Marina Ratta

Leber's hereditary optic neuropathy Biochemical effect of 11778/ND4 and 3460/ND1 mutations and correlation with the mitochondrial genotype

To clarify the bioenergetic relevance of mtDNA mutations in Lebers hereditary optic neuropathy (LHON), we investigated affected individuals and healthy carriers from six Italian LHON families harboring the 11778/ ND4 and the 3460/ND1 mtDNA mutations. The enzymatic activities of mitochondrial complex I and its sensitivity to the potent inhibitors rotenone and rolliniastatin-2 were studied in mitochondrial particles from platelets, in correlation with mtDNA analysis of platelets and leukocytes. In platelets homoplasmic for mutant mtDNA, both 11778/ND4 and 3460/ND1 mutations induced resistance to rotenone and the 3460/ND1 mutation also provoked a marked decrease in the specific activity of complex I. Individuals heteroplasmic in platelets for either mutation showed normal biochemical features, indicating functional complementation of wild-type mtDNA. There was no correlation between the clinical status and mtDNA homoheteroplasmy in platelets, but the biochemical features correlated with the mitochondrial genotype of platelets. In some cases, the degree of mtDNA heteroplasmy differed in platelets and leukocytes from the same individual with a prevalence of wild-type mtDNA in the platelets. These results imply that biochemical studies on mitochondrial diseases should always be integrated with mtDNA analysis of the same tissue investigated and also suggest that the mtDNA analysis on the leukocyte fraction, as usually performed in LHON, does not necessarily reflect the mutant genotype level of other tissues. The differential tissue heteroplasmy may be more relevant than previously thought in determining disease penetrance.

Functional alterations of the mitochondrially encoded ND4 subunit associated with Leber's hereditary optic neuropathy

Lebers hereditary optic neuropathy (LHON) is a maternally inherited disease associated with point mutations in mitochondrial DNA. The most frequent of these mutations is the G‐to‐A substitution at nucleotide position 11,778 which changes an evolutionarily conserved arginine with a histidine at position 340 in subunit ND4 of NADH: ubiquinone reductase (respiratory complex I). We report that this amino acid substitution alters the affinity of complex I for the ubiquinone substrate and induces resistance towards its potent inhibitor rotenone in mitochondria of LHON patients. Such changes could reflect a substantial loss in the energy conserving function of NADH: ubiquinone reductase and thus explain the pathological effect of the ND4/11,778 mutation.

Generation and functional characterization of human dendritic cells derived from CD34 ˛ cells mobilized into peripheral blood: comparison with bone marrow CD34 ˛ cells

Dendritic cells (DCs) are the most powerful professional antigen‐presenting cells (APC), specializing in capturing antigens and stimulating T‐cell‐dependent immunity. In this study we report the generation and characterization of functional DCs derived from both steady‐state bone marrow (BM) and circulating haemopoietic CD34+ cells from 14 individuals undergoing granulocyte colony‐stimulating factor (G‐CSF) treatment for peripheral blood stem cells (PBSC) mobilization and transplantation. Clonogenic assays in methylcellulose showed an increased frequency and proliferation of colony‐forming unit‐dendritic cells (CFU‐DC) in circulating CD34+ cells, compared to that of BM CD34+ precursors in response to GM‐CSF and TNF‐α with or without SCF and FLT‐3L. Moreover, peripheral blood (PB) CD34+ cells generated a significantly higher number of fully functional DCs, as determined by conventional mixed lymphocyte reactions (MLR), than their BM counterparts upon different culture conditions. DCs derived from mobilized stem cells were also capable of processing and presenting soluble antigens to autologous T cells for both primary and secondary immune response. Replacement of the early‐acting growth factors SCF and FLT‐3L with IL‐4 at day 7 of culture of PB CD34+ cells enhanced both the percentage of total CD1a+ cells and CD1a+CD14− cells and the yield of DCs after 14 d of incubation. In addition, the alloreactivity of IL‐4‐stimulated DCs was significantly higher than those generated in the absence of IL‐4. Furthermore, autologous serum collected during G‐CSF treatment was more efficient than fetal calf serum (FCS) or two different serum‐free media for large‐scale production of DCs. Thus, our comparative studies indicate that G‐CSF mobilizes CD34+ DC precursors into PB and circulating CD34+ cells represent the optimal source for the massive generation of DCs. The sequential use of early‐acting and intermediate‐late‐acting colony‐stimulating factors (CSFs) as well as the use of autologous serum greatly enhanced the growth of DCs. These data may provide new insights for manipulating immunocompetent cells for cancer therapy.

Stem Cell Factor and FLT3-Ligand Are Strictly Required to Sustain the Long-Term Expansion of Primitive CD34+DR− Dendritic Cell Precursors

We studied cytokine-driven differentiation of primitive human CD34+HLA-DR− cells to myeloid dendritic cells (DC). Hemopoietic cells were grown in long-term cultures in the presence of various combinations of early acting cytokines such as FLT3-ligand (FLT3-L) and stem cell factor (SCF) and the differentiating growth factors GM-CSF and TNF-α. Two weeks of incubation with GM-CSF and TNF-α generated fully functional DC. However, clonogenic assays demonstrated that CFU-DC did not survive beyond 1 wk in liquid culture regardless of whether FLT3-L and/or SCF were added. FLT3-L or SCF alone did not support DC maturation. However, the combination of the two early acting cytokines allowed a 100-fold expansion of CFU-DC for >1 month. Phenotypic analysis demonstrated the differentiation of CD34+DR− cells into CD34−CD33+DR+CD14+ cells, which were intermediate progenitors capable of differentiating into functionally active DC upon further incubation with GM-CSF and TNF-α. As expected, GM-CSF and TNF-α generated DC from committed CD34+DR+ cells. However, only SCF, with or without FLT3-L, induced the expansion of DC precursors for >4 wk, as documented by secondary clonogenic assays. This demonstrates that although GM-CSF and TNF-α do not require additional cytokines to generate DC from primitive human CD34+DR− progenitor cells, they do force terminal differentiation of DC precursors. Conversely, FLT3-L and SCF do not directly affect DC differentiation, but instead sustain the long-term expansion of CFU-DC, which can be induced to produce mature DC by GM-CSF and TNF-α.

Efficient presentation of tumor idiotype to autologous T cells by CD83(+) dendritic cells derived from highly purified circulating CD14(+) monocytes in multiple myeloma patients.

To generate mature and fully functional CD83(+) dendritic cells derived from circulating CD14(+) cells highly purified from the leukapheresis products of multiple myeloma patients.CD14(+) monocytes were selected by high-gradient magnetic separation and differentiated to immature dendritic cells with granulocyte-macrophage colony-stimulating factor and interleukin-4 for 6-7 days and then induced to terminal maturation by the addition of tumor necrosis factor-alpha or stimulation with CD40 ligand. Dendritic cells were characterized by immunophenotyping, evaluation of soluble antigens uptake, cytokine secretion, capacity of stimulating allogeneic T cells, and ability of presenting nominal antigens, including tumor idiotype, to autologous T lymphocytes. Phenotypic analysis showed that 90% +/- 6% of cells recovered after granulocyte-macrophage colony-stimulating factor and interleukin-4 stimulation expressed all surface markers typical of immature dendritic cells and demonstrated a high capacity of uptaking soluble antigens as shown by the FITC-dextran assay. Subsequent exposure to maturation stimuli induced the downregulation of CD1a and upregulation of CD83, HLA-DR, costimulatory molecules and induced the secretion of large amounts of interleukin-12. Mature CD83(+) cells showed a diminished ability of antigen uptake whereas they proved to be potent stimulators of allogeneic T cells in a mixed lymphocyte reaction. Monocyte-derived dendritic cells, pulsed before the addition of maturation stimuli, were capable of presenting soluble proteins such as keyhole limpet hemocyanin and tetanus toxoid to autologous T cells for primary and secondary immune response, respectively. Conversely, pulsing of mature (CD83(+)) dendritic cells was less efficient for the induction of T-cell proliferation. More importantly, CD14(+) cells-derived dendritic cells stimulated autologous T-cell proliferation in response to a tumor antigen such as the patient-specific idiotype. Moreover, idiotype-pulsed dendritic cells induced the secretion of interleukin-2 and gamma-interferon by purified CD4(+) cells. T-cell activation was better achieved when Fab immunoglobulin fragments were used as compared with the whole protein. When dendritic cells derived from CD14(+) cells from healthy volunteers were analyzed, we did not find any difference with samples from myeloma patients as for cell yield, phenotypic profile, and functional characteristics. These studies demonstrate that mobilized purified CD14(+) cells represent the optimal source for the production of a homogeneous cell population of mature CD83(+) dendritic cells suitable for clinical trials in multiple myeloma.

SELECTION AND TRANSPLANTATION OF AUTOLOGOUS HEMATOPOIETIC CD34+ CELLS FOR PATIENTS WITH MULTIPLE MYELOMA

Here we review our recent experience addressing the issue of positive selection and transplantation of hematopoietic CD34+ cells to reduce neoplastic contamination in peripheral blood (PB) autografts from patients with multiple myeloma (MM). We evaluated PB samples from 30 pretreated MM patients following the administration of high dose cyclophosphamide (Cy; 7g/m2 or 4g/m2) and granulocyte-colony stimulating factor (G-CSF), for collection of circulating stem cells (PBSC) to support hematopoietic reconstitution following myeloablative radio-chemotherapy. Twenty six patients showed adequate mobilization of CD34+ progenitor cells and were submitted to PBSC collection. Circulating hematopoietic CD34+ cells were highly enriched by avidin-biotin immunoabsorption, cryopreserved, and used to reconstitute BM function after myeloablative therapy in 13 patients. The median purity of the enriched CD34+ cell population was 89.5% (range 51-94%) with a 75-fold increase compared to the pretreatment samples. The median overall recovery of CD34+ cells and CFU-GM was 58% (range 33-95%) and 45% (range 7-100%), respectively. Positive selection of CD34+ cells resulted in 2.5-3 log of plasma cells and CD19+ B-lineage cells depletion as determined by immunofluorescence studies, although DNA analysis of CDR III region of IgH gene demonstrated the persistence of minimal residual disease (MRD) in 5 out of 6 patient samples studied. Myeloma patients were reinfused with enriched CD34+ cells after myeloablative therapy consisting of total body irradiation (TBI, 1000 cGy) and high dose Melphalan (140 mg/m2) or Melphalan (200 mg/m2) alone. They received a median of 5 x 10(6) CD34+ cells/kg and showed a rapid reconstitution of hematopoiesis: the median time to 0.5 x 10(9) neutrophils, 20 and 50 x 10(9) platelets/L of PB was 10, 11 and 12 days, respectively. When we analyzed the immunological reconstitution of this group of patients, we observed a rapid and full recovery of total lymphocyte and NK cell count, although the absolute CD4+ cell count was lower than pretreatment level. These results, as well as other clinically significant parameters, did not significantly differ from those of patients (=13) receiving unmanipulated PBSC following the same pretransplant conditioning regimen. The results of this trial demonstrate that positive selection of CD34+ cells reduces the contamination of myeloma cells from the apheresis products up to 3 log and provides a cell suspension capable of restoring a normal hematopoiesis after a TBI-containing conditioning regimen. Based on this pilot trial, we have recently started a clinical study involving a double autotransplant, conditioned with melphalan (200 mg/m2) followed by melphalan (140 mg/m2) and busulphan (14 mg/kg), supported by the reinfusion of highly purified CD34+ cells.

BIOLOGICAL CHARACTERIZATION OF CD34+ CELLS MOBILIZED INTO PERIPHERAL BLOOD

We evaluated 18 acute myeloblastic leukaemia (AML) and myelodysplastic syndrome (MDS) patients with abnormal karyotype at diagnosis who underwent peripheral blood stem cell (PBSC) transplantation. To evaluate the presence of residual tumour cells, bone marrow (BM) samples and PBSC collections were analysed by cytogenetics and in selected cases also by fluorescence in situ hybridisation (FISH) and molecular studies. All patients were considered to be in morphologic and cytogenetic complete remission (CR) at the time of mobilisation. Seven patients showed neoplastic cells in PBSC harvest and/or BM specimen before reinfusion. Cytogenetic studies revealed contamination in apheretic collections in one patient only, while three patients had BM but not PBSC contamination. Three more patients had leukaemic cells both in the BM and PBSC. All but one (with only BM contamination) of these patients relapsed within 9 months. However, five more patients relapsed after transplantion: in four cases there was no cytogenetic sign of contamination either in PBSC or BM cells and in one case no molecular evidence was revealed either. This study suggests that, whereas the presence of leukaemic cells in autologous grafts correlates with a poor prognosis, the lack of detection of tumour cells is not always predictive of long-term disease-free survival. More importantly, PBSC collections from AML patients are not contaminated by leukaemic cells if the BM is disease-free.

T cell alloreactivity induced by normal G-CSF-mobilized CD34+ blood cells

In this study, the hypothesis that a subset of granulocyte colony-stimulating factor (G-CSF)-mobilized CD34+ blood cells may actively induce an allogeneic T cell response in vitro was tested. Circulating CD34+ cells were purified to ⩾98% by high gradient magnetic separation and then analyzed for the coexpression of HLA-DR, the common β-chain of the leukointegrin family CD18 and costimulatory molecules CD80 (B7–1) and CD86 (B7–2). These antigens were expressed on average on: 94.9 ± 2.5%, 64.4 ± 15.4%, 0% and 1.9 ± 1.2% CD34+ blood cells, respectively. Irradiated CD34+ cells induced a high proliferative response of allogeneic, but not autologous, purified CD4+ and CD8+ T cells in primary mixed leukocyte culture (MLC). An average three-fold lower CD4+ and CD8+ T cell response was induced by mononuclear cells from G-CSF-treated donors. A lower frequency of allostimulating cells among mononuclear cells rather than among CD34+ cells in the apheresis was documented by limiting dilution assay (LDA). As previously observed with marrow, sorted CD34+/CD18+ cells induced the proliferation of allogeneic T cells in MLC, while CD34+/CD18− cells, which were >94% HLA-DR+ and contained both committed (CFU-C) and early (LTC-IC) hematopoietic progenitors, stimulated allogeneic T cells poorly. Three-color staining cytofluorimetry indicated that expression of CD80 and CD86 were upregulated in 6.9 ± 4.9 and 10.7 ± 2.6% CD34+ blood cells respectively, after 24–30 h of culture with autologous or allogeneic mononuclear cells, or with CD4+, or CD8+ T cells, but not with medium alone. Moreover, the upregulation of CD86 was observed on CD34+/CD18+ rather than on CD34+/CD18− cells after 30 h in MLC. Blocking experiments demonstrated that preincubation of stimulator and responder cells with anti-CD80 plus anti-CD86 monoclonal antibodies induced a 84 ± 8% inhibition of CD34+ cell allostimulating activity after 6 days in primary MLC. These results suggest that G-CSF-mobilized CD34+ hematopoietic progenitors with alloantigen presenting function express CD18 and may upregulate CD80 and CD86 upon interaction with T cells. Since activation of B7 costimulatory molecules represents an active costimulatory pathway on G-CSF-mobilized CD34+ cells, the blockade of these molecules or, alternatively, the use of selected non-immunogenic CD34+/CD18− blood stem cells may represent a new strategy for reducing graft rejection and overcoming HLA barriers in allogeneic stem cell transplantation.

Selective expansion of normal haemopoietic progenitors from chronic myelogenous leukaemia marrow.

CD34+ and CD34+ DR− cells from the bone marrow (BM) of chronic‐phase chronic myelogenous leukaemia (CML) patients at diagnosis were tested for their colony‐forming ability in response to early and intermediate‐late colony stimulating factors (CSFs). Molecular analysis revealed that 55.6 ± 9% SD of CD34+DR− colonies, in which actin and ABL mRNA were detectable, expressed the product of the BCR‐ABL gene. The percentage and the clonogenic efficiency of CML DR− cells were significantly lower than those of comparable DR− cells from normal donors. However, clonogenic assays using recombinant human CSFs demonstrated a remarkable proliferation of CML cells when stimulated by SCF, IL‐11 and IL‐3, used as single factors in the presence of erythropoietin (EPO) and was almost entirely due to erythroid progenitors. Conversely, optimal stimulation of CD34+DR− cells from normal donors required co‐incubation with three or more CSFs. Stroma‐noncontact long‐term cultures were then established in the presence of exogenous CSFs and human irradiated allogeneic stromal layers or the murine stromal cell line M2‐10B4, engineered to produce G‐CSF and IL‐3. In these cultures the combination of SCF and IL‐3 induced a 25.4 ± 5 SD, 40 ± 6 SD and 20.5 ± 6 SD fold increase of colony‐forming unit cells (CFU‐C), at weeks 2, 4 and 5, respectively. At the same time‐points the number of primitive long‐term culture initiating cells (LTC‐IC) showed a 4 ± 2 SD, 3.3 ± 1.5 SD and 2.3 ± 1 SD fold increase compared to baseline values. BCR‐ABL mRNA analysis of single colonies demonstrated that 27 ± 9% SD and 7 ± 3% SD CFU‐C at weeks 4 and 5, respectively, expressed the fusion gene, whereas leukaemic LTC‐IC disappeared from the culture by week 2. These results suggest that leukaemic CD34+DR− cells have a different pattern of response to CSFs than normal cells. In addition, we established culture conditions which allow selective expansion of benign haemopoietic cells coexisting with leukaemic progenitors.