Basophils Trigger Fibroblast Activation in Cardiac Allograft Fibrosis Development.

Fibrosis is a major component of chronic cardiac allograft rejection. Although several cell types are able to produce collagen, resident (donor-derived) fibroblasts are mainly responsible for excessive production of extracellular matrix proteins. It is currently unclear which cells regulate production of connective tissue elements in allograft fibrosis and how basophils, as potential producers of profibrotic cytokines, are involved this process. We studied this question in a fully MHC-mismatched model of heart transplantation with transient depletion of CD4(+) T cells to largely prevent acute rejection. The model is characterized by myocardial infiltration of leukocytes and development of interstitial fibrosis and allograft vasculopathy. Using depletion of basophils, IL-4-deficient recipients and IL-4 receptor-deficient grafts, we showed that basophils and IL-4 play crucial roles in activation of fibroblasts and development of fibrotic organ remodeling. In the absence of CD4(+) T cells, basophils are the predominant source of IL-4 in the graft and contribute to expansion of myofibroblasts, interstitial deposition of collagen and development of allograft vasculopathy. Our results indicated that basophils trigger the production of various connective tissue elements by myofibroblasts. Basophil-derived IL-4 may be an attractive target for treatment of chronic allograft rejection.

Protective function of pirfenidone and everolimus on the development of chronic allograft rejection after experimental lung transplantation.

Long-term survival of lung allografts is limited by chronic rejection (CR). Oxidative stress (OxS) plays a central role in the development of CR. We investigated the influence of pirfenidone (alone or in combination with everolimus) on OxS and CR. A rat model of left lung allo-transplantation (F344-to-WKY) was used to evaluate the effects of pirfenidone alone [0,85% in chow from postoperative day (POD) -3 to 20/60] and in combination with everolimus [2,5 mg/kg bw daily from POD 7 to 20/60]. Allografts of non-treated animals, everolimus treated animals and right, non-transplanted lungs were used as references. Immunohistology of myeloperoxidase (MPO), haemoxygenase-1 (HO-1), iron and platelet-derived-growth-factor-receptor-alpha (PDGFR-a) were performed. On POD 20, all groups showed severe acute rejection (ISHLT A3-4/B1R-B2R). Groups treated with pirfenidone showed a lower interstitial inflammatory infiltration and a lower participation of highly fibrotic degenerated vessels (ISHLT-D2R). In the long term follow up (POD 60), pirfenidone alone significantly reduced chronic airway rejection (ISHLT-C; p≤0.05), interstitial fibrosis (IF; p≤0.05), content of collagen (p≤0.05), expression of PDGFR-a (p≤0.05) and the deposition of iron (p≤0.05). All groups treated with pirfenidone showed a high expression of the cytoprotective enzyme HO-1 (p≤0.05). The additional application of everolimus resulted in a significant decrease of chronic airway rejection (ISHLT-C; p≤0.05), vasculopathy (ISHLT; p≤0.05) and IF (p≤0.05). In conclusion, early application of pirfenidone inhibited the progression of CR by its anti-fibrotic and anti-oxidative properties. The additional application of an m-TOR-inhibitor increased the anti-fibrotic effects of pirfenidone which resulted in a reduction of CR after experimental LTx.

Synergism of imatinib mesylate and everolimus in attenuation of bronchiolitis obliterans after rat LTX.

Bronchiolitis obliterans (BO) is a progressive and fatal disease after lung transplantation (LTX). Dysregulated growth factor-induced proliferation of myofibroblasts seems to be responsible for the development of BO. The aim was to confirm the efficacy of both inhibitors of receptor tyrosine kinases (RTKI) and of mammalian target of rapamycin (mTORI) after rat LTX. We used a rat model of left lung allo-transplantation (F344-to-WKY) to evaluate the effect of imatinib (RTKI; 20 mg/kg/day; postoperative day (POD) 0-100) alone or in combination with everolimus (mTORI; 2.5 mg/kg/day; POD 14-100). Non-treated animals were the reference. In non-treated rats, acute rejection (AR) peaked between POD 20 and 30 (19/19) and ended in chronic rejection (CR) on POD 60/100 (12/12). Imatinib alone did not prevent AR (6/6), but attenuated the degree of degenerated bronchioles on POD 30 (non-treated, 57%; imatinib, 4%), and increased the allografts free of CR on POD 60/100 (3/12). A combination of imatinib and everolimus significantly reduced AR, attenuated fibrotic degenerated bronchioles (5%) and vessels (non-treated, 24%; combination therapy, 11%) on POD 30, and reduced fibrotic degenerated vessels (non-treated, 97%; combination therapy, 43%) and bronchioles (non-treated, 88%; combination therapy, 34%) on POD 60/100. Fifty percent of the animals were completely free of BO and vasculopathy. In conclusion, co-application of RTKI and mTORI attenuated the development of BO and vasculopathy. Thus, imatinib might be an interesting therapeutic approach after LTX.

Inhibition of innate co-receptor TREM-1 signaling reduces CD4(+) T cell activation and prolongs cardiac allograft survival.

The innate receptor "triggering-receptor-expressed-on-myeloid-cells-1" (TREM-1) enhances downstream signaling of "pattern recognition receptor" (PRR) molecules implicated in inflammatory responses. However the mechanistic role of TREM-1 in chronic heart rejection has yet to be elucidated. We examined the effect of TREM-1(+) antigen-presenting cells (APC) on alloreactive CD4(+) lymphocytes. Bm12 donor hearts were transplanted into wild-type MHC-class-II-mismatched C57BL/6J recipient mice. Progressive allograft rejection of bm12-donor hearts with decreased organ function, severe vasculopathy and allograft fibrosis was evident within 4 weeks. TREM-1(+) CD11b(+) MHC-II(+) F4/80(+) CCR2(+) APC and IFNγ-producing CD4(+) cells were detected during chronic rejection. Peptide inhibition of TREM-1 attenuated graft vasculopathy, reduced graft-infiltrating leukocytes and prolonged allograft survival, while being accompanied by sustained low levels of CD4(+) and CD8(+) cell infiltration. Remarkably, temporary inhibition of TREM-1 during early immune activation was sufficient for long-term allograft survival. Mechanistically, TREM-1 inhibition leads to reduced differentiation and proliferation of IFNγ-producing Th1 cells. In conclusion, TREM-1 influences chronic heart rejection by regulating the infiltration and differentiation of CD4(+) lymphocytes.

Early administration of FTY720 prevents chronic airway as well as vascular destruction in experimental rat lung transplantation.

BACKGROUND: Chronic rejection (CR) in terms of bronchiolitis obliterans (BO) and vascular sclerosis (VS) still represents the major obstacle for pulmonary graft survival in the medium and long term course after lung transplantation (LTX). Aside from nonspecific stimuli, early acute rejection (AR) seems to be causative especially in cases of a late diagnosis or inadequate treatment. This study investigated the effects of FTY720, a new immunosuppressant that promotes lymphocyte sequestration into lymph nodes and Peyer's patches, on the development of CR after experimental LTX. METHODS: A total of 50 rats underwent allogenic (F344-to-WKY) and syngenic (WKY-to-WKY) left LTX. Group 1 animals had no treatment. Group 2 animals were administered FTY720 (3 mg/kg body weight per day) at the maximum time of AR (day 14) and continued up to day 100 after LTX. Group 3 animals were treated with the same dosage of FTY720 from day 0 to 100. The grades of AR and CR were classified according to the criteria of the International Society for Heart and Lung Transplantation. RESULTS: Within 14 days after allogenic LTX, all nontreated rats developed early AR followed by severe CR with VS and BO. Similar data were observed for FTY720 treatment of existing AR (group 2). Only early administration of FTY720 (at the time of LTX) significantly reduced the proportion of animals with severe acute vascular rejection (P < .001). However, all of these allografts showed high-grade acute airway inflammation. After long-term application, the chronic inflammatory response was absent; none of the allografts developed BO and VS. CONCLUSION: Only application of FTY720 immediately after LTX prevented lymphocyte recirculation and lung injury.

Relevance of maintenance triple-drug immunosuppression to bridle the amplification of rat cytomegalovirus infection after experimental lung transplantation.

Immunosuppressive therapy required to treat rejection after lung transplantation (LTx) contributes significantly to the pathogenesis of cytomegalovirus (CMV) infection and disease. In a weak allogeneic left LTx model in the rat (Fisher 344 [F344] to Wistar Kyoto [WKY] rats) we analyzed the influence of acute CMV infection on postoperative day (POD) 3, with application of standard triple-drug immunosuppression (TD-IS) (cyclosporin A, azathioprine, prednisolone) on late outcome after LTx. Native right lungs and syngeneic grafts (WKY to WKY) served as controls. Rats were sacrificed on POD 15, 30, 60, and 100. TD-IS completely prevented acute and chronic rejection in non-infected rats. Allografts of CMV-infected rats treated with TD-IS showed only mild perivascular infiltrations in 6/10 rats (POD 15 and 30), which persisted up to POD 100 in 4/10 rats. In the long-term course, mild isolated interstitial and alveolar changes were found in 40% of these animals. In conclusion, rat CMV infection partially neutralized the immunosuppressive effect of TD-IS. However, an amplification of CMV infection under TD-IS can be controlled and does not result in fatal outcome.

Lung transplantation in the fischer 344-wistar kyoto strain combination is a relevant experimental model to study the development of bronchiolitis obliterans in the rat.

Chronic allograft rejection and bronchiolitis obliterans (BO) limited successful long-term outcome after lung transplantation (LTX). Reliable animal models are needed to study the pathogenesis of BO and to develop effective therapeutic strategies. The relevance of an available experimental LTX model without immunosuppression-the Fischer (F344)-Wistar Kyoto (WKY) rat strain combination-was analyzed. The kinetics of acute and chronic rejection and respective graft histopathology were described in the F344-to-WKY rat strain combination after allogeneic LTX. A modified classification of chronic lung allograft rejection was introduced to describe obliterative changes in the airways after rat LTX. Animals from Harlan Winkelmann (HW) and Charles River (CR) were examined. Within 14 days after LTX, allografts showed moderate to severe acute vascular and bronchiolar inflammation. In contrast to rats from CR, animals from HW showed a delayed acute rejection process. Mid-term phase after LTX (days 20-40) presented a "borderline form" consisting of both acute and first signs of chronic airway rejection. On postoperative day (POD) 60, first signs of airway remodelling and distinct BO were diagnosed in 80% of animals from HW. At the same time, the allografts with BO-like lesions increased up to 100% in rats from CR. The F344-to-WKY rat LTX model allows detailed assessment of all features of acute and chronic pulmonary rejection representing a clinically relevant model. However, due to breeding differences resulting in various sublines of the same rat strain, the source and husbandary history of the animals is important for analysis of immuno-mediated processes.

Successful use of a percutaneous miniaturized extracorporeal life support system as a bridge and assistance to left ventricular assist device implantation in a patient with severe refractory cardiogenic shock.

We present a 51-year-old man with cardiogenic shock in whom a percutaneous extracorporeal life support system (ECLS) was inserted to restore cardiopulmonary stability. After successful stabilization, a left ventricular assist device was implanted, using the ECLS without switching to a conventional cardiopulmonary bypass system to reduce its side effects.

Impact of the Eurotransplant high-urgency heart allocation system on the outcome of transplant candidates in Germany.

BACKGROUND: In 2000, the Eurotransplant Foundation changed the allocation criteria following the enactment of the German Transplantation Law (GTL). Our study investigated the impact of the new allocation system on outcomes after heart transplantation (HTx) in Germany. METHODS: We compared 2 cohorts of patients who underwent HTx at our institution in two different 3-year periods before (Group 1: 01/1995-12/1997) and after (Group 2: 01/2003-12/2005) implementation of the new system. RESULTS: An increase in the ratio of HTx performed on an urgency basis was found in Group 2 (8.3 % vs. 87.2 %; P < 0.001). The median waiting time and the ischemia time were significantly lower in Group 1 ( P < 0.05). Postoperatively, the length of ICU stay was significantly higher in Group 2 ( P = 0.04). CONCLUSION: The new allocation system decreased the proportion of local and regional organ harvesting. It generates a higher ischemia time without increasing the number of transplantations and without improving the clinical outcome after HTx. The severity of heart failure, rate of secondary organ impairment, and comorbidity is markedly elevated in patients waiting for HTx on HU.

C2 therapeutic drug monitoring of cyclosporine is a safe and feasible method in de novo heart transplant patients.

BACKGROUND: The narrow therapeutic window of cyclosporine (CsA) requires close therapeutic drug monitoring (TDM). While C2-TDM has been established after renal and liver transplantations, clinical experience is limited for patients after de novo heart transplantation (HTX). PATIENTS AND METHODS: In a retrospective study, we investigated 40 patients undergoing HTX; 34 patients received induction therapy using antithymocyte globulin (ATG Mérieux). Immunosuppression was administered with CsA (Sandimmun-Optoral), with dosages adjusted according to C2 levels (800-1100 ng/mL during the first 6 months and reduced to 400-600 ng/mL from the beginning of the first year). At different times TI (months 1-3). TII (months 12-14) TIII (months 24-26), and TIV (months 34-36), we obtained measures of acute cellular rejections (ARs), cytomegalovirus (CMV) infections, creatinine, and safety laboratory parameters. RESULTS: The cumulative survival was 95% after 1 year, and 88% after 3.8 years. Eight ARs were diagnosed at a mean of 7.6 months after HTX in 7 patients. Twenty-four CMV infections/reactivations were verified. In 10 cases, treatment was started because of clinical symptoms. The mean creatinine values significantly rose in the early postoperative phase (TI: 1.23+/-0.47 mg/dL, TII: 1.49+/-0.41 mg/dL; P<.0001). Thereafter the creatinine values declined; however, this was not statistically significant (TIII: 1.38+/-0.57 mg/dL, TIV: 1.15+/-0.30 mg/dL). All other safety parameters showed no significant changes. CONCLUSIONS: C2 allows individualization of immunosuppression with reduced CsA toxicity, but without loss in safety among de novo patients after HTX. We obtained freedom from severe AR, a low number of CMV infections, and excellent patient survival.

Influence of intensified medical treatment and organ allocation on outcome of transplant candidates.

BACKGROUND: To test how the organization of a pre-transplant clinic and changes in organ allocation modus influence the survival of potential transplant candidates, the survival of patients referred for transplant evaluation between 4/93 and 4/96 (group A) was compared to that of patients referred from 5/96 to 7/00(group B). METHODS: After screening for transplant indication, group A was followed by the referring physician up to transplantation or 3-month reevaluation. Group B was closely followed by a specialized heart-failure clinic. Group A was transplanted according to Eurotransplant criteria, with waiting time being the strongest priority criterion. Due to an allocation partnership off our transplant centers, group B could be transplanted according to medical urgency regardless of waiting time. RESULTS: Overall one-year survival after referral was 69.8% for group A vs. 91 %for group B (p <0.0001). Transplantation within 1 year was required in more group A than group B patients (34% vs. 23%)with worse one-year post-transplant survival in group A (82%vs. 93%). CONCLUSIONS: Intensified treatment by a specialized heart failure program and an allocation system that allows for preferred transplantation of the 'sickest' patient improved over-all survival of transplant candidates and reduced the percentage of patients requiring transplantation.

A case of fetal parvovirus B19 myocarditis, terminal cardiac heart failure, and perinatal heart transplantation.

We report a case of fetal cardiomegaly secondary to myocarditis as a result of intrauterine parvovirus B19 infection. The fetus was delivered through caesarean section because of increasing deterioration of cardiac function at 33 + 3 weeks with reverse flow in the ductus venosus. Four weeks later, a cardiac transplantation was carried out because of therapy-resistant dilative cardiomyopathy. This case shows that fetal parvovirus B19 infection may occur without anemia and myocarditis and does not always result in spontaneous reformation of a dilated heart and normal recovery. It may become the determining prognostic factor for the child.

Topical amphotericin B application in severe bronchial aspergillosis after lung transplantation: report of experiences in 3 cases.

Ulcerative tracheobronchial aspergillosis after lung transplantation (ltx) may lead to bronchial-pulmonary artery fistula that results in fatal bleeding. We report our early experience with combined systemic, aerolized and topical application of amphotericin B in 3 cases of bronchial aspergillosis after ltx. Two patients are still alive, but 1 died of bleeding from a fistula between the left upper lobe bronchus and the pulmonary artery. Aspergillosis in the second patient resolved with minimal stenosis of the left main and the left upper lobe bronchus, and the third patient developed an anastomotic stenosis that was successfully dilated.

Detection of CMV pneumonitis after lung transplantation using PCR of DNA from bronchoalveolar lavage cells.

BACKGROUND: Cytomegalovirus (CMV) is known as a common pathogen causing infections after lung transplantation. Rapid diagnosis of CMV infection is important for the initiation of a specific treatment. OBJECTIVE: Evaluation of methods for the rapid diagnosis of CMV pneumonitis. METHODS: The detection rates of CMV DNA in bronchoalveolar lavage (BAL) and bronchial brushes by polymerase chain reaction (PCR), of viral antigens (p52 and IE1) in BAL and of pp65 antigen in peripheral blood leukocytes were compared to the clinical status after lung transplantation. In 28 patients, 105 BAL, 96 blood samples and 14 brushes were analyzed. RESULTS: In 6 patients, a total of eight episodes of CMV pneumonitis occurred. Five of the 6 with positive CMV antigens in BAL (p52 or IE1) showed signs of CMV pneumonitis. All episodes of CMV pneumonitis were detected by the PCR of BAL cells. Fourteen samples positive for CMV pp65 antigen in blood were negative in BAL PCR. In these cases, no clinical signs of pulmonary CMV infection occurred. Overall sensitivity, specificity, and positive and negative predictive values for the detection of CMV pneumonitis by PCR of BAL cells were 100, 98.9, 88.9 and 100%, respectively. In brush samples, PCR did not provide additional information to the results of the PCR of BAL cells. CONCLUSIONS: PCR of DNA from BAL cells is suitable for reliable and rapid detection of CMV pneumonitis.

Immunoglobulin A and secretory immunoglobulin A in the bronchoalveolar lavage from patients after lung transplantation.

Secretory immunoglobulin A (sIgA) is the most important Ig on mucosal surfaces. In bronchoalveolar lavage (BAL) fluid, sIgA is mainly produced by bronchus-associated lymphoid tissue (BALT). The presence of pre-formed antibodies against donor tissue in kidney transplantation is associated with hyperacute rejection, indicating a humoral (antibody-mediated) reaction. In heart and liver transplantation, humoral rejection has also been documented. The goal of this study was to evaluate the role of IgA in patients after lung transplantation. An enzyme-linked immunosorbent assay was established to determine the levels of sIgA, IgA, and total protein in the lavage. IgA and sIgA were both detectable in BAL from transplanted lungs. IgA and sIgA levels were both higher during episodes of infection than during episodes of rejection or during the control episodes. The level of IgA during episodes of rejection equaled the level of IgA during the control episodes. The level of sIgA was significantly decreased during episodes of acute rejection (1.8 +/- 1.0 microg/mL) when compared with the control (7.2 +/- 1.0 microg/mL; p = 0.013). This study demonstrates that BALT retains the ability to produce Ig even after lung transplantation. The levels of IgA and sIgA and their ratio do not contribute to the differentiation between rejection and infection in lung-transplanted patients.

Surfactant replacement in reperfusion injury after clinical lung transplantation.

BACKGROUND: Reperfusion injury remains a significant risk factor in the immediate postoperative course after lung transplantation. We report on our initial clinical experience of surfactant replacement in reperfusion injury after clinical lung transplantation. METHODS AND RESULTS: In 31 consecutive patients, lung (8 single lung, 16 bilateral lung) or heart-lung (7) transplantation was performed. In 6 patients, severe reperfusion injury developed and was treated with continuously nebulized surfactant. Compliance of the allograft increased 40 +/- 25 % within 3 h following treatment with surfactant. Alveolar arterial oxygen gradient decreased by 23 +/- 11 % after 3 h and by 35 +/- 20 % after 6 h. Normal graft function was reestablished within 1-3 days after transplantation. All treated recipients were extubated until the 6th postoperative day. The 30-day mortality for the 31 recipients was 3.3 %, the 1-year survival 84 %. CONCLUSIONS: Surfactant replacement may become a clinical method for treatment of reperfusion injury after lung transplantation.

Development of obliterative bronchiolitis after allogeneic rat lung transplantation: implication of acute rejection and the time point of treatment.

BACKGROUND: Chronic allograft failure represents the major cause of late morbidity and mortality after solid organ transplantation. Despite the pathological and clinical changes of this disease being well-described, the etiology and the causative factors are still under discussion. Several clinical, as well experimental studies, emphasize the significance of acute rejection. In rat model of left lung allo-transplantation (F344-to-WKY) the influence of acute rejection (AR) on the development of chronic rejection (CR) was studied. METHODS: In Group I (n = 25) no immunosuppression was used, while methylprednisolone (MP) (10 mg/kg) was applied in Group II (n = 20) in the early phase of AR on postoperative Days 9, 10, 11 and in Group III (n = 20) during AR on Day 14, Day 15, Day 16. The rats were sacrificed on Day 5, Day 15/20, Day 30, Day 60, Day 100 and following HE-staining the extend of AR as well CR was graded according to the working formulation of The International Society of Heart and Lung Transplantation. RESULTS: In Group I, AR was found at Day 15 and Day 30 which resolved spontaneously and resulted in CR on Day 60 and Day 100. In Group II, signs of AR were less evident on Day 20, while mild AR persisted on Day 30 and Day 60. On Day 100, normal lung structure was found in all rats. The recipients of Group III showed decreased signs of AR in the early course, however, severe CR was found on Day 60 and Day 100. Extensive airway inflammation with destruction of the subepithelial layer of the smaller airways resulted in severe early obliterative bronchiolitis. CONCLUSIONS: Untreated severe AR in the early course after lung transplantation results in CR in the F344-to-WKY model. Preventive treatment with MP during the early phase of AR clearly diminishes the degree of AR and the graft recovers completely without any evidence of CR. Late application of steroids during the zenith of AR is successful to control the extent of AR, however, it fails to prevent CR.