Quality control on mononuclear cells collected for extracorporeal photochemotherapy: comparison between two UV-A irradiation devices.

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.

Long-term safety and efficacy of autologous platelet lysate drops for treatment of ocular GvHD.

Current ocular GvHD (oGvHD) treatments are suboptimal. We investigated the safety and efficacy of long-term continuous treatment with autologous platelet lysate (PL) drops in patients with oGvHD Dry Eye Syndrome (DES) score 2-3 refractory to topical conventional therapy. Ophthalmic evaluation was performed at 6 month intervals. Symptoms were assessed using the Glaucoma Symptom Scale (GSS). Patients were defined 'responders' when showing a reduction at least one grade on National Institutes of Health Eye Score from baseline at the 6 month visit. Thirty-one patients were included, and 16 (51%) completed 36 months of follow-up (range 6.5-72.7). At 6 months all patients were classified as responders: median GSS symptom score decreased from 70 to 41 (33 at 36 months), median GSS function score reduced from 68 to 46 (33 at 36 months) (all P<0.001). Median Tear Break Up Time improved from 3 to 6 s after 6 months and was maintained over time. All signs improved at 6 and 36 months (clinical and statistical significance). No severe adverse events occurred. Long-term treatment with PL drops is secure and effective for oGvHD and can be an efficient therapy option from initial stages of oGvHD to prevent permanent ocular impairment and improving quality of life.

A cross-sectional study on vision-related quality of life in patients with ocular GvHD.

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.

Impact of leukapheresis cell composition on immunomagnetic cell selection with the Baxter Isolex 300i device: a statistical analysis.

Immunomagnetic cell selection (ICS) of CD34(+) cells is increasingly adopted in allogeneic and autologous transplant settings. Because many variables can affect the final results of ICS, we focused our study toward the influence exerted by the leukapheresis (LKF) cell composition on recovery, purity, and log of T and B depletion of the immunoselected cells. A total of 39 consecutive CD34(+) ICS were performed with the Isolex 300i (Baxter) device on 39 LKF from 9 HLA haploidentical donors and 20 patients. Flow cytometric analysis was performed both on the leukapheresis content and on the immunoselected cells. The statistical analysis was performed utilizing the Pearson's correlation test and the Mann-Whitney U test. The median purity and recovery of the immunoselected CD34(+) cells were 95.3% (IR: 93.0-99.0) and 55.1% (IR: 41.8-68.2), respectively. The median log of T and B depletion were 3.87 (IR: 3.5-4.3) and 2.9 (IR: 2.5-3.5), respectively. Our data indicate that not only the CD34(+) cell load but also the ratio among the cells belonging to the starting fraction can influence the results of ICS. LKF collection protocols have to be addressed to collect an high number of CD34(+) cells (>500 x 10(6)) without taking care of the contaminating cells when the Baxter Isolex 300i device is employed.

"Sponge-like" dressings based on biopolymers for the delivery of platelet lysate to skin chronic wounds.

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.

Autologous platelet lysate for treatment of refractory ocular GVHD.

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.

Optimization of in vitro expansion of human multipotent mesenchymal stromal cells for cell-therapy approaches: further insights in the search for a fetal calf serum substitute.

There is great interest in mesenchymal stromal cells (MSCs) for cell-therapy and tissue engineering approaches. MSCs are currently expanded in vitro in the presence of fetal calf serum (FCS); however, FCS raises concerns when used in clinical grade preparations. The aim of this study was to evaluate whether MSCs expanded in medium supplemented with platelet-lysate (PL), already shown to promote MSC growth, are endowed with biological properties appropriate for cell-therapy approaches. We confirm previously published data showing that MSCs expanded in either FCS or PL display comparable morphology, phenotype, and differentiation capacity, while PL-MSCs were superior in terms of clonogenic efficiency and proliferative capacity. We further extended these data by investigating the immune-regulatory effect of MSCs on the alloantigen-specific immune response in mixed lymphocyte culture (MLC). We found that MSCs-PL are comparable to MSCs-FCS in their capacity to: (i) decrease alloantigen-induced cytotoxic activity; (ii) favor differentiation of CD4+ T-cell subsets expressing a Treg phenotype; (iii) increase early secretion of IL-10 in MLC supernatant, as well as induce a striking augmentation of IL-6 production. As compared with MSCs-PL, MSCs-FCS were more efficient in suppressing alloantigen-induced lymphocyte subset proliferation and reducing early IFNgamma-secretion. Resistance to spontaneous transformation into tumor cells of expanded MSCs was demonstrated by molecular karyotyping and maintenance of normal morphology/phenotype after prolonged in vitro culture. Our data support the immunological functional plasticity of MSCs and suggest that MSCs-PL can be used as an alternative to MSCs-FCS, although these latter cells might be more suitable for preventing/treating alloreactivity-related immune complications.

Extracorporeal photochemotherapy for treatment of acute and chronic GVHD in childhood.

BACKGROUND: Extracorporeal photochemotherapy (EPC) has recently been proposed for the treatment of adults with either acute or chronic GVHD. However, data on children given this therapy are scarce. A Phase I-II study was carried out on EPC in children experiencing GVHD after allogeneic transplantation of HPCs. STUDY DESIGN AND METHODS: Nine patients with steroid-resistant, grade II-IV acute GVHD and 14 with chronic GVHD, all of whom had been refractory to at least one line of treatment, were enrolled in this study and analyzed. The median age was 10.3 years (range, 5.4-18.1), and the median body weight was 35 kg (range, 17-89). RESULTS: Seven of the nine patients with acute GVHD showed a response to EPC, whereas the disease progressed in the remaining two children (both with skin, gastrointestinal, and liver GVHD), and they died of grade IV acute GVHD. Among the seven children who responded to EPC, it was possible to completely discontinue immunosuppressive treatment in three. In the 14 children with chronic GVHD, 4 and 5 patients experienced complete and partial response to EPC, respectively, whereas the remaining 5 patients, all with extensive chronic GVHD, had stable disease or disease that progressed during EPC. Among these latter 5 patients, 3 died. In 6 of the 9 patients with chronic GVHD responding to EPC, immunosuppressive therapy was discontinued. CONCLUSION: EPC is safe, feasible, and effective in children with either acute or chronic GVHD occurring after an allograft.

Feasibility and safety of a new technique of extracorporeal photochemotherapy: experience of 240 procedures.

BACKGROUND AND OBJECTIVE: 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. DESIGN AND METHODS: 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. RESULTS: 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. INTERPRETATION AND CONCLUSIONS: 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.