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CLAD (Chronic Lung Allograft Dysfunction) remains a serious complication following lung transplantation. Several clinical evidence illustrate that a fraction of ECP (ExtraCorporeal Photoapheresis) treated patients whit CLAD improve/stabilize their graft function. In spite of that, data concerning molecular mechanism of this therapy are still unknown. In our previous study, we observed a significant downregulation of circulating hsa-miR-155-5p, hsa-miR-146a-5p and hsa-miR-31-5p in BOS patients at ECP start when compared to healthy subjects. In responders, increased miR-155-5p and decreased miR-23b-3p expression levels at 6 months were found. The aim of the current study was to assess the epigenetic regulation behind the effects of ECP and to investigate its effect on Peripheral Blood Mononuclear cells (PBMCs) in term of differential miRNAs expression and growth factors and cytokines released. PBMCs obtained from leuko-apheresis of 10 patients with established BOS, prior to the start and after the completion of the X cycles of ECP treatment, were cultured for 48h in complete RPMI medium alone or in presence of PHA/LPS. miR-146a-5p, miR-155-5p, miR-31-5p, miR-181a-5p, miR-142-3p, miR-16-5p and miR-23b-5p expression levels in PBMCs-exosomes were evaluated by qRT-PCR. Interleukine-6 (IL-6), IL-10, IL-8, CTGF, TGFβ, VEGF, PDGF and FGF levels in supernatant of treated/untreated PBMCs were evaluated by ELISA. We observed a significant up-regulation of miR-146a-5p and miR-155-5p in exosome of untreated PBMCs after X cycle compared to pre-ECP levels. The same results were observed for both miRNAs levels in exosome derived from PBMCs stimulated with PHA. Among all cytokines and growth factors, only CTGF levels resulted decreased in supernatant of untreated PBMCs after X cycle-ECP (p=0.041). In conclusion, according to our previous evidence, the upregulation of miR-146a and miR-155 after ECP treatment is due to their release by PBMCs in exosomes. Both miRNAs are known to regulate the immune system and tolerance via T regulatory cells modulation. Interestingly, ECP seems also to regulate CTGF release by PBMC thus affecting fibro-genesis. However, further study are necessary to confirm these findings and clarify deeper ECP related mechanisms. [ABSTRACT FROM AUTHOR]

Background and objectives Several transplantation outcomes have been shown to be associated with the infused bone marrow cell dose/kg of the recipient's body weight. The donor bone marrow density is directly related to the infused cell dose. The aim of the present study was to identify donor-related variables that are associated with high donor bone marrow density. Materials and methods We retrospectively analysed the predictive factors of high marrow density in 65 consecutive HLA-haploidentical bone marrow donors harvested at our centre between 2009 and 2013. Results Body mass index ( BMI) and peripheral white blood cell ( WBC) count were directly associated with bone marrow density (regression coefficient β = 5·33 and β = 2·93, respectively; P < 0·01). The likelihood of obtaining a collection with a high density was first predicted using BMI ( BMI ≥30, mean density = 25·8 TNC/ml × 106). Second, donors with a BMI <30 were split into two groups according to peripheral WBC count ( WBC <8 × 103/mm3: mean density = 18·4 TNC/ml × 106; WBC ≥8 × 103/mm3: mean density = 23·1 TNC/ml × 106). We also observed that the density of the first collected bag directly correlated with the overall density ( R2 = 0·69, P < 0·01). Conclusion The donor-related features BMI and WBC count affect the cell quantity obtainable with the harvest and should be taken into account when choosing the donor. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are considered important modulators of the immune system and have been associated with several diseases. Available clinical evidence suggests improvement or stabilization of lung function in BOS patients treated by extracorporeal photopheresis (ECP). However, few studies have explored the epigenetic and molecular regulation of this therapy. In particular, no studies have addressed miRNAs expression during ECP treatment in BOS. The aim of the study was to evaluate whether a specific set of miRNAs, known to be involved in immune regulation, showed a significant dysregulation during ECP. Total RNA was isolated from serum of BOS patients prior to start ECP treatment and after 6 months. The expression profiles of different miRNAs were obtained by qPCR. Patients whose FEV 1 declined less than 10% with respect to basal value were classified as responders. Twenty-six BOS patients receiving ECP and 17 healthy subjects were included in the analysis. Among the studied miRNAs, we observed a significant down-regulation of circulating hsa-miR-155-5p (p<0.0001), hsa-miR-146a-5p (p<0.0001) and hsa-miR-31-5p (p =0.015) in BOS patients at ECP start when compared to healthy subjects. In order to investigate differential miRNAs expression associated with ECP response, miRNAs levels were compared among responders and non-responders. None of the tested miRNAs was significantly different in the two subpopulations prior to ECP initiation. In responders, paired analysis showed increased expression levels of hsa-miR-155-5p after 6-months treatment (p<0.0001). ECP also induced a significant reduction in hsa-miR-23b-3p expression levels (p<0.0001), although its levels were not significantly dysregulated at baseline compared to healthy controls. The significant down regulation of miR-155 and miR-146a in patients experiencing BOS at ECP start is in line with a low level of immunologic tolerance towards the allograft. Of note, ECP induces an increase of miR-155p among responders. Variation of miR-23b during treatment is more controversial, possibly related to a modulation of fibrogenic molecular pathways. [ABSTRACT FROM AUTHOR]

Abstract: The aim of the present work was the development of sponge-like dressings, obtained by freeze-drying, based on chitosan glutamate and sodium hyaluronate for platelet lysate (PL) delivery to chronic skin wounds. A first phase of the research focused on the choice of the best dressing composition to obtain formulations endowed with the desired mechanical and hydration properties. In particular glycine amount (cryoprotectant agent), and water content were considered as formulation variables. The addition of glycerophosphate, used to solubilize chitosan at pH close to neutrality, was also investigated. In the second phase of the research, dressings were loaded with different amounts of PL. The influence of freeze-drying process and of excipients on the biological activity of platelet growth factors was investigated by means of a cell proliferation test using human fibroblasts. PDGF AB (platelet derived growth factor) content was assayed by means of ELISA test. Depending on composition, dressings showed different mechanical and hydration properties that make them suitable to wounds with different exudate amounts. Both freeze-drying process and excipients employed did not disturb the activity of platelet growth factors. The dressings loaded with platelet lysate were characterized by % proliferation values on fibroblast cell comparable to those observed for the fresh hemoderivate. The PDGF AB assay confirmed the results obtained from cell proliferation test. [Copyright &y& Elsevier]

Current treatment of ocular GVHD (oGVHD), represented by systemic immunosuppressive regimens and local therapies (mainly artificial tears and corticosteroids), gives unsatisfactory results. We investigated the safety and efficacy of autologous plasma rich in PDGFs to treat oGVHD unresponsive to standard medications. A total of 23 patients with refractory oGVHD (grade II-IV) unresponsive to standard therapy were treated with autologous plasma rich in PDGFs eye drops (PRGD) four times/day for 6 months. Symptoms and signs (best visual acuity, Schirmer's test and tear break up time (TBUT), evaluation of the anterior segment and fluorescein and lissamine staining) were always assessed by the same ophthalmologist. Patients were defined as 'responders' when showing improvement for total complaints and at least one sign. At 30 days of treatment, 17 patients (73.9%) were classified as responders. The symptom that improved most was photophobia (improved in 19 patients, 82.6%). TBUT improved in 20 patients (86.9%) and anterior segment score in 19 patients (82.6%). Response was maintained over time. No serious adverse events occurred. PRGD proved to be safe and effective in treating oGVHD and may be a valid treatment option from the early stages of the disease to avoid irreversible ocular damage. [ABSTRACT FROM AUTHOR]

Background The adoptive transfer of ex vivo-induced tumor-specific T-cell lines provides a promising approach for cancer immunotherapy. We have demonstrated previously the feasibility of inducing in vitro long-term anti-tumor cytotoxic T-cell (CTL) lines directed against different types of solid tumors derived from both autologous and allogeneic PBMC. We have now investigated the possibility of producing large amounts of autologous anti-tumor CTL, in compliance with good manufacturing practices, for in vivo use. Methods Four patients with advanced solid tumors (two sarcoma, one renal cell cancer and one ovarian cancer), who had received several lines of anticancer therapy, were enrolled. For anti-tumor CTL induction, patient-derived CD8-enriched PBMC were stimulated with DC pulsed with apoptotic autologous tumor cells (TC) as the source of tumor Ag. CTL were then restimulated in the presence of TC and expanded in an Ag-independent way. Results Large amounts of anti-tumor CTL (range 14-20×109), which displayed high levels of cytotoxic activity against autologous TC, were obtained in all patients by means of two-three rounds of tumor-specific stimulation and two rounds of Ag-independent expansion, even when a very low number of viable TC was available. More than 90% of effector cells were CD3+ CD8+ T cells, while CD4+ T lymphocytes and/or NK cells were less than 10%. Discussion Our results demonstrate the feasibility of obtaining large quantities of anti-tumor specific CTL suitable for adoptive immunotherapy approaches. [ABSTRACT FROM AUTHOR]

Abstract: Extracorporeal photopheresis (ECP) has been proposed as a possible alternative therapy for patients with bronchiolitis obliterans syndrome (BOS), with some evidence of efficacy. Although the mechanism by which ECP exerts its protective effects remains to be determined, two recent studies suggest that the modulation of transplant immune rejection may depend on the capacity to increase the number of peripheral T-regulatory (Treg) cells. We evaluated the effect of ECP treatment on the number of naturally occurring CD4+CD25+ Treg cells in the peripheral blood of six lung transplant recipients: in five cases after failure of augmented or changed immunosuppression for BOS, and in one case owing to persistent acute rejection in a patient who contracted chronic hepatitis C viral infection after lung transplant. A functional stabilization was observed in three of our five patients with BOS, which was accompanied by a slight increase or stabilization of the number of peripheral blood CD4+CD25high cells with in vitro features of Treg cells. On the contrary, two patients with BOS who did not experience graft functional stabilization also showed a decline in the peripheral Treg subset. In the last patient Treg cell kinetics showed stabilization during the first 5 months of ECP treatment when lung function remained stable and graft histology normalized but showed a subsequent decrease, predating BOS diagnosis. In all, our results indicate that ECP may modulate peripheral Treg cell number but the time course of peripheral Treg cells varies according to graft function. [Copyright &y& Elsevier]

We hereby report our real-life experience with extracorporeal photopheresis (ECP) for CLAD. This was an observational retrospective study on LuTx adult patients who received a diagnosis of chronic lung allograft dysfunction (CLAD) from January 2008 to December 2018. They were referred to an ECP Service on a regional basis. Both automated closed and open loop devices were used. Over the period considered, 42 patients were diagnosed with CLAD, 10 (24%) restrictive allograft syndrome (RAS) and 32 BOS. Of those 42, 6 individuals were considered ineligible to initiate ECP due to their excessively compromised conditions; they all died soon after (less than 6 months). The rest (36 patients) were scheduled to receive ECP treatments. Median time from CLAD diagnosis to ECP initiation was 2 (1; 4) months. Median rate of FEV1 decline in the first 6 months was -13 mL/month; however, 11 (31%) did not respond (FEV1 decreased > 10% from ECP initiation). 14 (38%) patients were hospitalized at least once for respiratory infection. The majority of treated patients (21, 58%) reported a good quality of life and worked or were fit to work. 6 patients died, 5 of CLAD-related death. 3 individuals received a re-transplant, and 2 continued with ECP even afterwards. Given also that the majority of patients at our centre are affected by cystic fibrosis (24, 58% of this cohort), one of the major problems with performing ECP was represented by the lack of good vascular access; 4 patients needed a long term catheter insertion. In one of these patients early termination of treatment was decided after a sepsis due to S. epidermidis starting from her Tesio access. No other significant adverse event occurred. No difference was observed between closed and open loop devices. Our data seem to further support the efficacy and safety of ECP to treat CLAD, not only in terms of pulmonary function but also of survival and quality of life. Vascular access related problems were the major issue. Several limits to be acknowledged: retrospective nature, single centre, no control group. [ABSTRACT FROM AUTHOR]

Transient T cell immunodeficiency is a common complication following hematopoietic stem cell transplantation. In breast cancer patients transplanted with autologous peripheral blood progenitor cells (PBPC) harvested after cytotoxic treatment with either cyclophosphamide or epirubicin plus paclitaxel, we evaluated T cells infused in grafts and in peripheral blood during the early reconstitution phase. We found that PBPC grafts harvested after treatment with epirubicin plus paclitaxel contained substantially larger numbers of T cells with less altered composition than after cyclophosphamide. Three months after high-dose cytotoxic chemotherapy, the numbers and the kinetics of circulating naive T cells, but not of memory and CD28- T cells, correlated positively with the number of naive T cells infused PBPC grafts. Finally, retrospective analysis of two cohorts of patients transplanted in different clinical settings with PBPC grafts harvested following cyclophosphamide or epirubicin plus paclitaxel showed apparently different susceptibilities to develop endogenous varicella zoster virus reactivation in the first year after high-dose cytotoxic chemotherapy. On the whole, these data indicate that number and composition of T cells in PBPC grafts vary according to the former cytotoxic therapy, and suggest that autologous transfer of T cells may accelerate the early T cell reconstitution phase and possibly ameliorate immune competence in patients rendered lymphopenic by high-dose chemotherapy. [ABSTRACT FROM AUTHOR]

The objective of this study was to evaluate the cycling status of cord blood (CB)-derived colony-forming cells (CFC) and long-term culture-initiating cells (LTC-IC), and their recruitment into the S-phase of the cell cycle. By using the cytosine arabinoside (Ara-C) suicide approach, we found that only small proportions of both CFC and LTC-IC were in the S-phase of the cell cycle. These estimates were confirmed by flow cytometric DNA analysis, which showed that 96 ± 2% of CB-derived CD34+ cells were in G[sub 0]/G[sub 1] and only 1.6 ± 0.4% in the S-phase. Staining of CD34+ cells with an antistatin monoclonal antibody, a marker of the G[sub 0] phase, indicated that among CD34+ cells with a flow cytometric DNA content typical of the G[sub 0]/G[sub 1] phase 68 ± 7% of cells were in the G[sub 0] phase of the cell cycle. Incubation (24 h) with interleukin 3 (IL-3), recombinant human stem cell factor (SCF) and granulocyte colony-stimulating factor (G-CSF) significantly increased the proportion of cells in the S-phase for both CFC and LTC-IC without inducing any loss in numbers. Flow cytometric DNA analysis also showed an increase in CD34+ cells in the S-phase upon continuous exposure to these cytokines. Our findings indicate that: (i) very few CB-derived CFC or LTC-IC were in the S-phase of the cell cycle; (ii) a substantial amount of CD34+ cells with a flow cytometric DNA content typical of the G[sub 0]/G[sub 1] fraction was cycling, as found in the G[sub 1] phase of the cell cycle; and (iii) 24-h incubation with IL-3, SCF and G-CSF could drive a proportion of progenitor cells into the S-phase without reducing their number. These data might be useful for gene transfer protocols and the ex vivo expansion of CB-derived progenitor cells. [ABSTRACT FROM AUTHOR]

Recently, MacoPharma released a new UV-A cell irradiator device (Macogenic G2) for extracorporeal photopheresis ( ECP), smaller and lighter than the Macogenic G1 but with no integrated cooling system. We compared the two devices at different working temperatures (G1 at standard irradiation temperature - 21°C - and G2 set by purpose at 34°C) in patients affected with chronic graft-versus-host disease and chronic lung allograft dysfunction treated by ECP. We demonstrate that both G1 and G2 devices are efficient in inducing the inhibition of lymphocytic proliferation and mononuclear cells apoptosis after 48 h even when G2 is set at higher-than-standard temperature. [ABSTRACT FROM AUTHOR]

Ocular GvHD affects about 40-60% of patients receiving bone marrow transplantation. Ocular complaints worsen quality of life (QoL), which, besides survival time, is a primary end point in a patient's follow-up. The aim of our study was to assess the ocular surface status and vision-related QoL (VRQoL) and explore the potential determinants in VRQoL in patients with chronic GvHD with ocular involvement. In this cross-sectional study, we investigated 40 patients with ocular GvHD after allogeneic hematopoietic stem cell transplantation assessing ocular symptoms and signs, VRQoL and ophthalmologic parameters. The median age was 52.1 years; 32.5% were females. Most of them presented a multiple organ involvement. Ophthalmological parameter examinations were on average abnormal. Corneal staining was severe/very severe in 25%; conjunctival staining in 10% of subjects. The worse QoL scores were on 'general vision', 'ocular pain', 'vision-specific mental health' and 'vision-specific role difficulties'. Both symptoms and sign scores indicate poor VRQoL. A lower VRQoL was related to schooling level, job position, underlying disease and extracorporeal photopheresis. Corneal staining, Schirmer and tear film breakup time were negatively associated to visual function-related subscales. An accurate ophthalmological and VRQoL assessment should be mandatory for a long time to promptly recognize early signs of ocular suffering, and to prevent irreversible ocular complications. [ABSTRACT FROM AUTHOR]

Bone marrow (BM) and/or peripheral blood progenitor cells (PBPC) given after high-dose chemo-radiotherapy are commonly cryopreserved. Re-infusion of the thawed product can cause cardiovascular and other complications. We compared two groups of adult patients receiving autologous BM or PBPC transplant to assess the incidence of adverse events occurring during infusion. Fifty-one patients received BM, and 75 PBPC. The two groups were comparable in respect of age, total volume infused, quantity of dimethylsulfoxide (DMSO) and number of polymorphonuclear neutrophils. Patients receiving PBPC had a higher number of nucleated cells per kg of body weight; those in the BM group received a significantly greater quantity of red cells. Non-cardiovascular complications occurred in 19% and 8% of patients rescued by BM and PBPC respectively. The incidence of hypertension was 21% in the BM and 36% in the PBPC group. Asymptomatic hypotension was more frequent in PBPC patients (P < 0.001). bradyarrhythmia was noticed in two of 75 pbpc patients and in 14 of 51 bm patients (P < 0.001). in the former group one patient had heart block; he died of renal failure 10 days later. bradycardia and hemoglobinuria were more common in patients receiving bm where a higher concentration of red cells was present (P < 0.001). since bradyarrhythmias may be a life-threatening complication we advise continuous careful monitoring during infusion of thawed bm. the strong correlation between bradycardia and red blood cell contamination suggests the use of purified products with a very low red cell content. [ABSTRACT FROM AUTHOR]

Sensitive detection of circulating epithelial cancer cells might have important therapeutic and prognostic implications in patients with breast cancer (BC) receiving high-dose chemotherapy and PBSC support. We have compared the specificity and sensitivity of the recently developed ‘one tube’ reverse transcriptase PCR (RT-PCR) assay with the more widely used nested RT-PCR method for detection of cytokeratin 19 (CK19)-positive cells. The analysis of 30 control samples provides evidence that one tube RT-PCR is highly specific in contrast to the nested method which showed 23% false positive results. The sensitivity of both techniques to detect tumour contamination was 10-6. PBSC harvests from 45 BC patients were tested with both RT-PCR methods and the results were compared with immunocytochemistry (ICC). The five samples found positive by ICC were also positive by one tube RT-PCR; in addition, 11 more samples were positive by one tube RT-PCR analysis. The greater number of PBSC found positive by one tube RT-PCR might be due to the larger number of cells analysed. We conclude that one tube RT-PCR is sensitive and reveals no false positive results. This method is less time consuming than the nested one, technically simpler and should be considered for tumour cell detection. [ABSTRACT FROM AUTHOR]

Extracorporeal photopheresis (ECP) has emerged as a promising treatment after lung transplantation (LTx) and several researches data suggest slowing or cessation of disease progression after treatment. So far, few studies have explored the molecular regulation and to date no studies have addressed how specific is miRNA expression change during ECP. The aim of the study was to analyze the expression of miRNAs potentially involved ECP response after LTx, and in particular, their changes in responding and non-responding patients to this therapy. LTx patients underwent ECP therapy by off-line methods according to internal protocols. Plasma were drawn pre-ECP and after 3 and 6 months of treatment. We include 12 patients who have undergone transplantation and developed BOS. Patients whose FEV1 declined less than 10% with respect to basal value were classified as responders. The selection of miRNAs included for the study derives from a previous research which showed miRNAs potentially involved in graft versus host disease; from which a possible involvement of miR-22-5p, miR-34a-5p, miR-148a-3p, miR-505-3p was shown. In addition, we evaluated miR-21a-5p expression levels, given its widely demonstrated involvement in literature. We quantified candidate miRNAs using by qRT-PCR. Among analyzed micro-RNAs not significant change was detectable for all miRNAs considered in patients with respect to controls. Furthermore, not appear to be variations in the post-treatment levels for miR-22-5p, miR-34a-5p, miR-148a-3p, miR-505-3p after 3 and 6 months. On the contrary, a significant decrease of miR-21a-5p at 6 months was documented among responders patients compared to non-responders. This study identifies miR-21a-5p as a mechanism associated to ECP suggesting that it can provide not only important clues to pathogenesis but it is also implicated in the mechanisms of response to ECP therapy in BOS. [ABSTRACT FROM AUTHOR]