Peripheral CD19+CD24highCD38high B-regulatory cells in lung transplant recipients.

BACKGROUND: The role of CD19+CD24highCD38high B-regulatory cells in solid-organ Transplant (Tx) in acceptance are still scarce. In previous studies on kidney transplant recipients may suggest a protective role of this cell subtype in graft tolerance and the existence of a cross talk between B-and T-regulatory clones. In lung transplantation, the role of B-regulatory cells has never been investigated. In a murine tracheal transplantation model, this subset seems able to prevent tracheal obliteration when in combination with rapamycin. Aim of this study is to analyze peripheral CD19+CD24highCD38high B-reg cells counts in a cohort of lung recipients, their association with several clinical and pharmacological variables and their possible association with T regulatory cell. METHODS: From Jan 2009 to Dec 2014, 117 lung Tx recipients were submitted to an immunological follow up I-FU(median: 108.7 months (6.7-310.5)). Immunological follow up consisted of a complete blood peripheral immuno-phenotype, inclusive of CD19+CD24highCD38high B-cells (globally 1106 determinations). We tested the association between B-reg and relevant variables by linear or regression models for repeated measures, adjusting for time from Tx. RESULTS: Among all variables analyzed at multivariate analysis: chronic rejection (OR - 0.19, p = .039), use of Mycophenolate (OR - 0.38, p < .001) and the presence of a concomitant pulmonary infection of S. aureus (OR 0.66, p = .002) and A. fumigatus (OR 0.50, p = .009) were significantly associated to B-reg cell. No significant correlation between CD19+CD24highCD38high B-reg cells and T-reg cells counts was found in our cohort. CONCLUSIONS: Our present data highlight, for the first time, that this cell subset might participate in long-term lung graft acceptance mechanisms.

A Pilot Study to Investigate the Balance between Proteases and α1-Antitrypsin in Bronchoalveolar Lavage Fluid of Lung Transplant Recipients.

: The neutrophilic component in bronchiolitis obliterans syndrome (BOS, the main form of chronic lung rejection), plays a crucial role in the pathogenesis and maintenance of the disorder. Human Neutrophil Elastase (HNE), a serine protease responsible of elastin degradation whose action is counteracted by α1-antitrypsin (AAT), a serum inhibitor specific for this protease. This work aimed to investigate the relationship between HNE and AAT in bronchoalveolar lavage fluid (BALf) from stable lung transplant recipients and BOS patients to understand whether the imbalance between proteases and inhibitors is relevant to the development of BOS. To reach this goal a multidisciplinary procedure was applied which included: (i) the use of electrophoresis/western blotting coupled with liquid chromatography-mass spectrometric analysis; (ii) the functional evaluation of the residual antiprotease activity, and (iii) a neutrophil count.

Analysis of long term CD4+CD25highCD127- T-reg cells kinetics in peripheral blood of lung transplant recipients.

BACKGROUND: The role of CD4+CD25highCD127- T-reg cells in solid-organ Transplant (Tx) acceptance has been extensively studied. In previous studies on kidney and liver recipients, peripheral T-reg cell counts were associated to graft survival, while in lung Tx, there is limited evidence for similar findings. This study aims to analyze long term peripheral kinetics of T-reg-cells in a cohort of lung recipients and tests its association to several clinical variables. METHODS: From jan 2009 to dec 2014, 137 lung Tx recipients were submitted to an immunological follow up (median: 105.9 months (6.7-310.5)). Immunological follow up consisted of a complete blood peripheral immuno-phenotype, inclusive of CD4+CD25highCD127- T and FOXP3+ cells. We tested the association between T-reg and relevant variables by linear OR regression models for repeated measures, adjusting for time from Tx. Also, by ordered logistic models for panel data, the association between Chronic Lung Allograft Dysfuncton (CLAD) onset/progression and T-reg counts in the previous 3 months was tested. RESULTS: Among all variables analyzed at multivariate analysis: Bronchiolitis Obliterans Syndrome (OR -6.51, p < 0.001), Restrictive Allograft Syndrome (OR -5.19, p = 0.04) and Extracorporeal photopheresis (OR -5.65, p < 0.001) were significantly associated to T-reg cell. T-reg cell counts progressively decreased according to the severity of CLAD. Furthermore, patients with higher mean T-reg counts in a trimester had a significantly lower risk (OR 0.97, p = 0.012) of presenting CLAD or progressing in the graft dysfunction in the following trimester. CONCLUSIONS: Our present data confirm animal observations on the possible role of T-reg in the evolution of CLAD.

Bioengineered gold nanoparticles targeted to mesenchymal cells from patients with bronchiolitis obliterans syndrome does not rise the inflammatory response and can be safely inhaled by rodents.

The use of gold nanoparticles (GNPs) as drug delivery system represents a promising issue for diseases without effective pharmacological treatment due to insufficient local drug accumulation and excessive systemic toxicity. Bronchiolitis obliterans syndrome (BOS) represents about 70% of cases of chronic lung allograft dysfunction, the main challenge to long-term lung transplantation. It is believed that due to repeated insults to epithelial bronchiolar cells local inflammatory response creates a milieu that favors epithelial-mesenchymal transition and activation of local mesenchymal cells (MCs) leading to airway fibro-obliteration. In a previous work, we engineered GNPs loaded with the mammalian target of rapamycin inhibitor everolimus, specifically decorated with an antibody against CD44, a surface receptor expressed by primary MCs isolated from bronchoalveolar lavage of BOS patients. We proved in vitro that these GNPs (GNP-HCe) were able to specifically inhibit primary MCs without affecting the bronchial epithelial cell. In the present work, we investigated the effect of these bioengineered nanoconstructs on inflammatory cells, given that a stimulating effect on macrophages, neutrophils or lymphocytes is strongly unwanted in graft airways since it would foster fibrogenesis. In addition, we administered GNP-HCe by the inhalatory route to normal mice for a preliminary assessment of their pulmonary and peripheral (liver, spleen and kidney) uptake. By these experiments, an evaluation of tissue toxicity was also performed. The present study proves that our bioengineered nanotools do not rise an inflammatory response and, under the tested inhalatory conditions that were used, are non-toxic.

Clinical utility of CD4+ function assessment (ViraCor-IBT ImmuKnow test) in lung recipients.

The ImmuKnow assay measures cell-mediated immunity, quantifying ATP production from peripheral blood CD4+T-cells in solid-organ transplant patients who undergo immunosuppressive therapy. We aimed to measure functional immunity in lung transplant recipients and correlate Immuknow values with immunosuppression levels, presence of chronic lung allograft dysfunction (CLAD) and infections. We evaluated 61 lung recipients who underwent follow-up for lung transplantation between 2010 and 2014. Rejection and infection were retrospectively analyzed. The association between over-immunosuppression and a number of predictors was assessed by means of univariate and multivariate logistic regression models. 71 out of 127 samples (56%) showed an over-immunosuppression with an ImmuKnow assay mean level of 112.92ng/ml (SD±58.2), vs. 406.14ng/ml (SD±167.7) of the rest of our cohort. In the over-immunosuppression group we found 51 episodes of infection (71%) (OR 2.754, 95% CI 1.40-5.39; P-value 0.003). In the other group, only 25 samples (44%) were taken during an infectious episode. The mean absolute ATP level was significantly different between patients with or without infection (202.38±139.06ng/ml vs. 315.51±221.60ng/ml; P<0.001). RAS (Restrictive allograft syndrome) was associated to low ImmuKnow level (P<0.001). These results were confirmed by the multivariate analysis. The ImmuKnow assay levels were significantly lower in infected lung transplant recipients compared with non-infected recipients and in RAS patients.

Antibody-engineered nanoparticles selectively inhibit mesenchymal cells isolated from patients with chronic lung allograft dysfunction.

AIMS: Chronic lung allograft dysfunction represents the main cause of death after lung transplantation, and so far there is no effective therapy. Mesenchymal cells (MCs) are primarily responsible for fibrous obliteration of small airways typical of chronic lung allograft dysfunction. Here, we engineered gold nanoparticles containing a drug in the hydrophobic section to inhibit MCs, and exposing on the outer hydrophilic surface a monoclonal antibody targeting a MC-specific marker (half-chain gold nanoparticles with everolimus). MATERIALS & METHODS: Half-chain gold nanoparticles with everolimus have been synthesized and incubated with MCs to evaluate the effect on proliferation and apoptosis. RESULTS & DISCUSSION: Drug-loaded gold nanoparticles coated with the specific antibody were able to inhibit proliferation and induce apoptosis without stimulating an inflammatory response, as assessed by in vitro experiments. CONCLUSION: These findings demonstrate the effectiveness of our nanoparticles in inhibiting MCs and open new perspectives for a local treatment of chronic lung allograft dysfunction.