Elisabetta Zappone

EXPRESSION OF CXCR4, THE RECEPTOR FOR STROMAL CELL-DERIVED FACTOR-1 ON FETAL AND ADULT HUMAN LYMPHOHEMATOPOIETIC PROGENITORS

Stromal cell‐derived factor‐1 (SDF‐1) is a CXC chemokine produced by stromal cells that acts as a chemoattractant for human CD34+ progenitor cells. We investigated the expression of CXCR4, the receptor for SDF‐1, on CD34+ cells from different hematopoietic sites and developmental stages. CXCR4 was detected by flow cytometry on 37 % of fetal bone marrow (BM) [gestation weeks (gw) 14 – 23] and 40 % of adult BM CD34+ cells. Interestingly, in fetal liver CD34+ cells, CXCR4 was expressed at lower levels at later stages (9 %, gw 20 – 23) compared to early stages of development (39 %, gw 7.5 – 18), suggesting a development‐related change in the migratory capacity of progenitors. CXCR4 was detected at similar levels on both phenotypically primitive and committed progenitors from fetal and adult sites. However, B cell lineage progenitor and precursor cells expressed CXCR4 at the highest density (80 % of BM CD34+/CD10+ pro‐B cells are CXCR4+). CXCR4 was also expressed in the fetal thymus in early T cell precursors and found to be down‐regulated during T cell maturation. Finally, we found that stem cell factor, alone or in combination with other cytokines, can up‐modulate CXCR4 expression on CD34+ cells by three‐ to fourfold. In conclusion, our results suggest that CXCR4 may play an important role in the local and systemic trafficking of human CD34+ cells as well as in human B lymphopoiesis and that its expression can be modulated by cytokines.

Immunological Outcome in Haploidentical-HSC Transplanted Patients Treated with IL-10-Anergized Donor T Cells.

T-cell therapy after hematopoietic stem cell transplantation (HSCT) has been used alone or in combination with immunosuppression to cure hematologic malignancies and to prevent disease recurrence. Here, we describe the outcome of patients with high-risk/advanced stage hematologic malignancies, who received T-cell depleted (TCD) haploidentical-HSCT (haplo-HSCT) combined with donor T lymphocytes pretreated with IL-10 (ALT-TEN trial). IL-10-anergized donor T cells (IL-10-DLI) contained T regulatory type 1 (Tr1) cells specific for the host alloantigens, limiting donor-vs.-host-reactivity, and memory T cells able to respond to pathogens. IL-10-DLI were infused in 12 patients with the goal of improving immune reconstitution after haplo-HSCT without increasing the risk of graft-versus-host-disease (GvHD). IL-10-DLI led to fast immune reconstitution in five patients. In four out of the five patients, total T-cell counts, TCR-Vβ repertoire and T-cell functions progressively normalized after IL-10-DLI. These four patients are alive, in complete disease remission and immunosuppression-free at 7.2 years (median follow-up) after haplo-HSCT. Transient GvHD was observed in the immune reconstituted (IR) patients, despite persistent host-specific hypo-responsiveness of donor T cells in vitro and enrichment of cells with Tr1-specific biomarkers in vivo. Gene-expression profiles of IR patients showed a common signature of tolerance. This study provides the first indication of the feasibility of Tr1 cell-based therapy and paves way for the use of these Tr1 cells as adjuvant treatment for malignancies and immune-mediated disorders.

Characteristics of a ferritin-binding protein present in human serum

Summary. The ferritin present in human serum differs from the ferritins found in tissues and other body fluids in having negligible proportions of H subunits. This has been related to the possible presence of binding factors which would form complexes with H‐subunit containing ferritins and thereby determine their rapid clearance and/or interference with immunoassays (serum inhibition’). In this work we have tried to identify and characterize these binding factors. Dotting and blotting experiments demonstrated an interaction between tissue ferritins and human serum. This was stronger with human heart and recombinant H‐type ferritin obtained from E. coli than with human liver ferritin. The serum binder appeared to be a glycoprotein migrating in the beta‐2 region and with a molecular weight of about 200 000 and pi between 4 and 5. Two different approaches to purification of the ferritin‐binding protein yielded enriched fractions containing also the complement proteins C3 and C4, the plasma protease inhibitor alpha‐2‐macroglobulin, and immunoglobulins. These in vitro findings may have physiological relevance.

Immunological reactivity of serum ferritin in patients with malignancy.

Serum ferritin has been suggested as a tumor marker in the diagnosis of certain malignancies and for following the activity or dissemination of the malignant process. Since neoplastic tissues generally contain more acidic isoferritins than their normal tissue counterparts, it has also been suggested that the specific assay of such isoferritins in serum may be of particular value in the diagnosis of malignancy. In this work, we have evaluated ferritin concentration in the serum of normal subjects and patients with acute nonlymphocytic leukemia, Hodgkins disease, breast cancer and lung cancer by simultaneously using three different immunoassays: (a) an immunoradiometric assay based on polyclonal antibodies against human liver (basic, L-subunit rich) ferritin, (b) a radioimmunoassay based on polyclonad antibodies against HeLa cell (acidic, H-subunit rich) ferritin, and (c) an immunoradiometric assay based on the monoclonal antibody 2A4 raised against human heart (acidic, H-subunit rich) ferritin. Most of the patients studied had increased values for liver-type ferritin in the absence of increased iron stores. Binding of serum ferritin to concanavalin A did not prove to be useful in distinguishing a tumor-specific basic isoferritin. The HeLa ferritin assay was found to be less specific than the heart ferritin assay in the detection of acidic isoferritins, and did not provide any advantage over the liver assay in detecting the increased levels of serum ferritin associated with malignant disease. Heart-type ferritin was found in one-fifth of normal sera and 64 % of sera from patients with malignancy. Values were very low compared with those for basic ferritin, ranging from less than 0.1 to 17 % of total serum ferritin (geometric mean value 1.3 %) in patients with malignancy. These findings indicate that at present there is little application for serum ferritin immunoassays based on antibodies to HeLa cell or heart ferritin in the diagnosis or monitoring of malignant disease. This seems to be due to the presence in human serum of biding factors which are responsible for the rapid clearance of acidic isoferritins from the circulation. The serum concentration of basic ferritin, however, can be useful in the diagnosis and management of some malignancies, and it is possible that studies on cell isoferritins can be important in biologic monitoring of neoplastic disorders. It should also be noted that the increased levels of serum ferritin found in patients with malignancy can exert adverse effects on the host immune response and perhaps an inhibitory effect on hematopoiesis.

Qualitative and quantitative polymerase chain reaction detection of the residual myeloma cell contamination after positive selection of CD34+ cells with small‐ and large‐scale Miltenyi cell sorting system

Summary.  The purging efficacy of the Miltenyi sorting system was evaluated by qualitative and TaqMan quantitative polymerase chain reaction (PCR) in myeloma patients, using immunoglobulin genes. After small‐scale selection, qualitative PCR showed that in 6 of 12 leukaphereses myeloma cells were no longer detectable. Envisaging a possible clinical application, the leukaphereses from three patients underwent large‐scale selection. Qualitative PCR showed that myeloma cells were still detectable. Quantitative PCR, performed in two patients, showed a tumour depletion of␣1 and 2 logs respectively. Although numbers are small, the␣promising results obtained with small‐scale selection were␣not reproduced in large‐scale experiments.

A screen for RAS mutations in individuals at risk of secondary leukaemia due to occupational exposure to petrochemicals

Occupational exposure to petrochemicals, in particular benzene, has been identified as a risk factor in the development of acute leukaemia. A cohort of exposed (n = 44) and non-exposed individuals (n = 19) from the same petrochemical installation were screened by polymerase chain reaction (PCR) followed by oligonucleotide hybridization (ONH) for the presence of mutations in the H, K, and NRAS cellular proto-oncogenes. A KRAS mutation was detected in one individual from the exposed group who was haematologically normal at the time of sampling. The presence of this mutation was confirmed by nude mouse tumorigenicity assay and positively identified as a K13 Gly-Asp substitution by cloning and sequencing.