Characterization of NKG2-A/-C, Kir and CD57 on NK Cells Stimulated with pp65 and IE-1 Antigens in Patients Awaiting Lung Transplant

Introduction: Cytomegalovirus (CMV) is the leading opportunistic infection in lung transplant (LTx) recipients. CMV is associated with graft failure and decreased survival. Recently, new antiviral therapies have been proposed. The present study aimed to investigate NK and T cell subsets of patients awaiting LTx. We analyzed the cellular populations between reactive and non-reactive QuantiFERON (QF) CMV patients for the prediction of immunological response to infection. Methods: Seventeen pre-LTx patients and 15 healthy controls (HC) have been enrolled. QF and IFN-gamma ELISA assay detections were applied. NK cell subsets and T cell and proliferation assay were detected before and after stimulation with pp-65 and IE-1 CMV antigens after stratification as QF+ and QF-. Furthermore, we quantified the serum concentrations of NK- and T-related cytokines by bead-based multiplex analysis. Results: CD56(br)CD16(low)NKG2A(+)KIR(+) resulted in the best discriminatory cellular subsets between pre-LTx and HC. Discrepancies emerged between serology and QF assay. Better proliferative capability emerged from patients who were QF+, in particular in CD8 and CD25-activated cells. CD56(br)CD16(low), adaptive/memory-like NK and CD8Teff were highly increased only in QF+ patients. Conclusions: QF more than serology is useful in the detection of patients able to respond to viral infection. This study provides new insights in terms of immunological responses to CMV in pre-LTX patients, particularly in NK and T cells biology

Ciprofloxacin carrier systems based on hectorite/halloysite hybrid hydrogels for potential wound healing applications

The design of multifunctional nanomaterials which can help the healing processes of skin, preventing the bacterial infections, is crucial for the development of suitable therapy for the treatment of chronic lesions. The use of clay minerals in wound healing applications is well documented since the prehistoric period and offers several advantages due to their intrinsic properties. Herein, we report the development of ciprofloxacin carrier systems based on hectorite/halloysite (Ht/Hal) hybrid hydrogels for potential wound healing applications. To achieve this objective firstly the ciprofloxacin molecules were loaded onto Hal by a supramolecular and covalent approach. The so obtained fillers were thoroughly investigated by several techniques and at molecular level by means of quantum mechanics calculations along with empirical interatomic potentials. Afterwards the modified Hal were used as filler for Ht hydrogels. The introduction of modified Hal, in hectorite hydrogel, helps the gel formation with an improvement of the rheological properties. The in vitro kinetic release from both the fillers and from the hybrid hydrogels was studied both at skin's pH (5.4) and under neutral conditions (pH 7.4); in addition, the factors controlling the ciprofloxacin release process were determined and discussed. Finally, the in vitro biocompatibility of the Hal fillers was evaluated by means of cytotoxic assays and laser scanning confocal microscopy on normal human dermal fibroblasts.The work was carried out in the frame of the PON “AIM: Attrazione e Mobilità Internazionale” No. 1808223-1 project. Authors are thankful to H.A. Duarte for providing atomic coordinates of halloysite, to the CSIC Computational Center and the University of Granada Computation Center for computation facilities, and the Andalusian project grants RNM-1897 and P18-RT-3786 , and the Spanish MINECO projects , PCIN-2017-098 , FIS2016-77692-C2-2-P and CGL2016-80833-R , for the financial support of this research

Markers of Bronchiolitis Obliterans Syndrome after Lung Transplant: Between Old Knowledge and Future Perspective

Bronchiolitis obliterans syndrome (BOS) is the most common form of CLAD and is characterized by airflow limitation and an obstructive spirometric pattern without high-resolution computed tomography (HRCT) evidence of parenchymal opacities. Computed tomography and microCT analysis show abundant small airway obstruction, starting from the fifth generation of airway branching and affecting up to 40–70% of airways. The pathogenesis of BOS remains unclear. It is a multifactorial syndrome that leads to pathological tissue changes and clinical manifestations. Because BOS is associated with the worst long-term survival in LTx patients, many studies are focused on the early identification of BOS. Markers may be useful for diagnosis and for understanding the molecular and immunological mechanisms involved in the onset of BOS. Diagnostic and predictive markers of BOS have also been investigated in various biological materials, such as blood, BAL, lung tissue and extracellular vesicles. The aim of this review was to evaluate the scientific literature on markers of BOS after lung transplant. We performed a systematic review to find all available data on potential prognostic and diagnostic markers of BOS

Bronchoalveolar-Lavage-Derived Fibroblast Cell Line (B-LSDM7) as a New Protocol for Investigating the Mechanisms of Idiopathic Pulmonary Fibrosis

Background: The use of BAL to study ILDs has improved our understanding of IPF pathogenesis. BAL fluid is routinely collected and can be considered a clinical and research tool. The procedure is well tolerated and minimally invasive. No specific cell lines from BAL or immortalized cell lines from IPF patients are available commercially. A method to quickly isolate and characterize fibroblasts from BAL is an unmet research need. Materials and methods: Here we describe a new protocol by which we isolated a cell line from IPF. The cell line was expanded in vitro and characterized phenotypically, morphologically and functionally. Results: This culture showed highly filamentous cells with an evident central nucleus. From the phenotypic point of view, this cell line displays fibroblast/myofibroblast-like features including expression of alpha-SMA, vimentin, collagen type-1 and fibronectin. The results showed high expression of ROS in these cells. Oxidative stress invariably promotes extracellular matrix expression in lung diseases directly or through over-production of pro-fibrotic growth factors. Conclusions: Our protocol makes it possible to obtain fibroblasts BAL that is a routine non-invasive method that offers the possibility of having a large sample of patients. Standardized culture methods are important for a reliable model for testing molecules and eventual novel development therapeutic targets

CD103 Expression on Regulatory and Follicular T Cells in Lymph Nodes, Bronchoalveolar Lavage Fluid and Peripheral Blood of Sarcoidosis Patients

(1) Background: Sarcoidosis is a chronic multisystem disorder of unknown aetiology, driven by a T-cell mechanism allowing T-cell attachment and transmigration through the endothelium, and endorsed by the expression of an integrin alpha-E beta-7 (CD103). This study aimed to analyse the different distribution and compartmentalisation of CD103 expression on T cell subsets in BAL, peripheral blood mononuclear cells (PBMC) and lymph nodes (LLN) from sarcoidosis patients. (2) Patients: We consecutively and prospectively enrolled 14 sarcoidosis patients. We collected PBMC, LLN and BAL at the same time from all patients. Through flow cytometric analysis, we analysed the expression of CD103 on regulatory and follicular T cell subsets. (3) Results: All patients were in radiological Scadding stage II. The multivariate analysis found that the variables which most influenced the peripheral blood compartment were high CD8+ and low ThReg, CD8+CD103+ and Tfh cell percentages. A principal component analysis plot performed to distinguish LLN, BAL and PBMC showed that they separated on the basis of CD4+, CD4+CD103+, CD8+, CD8+CD103+, TcEffector, TcNaive, ThNaive, ThEffector, Threg, ThregCD103+, Tfh, TcfCXC5+ and CD4+CD103+/CD4+ with 65.96% of the total variance. (4) Conclusions: Our study is the first to report a link between the imbalance in circulating, alveolar and lymph node CD8+ and CD8+CD103+ T cells, ThReg, Tfh and ThNaive and the CD103+CD4+/CD4+ T cell ratio in the development of sarcoidosis. These findings shine a spotlight on the pathogenesis of sarcoidosis and may offer new predictors for diagnosis. Our study provides additional understanding for a personalised, and hopefully more effective treatment of sarcoidosis

Krebs von den Lungen-6 as Disease Severity Marker for COVID-19 Patients: Analytical Verification and Quality Assessment of the Tosoh AIA-360 Compared to Lumipulse G600II

Background: Krebs von den Lungen-6 (KL-6) has been proposed as a disease severity marker of COVID-19. All research articles reported the KL-6 assay detected through Fujirebio reagents by Lumipulse G600/G1200 instrument. In the present study, KL-6 assay was analysed through Tosoh AIA-360 and compared with analytical results by Lumipulse G600 in a population of COVID-19 patients. Materials and methods: Sixty-four patients (median age, IQR 67 (58–76) years), all hospitalized for COVID-19 interstitial pneumonia at Siena COVID Unit. KL-6 was measured by two methods, chemiluminescence enzyme immunoassay (CLEIA) and fluorescent enzyme immunoassay (FEIA) method by Lumipulse G600 II and AIA 360 systems, respectively. Results: KL-6 concentrations evaluated by Lumipulse G600II were significantly higher in severe than those in non-severe patients (p < 0.0001) as well as evaluating by AIA360 (p < 0.0001). Receiver operating curve (ROC) curve analysis showed that KL-6 concentrations, by Lumipuse G600II, distinguished severe from non-severe COVID-19 patients with an area under the curve (AUC) of 99.8% and the best cut-off value was 448 U/mL. AUROC between severe and non-severe COVID-19 patients using T0 KL-6 concentrations by AIA360 was 97.4% and the best cut-off value was 398 U/mL. According to T0 KL-6 concentrations in COVID-19 patients, Bland–Altman difference analysis revealed a mean bias of 78 ± 174.8; while using T1 KL-6 concentrations in COVID-19 patients, Bland–Altman difference analysis revealed a mean bias of 48 ± 126 (95% limits of agreement −199–295) between the Lumipulse G600 II and the AIA360 systems. Conclusions: In conclusion, our study demonstrated that CLEIA and FEIA methods for serum KL-6 detection are comparable and reliable. KL-6 was confirmed as an easily detectable and effective biomarker to identify severe COVID-19 patients

Immune checkpoint analysis of T-cell responses to pp65 and IE-1 antigens in end-stage lung diseases

Lung transplant (LTX) patients are at high risk of cytomegalovirus (CMV) infection, which is often associated with high mortality and morbidity. Reactivation of CMV causes cell injury due to the cytopathic effect of viral replication and triggering of T cell immunity. The aim of this study was to compare expression of immune checkpoints (ICs) (PD-1, CTLA-4, LAG-3 and TIGIT) in CD4, CD8 and CD56 and activation markers CD137, CD154 and CD69 of end-stage patients awaiting lung transplant. Eighteen pre-LTX positive for anti-CMV IgG titres and 18 healthy subjects were enrolled. IC and activation markers have been evaluated through flow cytometric analysis in HC and pre-LTX patients. Reactive (QF+) and unreactive (QF−) patients were stratified according to QuantiFERON-CMV assays. ICs' and activation markers' expression were determined before and after in vitro stimulation with pp-65 and IE-1 antigens. Lower expression of PD-1 was observed in CD4 and CD8 cells of pre-LTX patients than controls, whereas CTLA4 appeared upregulated in CD56 and CD8 cells. TIGIT is increased on the surface of CD4, CD8 and NK cells after peptide stimulation in QF-negative patients and PD-1 is only downregulated after stimulation in the QF-positive patients. This study provides new evidence of immune dysregulation in patients with end-stage lung disorders, particularly in relation to immune checkpoint cell biology. The change in QF+ mostly happens on cytotoxic cells NK and CD8, while the changes in QF− were observed in adaptive immune cells, including CD4 and CD8. © 2023 The Scandinavian Foundation for Immunology

Ultrasonography tight control and monitoring in Crohn's disease during different biological therapies: a multicenter study

Background & Aims: Bowel ultrasonography (BUS) is a noninvasive tool for evaluating bowel activity in Crohn's disease (CD) patients. Aim of our multicenter study was to assess whether BUS helps to monitor intestinal activity improvement/resolution following different biological therapies. Methods: Adult CD patients were prospectively enrolled at 16 sites in Italy. Changes in BUS parameters [i.e. bowel wall thickening (BWT), lesion length, echo pattern, blood flow changes and transmural healing (TH: normalization of all BUS parameters)] were analyzed at baseline and after 3, 6 and 12 months of different biological therapies. Results: One hundred eighty-eight out of 201 CD patients were enrolled and analyzed (116 males [62%]; median age 36 years). Fifty-five percent of patients were treated with adalimumab, 16% with infliximab, 13% with vedolizumab and 16% with ustekinumab. TH rates at 12 months were 27.5% with an NNT of 3.6. TH at 12 months after adalimumab was 26.8%, 37% after infliximab, 27.2% after vedolizumab and 20% after ustekinumab. Mean BWT improvement from baseline was statistically significant at 3 and 12 months (P <.0001). Median Harvey-Bradshaw index, C-reactive protein and fecal calprotectin decreased after 12 months from baseline (P <.0001). Logistic regression analysis showed colonic lesion was associated with a higher risk of TH at 3 months and a greater BWT at baseline was associated with a lower risk of TH at 3 months [P =.03 (OR 0.70, 95% CI 0.50-0.97)] and 12 months [P =.01 (OR 0.58, 95% CI 0.38-0.89)]. At 3 months therapy optimization during the study was the only independent factor associated with a higher risk of no ultrasonographic response [P =.02 (OR 3.34, 95% CI 1.18-9.47)] and at 12 months disease duration [P =.02 (OR 3.03, 95% CI 1.15-7.94)]. Conclusions: Data indicate that BUS is useful to monitor biologics-induced bowel activity improvement/resolution in CD