastic IP(3) receptor channels (IP(3)Rs) controlling the release of Ca(2+) from the endoplasmic reticulum. IP(3)Rs are activated by a small rise of the cytosolic Ca(2+) concentration and inhibited by large concentrations. Buffering of cytosolic Ca(2+) shapes global Ca(2+) transients. Here we use a model to investigate the effect of buffers with slow and fast reaction rates on single release spikes. We find that, depending on their diffusion coefficient, fast buffers can either decouple clusters or delay inhibition. Slow buffers have little effect on Ca(2+) release, but affect the time course of the signals from the fluorescent Ca(2+) indicator mainly by competing for Ca(2+). At low [IP(3)], fast buffers suppress fluorescence signals, slow buffers increase the contrast between bulk signals and signals at open clusters, and large concentrations of buffers, either fast or slow, decouple clusters.

Bridge to thoracic organ transplantation in patients with pulmonary arterial hypertension using a pumpless lung assist device.

We describe a novel technique of pumpless extracorporeal life support in four patients with cardiogenic shock due to end-stage pulmonary hypertension (PH) including patients with veno-occlusive disease (PVOD) using a pumpless lung assist device (LAD). The device was connected via the pulmonary arterial main trunk and the left atrium, thereby creating a septostomy-like shunt with the unique addition of gas exchange abilities in parallel to the lung. Using this approach, all four patients were successfully bridged to bilateral lung transplantation and combined heart-lung transplantation, respectively. Although all patients presented in cardiogenic shock, hemodynamic unloading of the right ventricle using the low-resistance LAD stabilized the hemodynamic situation immediately so that no pump support was subsequently required.

Intentional ABO-incompatible lung transplantation.

We report on a case of intentional blood group incompatible lung transplantation. A blood group O cystic fibrosis patient was mechanically ventilated and put on interventional lung assist for severe respiratory decompensation. Since timely allocation of a blood group O donor lung was impossible, an AB deceased donor lung rescue allocation was accepted and the transplant performed using a pre-, peri- and postoperative antibody depletion protocol including plasmapheresis, ivIg administration, rituximab and immunoadsorption. Nine months after the transplant the patient is at home and well.

Induction and inhibition of Friend erythroleukemia cell differentiation by pyrimidine analogs: analysis of the requirement for intracellular accumulation and incorporation into DNA.

Alkyldeoxyuridines which differ from thymidine by a C5 substitution of straight or branched alkyl chains of two to six carbon atoms have been tested for their ability to be taken up, phosphorylated, and incorporated into DNA. Analysis of the uptake of 5-ethyl-2'-deoxyuridine and 5-propyl-2'-deoxyuridine (n-PrdU)--similar to both thymidine and 5-bromo-2'-deoxyuridine--indicates that transport is dependent upon a functional cellular thymidine kinase. All of the aforementioned pyrimidines with the exception of n-PrdU are phosphorylated to the triphosphate and incorporated into DNA. The homologs 5-iso-propyl-2'-deoxyuridine (iso-PrdU) and 5-hexyl-2'-deoxyuridine are neither transported into the cell, phosphorylated, nor incorporated into DNA. These analogs were tested (i) for their ability to induce in the absence of dimethyl sulfoxide and (ii) to determine whether they enhance or inhibit dimethyl sulfoxide-induced differentiation of Friend erythroleukemia cells. Inhibition of erythroid differentiation appears to require the incorporation of thymidine analogs into DNA, and thus only 5-ethyl-2'-deoxyuridine and 5-bromo-2'-deoxyuridine were effective in inhibiting dimethyl sulfoxide-induced differentiation. The observation that iso-PrdU, and to a lesser extent n-PrdU and 5-propyldeoxyuridine monophosphate, induce differentiation under conditions in which they are not detectable intracellularly is strong evidence that this class of inducer acts at the cell membrane.