Immunoglobulins G and lysozyme concentrations in canine fetal fluids at term of pregnancy.

In the dog, the endotheliochorial placenta allows only the 5% to 10% transfer of maternal antibodies to the fetus, but the timing and the factors influencing the immunoglobulin G (IgG) transplacental transport were not fully investigated. The aims of the present study were the following: (1) to assess the presence of both IgG and lysozyme in amniotic and allantoic fluids collected from fully developed and viable newborn puppies born by elective cesarean section at term and possible correlations between amniotic and allantoic IgG and lysozyme levels; (2) to verify possible differences in IgG and lysozyme concentrations between the two fluids; and (3) to detect possible differences in IgG and lysozyme fetal fluid levels in relation to the maternal breed body size and parity, as well as to the neonatal gender. The study, performed on 41 purebred bitches submitted to elective cesarean section at term, enrolled 142 puppies, 74 males and 68 females, born mature, viable, without gross malformations, and with a normal weight. At surgery, a total of 129 amniotic and 84 allantoic samples were collected for IgG and lysozyme analysis. Class G immunoglobulins and lysozyme were detected in both fluids, but IgG concentrations were higher (P < 0.01) in amniotic fluid. Moreover, a significant positive correlation (P < 0.01) between IgG amniotic and allantoic levels, but not for lysozyme, was observed. A significant effect of the maternal parity (P < 0.05), but not of the breed body size, on the amniotic IgG concentrations was found, whereas the newborn gender was not associated to different IgG or lysozyme amniotic or allantoic levels. Given the significant contributions of fetal fluids to fetal and neonatal health, the results reported that the amniotic and allantoic fluids play a role in the immune protection of the fetus/newborn also in canine species. However, additional research is needed to better elucidate both the origin of IgG and lysozyme and the factors influencing the wide interindividual variations.

Sudden death in a dog after doxorubicin chemotherapy.

A case is reported of fatal cardiomyopathy in an 8-year-old female German Shepherd after standard chemotherapy with doxorubicin for splenic hemangiosarcoma. The main gross lesion was a moderate bilateral cardiac ventricular dilation with diffusely pale myocardium. Histological analysis revealed severe multifocal vacuolar degeneration of cardiomyocytes, myocytolysis, myofibril loss, myocardial fibrosis, and edema. Myocardial fiber vacuolization and myocytolysis were highly suggestive of doxorubicin cardiotoxicity.

Indeterminate cell histiocytosis in association with later occurrence of acute myeloblastic leukaemia.

Indeterminate cell histiocytosis (ICH) is a proliferation of indeterminate CD1a+, CD68+, S100+ and CD207- dermal dendritic cells. We describe a 39-year-old man who developed diffuse ICH and, 6 years later, acute myeloblastic leukaemia (AML). He was treated with cyclophosphamide, etoposide and vinblastine until 2003. In August 2004, he presented dyspnoea, hyperpyrexia and infiltration of the lung parenchyma, compatible with an AML invasion, and died after a course of induction chemotherapy. Cytomorphology and immunophenotype analyses suggested an ICH clonal evolution. The leukaemogenic role of etoposide is discussed. ICH has previously been reported in association with B-cell malignancy, but only one case has shown systemic progression.

Sensitivity of human multiple myelomas and myeloid leukemias to the proteasome inhibitor I.

The proteasome inhibitor PSI is potently cytotoxic in vitro against human chronic myeloid leukemia (CML) and acute myeloid leukemias (AML). Here, we have tested proteasome inhibitor I (PSI) in a panel of 11 human multiple myeloma (MM) cell lines and found that it has antiproliferative activity, with an IC50 between 4.5 and 557 nM at 48 h. PSI potentiated the toxicity of a number of chemotherapeutic agents in myeloid leukemia but not in MM cell lines, while in combination with therapeutic proteasome inhibitor PS-341 (Bortezomib) it had a synergistic effect. PSI suppressed the growth of AML cell lines more effectively than PS-341. CFU-GM colony assays revealed that CD34+ bone marrow progenitors from CML and AML patients were more sensitive to PSI than those from normal subjects (IC50: 5, 15 and 50 nM for AML, CML and normal, respectively). Moreover, the growth of normal primitive progenitors (LTC-IC) was unaffected by 15 nM PSI (P=0.576). PSI-induced cell death required RNA transcription and protein synthesis, but not DNA replication, was accompanied by the upregulation of Bcl-2 and modest reduction of Bax and Bcl-XL proteins, and involved the activation of caspases 2, 3, 7 and 8. These findings lend additional support to preclinical investigations with PSI.

Ultrastructural features of CD34+ hematopoietic progenitor cells from bone marrow, peripheral blood and umbilical cord blood.

Hematopoietic progenitor cells from different sources have been widely characterized, but their ultrastructural morphology has never been described in detail. In this study, imunomagnetically separated CD34+ cells from normal bone marrow (BM), mobilized peripheral blood (PBSC) and human umbilical cord blood (CB) were studied by transmission electron microscopy (TEM) using a cytochemical method which reveals endogenous myelo-peroxidase (MPO) activity. This technique is particularly suited for detecting early signs of the myeloid commitment. The CD34+ cells from PBSC were morphologically very homogeneous and 94.7+/-4.5% of these cells were MPO-: these ultrastructural features are generally considered typical of immature cells. The CD34+ BM cells were instead more heterogeneous, with 24.6+/-7.4% showing intense MPO activity. The ultrastructural characteristics of CB cells fell between those observed in PBSC and BM, but there was a high percentage of morphologically immature cells with no evidence of MPO activity (about 83%). The number of apoptotic cells within samples from different sources was also examined both by TEM and flow cytometry. The percentage of apoptotic cells was 0.7% in PBSC, 2.3% in BM, 2.9% in CB from vaginal delivery and 11.6% in CB from cesarean section. These observations confirm the relative phenotypic immaturity of CB in comparison with BM cells; they also suggest that CB collected after cesarean section may be associated with reduced stem cells viability.

Differentiation and expansion of endothelial cells from human bone marrow CD133(+) cells.

We report a method of purifying, characterizing and expanding endothelial cells (ECs) derived from CD133(+) bone marrow cells, a subset of CD34(+) haematopoietic progenitors. Isolated using immunomagnetic sorting (mean purity 90 +/- 5%), the CD133(+) bone marrow cells were grown on fibronectin-coated flasks in M199 medium supplemented with fetal bovine serum (FBS), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and insulin growth factor (IGF-1). The CD133(+) fraction contained 95 +/- 4% CD34(+) cells, 3 +/- 2% cells expressing VEGF receptor (VEGFR-2/KDR), but did not express von Willebrand factor (VWF), VE-cadherin, P1H12 or TE-7. After 3 weeks of culture, the cells formed a monolayer with a typical EC morphology and expanded 11 +/- 5 times. The cells were further purified using Ulex europaeus agglutinin-1 (UEA-1)-fluorescein isothiocyanate (FITC) and anti-FITC microbeads, and expanded with VEGF for a further 3 weeks. All of the cells were CD45(-) and CD14(-), and expressed several endothelial markers (UEA-1, VWF, P1H12, CD105, E-selectin, VCAM-1 and VE-cadherin) and typical Weibel-Palade bodies. They had a high proliferative potential (up to a 2400-fold increase in cell number after 3 weeks of culture) and the capacity to modulate cell surface antigens upon stimulation with inflammatory cytokines. Purified ECs were also co-cultivated with CD34(+) cells, in parallel with a purified fibroblastic cell monolayer. CD34(+) cells (10 x 10(5)) gave rise to 17,951 +/- 2422 CFU-GM colonies when grown on endothelial cells, and to 12,928 +/- 4415 CFU-GM colonies on fibroblast monolayers. The ECs also supported erythroid blast-forming unit (BFU-E) colonies better. These results suggest that bone marrow CD133(+) progenitor cells can give rise to highly purified ECs, which have a high proliferative capacity, can be activated by inflammatory cytokines and are superior to fibroblasts in supporting haematopoiesis. Our data support the hypothesis that endothelial cell progenitors are present in adult bone marrow and may contribute to neo-angiogenesis.

The apoptogenic response of human myeloid leukaemia cell lines and of normal and malignant haematopoietic progenitor cells to the proteasome inhibitor PSI.

Degradation of several intracellular proteins involved in cell cycle control and tumour growth is regulated by the ubiquitin-dependent multicatalytic protease complex (proteasome). We report that proteasome inhibitor Z-Ile-Glu(OtBu)-Ala-Leucinal (PSI) was cytotoxic on most human myeloid leukaemia cell lines at IC50 doses ranging from 5 to 25 nmol/l. Additionally, PSI pre-treatment enhanced cytotoxicity by taxol and cisplatinum. PSI was more active on leukaemic than on normal CD34(+) bone marrow progenitors because the 50% growth inhibition of colony-forming unit granulocyte macrophage (CFU-GM) from cases of chronic myelogenous leukaemia (CML) and normal subjects was achieved by 15 nmol/l and 50 nmol/l PSI respectively. PSI killed cells by apoptosis as revealed by ultrastructural changes, nuclear DNA fragmentation, cleavage of poly (ADP-ribose) polymerase (PARP) and of beta-catenin, and was antagonized by ectopic expression of Bcl-2 but not by inactivating mutations of p53. This event was associated with a slight accumulation of Bcl-2, a decrease of Bax but no changes in Bcl-X(L) protein expression at any time point. In Ph(+) cell lines BCR-ABL protein was only down-regulated after 48 h of treatment with 10 nmol/l PSI. Altogether, these results indicate that PSI, alone or in association with other cytotoxic agents, has anti-tumour activity against myeloid malignancies and is more effective on leukaemic than on normal haematopoietic progenitor cells.

Functional differences between dendritic cells derived from CD34+ bone marrow and peripheral blood stem cells.

BACKGROUND AND OBJECTIVE: It has been previously demonstrated that dendritic cells (DCs) are characterized by an immature stage with high antigen internalization capacity, followed by a mature stage with predominantly immunostimulatory ability. The shift from the immature to the mature state can be induced in vitro by the addition of tumor necrosis factor-a (TNFa). The aim of our study was to investigate the maturation steps of DCs obtained from CD34(+) cells from peripheral blood stem cells (PBSC) and bone marrow (BM). DESIGN AND METHODS: DCs were generated in vitro from PBSC and BM CD34(+) selected cells. The endocytic activity of the cells was measured by means of dextran-FITC uptake and alloreactivity evaluated with mixed leukocyte reactions. Immunophenotypic analysis was performed by flow cytometry. RESULTS: We observed that DCs from PBSC, in contrast to the BM derived DCs, were never able to take up soluble antigens. Mixed leukocyte reactions (MLR) performed both on PBSC and BM CD34(+) derived DCs showed an allo-stimulatory activity comparable to normal controls at day 10, but significantly higher at day 14 after the addition of TNFa. Immunophenotypic analysis showed typical dendritic markers in all the samples and, after treatment with TNFa, enhanced expression of co-stimulatory molecules. INTERPRETATION AND CONCLUSIONS: Our data seem to indicate that, in our culture conditions, BM-derived DCs could be efficiently used for pulsing with specific peptides, while PBSC-derived DCs, being functionally mature, should be more suitable for gene therapy.

In vitro effect of clozapine on hemopoietic progenitor cells.

BACKGROUND AND OBJECTIVE: Clozapine is a diabenzodiazepine derivative characterized by a high therapeutic index in schizophrenic patients resistant to traditional neuroleptic drugs, because of the rarity of any extrapyramidal side effects, and its particular hematologic toxicity. According to the international literature, clozapine-induced neutropenia occurs mainly during the first 4-6 months of treatment, and its incidence decreases considerably over time. This neutropenic effect is not dose-dependent and normally clears up after drug discontinuation, although it may evolve into agranulocytosis. The aim of this study is to evaluate the in vitro toxic effect of clozapine and N-desmethylclozapine on both committed and immature human hematopoietic progenitor cells. DESIGN AND METHODS: Cytotoxic assays were performed in vitro on normal human bone marrow samples treated with clozapine or with its metabolite N-desmethylclozapine. The clonogenic potential after treatment with both compounds was assessed on low density mononuclear cells (LD-MNC), purified CD34+ cells, cytokine driven liquid cultures and long term culture initiating cell (LTC-IC). RESULTS: Clozapine and N-desmethylclozapine had a dose-dependent inhibitory effect on in vitro growth of CFU-GM and BFU-E from normal bone marrow. The two drugs had toxic effects on purified CD34+ progenitor cells but no significant effect on LTI-IC. INTERPRETATIONS AND CONCLUSIONS: Our data indicate a cytotoxic effect, which is more pronounced with N-desmethylclozapine and at high doses, on the committed progenitor cell compartment but not on primitive hematopoietic cells. Furthermore, our data show that clozapine and N-desmethylclozapine have a direct effect on treated cells and do not induce apoptotic death.

Expansion of dendritic cells derived from human CD34+ cells in static and continuous perfusion cultures.

We examined the effects of different cytokine combinations and culture conditions on the expansion and modulation of cell surface antigens of CD34+ derived dendritic cells (DCs), the most efficient antigen-presenting cells capable of stimulating resting T cells in the primary immune response. Cells with a dendritic morphology and expressing HLA-DR, CD1a, S100 and CD83 were maximally expanded under serum-free conditions with the addition of SCF, GM-CSF, TNF-alpha, TGF-beta and Flt-3 ligand (fold increase of CD1a+ cells = 102 +/- 32 after 2 weeks of culture). CD34+ cells were also grown under continuous flow conditions in an artificial capillary system: after 14d of culture, the expansion in the total cell number was lower than that of the static cultures (3.3 +/- 2 v 18.9 +/- 4) but the percentage of CD1a+/CD83+/ CD80+ cells was considerably higher, whereas the CD14+ cells were significantly reduced (8.9 +/- 2 v 26 +/- 13). In continuous perfusion cultures, low levels of DC precursors and of LTC-IC were still present up to day 14. The DCs generated under flow conditions stimulated the mixed leucocyte reaction (MLR) more than the cells grown in static cultures. By electron microscopy, cells grown in the continuous flow system showed an increased number of large cells with numerous dendritic processes and abundant multilamellar complexes. The cells expanded under these conditions were sorted on the basis of their light-scatter properties into two fractions: one containing a predominance of CD1a+/S100+/ CD8 3+/CD80+/CD14- 'large cells' with great internal complexity (mature DCs); the second including 'small cells' either CD33+/CD14+, CD33+/CD15+ or CD33+/CD13-/CD14. The DCs generated and selected with this method are therefore particularly well suited for immunotherapeutic protocols.

Haematopoietic abnormalities after autologous stem cell transplantation in lymphoma patients.

Haematopoietic reconstitution after autologous stem cell transplantation (ASCT) was evaluated at different times in 26 lymphoma patients. All of the patients showed a significant decrease in the number of both committed (CFU-C) and more primitive progenitor cells (LTC-IC). The expansion of bone marrow progenitor cells in a 'stroma-free' long-term liquid culture system supplemented with SCF, IL-3, IL-6 and GM-CSF from 19 transplanted patients was significantly reduced compared to normal controls. The stromal cell compartment, evaluated by means of a CFU-F assay, was also greatly reduced. The number of haematopoietic and stromal cell progenitors was, nevertheless, very similar to their pre-transplant values. Bone marrow histology, which was evaluated at different times after transplant, showed an increase in reticulin fibres, the dilatation of parenchymal sinusoids and some morphological evidence of trilineage dysplasia in 11 patients; however, the same abnormalities were seen in the majority of pre-transplant samples. No cytogenetic abnormalities were observed in 15 patients before transplant, but four subsequently developed persistent clonal karyotypic alterations and five showed non-clonal abnormalities that generally disappeared over time. Our data suggest that both the stromal and the haematopoietic compartments are somehow damaged after ASCT for lymphoma; however, these defects generally pre-exist the transplant conditioning regimen and seem to become less pronounced over time.

Response of myelodysplastic syndrome bone marrow cells to multiple cytokine stimulation in liquid cultures: an in situ hybridization study.

BACKGROUND AND OBJECTIVE: Myelodysplastic syndrome progenitor cells can be grown and expanded in long term bone marrow liquid cultures in the presence of multiple cytokines. In this study we investigated the pattern of differentiation and response to growth factors in six cases of myelodysplastic syndrome (MDS) with well-defined cytogenetic abnormalities by means of conventional cytogenetics and fluorescence in situ hybridization (FISH). METHODS: Bone marrow cells were grown in stroma-free liquid cultures in the presence of SCF, IL-3, IL-6 and GM-CSF. RESULTS: IN three cases a CFU-GM expansion comparable to normal controls was observed, together with a decrease or increase of cells with abnormal karyotype. Two cases showed no response to growth factor stimulation, morphological signs of terminal myeloid differentiation and increase (one case) or decrease (one case) in the percentage of abnormal FISH signals along the cultures. In one additional case, while CFU-C expansion was present, clearcut leukemic transformation was observed in the culture, together with a sharp decrease in the percentage of abnormal FISH signals, indicating a leukemic transformation of MDS progenitor cells with a normal karyotype. INTERPRETATION AND CONCLUSIONS: Our data indicate that FISH analysis is generally a poor indicator of clonality in MDS; nevertheless, determining the kinetics of cytogenetically abnormal clones in liquid bone marrow cultures may provide insight as to the growth abnormalities of MDS progenitor cells and may be useful prior to in vivo growth factor administration.

Functional and morphological characterization of immunomagnetically selected CD34+ hematopoietic progenitor cells.

We evaluated the potential of immunomagnetically selected (miniMACS) progenitor cells to give rise to colony-forming cells and their precursors, detected as long-term culture-initiating cells (LTC-IC), as well as their capacity to expand in liquid cultures. A 90% mean purity, a 43.2% yield and a 55.8-fold enrichment were achieved from normal bone marrow. When corrected for enrichment, the mean number of committed progenitor cells and the frequency of LTC-IC (evaluated by means of limiting dilution assay [LDA]) were not statistically different in low density mononuclear cells or in the CD34-enriched fractions. In five cases CD34+ selected cells grown in a stroma-free long-term bone marrow culture system with the addition of stem cell factor, interleukin 3, interleukin 6 and GM-CSF every 48 h, showed a 15 (+/- 15) and 31 (+/- 21) mean colony forming unit-granulocyte/macrophage fold increase on cultures at days 7 and 14. However, when corrected for enrichment, the expansion capability of these cells was significantly lower than that of the unseparated fraction, particularly after the first week. Immediately after separation, electron microscopy revealed that the CD34+ selected fraction contained more than 45% of well-differentiated myeloid cells (MPO+), with iron beads preferentially clustered at one pole of the cell surface and sometimes already endocytosed in pinocytic vesicles. After 24 h and 48 h incubation at 37 degrees C, the majority of the cells showed no iron particles, but about 30% of the cells were iron-labeled phagocytic cells. The percentage of apoptotic cells with internalized iron was negligible. These data show that immunomagnetically separated CD34+ cells may have a slightly impaired short-term expansion capability, but give rise to both committed and more primitive progenitor cells. During the separation, the iron beads are internalized, rapidly processed in the cytoplasm and do not seem to interfere with in vitro growth.

Response of myelodysplastic syndrome marrow progenitor cells to stimualtion with cytokine combinations in a stroma-free long-term culture system.

The effect of an ex vivo expansion culture system using multiple cytokine combinations was evaluated in 38 cases of myelodysplastic syndrome (MDS) with the aim of overcoming the defective in vitro growth of haemopoietic progenitor cells. A combination of four growth factors (GF) including SCF, IL-3, IL-6 and GM-CSF was identified as the optimal combination for expanding clonogenic progenitor cells in MDS bone marrow liquid cultures. The cultures of 50% of the patients (19/38) responded to GF stimulation (mean CFU-GM fold increase 15.65+/-48 at week 4) and showed morphological features of normal and/or dysplastic myeloid differentiation. In 12/38 cases (31%), complete unresponsiveness to multiple cytokine stimulation was observed; a small number of patients (7/38) showed progressive leukaemic growth along the cultures with the presence of 100% immature blasts at week 4. GM-CSF and c-kit receptors, analysed by immuno-histochemistry in 10 patients, were over-expressed in responding patients and either lacking or down-regulated in non-responders. Fluorescence in situ hybridization (FISH) analysis of cultured interphase cells of nine patients (trisomy 8 in eight patients) showed a clear-cut increase in the percentage of cells with three signals in the two responding patients, thus indicating the expansion of a MDS clone. Multiple cytokine liquid cultures seem to be able to override the refractoriness of MDS progenitor cells to GF stimulation in many cases, revealing a heterogeneity which may have prognostic implications and should be considered in ex-vivo and in vivo clinical trials with cytokine combinations.

The effect of interleukin-12 in ex-vivo expansion of human haemopoietic progenitors.

We evaluated progenitor cell proliferation in cultures supplemented by different cytokine combinations in the presence or absence of IL-12. In cultures of low density cells, cytokine combinations including IL-12 were associated to a greater proliferation (up to 6.7 +/- 2.5 CFU-GM fold expansion). However, in cultures of purified CD34+ cells the more efficient cytokine combination (147 +/- 49 CFU-GM fold expansion) was SCF, IL-3, IL-11 and MIP-1 alpha, and the addition of IL-12 did not further enhance expansion of progenitors. These results indicate that accessory cells, lost in CD34+ cell purification, could be in part responsible for IL-12 effect on progenitor cell proliferation. In CD34+ cell cultures the addition of IL-12 led to CD19 mRNA generation, suggesting that IL-12 acts on haemopoietic cells with both myeloid and lymphoid potential.

Effects of recombinant human stem cell factor (rh-SCF) on colony formation and long-term bone marrow cultures (LTBMC) in patients with myelodysplastic syndromes.

Stem cell factor (SCF), the ligand of the c-kit receptor, is a potent enhancing cytokine for haematopoietic cells in the presence of IL-3, GM-CSF and erythropoietin (Epo). In the clonogenic assays of 63 MDS patients, the addition of rh-SCF + GM-CSF and/or IL-3 induced a significant increase (p < 0.001) in the number and size of CFU-GM. Never reaching the levels of controls, this increase was seen in all FAB subtypes, but particularly in RA. There was no significant increase in cluster formation, even in RAEB or RAEBt. Rh-SCF (10 ng/ml) led to mean increases of up to 26 times in the number of Epo-dependent BFU-E colonies, particularly in RA (p < 0.001) and RAEB (p < 0.05). Individual responses varied widely (especially in RA) from no response to supranormal levels. Added to the weekly refeed of 37 MDS LTBMC, SCF (10 ng/ml) induced only a 7% mean increase in both cell output and the number of clonogenic cells recovered in the supernatant. Immunohistochemical examination of the supernatant showed significant increases in differentiating myeloid cells in all examined cases, and in erythroid cells in 3 cases; blast cells increased in only 3 cases. These data suggest that rh-SCF is capable of at least partially reversing defective MDS myeloid haematopoiesis, and leads no overt risk of leukaemic transformation. Its potent effect on erythroid cells is encouraging for future clinical applications in patients, particularly if they are selected by means of in vitro tests.