New strategies in hematopoietic stem cell transplantation: G-CSF-mobilized unprocessed whole blood

Transplantation of mobilized peripheral blood stem cells (PBSC) for rescue of bone marrow function after high-dose chemo-/radiotherapy is widely used in hematologic malignancies and solid tumors. Mobilization of stem cells to the peripheral blood can be achieved by cytokine treatment of the patients. The main advantage of autologous PBSC transplantation over bone marrow transplantation is the faster recovery of neutrophil and platelet counts. The threshold number of PBSC required for adequate rescue of bone marrow is thought to be about 2 x 106 CD34+ cells/kg, if the stem cells are collected by leukapheresis and subsequently cryopreserved. We show that this critical number could be further reduced to as few as 0.2 x 106 cells/kg. In 30 patients with multiple myeloma and 25 patients with bad risk lymphoma 1 liter of granulocyte colony-stimulating factor (G-CSF)-mobilized unprocessed whole blood (stored at 4oC for 1-3 days) was used for transplantation. Compared to a historical control group, a significant reduction in the duration of neutropenia, thrombocytopenia and the length of hospital stay was documented. Furthermore, the effect of stem cell support was reflected by a lower need for platelet and red cell transfusions and a reduced antibiotic use. Considering the data as a whole, a cost saving of about 50% was achieved. To date, this easy to perform method of transplantation is only feasible following high-dose therapies that are completed within 72 h, since longer storage of unprocessed blood is accompanied by a substantial loss of progenitor cell function. Ongoing investigations include attempts to prolong storage times for whole blood

Relationship of cytokines and cytokine signaling to immunodeficiency disorders in the mouse

The contributions of cytokines to the development and progression of disease in a mouse model of retrovirus-induced immunodeficiency (MAIDS) are controversial. Some studies have indicated an etiologic role for type 2 cytokines, while others have emphasized the importance of type 1 cytokines. We have used mice deficient in expression of IL-4, IL-10, IL-4 and IL-10, IFN-g, or ICSBP - a transcriptional protein involved in IFN signaling - to examine their contributions to this disorder. Our results demonstrate that expression of type 2 cytokines is an epiphenomenon of infection and that IFN-g is a driving force in disease progression. In addition, exogenously administered IL-12 prevents many manifestations of disease while blocking retrovirus expression. Interruption of the IFN signaling pathways in ICSBP-/- mice blocks induction of MAIDS. Predictably, ICSBP-deficient mice exhibit impaired responses to challenge with several other viruses. This immunodeficiency is associated with impaired production of IFN-g and IL-12. Unexpectedly, however, the ICSBP-/- mice also develop a syndrome with many similarities to chronic myelogenous leukemia in humans. The chronic phase of this disease is followed by a fatal blast crisis characterized by clonal expansions of undifferentiated cells. ICSBP is thus an important determinant of hematopoietic growth and differentiation as well as a prominent signaling molecule for IFNs

Effect of all-trans retinoic acid on newly diagnosed acute promyelocytic leukemia patients: results of a Brazilian center

Thirty-seven patients with acute promyelocytic leukemia (APL) were treated with all-trans retinoic acid (ATRA). Patients received 45 mg m-2 day-1 po of ATRA until complete remission (CR) was achieved, defined as: a) presence of less than 5% blasts in the bone marrow, with b) white blood cells >103/mm3, c) platelets >105/mm3 and d) hemoglobin concentration >8 g/dl, with no blood or platelet transfusions. Thirty-one (83.7%) patients achieved CR by day 50, and 75% of these before day 30. Correction of the coagulopathy, achieved between days 2 and 10 (mean, 3 days), was the first evidence of response to treatment. Only one patient had been previously treated with chemotherapy and three had the microgranular variant M3 form. Dryness of skin and mucosae was the most common side effect observed in 82% of the patients. Thrombosis, hepatotoxicity and retinoid acid syndrome (RAS) were observed in 7 (19%), 6 (16%) and 4 (11%) patients, respectively. Thirteen (35%) patients had to be submitted to chemotherapy due to hyperleukocytosis (above 40 x 103/mm3) and six of these presented with new signs of coagulopathy after chemotherapy. Four (11%) patients died secondarily to intracerebral hemorrhage (IH) and two (5.4%) dropped out of the protocol due to severe ATRA side effects (one RAS and one hepatotoxicity). RAS and IH were related strictly to hyperleukocytosis. The reduced use of platelets and fresh frozen plasma probably lowered the total cost of treatment. We conclude that ATRA is an effective agent for inducing complete remission in APL patients.

The hematopoietic stroma

All blood cells are derived from a small common pool of totipotent cells, called hematopoietic stem cells. The process is strictly regulated by the hematopoietic microenvironment, which includes stromal cells, extracellular matrix molecules and soluble regulatory factors. Several experimental in vitro assays have been developed for the study of hematopoietic differentiation, and have provided valuable information on the stroma, which includes, among other cell types, macrophages, fibroblasts, adipocytes, and endothelial cells. The composition, ontogeny, and function in physiological as well as pathological conditions of stroma are discussed.

Collateral methotrexate resistance in cisplatin-selected murine leukemia cells

Resistance to anticancer drugs is a major cause of failure of many therapeutic protocols. A variety of mechanisms have been proposed to explain this phenomenon. The exact mechanism depends upon the drug of interest as well as the tumor type treated. While studying a cell line selected for its resistance to cisplatin we noted that the cells expressed a >25,000-fold collateral resistance to methotrexate. Given the magnitude of this resistance we elected to investigate this intriguing collateral resistance. From a series of investigations we have identified an alteration in a membrane protein of the resistant cell as compared to the sensitive cells that could be the primary mechanism of resistance. Our studies reviewed here indicate decreased tyrosine phosphorylation of a protein (molecular mass = 66) in the resistant cells, which results in little or no transfer of methotrexate from the medium into the cell. Since this is a relatively novel function for tyrosine phosphorylation, this information may provide insight into possible pharmacological approaches to modify therapeutic regimens by analyzing the status of this protein in tumor samples for a better survival of the cancer patients.

The pathogenesis of tropical spastic paraparesis/human T-cell leukemia type I-associated myelopathy

Tropical spastic paraparesis/human T-cell leukemia type I-associated myelopathy (TSP/HAM) is caused by a human T-cell leukemia virus type I (HTLV-I) after a long incubation period. TSP/HAM is characterized by a chronic progressive paraparesis with sphincter disturbances, no/mild sensory loss, the absence of spinal cord compression and seropositivity for HTLV-I antibodies. The pathogenesis of this entity is not completely known and involves a multivariable phenomenon of immune system activation against the presence of HTLV-I antigens, leading to an inflammatory process and demyelination, mainly in the thoracic spinal cord. The current hypothesis about the pathogenesis of TSP/HAM is: 1) presence of HTLV-I antigens in the lumbar spinal cord, noted by an increased DNA HTLV-I load; 2) CTL either with their lytic functions or release/production of soluble factors, such as CC-chemokines, cytokines, and adhesion molecules; 3) the presence of Tax gene expression that activates T-cell proliferation or induces an inflammatory process in the spinal cord; 4) the presence of B cells with neutralizing antibody production, or complement activation by an immune complex phenomenon, and 5) lower IL-2 and IFN-gamma production and increased IL-10, indicating drive to a cytokine type 2 pattern in the TSP/HAM subjects and the existence of a genetic background such as some HLA haplotypes. All of these factors should be implicated in TSP/HAM and further studies are necessary to investigate their role in the development of TSP/HAM.

Acute promyelocytic leukemia: the study of t(15;17) translocation by fluorescent in situ hybridization, reverse transcriptase-polymerase chain reaction and cytogenetic techniques

Acute promyelocytic leukemia (AML M3) is a well-defined subtype of leukemia with specific and peculiar characteristics. Immediate identification of t(15;17) or the PML/RARA gene rearrangement is fundamental for treatment. The objective of the present study was to compare fluorescent in situ hybridization (FISH), reverse transcriptase-polymerase chain reaction (RT-PCR) and karyotyping in 18 samples (12 at diagnosis and 6 after treatment) from 13 AML M3 patients. Bone marrow samples were submitted to karyotype G-banding, FISH and RT-PCR. At diagnosis, cytogenetics was successful in 10 of 12 samples, 8 with t(15;17) and 2 without. FISH was positive in 11/12 cases (one had no cells for analysis) and positivity varied from 25 to 93% (mean: 56%). RT-PCR was done in 6/12 cases and all were positive. Four of 8 patients with t(15;17) presented positive RT-PCR as well as 2 without metaphases. The lack of RT-PCR results in the other samples was due to poor quality RNA. When the three tests were compared at diagnosis, karyotyping presented the translocation in 80% of the tested samples while FISH and RT-PCR showed the PML/RARA rearrangement in 100% of them. Of 6 samples evaluated after treatment, 3 showed a normal karyotype, 1 persistence of an abnormal clone and 2 no metaphases. FISH was negative in 4 samples studied and 2 had no material for analysis. RT-PCR was positive in 4 (2 of which showed negative FISH, indicating residual disease) and negative in 2. When the three tests were compared after treatment, they showed concordance in 2 of 6 samples or, when there were not enough cells for all tests, concordance between karyotype and RT-PCR in one. At remission, RT-PCR was the most sensitive test in detecting residual disease, as expected (positive in 4/6 samples). An incidence of about 40% of 5' breaks and 60% of 3' breaks, i.e., bcr3 and bcr1/bcr2, respectively, was observed.

In vitro cytotoxicity of the LDE: daunorubicin complex in acute myelogenous leukemia blast cells

Acute myelogenous leukemia (AML) blast cells show high-affinity degradation of low-density lipoprotein (LDL), suggesting an increased expression of cellular LDL receptors. LDE is a lipid microemulsion easily synthesized in vitro which is known to mimic the metabolic pathway of LDL. We used LDE as a carrier for daunorubicin and assayed the cytotoxicity of the complex using AML blast cells since RT-PCR analysis showed that AML cells express LDL receptor mRNA. The LDE:daunorubicin complex killed 46.7% of blast cells and 20.2% of normal bone marrow cells (P<0.001; Student t-test). Moreover, this complex destroyed AML blast cells as efficiently as free daunorubicin. Thus, LDE might be a suitable carrier of chemotherapeutic agents targeting these drugs to neoplastic cells and protecting normal tissues.

Dipeptidyl peptidase IV (CD26) activity in the hematopoietic system: differences between the membrane-anchored and the released enzyme activity

Dipeptidyl peptidase IV (DPP-IV; CD26) (EC 3.4.14.5) is a membrane-anchored ectoenzyme with N-terminal exopeptidase activity that preferentially cleaves X-Pro-dipeptides. It can also be spontaneously released to act in the extracellular environment or associated with the extracellular matrix. Many hematopoietic cytokines and chemokines contain DPP-IV-susceptible N-terminal sequences. We monitored DPP-IV expression and activity in murine bone marrow and liver stroma cells which sustain hematopoiesis, myeloid precursors, skin fibroblasts, and myoblasts. RT-PCR analysis showed that all these cells produced mRNA for DPP-IV. Partially purified protein reacted with a commercial antibody to CD26. The K M values for Gly-Pro-p-nitroanilide ranged from 0.43 to 0.98 mM for the membrane-associated enzyme of connective tissue stromas, and from 6.76 to 8.86 mM for the enzyme released from the membrane, corresponding to a ten-fold difference, but only a two-fold difference in K M was found in myoblasts. K M of the released soluble enzyme decreased in the presence of glycosaminoglycans, nonsulfated polysaccharide polymers (0.8-10 µg/ml) or simple sugars (320-350 µg/ml). Purified membrane lipid rafts contained nearly 3/4 of the total cell enzyme activity, whose K M was three-fold decreased as compared to the total cell membrane pool, indicating that, in the hematopoietic environment, DPP-IV activity is essentially located in the lipid rafts. This is compatible with membrane-associated events and direct cell-cell interactions, whilst the long-range activity depending upon soluble enzyme is less probable in view of the low affinity of this form.

Acute myeloid leukemia in elderly patients: experience of a single center

Acute myeloid leukemia (AML) is a disease predominantly of older adults. Treatment of AML in the elderly is complicated not only by comorbidities but also by the high prevalence of poor prognosis markers. Thirty-one consecutive unselected patients with AML older than 60 years (representing 33% of all AML cases diagnosed at our institution during the same period) were followed over a period of 5 years (1997-2002). A high incidence of AML with multilineage dysplasia (45%) and no favorable cytogenetic abnormalities but 62% intermediate and 38% unfavorable karyotypes were found. Sixteen patients (52%) were selected for induction of intensive cytotoxic treatment and complete remission was achieved only by some of these intensively treated patients (7 of 16). Of these, 3 remained alive without disease (median: 11 months), 1 patient died shortly after complete remission, and 3 patients relapsed and died from refractory disease. Only 1 patient that was refractory to intensive cytotoxic treatment remained alive with disease under supportive care. Fifteen patients (48%) were managed with palliative/supportive care: 7 received palliative treatment and supportive care, 8 received supportive care only, and 4 patients remained alive with disease under supportive care (median: 9 months). Mortality rate was 74% and overall survival at two years was 12%. To the best of our knowledge, there is no previous report regarding elderly patients with AML in Brazilian subsets. The present data are similar to previously reported studies showing that elderly AML patients are not only older but also biologically distinct from younger AML patients, particularly in terms of the high incidence of poor prognostic karyotypes and resistance to therapy.

Immunological effects of donor lymphocyte infusion in patients with chronic myelogenous leukemia relapsing after bone marrow transplantation

Allogeneic bone marrow transplantation (alloBMT) is the only curative therapy for chronic myelogenous leukemia (CML). This success is explained by the delivery of high doses of antineoplastic agents followed by the rescue of marrow function and the induction of graft-versus-leukemia reaction mediated by allogeneic lymphocytes against host tumor cells. This reaction can also be induced by donor lymphocyte infusion (DLI) producing remission in most patients with CML who relapse after alloBMT. The immunological mechanisms involved in DLI therapy are poorly understood. We studied five CML patients in the chronic phase, who received DLI after relapsing from an HLA-identical BMT. Using flow cytometry we evaluated cellular activation and apoptosis, NK cytotoxicity, lymphocytes producing cytokines (IL-2, IL-4 and IFN-gamma), and unstimulated (in vivo) lymphocyte proliferation. In three CML patients who achieved hematological and/or cytogenetic remission after DLI we observed an increase of the percent of activation markers on T and NK cells (CD3/DR, CD3/CD25 and CD56/DR), of lymphocytes producing IL-2 and IFN-gamma, of NK activity, and of in vivo lymphocyte proliferation. These changes were not observed consistently in two of the five patients who did not achieve complete remission with DLI. The percent of apoptotic markers (Fas, FasL and Bcl-2) on lymphocytes and CD34-positive cells did not change after DLI throughout the different study periods. Taken together, these preliminary results suggest that the therapeutic effect of DLI in the chronic phase of CML is mediated by classic cytotoxic and proliferative events involving T and NK cells but not by the Fas pathway of apoptosis.

Photocytotoxicity of a 5-nitrofuran-ethenyl-quinoline antiseptic (Quinifuryl) to P388 mouse leukemia cells

Quinifuryl (MW 449.52), 2-(5'-nitro-2'-furanyl)ethenyl-4-{N-[4'-(N,N-diethylamino)-1'-methylbutyl]carbamoyl} quinoline, is a water soluble representative of a family of 5-nitrofuran-ethenyl-quinoline drugs which has been shown to be highly toxic to various lines of transformed cells in the dark. In the present study, the toxicity of Quinifuryl to P388 mouse leukemia cells was compared in the dark and under illumination with visible light (390-500 nm). Illumination of water solutions of Quinifuryl (at concentrations ranging from 0.09 to 9.0 µg/ml) in the presence of P388 cells resulted in its photodecomposition and was accompanied by elevated cytotoxicity. A significant capacity to kill P388 cells was detected at a drug concentration as low as 0.09 µg/ml. The toxic effect detected at this drug concentration under illumination exceeded the effect observed in the dark by more than three times. Moreover, the general toxic effect of Quinifuryl, which included cell proliferation arrest, was nearly 100%. Both dose- and time-dependent toxic effects were measured under illumination. The LC50 value of Quinifuryl during incubation with P388 cells was ~0.45 µg/ml under illumination for 60 min and >12 µg/ml in the dark. We have demonstrated that the final products of the Quinifuryl photolysis are not toxic, which means that the short-lived intermediates of Quinifuryl photodecomposition are responsible for the phototoxicity of this compound. The data obtained in the present study are the first to indicate photocytotoxicity of a nitroheterocyclic compound and demonstrate the possibility of its application as a photosensitizer drug for photochemotherapy.

The role of osteoblasts in regulating hematopoietic stem cell activity and tumor metastasis

Bone marrow stromal cells are critical regulators of hematopoiesis. Osteoblasts are part of the stromal cell support system in bone marrow and may be derived from a common precursor. Several studies suggested that osteoblasts regulate hematopoiesis, yet the entire mechanism is not understood. It is clear, however, that both hematopoietic precursors and osteoblasts interact for the production of osteoclasts and the activation of resorption. We observed that hematopoietic stem cells (HSCs) regulate osteoblastic secretion of various growth factors, and that osteoblasts express some soluble factors exclusively in the presence of HSCs. Osteoblasts and hematopoietic cells are closely associated with each other in the bone marrow, suggesting a reciprocal relationship between them to develop the HSC niche. One critical component regulating the niche is stromal-derived factor-1 (SDF-1) and its receptor CXCR4 which regulates stem cell homing and, as we have recently demonstrated, plays a crucial role in facilitating those tumors which metastasize to bone. Osteoblasts produce abundant amounts of SDF-1 and therefore osteoblasts play an important role in metastasis. These findings are discussed in the context of the role of osteoblasts in marrow function in health and disease.

Multicellular spheroids of bone marrow stromal cells: a three-dimensional in vitro culture system for the study of hematopoietic cell migration

Cell fate decisions are governed by a complex interplay between cell-autonomous signals and stimuli from the surrounding tissue. In vivo cells are connected to their neighbors and to the extracellular matrix forming a complex three-dimensional (3-D) microenvironment that is not reproduced in conventional in vitro systems. A large body of evidence indicates that mechanical tension applied to the cytoskeleton controls cell proliferation, differentiation and migration, suggesting that 3-D in vitro culture systems that mimic the in vivo situation would reveal biological subtleties. In hematopoietic tissues, the microenvironment plays a crucial role in stem and progenitor cell survival, differentiation, proliferation, and migration. In adults, hematopoiesis takes place inside the bone marrow cavity where hematopoietic cells are intimately associated with a specialized three 3-D scaffold of stromal cell surfaces and extracellular matrix that comprise specific niches. The relationship between hematopoietic cells and their niches is highly dynamic. Under steady-state conditions, hematopoietic cells migrate within the marrow cavity and circulate in the bloodstream. The mechanisms underlying hematopoietic stem/progenitor cell homing and mobilization have been studied in animal models, since conventional two-dimensional (2-D) bone marrow cell cultures do not reproduce the complex 3-D environment. In this review, we will highlight some of the mechanisms controlling hematopoietic cell migration and 3-D culture systems.

Immunophenotype of hematopoietic stem cells from placental/umbilical cord blood after culture

Identification and enumeration of human hematopoietic stem cells remain problematic, since in vitro and in vivo stem cell assays have different outcomes. We determined if the altered expression of adhesion molecules during stem cell expansion could be a reason for the discrepancy. CD34+CD38- and CD34+CD38+ cells from umbilical cord blood were analyzed before and after culture with thrombopoietin (TPO), FLT-3 ligand (FL) and kit ligand (KL; or stem cell factor) in different combinations: TPO + FL + KL, TPO + FL and TPO, at concentrations of 50 ng/mL each. Cells were immunophenotyped by four-color fluorescence using antibodies against CD11c, CD31, CD49e, CD61, CD62L, CD117, and HLA-DR. Low-density cord blood contained 1.4 ± 0.9% CD34+ cells, 2.6 ± 2.1% of which were CD38-negative. CD34+ cells were isolated using immuno-magnetic beads and cultured for up to 7 days. The TPO + FL + KL combination presented the best condition for maintenance of stem cells. The total cell number increased 4.3 ± 1.8-fold, but the number of viable CD34+ cells decreased by 46 ± 25%. On the other hand, the fraction of CD34+CD38- cells became 52.0 ± 29% of all CD34+ cells. The absolute number of CD34+CD38- cells was expanded on average 15 ± 12-fold when CD34+ cells were cultured with TPO + FL + KL for 7 days. The expression of CD62L, HLA-DR and CD117 was modulated after culture, particularly with TPO + FL + KL, explaining differences between the adhesion and engraftment of primary and cultured candidate stem cells. We conclude that culture of CD34+ cells with TPO + FL + KL results in a significant increase in the number of candidate stem cells with the CD34+CD38- phenotype.