Maria Dassi

Extracorporeal Photochemotherapy for the Treatment of Chronic Graft-Versus-Host Disease: Trend for a Possible Cell Dose-Related Effect?

Abstract:  Extracorporeal photochemotherapy (ECP) has been progressively introduced into the treatment of both acute and chronic graft‐versus‐host disease (cGvHD) over the last decade. Nevertheless, its mechanisms of action, as well as the optimal treatment schedule, have not yet been defined. We retrospectively analyzed 25 patients with cGvHD unresponsive to conventional treatments who underwent ECP from 1997 until 2005. The impact of various factors (such as treated and infused nucleated cells, time from transplantation and cGvHD onset, and time from cGvHD and ECP treatment) on the probability of no response to ECP was therefore investigated. A positive response to ECP was achieved in 80% of the patients, after a median of 19 ECP treatments (with a range of 8–38). Eighteen out of the 20 patients responsive to the treatment maintained their response for a median of 30 months. We mainly focused on clinical response and yield composition. The analysis on mononuclear cell (MNC) dose suggested that an increase of MNC dose/kg b.w. (body weight) induced a decrease in the odds of treatment failure, and that, if the MNC dose infused was at least 100 × 106/kg b.w. per ECP treatment, a more positive and longer‐lasting response was achieved. Moreover, the mean dose of treated and infused monocytes × 106/kg b.w./ECP did not account for a clear dose‐related effect. These findings may eventually result in a more patient‐tailored approach to ECP. Prospective multicenter trials should be designed to investigate the real impact of MNC dose on ECP responsiveness.

Platelet activation during plasma-reduced multicomponent PLT collection: a comparison between COBE Trima and Spectra LRS turbo cell separators

BACKGROUND:  The wide diffusion of multicomponent collection in donor apheresis has led to the yielding of different components, such as plasma‐reduced platelet‐pheresis at high PLT concentration. We investigated whether this collection modality could induce more PLT activation compared to standard plateletpheresis.

Pixantrone (BBR2778) reduces the severity of experimental allergic encephalomyelitis

Pixantrone is less cardiotoxic and is similarly effective to mitoxantrone (MTX) as an antineoplastic drug. In our study, pixantrone reduced the severity of acute and decreased the relapse rate of chronic relapsing experimental allergic encephalomyelitis (EAE) in rats. A marked and long-lasting decrease in CD3+, CD4+, CD8+ and CD45RA+ blood cells and reduced anti-MBP titers were observed with both pixantrone and MTX. In vitro mitogen- and antigen-induced T-cell proliferation tests of human and rodents cells evidenced that pixantrone was effective at concentrations which can be effectively obtained after i.v. administration in humans. Cardiotoxicity was present only in MTX-treated rats. The effectiveness and the favorable safety profile makes pixantrone a most promising immunosuppressant agent for clinical use in multiple sclerosis (MS).

Extracorporeal photochemotherapy: A safety and tolerability pilot study with preliminary efficacy results in refractory relapsing-remitting multiple sclerosis

Extracorporeal photochemotherapy (ECP) is an immunomodulating procedure consisting of autologous reinfusion of peripheral blood mononuclear cells (PBMC) after direct exposure to 8-methoxy-psoralen and UV-A. It has been described as a successful treatment for different T-cell-mediated diseases and preliminary results suggest that ECP might be effective in the treatment of relapsing–remitting multiple sclerosis, but does not significantly alter the course of the progressive form of MS. In this study, we report the safety data and some preliminary efficacy evidence obtained using ECP in the treatment of five patients with refractory relapsing-remitting (RR) MS: in most cases ECP induced a reduction in the relapse rate and an EDSS stabilisation, with an apparent general MRI stabilisation. In conclusion, our results confirm ECP safety and tolerability and suggest that this treatment might be useful as a therapeutic alternative in the subgroup of RRMS patients not responsive to or not eligible for traditional immunomodulating or immunosuppressive treatments.

Peripheral blood progenitor cell collection in chronic myeloid leukemia patients with complete cytogenetic response after treatment with imatinib mesylate

BACKGROUND:  Imatinib mesylate (IM) was introduced in chronic myeloid leukemia (CML) treatment in the late 1990s and substantially changed the therapeutic approach to the disease, by inducing complete cytogenetic response (CCR) in approximately 60 percent of cases. Nevertheless, some concerns exist about the duration of response to treatment and the onset of resistance to IM.

Reconstitution of lymphocyte subpopulations in children with inherited metabolic storage diseases after haematopoietic cell transplantation

We prospectively evaluated the reconstitution of lymphocyte subpopulations in nine children with lysosomal diseases who underwent 11 allogeneic haematopoietic cell transplants (HCTs) following CD34+ immunomagnetic enrichment, limited T‐cell addback and in vivo B‐cell depletion. Absolute lymphocyte count recovery was slow to cross the 5th percentile, occurring at a median of 10 months after HCT in patients with full chimaerism. Natural killer cells represented up to 90% of the total lymphoid population during the first 3 months. CD4+ lymphocyte recovery occurred 9–18 months after HCT. In most patients, CD8+ lymphocyte recovery was slow and comparable with that of CD4+ lymphocytes. The CD4+/CD8+ ratio normalised by 3–7 months after HCT in 50% of the patients. CD8+ lymphocyte recovery was enhanced in patients with viral reactivation. Reconstitution of B‐lymphocytes was particularly delayed in patients treated with rituximab. Declining chimaerism, rejection and viral reactivation were the most common problems in our series. Because of the unique graft manipulation, the pace of lymphocyte reconstitution was particularly slow, suggesting that these patients are at a significantly increased risk of infections for up to 2 years after HCT.

Isolation of monocytes from leukapheretic products for large-scale GMP-grade generation of cytomegalovirus-specific T-cell lines by means of an automated elutriation device

BACKGROUND: Dendritic cells (DC) act as antigen‐presenting cells in immune response–mediated mechanisms against malignant cells and/or viral or fungal pathogens. CD14+ monocytes have been so far isolated by techniques of plastic adherence or by using immunomagnetic methods. Here the effectiveness of a commercially available cell separation system (Elutra, Gambro BCT) in the separation of monocytes and the large‐scale production of cytomegalovirus (CMV)‐specific T‐cell lines were investigated.

Electrolyte monitoring in patients undergoing peripheral blood stem cell collection.

In recent years peripheral blood stem cell (PBSC) collection for allogeneic or autologous transplantation has experienced an increased use in the onco‐hematological setting. The latest generation cell separators allow a satisfactory and safe PBSC collection. Nevertheless, as in all therapeutic apheresis procedures, patients may experience procedure‐related side‐effects, mainly vasovagal reactions or symptoms related to hypocalcemia and/or hypomagnesemia. We investigated electrolyte changes in 18 patients, with a median age of 46 years (range 7–62), undergoing PBSC collection from January to April 1998. A significant decrease in total calcium in the final sample (9.65 ± 0.7 mg/dL) with respect to the basal one (9.2 ± 0.6 mg/dL, P < 0.05) was observed; also ionized calcium decreased markedly from the first sample drawn at +30 minutes: 1.22 ± 0.14 vs. 1.03 ± 0.15 mmol/L (P < 0.05), and a highly significant difference emerged when basal value were compared to the final value: 1.22 ± 0.14 vs. 0.94 ± 0.13 mmol/L (P < 0.0001). Similar findings affected potassium concentration: 4.1 ± 0.4 vs. 3.3 ± 0.3 mEq/L (P < 0.0001). Three out of eighteen patients (16.7%) reached a final potassium level <3.0 mEq/L, and eight out of eighteen (44.5%) showed a potassium concentration decrease >20% with respect to the basal value. A mild metabolic alkalosis occurred during the procedure: pH increased from 7.35 ± 0.02 to 7.43 ± 0.028 (P < 0.001), and plasma bicarbonate concentration increased from 27.48 ± 2.21 to 32.44 ± 2.52 mmol/L (P < 0.01). Sodium and chloride did not differ in the final sample with respect to the basal sample. None of our patients experienced clinically relevant side effects related to severe electrolyte changes (i.e., >20% with respect to the basal value). Because our current therapeutic schedules include patients older than 50 years in the PBSC collection and transplantation program and since it is well known that subclinical myocardial disease may occur in up to 4% of middle‐aged males, we suggest that patients aged 50 or older undergoing PBSC collection procedures be carefully monitored in order to identify significant electrolyte variation, especially if they present with low serum potassium levels. However, further investigation of larger patient series are needed to determine the clinical relevance of serum potassium changes during apheresis. J. Clin. Apheresis 14:14–17, 1999.

Extracorporeal photochemotherapy reduces the incidence of relapses in experimental allergic encephalomyelitis in DA rats

Sirs: The treatment of refractory multiple sclerosis is still an unsolved problem. Extracorporeal photochemotherapy (ECP) is an immunomodulating procedure which consists of the autologous reinfusion of UV-A irradiated peripheral blood mononuclear cells (PBMC) collected by apheresis and incubated with 8-methoxypsoralen (8-MOP) [5]. ECP is already routinely used in several human immuno-mediated disorders that are due to T-lymphocyte activity (i. e. acute and chronic graft-versushost disease, rejection in organ transplantation, T-cell cutaneous lymphoma) [1, 4–8, 14]. Photoactivated 8-MOP covalently binds to DNA, cell surface molecules and cytoplasmatic components of the irradiated cells and, although the exact mechanism of action is still unclear, it is evident that ECP triggers an immunomodulating effect also on the non-irradiated T-cells [5, 12]. ECP has been reported to be effective in two patients affected by relapsing-remitting MS [10], but not in chronic progressive MS [11]. Therefore, we performed this experiment using chronic relapsing experimental allergic encephalomyelitis (EAE), a reliable model of relapsing MS, in order to examine the effect of ECP and to evaluate whether this animal model is suitable for studying the mechanisms of action of ECP in experimental conditions. After footpad inoculation of syngenic whole myelin + Freund’s adjuvant (100 μg/100 μl) thirty-two adult female DA rats were randomly assigned to one of the two experimental groups (EAE alone or EAE+ECP, n = 16 each). ECP or sham treatment was performed four times during the course of EAE and the observation was prolonged for a period of 50 days in all the surviving animals. The experimental plan was approved by our institutional ethic committee and the procedures were performed according to the EU Council directive 86/609. PBMC were isolated on days 18, 25, 32 and 39 after inoculation from heparinized blood samples (2 ml) drawn from the retro-orbital plexus during anaesthesia. 8-MOP (Gerot Pharm, Austria) was added at 200 ng/ml final concentration, and then the PBMC sample was distributed in a sterile Petri dish and UV-A irradiated (365 nm wavelength) with a BIO-GENIC device (Vilber-Lourmat, France) at 2 joule/cm2 dose intensity for 10 min. After UV-A irradiation, PBMC were reinfused into the recipients EAE+ECP rats via the tail vein. Blood samples in the sham group rats were treated in the same way but the PBMC did not undergo UVA irradiation. The number of PBMC reinfused was not significantly different when the comparison was performed between groups and between each procedure (p > 0.05 by one-way ANOVA). Each animal received approximately 10 x 103–13 x 103 PBMC/procedure. The comparison in the overall incidence of EAE relapses observed in each rat (i. e. one point increase in the clinical score ranging from 0, i. e. normal, to 5, i. e. dead [2]) was the endpoint of the study. Five rats in the sham group (on days 14, 15, 19, 21 and 36 after inoculation) and four rats in the EAE+ECP group (on days 16, 17, 18, 22) died from the effect of EAE induction at different intervals from the inoculation while two rats in the EAE+ECP group died from anaesthesia overdose on day 18. The overall number of days of observation (i.e the sum of the days of survival in each rat) was 655 in the sham group (range 14–50 days of survival in individual rats) and 610 in the EAE+ECP group (range 16–50). In the EAE+ECP group the incidence of relapses was significantly lower than in the EAE group (fig. 1, p = 0.011 by Cox Hazard model). Moreover, although the incidence of relapses was very similar in the two groups until day 25 (i. e. until the second ECP or sham treatment), subsequently the relapse rate decreased in the EAEECP group and, remarkably, no animals in this group had an EAE relapse after the last ECP procedure (fig. 1), thus suggesting a cumulative immunomodulating effect of repeated ECP. The clinical score at the end of the experiment was similar in the rats of the 2 groups (median = 2, range 1–4 in both sham and EAE+ECP groups; mean = 2.35 in sham group vs. 2.06 in EAE+ECP group, p > 0.05 by the two-tailed Mann Whitney test). In a previous study in acute monophasic EAE in Lewis rats [3] we have already demonstrated that ECP is able to reduce the severity of the migration of circulating lymphocytes into the nervous system, an event which is in close relaLETTER TO THE EDITORS

Autologous purified peripheral blood SCT in childhood low-risk relapsed ALL

The treatment of childhood B-cell-precursor ALL after isolated-extramedullary or late relapse is controversial. Most approaches are based on chemotherapy or allogeneic transplantation. The aim of this report is to assess the long-term outcome of children with ‘low-risk’ relapsed ALL treated according to a prospective purified auto-transplantation protocol. From January 1997 to March 2004, at a single pediatric Center, 30 ALL consecutive children, lacking an HLA-identical sibling, were treated according to the autologous purified peripheral blood stem cell protocol after isolated-extramedullary (7) or late medullary (24) relapse. After the ‘DIAVE’ mobilizing regimen a median of 11.6 × 106CD34+/Kg (range 3.9–27.4) were collected. Leukaphereses were depleted by 99% of CD19+cells (range 98–100) by means of a double step immunological purification. The conditioning regimen included TBI. No early severe complications nor transplant-related deaths occurred; late effects, as expected, mostly consisted in endocrinological issues and were assessed at a median follow-up of 8.5 years. Five-year-EFS and survival were 68.5% (s.e. 7.9) and 85.7% (s.e. 5.9), respectively, for the 35 eligible patients and 70.0% (s.e. 8.4) and 86.7% (s.e. 6.2) for the 30 patients actually transplanted as per protocol. The outcome of this series favorably compares with historical data regarding both autologous transplantation and standard salvage chemotherapy.