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Importance: Convalescent plasma (CP) has been generally unsuccessful in preventing worsening of respiratory failure or death in hospitalized patients with COVID-19 pneumonia. Objective: To evaluate the efficacy of CP plus standard therapy (ST) vs ST alone in preventing worsening respiratory failure or death in patients with COVID-19 pneumonia. Design, setting, and participants: This prospective, open-label, randomized clinical trial enrolled (1:1 ratio) hospitalized patients with COVID-19 pneumonia to receive CP plus ST or ST alone between July 15 and December 8, 2020, at 27 clinical sites in Italy. Hospitalized adults with COVID-19 pneumonia and a partial pressure of oxygen-to-fraction of inspired oxygen (Pao2/Fio2) ratio between 350 and 200 mm Hg were eligible. Interventions: Patients in the experimental group received intravenous high-titer CP (≥1:160, by microneutralization test) plus ST. The volume of infused CP was 200 mL given from 1 to a maximum of 3 infusions. Patients in the control group received ST, represented by remdesivir, glucocorticoids, and low-molecular weight heparin, according to the Agenzia Italiana del Farmaco recommendations. Main outcomes and measures: The primary outcome was a composite of worsening respiratory failure (Pao2/Fio2 ratio

Background aims Because of their capacity to modulate the immune response and promote tissue repair, mesenchymal stromal cells (MSC) represent a potential novel treatment for autoimmune/inflammatory diseases, including Crohn's disease (CD). The aim of the study was in vitro characterization of MSC from active CD patients for future clinical application. Methods MSC from the bone marrow (BM) of seven CD patients (median age 32 years) were expanded ex vivo in the presence of 5% platelet lysate; cells were investigated for clonogenic efficiency, proliferative capacity, morphology, immunophenotype, differentiation potential, genetic stability and ability to suppress in vitro proliferation of both autologous and allogeneic lymphocytes to polyclonal mitogens. Results were compared with those of BM MSC of four healthy donors (HD). Results MSC were successfully expanded from all patients. Colony-forming unitfibroblast (CFU-F) frequency and proliferative capacity were comparable in CD and HD MSC. CD MSC showed typical spindle-shaped morphology and differentiated into osteoblasts, adipocytes and chondrocytes. Surface immunologic markers did not differ between CD and HD MSC, with the only exception of sizeable levels of HLA-DR at early culture passages [1284% at passage (P)1] in the former. CD MSC ceased their growth at variable passages (from P8 to P25) and entered senescence without any change in morphology/proliferation rate. Array-comparative genomic hybridization demonstrated that CD MSC do not show imbalanced chromosomal rearrangements. Both CD and HD MSC inhibited in vitro proliferation of lymphocytes to mitogens. Conclusions CD MSC show biologic characteristics similar to HD MSC and can be considered for anti-inflammatory and reparative cell therapy approaches in patients with refractory disease. © 2009 ISCT.

Extracorporeal photochemotherapy (ECP) is a therapeutic approach based on the biological effects of ultraviolet light (UV) - A and psoralens on mononuclear cells collected by apheresis. Recently, ECP has been under investigation as an alternative treatment for various immune and autoimmune diseases. The aim of this study was to evaluate the safety and feasibility of a new three-step ECP technique, in terms of reproducibility, acceptance, tolerability, and short and long term side effects.Seventeen patients affected by acute or chronic graft-versus-host disease (GvHD), pemphigus vulgaris, or interferon-resistant chronic hepatitis C and one patient being treated for prevention of heart transplant rejection underwent 240 ECP procedures. MNC collection and processing parameters were recorded, biological effects of UV-A/8 methoxy-psoralen (8-MOP) were evaluated, and short and long term side effects were monitored.At a mean follow up of 7 months (range 2-19) 240 ECP had been completed, a mean of 7,136 mL (range 1,998-10,591) of whole blood having beenprocessed per procedure. The mean of total nucleated cells collected per procedure was 6.5x109 (range 0.65-23.8), with a mean MNC percentage of 85% (41. 4-98%) in a mean final volume of 115.5 mL (37-160). No severe side effects were documented and no infectious episodes occurred throughout the course of the treatment.The new ECP technique was highly reproducible as regards the collection and each processing step. Short and long term side effects were mild. No increase in infectious episodes was recorded. All patients willingly underwent ECP, demonstrating an excellent tolerability for the procedure even after several courses.

In spite of the large number of procedures performed every year both in the peripheral blood stem cell transplantation (PBSCT) setting and for the treatment of acute and/or chronic GvHD by means of extracorpor eal photopheres is (ECP), there are still some unresolv ed issues about the optimal strategies (1) to perform PBSC mobilization/c ollection and (2) to treat GvHD patients unresponsive to immunosupp ressive therapies by means of ECP. To address these questions , two Italian scientific societies (SIdEM and GITMO), representing respectively physician working within apheresis units and clinical hematologis ts, committ ed to working groups of experts the developmen ts of consensus recomme ndations about the above quoted issues. 2. Methods

We evaluated the infusion-related toxicity of cryopreserved autologous circulating progenitor cells transplanted in 22 patients receiving high dose chemotherapy and stem cells transplantation for malignancy.Progenitor cells were collected following mobilization with chemotherapy plus filgrastim and stored in liquid nitrogen in the presence of 10% dimethylsulfoxide (DMSO). Before infusion of the graft, patients were medicated with mannitol, hydrocortisone and clorphenamine. The amount of DMSO infused as well as the number of dead and damaged cells were evaluated as possible cause of toxicity.Eleven patients (50%) experienced symptoms related to graft infusion, nausea and vomiting being the most common adverse events. Hypotension was documented in 3 patients (one of them developing transient bradycardia resolved with atropin administration) and one had hypertension with tachycardia. Other observed side effects were: chest tightness (2 pts), fever and chills (3 pts), associated with abdominal cramps (2 pts). 7 out of 8 (88%) patients infused with greater than 30 mL volume of DMSO experienced side-effects, the grade of toxicity being significantly less in those receiving lower amount (30 mL) of DMSO. Two out of 4 pts who received the highest number of dead cells (10 x 10(9)) developed toxicity.In our experience the infusion of cryopreserved peripheral blood progenitors caused minor to moderate toxicity in most cases and, when present, side effects were observed only during infusion. The amount of DMSO present in the graft is related to the grade of toxicity.

The use of circulating progenitor cell support following high-dose chemotherapy for malignancies decreases but does not entirely abolish platelet transfusion requirement. We investigated the feasibility of supporting the posttransplant thrombocytopenic phase exclusively with autologous platelets collected by apheresis and cryopreserved.25 patients underwent plateletpheresis during the platelet rebound occurring after high-dose cyclophosphamide. Autologous platelets were cryopreserved in 5% dimethylsulfoxide, thawed and transfused during the aplastic phase after the myeloablative regimen whenever clinically required.A single plateletpheresis was carried out in all patients, allowing the harvest of a platelet concentrate with a mean value of 7.7 x 10(11) platelets. No significant procedure- or transfusion-related side effects were recorded. Mean platelet recovery after freezing and thawing was 63% and the mean number of platelet reinfused was 4.8 x 10(11); 23 of 25 patients were fully supported with autologous platelets.Plateletpheresis performed in our selected group of patients was found to be a safe and effective procedure to collect large amounts of autologous platelets; the numbers obtained proved to be sufficient for the transfusion demand of almost all patients.

Concerns about the risk of transfusion therapy are driving towards new strategies which are designed to minimize exposure to allogeneic blood products. We aimed to find out whether it is possible to support the phase of thrombocytopenia following high-dose chemotherapy (HDC) and circulating progenitor cells (CPC) transplantation by autologous platelet concentrates (PC).PC were collected from 32 patients undergoing HDC and CPC transplantation for stage II/III breast cancer. A single plateletpheresis was performed at rebound after high-dose cyclophosphamide, when platelet count exceeded 250 x 10(9)/L. PC were cryopreserved in 5% DMSO after controlled-rate freezing and stored in liquid nitrogen. In vitro studies of cryopreserved platelets (aggregation, ATP release and change of mean platelet volume induced by EDTA) were performed. When platelet counts dropped below 20 x 10(9)/L following HDC (thiotepa 600 mg/m2, L-PAM 160 mg/m2) and CPC transplant (CD34+ cells5 x 10(6)/kg), PC were thawed in a 37 degrees C water bath, centrifuged to remove DMSO, resuspended in autologous plasma and reinfused within one hour.Large quantities of platelets were harvested in all patients (median 6.6 x 10(11), range 4.8-12.2). In vitro studies showed preserved platelet function as compared to both fresh platelets and standard PC. Twenty-eight out of 32 patients received autologous PC. At the time of transfusion most of the patients were febrile (38 degrees C) and had mucositisG2. The median number of platelets reinfused was 3.8 x 10(11) (range 2.0-8.1) with a median loss during the freeze-thaw-wash procedure of 37%. Autotransfusion was able to maintain platelet count above 20 x 10(9)/L in most patients, with a corrected count increment7.5 in 20 cases. Four patients required one additional allogeneic transfusion, two because of a poor increment and two due to a late-occurring epistaxis. No side effects related to PC infusion were recorded. Sixteen control patients who received the same HDC and a similar number of CD34+ cells required a total of 17 allogeneic PC units (1 patient did not require platelet transfusion).Our data demonstrate that large doses of autologous platelets can easily be collected and safely administered to support the period of thrombocytopenia in patients undergoing HDC and CPC transplantation. Autologous PC in these patients can abrogate the risks deriving from allogeneic platelet transfusion.

Between February 1995 and August 1997, 11 children (eight males, three females) aged 4-16 years (median 7 years) underwent allogeneic PBPC transplantation for treatment of hematological disorders. Seven patients with acute leukemia (n = 5 ALL, n = 1 AML) or lymphoma (n = 1) received primary allogeneic PBPC transplantation, four patients received a second allotransplantation for graft failure (n = 1 AML, n = 1 sickle cell anemia) or disease recurrence (n = 1 ALL, n = 1 MDS). Five donors were HLA-identical siblings, five were 0-1 antigen mismatched family members and one was a matched unrelated donor. Donors received G-CSF 10-12 microg/kg/day for 3-7 days, and underwent one or two leukaphereses. The median cell yield per donor expressed per kg of recipient body weight was as follows: mononucleated cells 10.8 x 10(8)/kg (range 4.7-21.2); CD34+ cells 8.6 x 10(6)/kg (range 3.2-22); CD3+ cells 3.7 x 10(8)/kg (range 2.7-7.5). All patients achieved an ANC0.5 x 10(9)/l after a median of 12 days (11-18). An unsupported platelet count50 x 10(9)/l was reached 15 days (13-21) after PBPC transplantation; four patients failed to reach this threshold. Acute GVHD (aGVHD) grades II to IV occurred in eight (73%) patients: seven of them experienced grade III-IV aGVHD. Seven patients evaluable for chronic GVHD (cGVHD) were scored as absent in five, limited in one and extensive in one patient. As of September 1997, six patients (55 %) were alive between 60 and 938 days post-transplant (median follow-up 274 days); four patients with malignancy were alive in CR after primary allotransplantation, two patients were alive after a second PBPC transplant. Five patients have died with the main causes of death being aGVHD (n = 3), ARDS (n = 1), relapse of the underlying disease (n = 1). In conclusion, despite the limited number of patients, these preliminary results indicate that PBPC may be considered as an alternative to bone marrow for allografting also in children.

We have recently reported the possibility of supporting the phase of severe thrombocytopenia after high-dose chemotherapy (HDC) and stem cell transplantation using 5% dimethylsulphoxide (DMSO)-cryopreserved autologous platelet concentrates (PCs). The aim of the present study was to evaluate the therapeutic potential of ThromboSol (a recently developed platelet storage solution) plus PCs cryopreserved in 2% DMSO in patients undergoing myeloablative chemotherapy and autologous transplantation. PCs were collected from 14 women with breast cancer by a single plateletapheresis and cryopreserved in ThromboSol/2% DMSO by either direct insertion in a -80 degrees C freezer or in liquid nitrogen after computer-controlled rate (CR) freezing. When required, PCs were thawed, centrifuged to remove the cryoprotectants and transfused. In vitro studies on thawed platelets showed loss of epitopes of surface glycoproteins and a marked reduction of functional activity compared with fresh platelets. Transfusion of CR-frozen PCs was associated with a mean 1 h corrected count increment (CCI) of 9.2 +/- 5.4 x 109/l and only one allogeneic PC was required in this group. In contrast, six out of seven patients required additional allogeneic transfusions in the -80 degrees C group (CCI = 2.7 +/- 1.4 x 109/l). ThromboSol-treated PCs have the ability to overcome thrombocytopenia if processed by a CR freezing protocol, but appear ineffective when frozen by direct placing at -80 degrees C.

Objective Progressive multifocal leukoencephalopathy (PML) is still burdened by high mortality in a subset of patients, such as those affected by hematological malignancies. The aim of this study was to analyze the safety and carry out preliminary evaluation of the efficacy of polyomavirus JC (JCPyV)-specific T cell therapy in a cohort of hematological patients with PML. Methods Between 2014 and 2019, 9 patients with a diagnosis of "definite PML" according to the 2013 consensus who were showing progressive clinical deterioration received JCPyV-specific T cells. Cell lines were expanded from autologous or allogenic peripheral blood mononuclear cells by stimulation with JCPyV antigen-derived peptides. Results None of the patients experienced treatment-related adverse events. In the evaluable patients, an increase in the frequency of circulating JCPyV-specific lymphocytes was observed, with a decrease or clearance of JCPyV viral load in cerebrospinal fluid. In responsive patients, transient appearance of punctate areas of contrast enhancement within, or close to, PML lesions was observed, which was interpreted as a sign of immune control and which regressed spontaneously without the need for steroid treatment. Six of 9 patients achieved PML control, with 5 alive and in good clinical condition at their last follow-up. Interpretation Among other novel treatments, T cell therapy is emerging as a viable treatment option in patients with PML, particularly for those not amenable to restoration of specific immunity. Neurologists should be encouraged to refer PML patients to specialized centers to allow access to this treatment strategy. ANN NEUROL 2021;89:769-779.

Traditionally, hematopoietic stem cell transplantation (HSCT) from both HLA-matched related and unrelated donors (UD) has been used for treating children with acute leukemia (AL) in need of an allograft. Recently, HLA-haploidentical HSCT after αβ T-cell/B-cell depletion (αβhaplo-HSCT) was shown to be effective in single-center studies. Here, we report the first multicenter retrospective analysis of 127 matched UD (MUD), 118 mismatched UD (MMUD), and 98 αβhaplo-HSCT recipients, transplanted between 2010 and 2015, in 13 Italian centers. All these AL children were transplanted in morphological remission after a myeloablative conditioning regimen. Graft failure occurred in 2% each of UD-HSCT and αβhaplo-HSCT groups. In MUD vs MMUD-HSCT recipients, the cumulative incidence of grade II to IV and grade III to IV acute graft-versus-host disease (GVHD) was 35% vs 44% and 6% vs 18%, respectively, compared with 16% and 0% in αβhaplo-HSCT recipients (P < .001). Children treated with αβhaplo-HSCT also had a significantly lower incidence of overall and extensive chronic GVHD (P < .01). Eight (6%) MUD, 32 (28%) MMUD, and 9 (9%) αβhaplo-HSCT patients died of transplant-related complications. With a median follow-up of 3.3 years, the 5-year probability of leukemia-free survival in the 3 groups was 67%, 55%, and 62%, respectively. In the 3 groups, chronic GVHD-free/relapse-free (GRFS) probability of survival was 61%, 34%, and 58%, respectively (P < .001). When compared with patients given MMUD-HSCT, αβhaplo-HSCT recipients had a lower cumulative incidence of nonrelapse mortality and a better GRFS (P < .001). These data indicate that αβhaplo-HSCT is a suitable therapeutic option for children with AL in need of transplantation, especially when an allele-matched UD is not available.

Patients undergoing allogeneic hematopoietic stem cell transplantation (HSCT) always require platelet transfusions, but the increase in platelet count is often less than expected. Since factors responsible for poor response to platelet transfusions in this clinical setting are largely unknown, we performed a prospective study in 87 consecutive children transplanted in a single institution. The mean 16-h corrected count increment (CCI) of 598 platelet transfusions was 5.76 +/- 8.32 x 10(9)/l. Both before and after HSCT, 13.8% of patients had antibodies against HLA and/or platelet-specific antigens. Univariate analysis identified 12 factors significantly associated with a lower post-transfusion CCI, but only four reached statistical significance in the multivariate analysis. These four factors were concomitant therapy with vancomycin, alloimmunization, use of an Autopheresis cell separator for preparation of platelet concentrates and cytomegalovirus infection. We, therefore, suggest that a better response to platelet transfusions could be obtained by choosing a suitable cell separator, by avoiding the use of vancomycin and by adopting measures that reduce alloimmunization and CMV infection. Moreover, screening patients for HLA and platelet-specific antibodies before HSCT would identify the majority of subjects who will develop alloimmune refractoriness after transplantation and would allow the search for a compatible donor in advance.

MSC expansion was successful in all cases; sustained complete closure (seven cases) or incomplete closure (three cases) of fistula tracks with a parallel reduction of Crohn's disease and perianal disease activity indexes (p < 0.01 for both), and rectal mucosal healing were induced by treatment without any adverse effects. The percentage of mucosal and circulating regulatory T cells significantly increased during the treatment and remained stable until the end of follow up (p < 0.0001 and p < 0.01, respectively). Furthermore, MSCs have been proven to affect mucosal T cell apoptotic rate. Objective: External fistulas represent a disabling manifestation of Crohn's disease with a difficult curability and a high relapse rate despite a large therapeutic armamentarium. Stem cell therapy is a novel and promising approach for treatment of chronic inflammatory conditions. We therefore investigated the feasibility, safety and efficacy of serial intrafistular injections of autologous bone marrow-derived mesenchymal stromal cells (MSCs) in the treatment of fistulising Crohn's disease. Patients and methods: We enrolled 12 consecutive outpatients (eight males, median age 32 years) refractory to or unsuitable for current available therapies. MSCs were isolated from bone marrow and expanded ex vivo to be used for both therapeutic and experimental purposes. Ten patients (two refused) received intrafistular MSC injections (median 4) scheduled every 4 weeks, and were monitored by surgical, MRI and endoscopic evaluation for 12 months afterwards. The feasibility of obtaining at least 50×106 MSCs from each patient, the appearance of adverse events, and the efficacy in terms of fistula healing and reduction of both Crohn's disease and perianal disease activity indexes were evaluated. In addition, the percentage of both mucosal and circulating regulatory T cells expressing FoxP3, and the ability of MSCs to influence mucosal T cell apoptosis were investigated. Results: MSC expansion was successful in all cases; sustained complete closure (seven cases) or incomplete closure (three cases) of fistula tracks with a parallel reduction of Crohn's disease and perianal disease activity indexes (p

Background: Mesenchymal stromal cells are employed in various different clinical settings in order to modulate immune response. However, relatively little is known about the mechanisms responsible for their immunomodulatory effects, which could be influenced by both the cell source and culture conditions. Design and Methods: We tested the ability of a 5% platelet lysate-supplemented medium to support isolation and ex vivo expansion of mesenchymal stromal cells from full-term umbilical-cord blood. We also investigated the biological/functional properties of umbilical cord blood mesenchymal stromal cells, in comparison with platelet lysate-expanded bone marrow mesenchymal stromal cells. Results: The success rate of isolation of mesenchymal stromal cells from umbilical cord blood was in the order of 20%. These cells exhibited typical morphology, immunophenotype and differentiation capacity. Although they have a low clonogenic efficiency, umbilical cord blood mesenchymal stromal cells may possess high proliferative potential. The genetic stability of these cells from umbilical cord blood was demonstrated by a normal molecular karyotype; in addition, these cells do not express hTERT and telomerase activity, do express p16ink4a protein and do not show anchorage-independent cell growth. Concerning alloantigen-specific immune responses, umbilical cord blood mesenchymal stromal cells were able to: (i) suppress T- and NK-lymphocyte proliferation, (ii) decrease cytotoxic activity and (iii) only slightly increase interleukin-10, while decreasing interferon-g secretion, in mixed lymphocyte culture supernatants. While an indoleamine 2,3-dioxygenase-specific inhibitor did not reverse mesenchymal stromal cell-induced suppressive effects, a prostaglandin E2-specific inhibitor hampered the suppressive effect of both umbilical cord blood- and bone marrowmesenchymal stromal cells on alloantigen-induced cytotoxic activity. Mesenchymal stromal cells from both sources expressed HLA-G. Conclusions: Umbilical cord blood- and bone marrow-mesenchymal stromal cells may differ in terms of clonogenic efficiency, proliferative capacity and immunomodulatory properties; these differences may be relevant for clinical applications. ©2009 Ferrata Storti Foundation.

This study aimed to ascertain whether extracorporeal photochemotherapy (ECP) is an effective treatment for paediatric patients with refractory graft-versus-host disease (GVHD). From January 1992 to December 2000, 77 children (median age 8.6 years) with either acute (n = 33) or chronic (n = 44) GVHD, resistant to conventional immunosuppressive therapy, were treated with ECP in four Italian paediatric hospitals. After ECP, acute GVHD involving skin, liver and gut responded completely in 76%, 60% and 75% of patients respectively. The 5-year overall survival was 69% for responding patients vs 12% for non-responders (P = 0.001). Among the 44 children with chronic GVHD, 15 (44%) showed a complete response and 10 (29%) a significant improvement after ECP. The 5-year overall survival was 96% for responders vs 58% for non-responders (P = 0.04). Our results suggest that ECP is an effective treatment that may be useful in paediatric patients with either acute or chronic GVHD who have failed to respond to standard immunosuppressive therapy.