Marina Podestà

STROMAL DAMAGE AS CONSEQUENCE OF HIGH-DOSE CHEMO/RADIOTHERAPY IN BONE MARROW TRANSPLANT RECIPIENTS

Bone marrow transplant (BMT) relies on the engraftment of donor hemopoietic precursors in the host marrow space. Colony forming units-fibroblasts (CFU-f), the precursor compartment for the osteogenic lineage, are essential to hemopoietic stem cell survival, proliferation and differentiation. We have studied CFU-f in donors (aged 5 months to 62 years) and in patients who had received allogeneic BMT (aged 2 months to 63 years). In donor marrows we found an inverse correlation between CFU-f frequency and age. In BMT recipients CFU-f frequencies were reduced by 60%-90% (p < 0.05) and the numbers did not recover up to 12 years after transplant. Stromal reconstitution to normal levels was found only in patients < 5 years old. In all patients studied CFU-f post-BMT were of host origin. Patients with low CFU-f levels displayed also a decreased bone mineral density (p < 0.05) and significantly reduced levels of long-term culture-initiating cells (LTC-IC) (p < 0.05). Our study demonstrates that the marrow stromal microenvironment is seriously and irreversibly damaged after BMT. Donor cells do not contribute to reconstitute the marrow microenvironment, whose residual CFU-fs remain of host origin.

Direct intrabone transplant of unrelated cord-blood cells in acute leukaemia: a phase I/II study

BACKGROUND Cord-blood transplants are associated with delayed or failed engraftment in about 20% of adult patients. The aim of this phase I/II study was to establish the safety and efficacy of a new administration route (intrabone) for cord-blood cells, measured by the donor-derived neutrophil and platelet engraftment. METHODS Adult patients with acute leukaemia, for whom an unrelated stem-cell transplantation was indicated and no suitable unrelated human leucocyte antigen (HLA)-matched donor had been identified, were included in the study and underwent a cord-blood transplant in San Martino Hospital, Genoa, Italy. Eight patients were in first complete remission, ten in second complete remission, and 14 had advanced-stage, refractory disease. HLA matching was 5/6, 4/6, and 3/6 for 9, 22, and one patient, respectively. Cord-blood cells were concentrated in four 5-mL syringes, and were infused in the superior-posterior iliac crest under rapid general anaesthesia. Median transplanted cell dose was 2.6 x 10(7)/kg (range 1.4-4.2). The primary endpoint was the probability of neutrophil and platelet recovery after intrabone cord-blood transplantantion. Secondary endpoints included the incidence of acute graft-versus-host disease, relapse, and overall survival. This trial is registered on the ClinicalTrials.gov website, number NCT 00696046. FINDINGS Between March 31, 2006, and Jan 25, 2008, 32 consecutive patients with acute myeloid leukaemia (n=20) or acute lymphoblastic leukaemia (n=12) underwent a cord-blood transplant (median age 36 years [range 18-66]). No complications occurred during or after the intrabone infusion of cells. Four patients with advanced-stage disease died within 12 days of the procedure. Median time to recovery of neutrophils in 28 patients (>/=0.5 x 10(9)/L) was 23 days (range 14-44) and median time to recovery of platelets in 27 patients (>/=20 x 10(9)/L) was 36 days (range 16-64). All patients were fully chimeric from 30 days after transplantation to the last follow-up visit, suggesting an early complete donor engraftment. No patient developed grade III-IV acute graft-versus-host disease. Causes of death were transplant related (n=5), infection (n=7), and relapse (n=4). 16 patients were alive and in haematological remission at a median follow-up of 13 months (range 3-23). INTERPRETATION Our preliminary data suggest that direct intrabone cord-blood transplantation overcomes the problem of graft failure even when low numbers of HLA-mismatched cord-blood cells are transplanted, thus leading to the possibility of use of this technique in a large number of adult patients.

Phenotypic and functional heterogeneity of human NK cells developing after umbilical cord blood transplantation: a role for human cytomegalovirus?

Natural killer (NK) cells play a crucial role in early immunity after hematopoietic stem cell transplantation because they are the first lymphocyte subset recovering after the allograft. In this study, we analyzed the development of NK cells after intrabone umbilical cord blood (CB) transplantation in 18 adult patients with hematologic malignancies. Our data indicate that, also in this transplantation setting, NK cells are the first lymphoid population detectable in peripheral blood. However, different patterns of NK-cell development could be identified. Indeed, in a group of patients, a relevant fraction of NK cells expressed a mature phenotype characterized by the KIR(+)NKG2A(-) signature 3-6 months after transplantation. In other patients, most NK cells maintained an immature phenotype even after 12 months. A possible role for cytomegalovirus in the promotion of NK-cell development was suggested by the observation that a more rapid NK-cell maturation together with expansion of NKG2C(+) NK cells was confined to patients experiencing cytomegalovirus reactivation. In a fraction of these patients, an aberrant and hyporesponsive CD56(-)CD16(+)p75/AIRM1(-) NK-cell subset (mostly KIR(+)NKG2A(-)) reminiscent of that described in patients with viremic HIV was detected. Our data support the concept that cytomegalovirus infection may drive NK-cell development after umbilical CB transplantation.

Multipotent mesenchymal stromal cells from amniotic fluid: solid perspectives for clinical application

Mesenchymal stromal cells are multipotent cells potentially useful in regenerative medicine. These cells are usually obtained from the bone marrow; however, the cell dose may be a critical factor, and alternative sources need to be explored. This study suggests that amniotic fluid represents a rich source of mesenchymal stromal cells. Background Mesenchymal stromal cells are multipotent cells considered to be of great promise for use in regenerative medicine. However, the cell dose may be a critical factor in many clinical conditions and the yield resulting from the ex vivo expansion of mesenchymal stromal cells derived from bone marrow may be insufficient. Thus, alternative sources of mesenchymal stromal cells need to be explored. In this study, mesenchymal stromal cells were successfully isolated from second trimester amniotic fluid and analyzed for chromosomal stability to validate their safety for potential utilization as a cell therapy product. Design and Methods Mesenchymal stromal cells were expanded up to the sixth passage starting from amniotic fluid using different culture conditions to optimize large-scale production. Results The highest number of mesenchymal stromal cells derived from amniotic fluid was reached at a low plating density; in these conditions the expansion of mesenchymal stromal cells from amniotic fluid was significantly greater than that of adult bone marrow-derived mesenchymal stromal cells. Mesenchymal stromal cells from amniotic fluid represent a relatively homogeneous population of immature cells with immunosuppressive properties and extensive proliferative potential. Despite their high proliferative capacity in culture, we did not observe any karyotypic abnormalities or transformation potential in vitro nor any tumorigenic effect in vivo. Conclusions Fetal mesenchymal stromal cells can be extensively expanded from amniotic fluid, showing no karyotypic abnormalities or transformation potential in vitro and no tumorigenic effect in vivo. They represent a relatively homogeneous population of immature mesenchymal stromal cells with long telomeres, immunosuppressive properties and extensive proliferative potential. Our results indicate that amniotic fluid represents a rich source of mesenchymal stromal cells suitable for banking to be used when large amounts of cells are required.

Extracellular cyclic ADP-ribose increases intracellular free calcium concentration and stimulates proliferation of human hemopoietic progenitors

Cyclic ADP‐ribose (cADPR) is a universal second messenger that regulates many calcium‐related cellular events by releasing calcium from intracellular stores. Since these events include enhanced cell proliferation and since the bone marrow harbors both ectoenzymes that generate cADPR from NAD+ (CD38 and BST‐1), we investigated the effects of extracellular cADPR on human hemopoietic progenitors (HP). Exposure of HP to 100 μM cADPR for 24 h induced a significant increase in colony output (P<0.01) and colony size (P<0.003). A horizontal expansion of HP, as demonstrated by a markedly increased replating efficiency in semisolid medium (up to 700 times compared to controls), was also observed, indicating that cADPR priming can affect cell growth for multiple generations over several weeks after exposure. Influx of extracellular cADPR into the cells was demonstrated, and a causal relationship between the functional effects and the increase of intracellular free calcium concentration induced by cADPR on HP was established through the use of specific antagonists. Similar effects on HP were produced by nanomolar concentrations of the nonhydrolyzable cADPR analog 3‐deaza‐cADPR. These data demonstrate that extracellular cADPR behaves as a cytokine enhancing the proliferation of human HP, a finding that may have biomedical applications for the ex vivo expansion of hemopoietic cells.—Podestà, M., Zocchi, E., Pitto, A., Usai, C., Franco, L., Bruzzone, S., Guida, L., Bacigalupo, A., Scadden, D. T., Walseth, T. F., De Flora, A., Daga, A. Extracellular cyclic ADP‐ribose increases intracellular free calcium concentration and stimulates proliferation of human hemopoietic progenitors. FASEB J. 14, 680–690 (2000)

In vivo mobilization of karyotypically normal peripheral blood progenitor cells in high-risk MDS, secondary or therapy-related acute myelogenous leukaemia

We have previously reported that mobilization of Philadelphia (Ph) chromosome‐negative progenitors is possible in a significant number of Ph1‐positive acute lymphoblastic leukaemia (ALL) and chronic myelogenous leukaemia (CML) patients. In this pilot study we employed the same approach for patients with RAEB‐t, secondary AML (sAML) and therapy‐related AML (t‐AML). All patients except one had double or complex cytogenetic abnormalities in marrow cells before mobilization therapy. All patients received an idarubicin‐containing regimen (mini‐ICE protocol) followed by rh‐G‐CSF and the first leukapheresis was performed as they were recovering from aplasia. In six out of nine patients the leukapheresis product was entirely karyotypically normal, combined with a significant number of CFU‐GM, CD34+ cells and LTC‐IC. Recovery time from mobilization therapy was short and no patient died as a result of the procedure. To date, three patients have undergone autografting using their karyotypically normal collections, of which two (sAML) are alive with karyotypically normal marrow a few months after autografting.

Severe Aplastic Anaemia: Correlation of in Vitro Tests with Clinical Response to Immunosuppression in 20 Patients

Summary. Colony formation in agar (CFU‐c) was studied in 20 patients with severe aplastic anaemia by three different assays: (1) cultures of light density untreated marrow cells; (2) cultures of marrow cells manipulated in order to enhance colony formation (pretreatment with antilymphocytic globulin, ALG, or 6‐methylprednisolone, 6‐MPr, T cell depletion, adherent cell (AC) depletion, depletion of both AC and T cells), and (3) co‐culture of putative suppressor T cells with autologous T‐depleted marrow cells.

Lymphocyte subsets recovery following allogeneic bone marrow transplantation (BMT): CD4+ cell count and transplant-related mortality

To assess the kinetics of lymphocyte subset recovery, 758 allografted patients were monitored by surface markers (CD3, CD4, CD8, CD56), with a 5-year follow-up. The donor was a matched sibling donor (MSD) (n=502) or an alternative donor (family mismatched or unrelated, AD) (n=256). The stem cell source was bone marrow for all patients. CD4+ cell recovery was influenced—in univariate analysis—by three factors: donor type, patient age and GvHD. This was not the case for CD8+ and CD56+ cells. The median CD4+ cell count on day +35 after HSCT was 86/μl. Patients achieving this CD4+ cell count had significantly lower transplant-related mortality (TRM) compared to patients who did not achieve this CD4+ cell count (20 vs 39%, P=0.00001), due to a lower risk of lethal infections (24 vs 47%, P=0.0003). In multivariate analysis MSD (RR 3.45, P=0.0001) and recipient age less than 16 years (RR 3.23, P=0.003) were significantly associated with a better CD4+ cell recovery. CD4+ counts on day +35 was predicted TRM (RR=1.97, P=0.0017) together with acute GvHD grade II–IV (RR 1.59, P=0.0097). No difference of TRM was observed for CD8+ and CD56+ cell counts.

The association of human mesenchymal stem cells with BMP-7 improves bone regeneration of critical-size segmental bone defects in athymic rats

Critical size segmental bone defects are still a major challenge in reconstructive orthopedic surgery. Transplantation of human mesenchymal stem cells (hMSC) has been proposed as an alternative to autogenous bone graft, as MSC can be expanded in vitro and induced to differentiate into bone-regenerating osteoblasts by several bone morphogenetic proteins (BMP). The aim of this study was to investigate whether the association of hMSC and BMP-7, with providing the necessary scaffold to fill the bone loss, improved bone regeneration in a rat model of critical size segmental bone defect, compared to treatment with either hMSC or BMP-7 and the matrix. In addition, we tested whether pre-treatment of hMSC with cyclic ADP-ribose (cADPR), an intracellular Ca2+ mobilizer previously shown to accelerate the in vitro expansion of hMSC (Scarfì S et al, Stem Cells, 2008), affected the osteoinductive capacity of the cells in vivo. X-ray analysis, performed 2, 10 and 16 weeks after transplantation, revealed a significantly higher score in the rats treated with hMSC and BMP-7 compared to controls, receiving either hMSC or BMP-7. Microtomography and histological analysis, performed 16weeks after transplantation, confirmed the improved bone regeneration in the animals treated with the association of hMSC and BMP-7 compared to controls. Pre-treatment with cADPR to stimulate hMSC proliferation in vitro did not affect the bone regenerating capacity of the cells in vivo. These results indicate that the association of in vitro expanded hMSC with BMP-7 provide a better osteoinductive graft compared to either hMSC or BMP-7 alone. Moreover, cADPR may be used to stimulate hMSC proliferation in vitro in order to reduce the time required to obtain a transplantable number of cells, with no adverse effect on the bone regenerating capacity of hMSC.

High dose bolus methylprednisolone for the treatment of acute graft versus host disease

SummaryNineteen patients with acute graft versus host disease (GvHD) following bone marrow transplantation (BMT) were treated with high dose bolus 6-methylprednisolone (BMPr), at the dose of 20 mg/kg/day i.v. for the first 3 days, 10 mg/kg/day i.v. for the following 4 days, and then at doses gradually tapered down to 1 mg/kg/day. All patients except one, who was given preventive BMPr 5 mg/kg/day i.v. on alternate days, were placed on preventive methotrexate therapy after BMT. Sixteen patients were grafted with an HLA matched, and three patients with an HLA mismatched marrow.Overall complete response rate in the HLA matched group was 43%, with an additional 50% showing a partial response. In the HLA mismatched group there were no responses and all three patients proved refractory to BMPr.With respect to organ involvement the complete and partial response rates were respectively 50% and 33% in the skin, 36% and 28% in the liver, 18% and 55% in the gut.Six of sixteen patients in the HLA matched group and none of the three in the HLA mismatched group are surviving. Thirteen patients died: nine patients for causes directly or indirectly related to GvHD, four of other causes (relapse, rejection, hemorrhage and idiopathic interstitial pneumonia). Side effects of BMPr consisted in hyperglicemia, and steroid associated gastritis in 2/3 of the patients, both of which responded well to conventional treatment.This study indicates that high dose BMPr is an effective form of treatment for established acute GvHD, and has no major side effects. The efficacy of BMPr is less clear in recipients of HLA mismatched grafts.