Background A group of patients with chronic interstitial pneumonia (IP) have an underlying autoimmune disease -typically a connective tissue disorder (CTD)-. In turn, autoimmune-related IP (AIP) processes are highly heterogenous as regards to their clinical presentation, extra-pulmonary features and risk of fibrotic progression. A better understanding of clinically-relevant disease phenotypes is needed in order to tailor therapeutics. Objectives MicroRNA are short non-coding RNAs which orchestrate multiple biological processes by regulating mRNAs via cleavage or translational repression. Circulating levels of miRNA molecules provide excellent information about pathogenic processes in complex diseases, help identify specific disease subgroups and therapeutic targets. Methods We have conducted NGS of small-noncoding RNA in patients with AIP enrolled in the NEREA register of AIP and being followed at any of the Special Multidisciplinary Clinics from the participating Centers. The study population comprised 29 subjects (24 cases, 5 controls; 93% women, 87% newly diagnosed patients). Sixty seven % of the patients were diagnosed with interstitial pneumonia with autoimmune features (IPAF) and all patients had a pulmonary dominant disease at the time of blood sampling. QIAGEN genomic services and a NextSeq (Illumina Inc.) instrument were used. For statistical comparisons, the sample was further segregated according to clinical diagnosis, radiographic pattern, evidence of fibrosis in HRCT, relative levels of Krebs von den Lungen (KL-)6 and type of autoantibodies (classified into those potentially-associated to vasculopathy, the ones typically associated to rheumatoid arthritis, and non-specific ANA). The association was considered true for a p level < 0.02 and a low false discovery rate (FDR), p < 0.1. DIANA tools including the Tarbase v8.0 were used to assess functional pathways with GO and KEGG analysis. Results As compared to the healthy controls, patients with IP showed higher levels of let-7i-5p (1.9-fold, p <0.01), miR-483-5p (3.5-fold, p <0.0005) and the miR-320 family. Fibrotic disease was associated to an up-regulation of the let-7 cluster, miR-151a-5p (2.04-fold, p < 0.01), miR-185-5p (2.4-fold, p 0.011), miR-320a-3p (2-fold, p 0.016), miR-320e (2-fold, p 0.016) and miR-423-5p (1.9-fold, p < 0.01). Besides, there was an up-regulation of both of let-7e-5pb levels (3.55-fold, <p 0.005) and miR-483-5p (4.78-fold, p <0.005) in patients with high KL6. On the other hand, patients with vasculopathy-related autoantibodies had a characteristic microRNA signature, with an up-regulation of the miR-10 and miR-30 families as well as levels of miR-320b (2.77-fold, p <0.005), miR-320c (3.71-fold, p < 0.005) and miR-320d (3.84-fold, p <0.005), in addition to miR-483-5p (4.2-fold, p < 0.005), miR-675-5p (7.74-fold, p < 0.005) and miR-1283 (7.40-fold, p < 0.005). The cluster dendogram showed association between miR-10a, miR-10b, miR320c and miR320d regulated processes, with an enrichment in the Hippo signalling, the TFGβ pathway and the fatty acid biosynthesis. Conclusion Or data suggests a common miRNA signature in patients with AIP, which includes 2 miRNA molecules, miR-483-5p and miR-320a, which have already been identified in in patients with CTD and IP. In addition, a specific microRNA pattern was found in those patients with a “SSc-like” autoantibodies, pointing to the existence of particular driving processes in this subtype of disease. Confirmation of these findings and validation of targets in a larger sample is warranted in order to advance to a pathophysiologic classification of AIP. Acknowledgements This work has been conducted with funding from a ISCIII AES2020 grant (PI20/00250) and a Boehringer Ingelheim External Research Grant 1199-0499 Disclosure of Interests None Declared.

Purpose Neutropenia is a frequently observed complication in solid organ transplant recipients as a result of immunosuppressive therapy and antimicrobial prophylaxis. The impact of neutropenia on the outcome of lung transplant recipients remains understudied.Our main objective was to analyse the frequency of neutropenia in the first 18 months after surgery.As a secondary objective, we assessed the association between the treatment of neutropenia with granulocyte colony-stimulating factor (GCSF) and overall survival (OS), acute rejection (AR) and chronic lung allograft dysfunction (CLAD). Methods We reviewed the outcomes in the first 18 months after surgery of 305 lung transplant recipients performed between 2009 and 2019. Neutropenia was categorised as mild (N 1000-1500), moderate (<1000) and severe (<500). Association of the use of GCSF with OS, AR and CLAD was performed using a Cox model, logistic regression and a Fine-Gray model, respectively. Results More than half of the patients included in this review 51.8% (158/305) developed at least one episode of neutropenia. In 107 patients (35.06%) neutropenia persisted in more than one period of three months follow-up. Neutropenia was detected in 267 of the 1554 follow-up visits (17.18%). The severity of was categorised as mild in 134 cases (50.19%), moderate in 100 (37.45%) and severe in 33 (12.36%). Treatment with GCSF was indicated in 71 patients (23%) and the mean doses per patients was 3.53 units (SD ± 4.2). We did not observe an association between neutropenia and the development of AR, CLAD or overall survival. However, there was higher mortality in patients who received GCSF compared to those who did not (HR 2.30 95%CI 1.32; 4.00, p=0.003) (Figure 1). Conclusion Neutropenia is very common in lung transplant recipients and may be persistent in more than one third of cases. In our series, the need for treatment with GCSF has been a marker of poor prognosis.

Purpose In a multicenter randomized clinical trial we evaluated the safety and efficacy of an intravenous immunoglobulin (IVIG) protocol to decrease the rate of reinfection in solid organ recipients with severe infections and secondary antibody deficiency. Methods Distribution: Heart 20, Lung 15, Kidney 5, Liver transplantation 4 were randomized. Patients with post transplant severe infections and secondary antibody deficiency (defined as IgG < 600 mg/dL) were included. IVIG protocol: Two doses of 15 grams (interval between doses 7-15 days) followed by another 3 doses of 20 grams (interval between doses 15-30 days) of a 5% IVIG product. 39 patients that completed the protocol were analysed [IVIG in combination with conventional antimicrobial therapy (n=21) versus conventional antimicrobial therapy alone (n=18)]. Specific antibodies were tested at inclusion in the clinical trial (V1) and 30-45 days after last IVIG dose (last-visit, V7) in a subgroup of patients to assess the kinetics of humoral immunity reconstitution. Results The primary outcome measure (rate of reinfection) was significantly lower in patients randomized to receive IVIG as compared with patients receiving only conventional antimicrobial therapy (28.6 vs 66.7%, chi-square test, p=0.017). Mean number of reinfections tended to be lower in IVIG group (0.47, interval 1-3, versus 0.89, interval 1-4, Mann-Whitney test, p=0.056). IVIG recipients tended to have lower IgG levels in V1 but disclosed significantly higher IgG concentrations at distinct visits during follow-up. Significantly higher levels of specific IgG anti-cytomegalovirus, IgG anti-clostridium difficile toxins A and B and IgG1 anti-tetanus toxoid antibodies was demonstrated at V7 in patients who received IVIG as compared with patient that were treated with antimicrobial therapy alone. A significant decrease of serum IL6 and BAFF levels (V1 versus V7) was demostrated in IVIG treated patients. IVIG infusions were well tolerated. Conclusion In a multicenter randomized clinical trial we have demonstrated that IVIG is associated with a lower rate of reinfection in solid organ transplantation with severe infection and secondary IgG hypogammaglobulinemia.

Background: The development of chronic lung allograph dysfunction (CLAD) is the leading limitation of long-term survival with good allograft function (LTS) after lung transplantation (LT). The aim of this study was to identify leukocyte subpopulations and to develop a gene model which allows the discrimination of LTS patients. Methods: The cell markers and mRNA expression levels were compared between LTS (n=30) and CLAD patients (n=30) using flow cytometry and microarray techniques. Gene classifiers were built using supervised machine learning methodology and the expression of selected classifier-genes was confirmed by RT-qPCR. Results: In LTS patients, the percentages of CD14highCD16- monocytes, CD56+CD16- NK cells, CD4-CD8- αβ T cell subset and CD62L+granulocytes were elevated whereas the percentage of Vδ1+γδ T cell subpopulation was significantly decreased. The computational process led to the identification of the 25 most relevant genes for LT recipients classification. LASSO method with the 25 classifier genes yielded the optimal results with an Area Under the Curve of 0.87 (sensitivity 0.79 and specificity 0.80). RT-qPCR analysis confirmed the differential expression of 17/18 of the genes selected from the microarrays. The combination of these markers in a model could ultimately improve classification performance. Conclusion: This study identifies a set of lymphocyte subsets and genes associated with LTS after LT which should be validated in an independent LT cohort. The combination of these markers may have utility as a medical tool for safe immunosuppression minimization in LT population. Study financed by ISC III (PI13/01076), FEDER, FUCAP, Astellas, Novartis and Chiesi.

In a randomized clinical trial we have preliminarily demonstrated that IVIG is associated with reconstitution of distinct specific antibodies and with a lower rate of re-infection in solid organ transplantation with severe infection and secondary antibody deficiency.

Purpose The identification of pre-transplant biomarkers to assess the risk of post transplant infections in lung transplantation is of interest. The anti-inflammatory cytokine interleukin-10 (IL-10) was investigated in this study. Higher IL-10 plasma levels have been associated with prolonged CMV clearance in lung recipients. In other clinical setings higher IL-10 levels have been found to be a predictor of clinical outcome in sepsis. In this study we evaluated the relationship between pre-transplant IL-10 serum concentration and the development of infections occurring after lung transplantation. Methods In a prospective follow-up multicenter study, 87 patients in 5 centers were included to identify potential biomarkers of risk for development of post transplant infections. 82 patients were transplanted. 41 patients (50%) developed at least one infection episode that required intravenous drug therapy during the first 6 months after transplantation. Pre-transplant IL-10 was measured with an enzyme-linked immunosorbent assay in blood. IL-10 levels were then correlated with the prevalence of infections. Results The distribution of post transplant infections was as follows: bacterial (67,35%), fungal (20,41%), viral (10,20%). Patients who developed infections disclosed significantly higher levels of pre-transplant serum IL-10: 43,26 ± 39,55 pg/ml vs. 21,70 ± 23,34 pg/ml; p 29 pg/ml were at higher risk of having post transplant infections (Logistic regression relative hazard 3.64 (p= 0.021; 95% confidence interval: 1.28- 10.87). Conclusion We demostrated that in lung recipients with pre-transplant higher levels of serum IL-10, the prevalence of post-transplant infections was higher which suggest a potential role of individual anti-inflammatory responses in the predisposition to infection.

Purpose Secondary Haemophagocytic syndrome (HPS) is an uncommon clinic entity, with rapid evolution and potential cause of multiorgan failure and death. The most common cause are to herpes viridae infections, Epstein-Barr virus and cytomegalovirus Objective: Description of the clinical, laboratory data, cause and treatment characteristics of lung transplant recipients whit HPS in our unit. Methods We retrospectively analyzed 4 cases of HPS after lung transplantation (2 male, 2 female; age 45-68 years), during a 10 year period. The clinical and laboratory diagnostic criterial according to HLH-2004 guidelines were: fever, splenomegaly, cytopenia of 2 cell lines or more (hemoglobin, platelets, and neutrophils), hypertriglyceridemia and/or hypofibrinogenemia, elevated ferritin, elevated interleukin-2 receptor, decreased natural killer cell activity, and hemophagocytosis in bone marrow (>5/8 criteria). Results HPS developed 1 - 48 months in the posttransplant period. Three patients presented with fever and two whith hepatosplenomegaly. Laboratory tests showed pancytopenia, hypofibrinogenemia, elevated LDH, hyperferritinemia and hemophagocytosis in bone marrow and prolongation prothrombin time in all patients. Two patients had Epstein-Barr virus infection, 1 had Hepatitis B infection and 1 unknown cause. Three of the four patients died despite aggressive supportive therapy. Conclusion We report here the largest series of HPS after lung transplantation. In our experience the most common cause was Epstein-Barr virus infection.

Purpose Non-invasive methods for early diagnosis of acute cellular rejection (ACR) are needed. Previous studies of other solid organ transplanted recipients have shown a relationship between the eosinophil count in peripheral blood (EOSp) and the presence of ACR. However, the relationship between this biomarker and ACR in lung transplant patients remains unclear.Objective: To asses the relationship between EOSp and the presence of allograft rejection in lung transplant recipients. Methods A retrospective clinical review of 363 transbronchial biopsies (BTB) performed in 194 lung transplant patients between 2014 and 2018. We analyzed: age, date of transplant, the presence and degree of ACR, and the simultaneous absolute number of EOSp. BTB indications were classified as: 1. Protocol BTB in the first month after transplant and after ACR treatment. 2. Decrease of lung function. 3. Other indications The relationship between the EOSp count and ACR was analyzed by the Chi-square test. We calculated the sensitivity, specificity of the EOSp count to diagnose ACR. The precision of the EOS count to predict ACR was analyzed using a receiver operating characteristic ROC curve. Results BTB showed ACR in 117 of 363 cases (32.2%). The most frequent ACR degree was A2 (53%). The EOSp count in patients with ACR was higher than non-ACR patients (197 vs. 92.7; p<0.001). ACR was found in 39 of the 62 (62.9%) BTB performed in patients with a EOSp count higher than 197 in comparison with 78 of 301 (25.9%) observed in patients with a lower count (p <0.001). The accuracy of BTB performed per protocol or lung function decrease was significantly increased if the simultaneous EOSp count was higher than 197 (figure 1). The diagnostic specificity of ACR in BTB performed by protocol and >197 EOSp was 91% and the area under the ROC curve was 0.724 with 95% confidence interval (CI) 0.636-0.813. Conclusion The monitoring of the EOSp count can be useful to increase the diagnostic yield for ACR of BTB performed by protocol or lung function deterioration. Non-invasive methods for early diagnosis of acute cellular rejection (ACR) are needed. Previous studies of other solid organ transplanted recipients have shown a relationship between the eosinophil count in peripheral blood (EOSp) and the presence of ACR. However, the relationship between this biomarker and ACR in lung transplant patients remains unclear.Objective: To asses the relationship between EOSp and the presence of allograft rejection in lung transplant recipients. A retrospective clinical review of 363 transbronchial biopsies (BTB) performed in 194 lung transplant patients between 2014 and 2018. We analyzed: age, date of transplant, the presence and degree of ACR, and the simultaneous absolute number of EOSp. BTB indications were classified as: 1. Protocol BTB in the first month after transplant and after ACR treatment. 2. Decrease of lung function. 3. Other indications The relationship between the EOSp count and ACR was analyzed by the Chi-square test. We calculated the sensitivity, specificity of the EOSp count to diagnose ACR. The precision of the EOS count to predict ACR was analyzed using a receiver operating characteristic ROC curve. BTB showed ACR in 117 of 363 cases (32.2%). The most frequent ACR degree was A2 (53%). The EOSp count in patients with ACR was higher than non-ACR patients (197 vs. 92.7; p<0.001). ACR was found in 39 of the 62 (62.9%) BTB performed in patients with a EOSp count higher than 197 in comparison with 78 of 301 (25.9%) observed in patients with a lower count (p <0.001). The accuracy of BTB performed per protocol or lung function decrease was significantly increased if the simultaneous EOSp count was higher than 197 (figure 1). The diagnostic specificity of ACR in BTB performed by protocol and >197 EOSp was 91% and the area under the ROC curve was 0.724 with 95% confidence interval (CI) 0.636-0.813. The monitoring of the EOSp count can be useful to increase the diagnostic yield for ACR of BTB performed by protocol or lung function deterioration.

Chronic Lung Allograft Dysfunction (CLAD) is the major limitation in long-term survival in lung transplant recipients. Some therapies such as extracorporeal photopheresis (ECP) and thymoglobulin (TG) have been described but there are few studies assesing comparative treatment efficacy.

Due to growing incidence of severe infections associated with secondary antibody deficiency (SAD), this risk factor is being explored as a potential new indication of intravenous immunoglobulin (IVIG) in settings such as solid organ transplantation, post-rituximab use in autoimmune diseases and after therapy of B-cell lymphomas. The clinical criteria for this IVIG indication is the presence of severe infections. The immunological criteria that is being explored by the European Medicines Agency is the presence of severe hypogammaglobulinemia (HGG: IgG below 400 mg/dL) with demonstration of failure to produce specific antibodies after vaccination. In the case of thoracic transplantation the majority of severe infections are concentrated during the first 6 months, a period in which vaccination is avoided in the majority of centers. Objective. To define the prevalence and components of severe SAD in thoracic transplantation.

Long-term survival after lung transplantation (LT) is limited by the development of chronic lung allograph dysfunction (CLAD). Despite this fact, a small number of lung transplant recipients are long-term survivors with a good allograft function (LTS). A study of this particular population could be a first step to search for transplant tolerance biomarkers that can lead to the reduction of immunosuppressive drugs in treatment plans and improve personalized medicine. The objective of this study was to identify leukocyte subpopulations as potential tolerance biomarkers in LTS after lung transplantation.

BACKGROUND:Hepatitis E virus (HEV) is one of the common causes of acute and chronic viral hepatitis with a global distribution. Genotypes 1 and 2 only affect humans and produce acute hepatitis epidemics in endemic regions (Asia, Africa). In nonendemic areas (America, Europe), genotypes 3 and 4 are considered a zoonosis and cause sporadic acute hepatitis. HEV has been described in solid organ transplant recipients; however, data on lung transplant patients are limited.OBJECTIVE:To present the first 3 cases of HEV infection in lung transplant recipients in our unit.CASE PRESENTATION:We report 3 cases of HEV infection in post-transplant patients presenting with symptoms and alterations in liver enzymes. All patients have no history of travel outside Spain prior to observing abnormalities in the liver function. Diagnoses were made with in-home polymerase chain reaction and enzyme-linked immunosorbent assay (IgG/IgM). The first patient was not treated and died of progressive hepatic disease, with postmortem diagnosis of HEV infection complications. The other 2 patients were treated with ribavirin after the diagnosis of HEV infection. Ribavirin was discontinued in 1 patient because of anemia necessitating red blood cell transfusions.CONCLUSIONS:HEV should be considered in the differential diagnosis of patients with abnormal liver enzymes after transplant. Early detection and treatment have implications in the prevention of liver failure and mortality. Large prospective seroprevalence studies of HEV in lung transplant patients are warranted to recognize the epidemiology of this infection in lung transplant recipients.

Acute allograft rejection is a problem in lung transplantation. More than a third of lung transplant recipients are treated for acute rejection in the first year after transplant. Growing evidence suggests that B cells may play a role in allograft rejection in solid organ transplantation. B cell activating factor, BAFF, plays a role in B cell survival, maturation and activation. To date, little is known of the role of BAFF in lung transplantation. We aimed to determine whether there was a correlation between BAFF concentration and acute rejection.

Cancer is the third most common cause of death among lung transplant (LT) recipients who survive for more than 1 year. The purpose of this study was to analyze the incidence and risk factors for cancer after LT in a Spanish cohort. The epidemiology and risk factors for cancer were retrospectively analyzed in LT recipients from 2 cities in Spain, Madrid and Barcelona. Of the 1353 LT patients initially included in the study, 125 (9.2%) developed cancer after a mean of 3.7 years. This frequency was 5-fold higher than in the general population. The most prevalent tumors were skin cancer (32%), lymphoproliferative disease (18%), and lung cancer (16.5%). In 4 patients, lung cancer was diagnosed on the day of the operation. The risk of cancer increased with age >55 year (hazard ratio [HR] 2.89 [1.64-5.09]; P<.001), in men (HR 2.8 [1.4-5.6]; P = .004), and in heavy smokers (>20 pack-years) (HR 2.94 [1.64-5.27]; P<.001). Other factors such as sun exposure were not found to be risk factors. In conclusion, prevalence of cancer is high in LT recipients in a Mediterranean country. Skin tumors, lymphoproliferative disease, and lung cancer are the most prevalent cancers. Age, male sex, and smoking were the main risk factors for cancer in this population.

s 24th European Conference on General Thoracic Surgery 29 May–1 June 2016, Naples, Italy

Background. In experimental models with von Willebrand disease pigs, plasma von Willebrand factor (vWF) was significantly increased after lung transplantation because lung endothelial cells strongly express vWF. However, these findings have not been confirmed in human beings. Methods. A 26-year-old man with mild vWF deficiency (FvW: antigen 39 IU/dL; FvW: ristocetin cofactor activity 44 IU/dL; factor VIII 99%; normal multimeric plasma vWF pattern) was referred to our institution and underwent bilateral lung transplantation for cystic fibrosis. The patient received factor VIII/vWF concentrate both during and after surgery without any relevant bleeding events. At hospital discharge, he was taking immunosuppression with oral tacrolimus, prednisone, and mycophenolate mofetil, which has continued until the present day (22 months after the procedure). Results. Plasma vWF level increased during the postoperative days, presumably due to endothelial injuries and the infusion of vWF concentrate. Laboratory tests at 5, 11, 14, and 22 months after lung transplantation demonstrated sustained normalization of all parameters. Conclusions. To our knowledge, this is the first reported case of von Willebrand deficiency corrected through lung transplantation.

Severe infections are frequent in lung recipients. The need to identify additional prognostic factors allowed us to evaluate the anti-polysaccharide IgG, IgA and IgM responses in 77 lung recipients immunized with the 23-valent pneumococcal polysaccharide (PS) vaccine. Anti-pneumococcal polysaccharide specific antibody concentrations (anti-PS23) are a surrogate for humoral immune response against T-cell independent antigens. A prospective follow-up multicenter study (5 centers in Spain, from 2009 through 2012) was performed. We used a new anti-PS23 IgA and IgM ELISA assay, which evaluates a global response to all 23 polysaccharides contained in the polysaccharide pneumococcal vaccine. Immunization was administered before transplantation. Assessment points: Baseline (pre-transplant), day 7 and day 30 post-transplant. Clinical follow-up: 6 months. Primary outcome: Severe infections defined as all infectious episodes that required IV antimicrobial therapy. Catheter associated infections and superficial wound infections were not included. Mean age: 53 (20-69 years). 52 men. Kinetics: As compared with baseline values, a decrease of anti-PS23 IgG, IgA and IgM antibody concentration was observed at day 7 and day 30 after transplantation (53, 19, 18 mg/dL; 18, 8, 9 mg/dL and 11, 7, 6 mg/dL, respectively). Outcome: The prevalence of severe infection was 46.4%. Risk factors: Anti-PS23 IgM concentration was lower in the baseline study in lung recipients who developed severe infections after transplantation as compared with lung recipients without severe infections (8±6 mg/dL vs. 15±17, Student´s T-test, p=0.05). Patients who disclosed anti-PPS IgM concentrations below 5 mg/dL in post transplant determinations (52.6%) had a higher incidence of severe infection after transplantation (65% vs. 38%, Chi-square test, p=0.038; Logistic regression, odds ratio 3.09, 95%CI 1.03-9.31, p=0.04). Specific anti-PPS23 antibody responses were not correlated to total IgG, IgA and IgM concentrations. Low IgM antibody response to pneumococcal vaccination has been previously demonstrated in adults with a low capacity to opsonize bacteria. Evaluation of specific IgM anti PPS23 responses after vaccination might represent a valuable tool to identify lung recipients at risk of severe infection.