Isolated Bone Marrow Research Articles

Abca7 Null Mice Retain Normal Macrophage Phosphatidylcholine and Cholesterol Efflux Activity despite Alterations in Adipose Mass and Serum Cholesterol Levels

Mutations in the A class of ATP-binding cassette transporters (ABCA) are causally implicated in three human diseases: Tangier disease (ABCA1), Stargadt's macular degeneration (ABCA4), and neonatal respiratory failure (ABCA3). Both ABCA1 and ABCA4 have been shown to transport lipids across cellular membranes, and ABCA3 may play a similar role in transporting pulmonary surfactant. Although the functions of the other 10 ABCA class transporters identified in the human genome remain obscure, ABCA7-transfected cells have been shown to efflux lipids in response to stimulation by apolipoprotein A-I. In an effort to elucidate the physiologic role of ABCA7, we generated mice lacking this transporter (Abca7-/- mice). Homozygous null mice were produced from intercrosses of heterozygous null mice at the expected Mendelian frequency and developed normally without any obvious phenotypic abnormalities. Cholesterol and phospholipid efflux stimulated by apolipoprotein A-I from macrophages isolated from wild type and Abca7-/- mice did not differ, suggesting that these activities may not be central to the physiological role of the transporter in vivo. Abca7-/- females, but not males, had significantly less visceral fat and lower total serum and high density lipoprotein cholesterol levels than wild type, gender-matched littermates. ABCA7 expression was detected in hippocampal and cortical neurons by in situ hybridization and in brain and white adipose tissue by Western blotting. Induction of adipocyte differentiation from 3T3 fibroblasts in culture led to a marked increase in ABCA7 expression. These studies suggest that ABCA7 plays a novel role in lipid and fat metabolism that Abca7-/- mice can be used to elucidate.

Primary and isolated anaplastic large cell lymphoma of the bone marrow

Anaplastic large cell lymphoma (ALCL) is a T-cell lymphoma in which the majority of patients present with advanced stage III or IV disease. Here we report a case of ALCL where bone marrow was the only site of disease, in a 60-year-old man with pyrexia and pancytopenia. The diagnosis of ALCL was made on detection of CD30-positive anaplastic cells in the bone marrow, together with prominent hemophagocytosis. Genetics confirmed the clonal nature of the disease and showed it to be anaplastic lymphoma kinase (ALK) negative. Primary isolated bone marrow ALCL should be considered in the diagnosis of pancytopenia associated with hemophagocytosis.

Prognostic factors for relapsed childhood acute lymphoblastic leukemia: Impact of allogeneic stem cell transplantation—a report from the Kyushu-Yamaguchi Children's Cancer Study Group

The treatment results of childhood acute lymphoblastic leukemia (ALL) with a first relapse were retrospectively analyzed to determine prognostic factors. In particular, an attempt was made to clarify whether stem cell transplantation (SCT) had any advantages over chemotherapy. Of the 407 children with ALL diagnosed between 1984 and 1996, 117 suffered from a relapse before December 1999. The patients were treated differently according to the protocols of each institution. The potential prognostic factors examined were: the time of initial diagnosis, gender, immunophenotype of leukemic blasts and the NCI-risk classification at initial diagnosis, the site of relapse, the time of relapse (early: within 18 months after diagnosis, intermediate: other than either early or late relapse, late: later than 6 months after the discontinuation of front-line chemotherapy), and the treatment after relapse (chemotherapy alone and SCT). A second complete remission (CR2) was achieved in 90 patients (77%) and thirty of them maintained CR2, thus resulting in an event-free survival rate (EFS) of 25.1% and an overall survival rate of 26.1%. The significant prognostic factors identified by a multivariate analysis included the time of relapse (EFS: early 16.2%, intermediate 23.9%, late 35.1%, P = 0.012) and the treatment after relapse (EFS: SCT 30.3%, chemotherapy 22.0%, P = 0.049). When patients with an isolated bone marrow relapse and continuous CR2 for more than 3 months were analyzed, the treatment in CR2 was the only independent prognostic factor (EFS: SCT 60.2%, chemotherapy 25.7%, P = 0.005). In children with ALL and a first relapse, the time of relapse and the treatment after relapse were found to be independent prognostic factors. Allogeneic SCT in CR2 showed significantly better results than chemotherapy in patients with an isolated bone marrow relapse.

Macrophages in streptozotocin-induced diabetic nephropathy: potential role in renal fibrosis

Renal fibrosis is central to the progression of diabetic nephropathy; however, the mechanisms responsible for fibroblast and matrix accumulation in this disease are only partially understood. Macrophages accumulate in diabetic kidneys, but it is unknown whether macrophages contribute to renal fibrosis. Therefore, we examined whether macrophage accumulation is associated with the progression of renal injury and fibrosis in type 1 diabetic nephropathy and whether macrophages exposed to the diabetic milieu could promote fibroblast proliferation. Kidney macrophages, renal injury and fibrosis were analysed in diabetic C57BL/6J mice at 2, 8, 12 and 18 weeks after streptozotocin injection. Isolated rat bone marrow macrophages were stimulated with diabetic rat serum or carboxymethyllysine (CML)-bovine serum albumin (BSA) to determine whether macrophage-conditioned medium could promote the proliferation of rat renal (NRK-49F) fibroblasts. Progressive injury and fibrosis in diabetic nephropathy was associated with increased numbers of kidney macrophages. Macrophage accumulation in diabetic mice correlated with hyperglycaemia (blood glucose, HbA1c levels), renal injury (albuminuria, plasma creatinine), histological damage and renal fibrosis (myofibroblasts, collagen IV). Culture supernatant derived from bone marrow macrophages incubated with diabetic rat serum or CML-BSA induced proliferation of fibroblasts, which was inhibited by pre-treating fibroblasts with interleukin-1 (IL-1) receptor antagonist or the platelet-derived growth factor (PDGF) receptor kinase inhibitor, STI-571. Kidney macrophage accumulation is associated with the progression of renal injury and fibrosis in streptozotocin-induced mouse diabetic nephropathy. Elements of the diabetic milieu can stimulate macrophages to promote fibroblast proliferation via IL-1- and PDGF-dependent pathways which may enhance renal fibrosis.

Hyperoxia Induces Defective Epo-Induced Signal Transduction and Loss of Expansion Capacity in Fanca- and Fancg-Deficient Erythroid Progenitors.

Fanconi anemia (FA) is characterized by chromosomal instability and bone marrow hypoplasia. To explore the mechanism of FA-related bone marrow failure, we studied erythropoietin (Epo)-dependent proliferation and differentiation of erythroid progenitors expanded from mice lacking the Fanca or Fancg gene. Erythroid progenitors can be expanded in serum-free medium supplemented with Epo, Stem Cell Factor (SCF) and glucocorticoids. The expansion capacity of Fanca−/− and Fancg−/− erythroid progenitors was reduced and the progenitors differentiated to mature erythrocytes instead after 10 days of culture. When Epo-induced signal transduction was compared between progenitors expanded for 6 days from fetal livers of Fanca−/−, Fancg−/− and wt littermates, Epo-induced phosphorylation of STAT5 and ERK1/2 appeared to be severely reduced and delayed in progenitors lacking Fanca or Fancg. Both in wt, Fanca−/− and Fancg−/− progenitors the phosphorylation of STAT5 and ERK1/2 increased with rising Epo-concentrations, but at low concentrations 4-fold more Epo was required to obtained comparable Stat5 phosphorylation in FancA−/− or FancG−/− cells, while efficient phosphorylation at high concentrations was never achieved in Fanca or Fancg-deficient cells. As a consequence, the regulation of Epo-target genes like p21WAF, Gilz, SOCS3, Spi2.1 and BclXL was impaired. In contrast to downstream signaling intermediates, Epo-induced phosphorylation of the Epo-receptor and its associated kinase JAK2 was not affected. Stem Cell Factor-induced c-Kit activation and downstream Erk1/2 phosphorylation was also equally efficient in wt, Fanca−/− and Fancg−/− progenitors. Thus, the signaling defect specifically seems to affect targets downstream of Jak2. Surprisingly, Epo-induced activation of Stat5 was unaffected in freshly isolated bone marrow cells. Comparison of Epo-induced Stat5 phosphorylation in erythroid progenitors cultured at atmospheric (20%) and physiologic (3%) oxygen revealed that Stat5 phosphorylation was only lost upon expansion of the progenitors at atmospheric (hyperoxic) conditions. Hyperoxia can damage many biomolecules among which DNA. In contrast to wt cells, Fanca−/− and Fancg−/− progenitors may not be able to repair DNA-damage induced by atmospheric oxygen. To examine whether increased DNA damage could impair Epo-induced signal transduction, we expanded erythroid progenitors from fetal livers of mice lacking the DNA-repair enzyme Ercc1. Ercc1 is directly involved in repair of DNA crosslinks, while a defective FA pathway renders cells hypersensitive to DNA-crosslinks. Epo-induced phosphorylation of Stat5 was impaired in Ercc1−/− progenitors, indicating that deficient repair of DNA damage, rather than FA-pathway specific mechanisms may cause the observed signaling defect. To substantiate this finding we treated wt and p53−/− progenitors with increasing concentrations of mitomycin C (MMC). Sublethal concentrations of MMC abrogated the renewal capacity and impaired Epo-induced phosporylation of Stat5 and Erk1/2 in wt but not in p53−/− erythroid progenitors. Together the data suggest that impaired progenitor expansion and Stat5 phosphorylation is not a specific aspect of cells lacking the fanconi pathway, but a general aspect of a p53-dependent response to genomic stress.

Outcome for Adolescent and Young Adults 16–21 years of age (AYA) with Acute Lymphoblastic Leukemia (ALL) Treated on the Children' s Cancer Group (CCG) 1961 Study.

AYA Patients 16–21 years of age with ALL are treated on both pediatric and adult ALL protocols. Recent publications have suggested that AYA patients treated on pediatric protocols have a significantly better outcome than those treated on adult protocols. From November 1996 to May 2002, 262 AYA ALL patients were entered on the CCG 1961 protocol. Treatment was based on the following observations from the previous CCG 1882 Protocol (1989–1995). 1) For patients with rapid early response (< = 25% blasts in a day seven bone marrow aspirate-RER), in the context of CCG modified BFM therapy (S-BFM), pre-symptomatic central nervous system therapy with intensive intrathecal (IT) methotrexate (MTX) alone provided equivalent results to standard IT MTX and cranial radiation (CRT). 2) For patients with slow early response (> than 25 blasts on day 7-SER), the augmented BFM (A-BFM) regimen produced a significant improvement in outcome compared to S-BFM. A-BFM featured a significant increase in dose intensity for vincristine (VCR), L-asparaginase (L-ASP), and steroid, the use of intravenous MTX without rescue in conjunction with VCR and L-ASP in interim maintenance (IM), and a second IM and delayed intensification (DI) phase. RER patients on 1961 were randomized to one of four arms; S-BFM, S-BFM with a second standard IM and DI phase, A-BFM with only 1 IM/DI phase, and full A-BFM. SER patients were randomized to A-BFM or A-BFM with Idarubicin/Cytoxan pulses added to the 2 DI phases. One hundred ninety patients had < 50K WBC (73.4%) and 21% had T cell immunophenotype. Seven patients had a t (9/22) and three patients had a t (4/11). Five year EFS for AYA patients treated on CCG 1961 was 68% ± 3.7% and the five-year survival was 77% ± 3.2%. The majority of events were isolated bone marrow relapses. For RER AYA patients, five year EFS for patients treated on the augmented intensity arms (N=87) was 83.2% compared to 60.8% for patients treated on the standard intensity arms (N=76) (P = .10). Within the augmented intensity arms, there was no difference in EFS for patients randomized to one or two DI phases. There was no difference in EFS for the 2 SER treatment arms. 5 year EFS was 78% for the Idarubicin/Cytoxan arm and 74% for the ABFM arm. Five year EFS for AYA patients with < 50K WBC was 73.2% (N=190) versus 54% for AYA patients with > 50K WBC. Unlike younger patients, there was no difference in outcome for males and females. Conclusions: 1) AYA patients with ALL have a 68% 5 year EFS when treated with BFM based chemotherapy. 2) WBC < 50K predicts for a favorable outcome. 3) Early augmented intensity therapy appears to improve outcome for AYA ALL patients showing a rapid response to induction therapy.

Long Term Survival Using Intensive Multiagent Chemotherapy for Relapses of Pediatric Acute Lymphoblastic Leukemia (ALL).

Relapses of childhood ALL that occur on therapy are associated with a dismal survival. To improve the prognosis for these patients, we developed an intensive multiagent chemotherapy protocol consisting of an induction with idarubicin, vincristine (VCR), dexamethasone (DEX), and peg-asparaginase (PEG). Consolidation included high-dose cytarabine (ARA-C), etoposide (VP-16), and PEG followed by VCR and methotrexate (MTX). After an interim maintenance (IM), induction and consolidation were repeated followed by maintenance therapy lasting two years. Maintenance and IM consisted of alternating two week cycles of VP-16, ARA-C, and PEG, with MTX ,VCR, DEX, and PEG. Between 1992 and 2002, 53 pts (32M, 21F) received treatment according to this protocol, 21 of whom were treated as part of the original study (Leahey AM et al., Med Ped Onco 34(5):313–8, 2000). Median time to relapse was 37 months from diagnosis (range 12–86 mos). Twenty-one pts were on therapy at relapse and 25 pts were <36 months from diagnosis. Relapses included isolated bone marrow (BM) (32 pts), BM and central nervous system (CNS) (9 pts), BM and testicular (3 pts), and extramedullary (EM) (9 pts). By present day criteria 26 pts were standard risk (SR), 23 were high risk (HR), and 4 were infants. Two patients died in induction, and 2 never achieved a second remission. All others achieved remission by the end of induction (92%). Five-year event-free survival (EFS) and overall survival (OS) are both 56% +/−7% (CI 41%–68%), at a mean of 47 months from relapse (range 0–141 mos). Patients with a first complete remission (CR1) duration <36 months have an EFS of 40% +/−10% (CI 21%–58%); >36 months CR1 is associated with an EFS of 70% +/−9% (CI 50%–84%). Of the events in all pts who initiated therapy,13 were from refractory/recurrent ALL (25%), and 10 pts died of toxicity (19%). Four pts died from chemotherapy induced toxicity (8%), and 6 died from transplant (BMT) related toxicities (11%). Fourteen pts in second remission proceeded to BMT at a mean of 5 months from relapse (range 4.5–8 mos), and 7 of these pts remain in CR2. Of the 39 pts who continued on chemotherapy, 6 pts (35%) with CR1<36 months remain in CR2 and 16 patients (57%) with CR1>36 months remain in CR2 or 3. The 19 pts who were treated on modern standard risk (SR) protocols (CCG-1881 to present) were more salvagable than their 20 high risk (HR) counterparts (CCG-1882 to present). Five year EFS for SR and HR pts is 67% +/−8% (CI 48%–80%) and 38% +/−12% (CI 16%–59%) respectively. Intensive rotating therapy with reinduction and reconsolidation improves EFS. Children with early EM relapses of ALL, CR1 > 36 months, and SR patients all have very good long term survival. Novel therapies need to be integrated into intensive relapse protocols for children with early BM relapse and children treated upfront on HR protocols. [Display omitted] [Display omitted]

A mouse model to study organ homing behaviour of haemopoietic progenitor cells reveals high selectivity but low efficiency of multipotent progenitors to home into haemopoietic organs.

To study the homing behaviour of an enriched multipotent primitive haemopoietic progenitor cell (HPC) population in mice, undifferentiated murine factor-dependent multipotent HPCs (FDCP-mix), stably transfected with the green fluorescence protein gene, were intravenously injected into congenic mice. After 2 or 24 h, cell suspensions were prepared from bone marrow, spleen, lung, liver, muscle, colon, kidney, brain or blood of the mice and analysed by flow cytometry. Using direct quantifiable determination of total HPC numbers homed per organ and a method to estimate the degree of organ contamination by HPC that were present in blood vessels within the organs before preparation, the highest absolute numbers of HPC were detected in the liver and lungs at 2 h but this was sharply decreased at 24 h, whereas HPC selectively accumulated in the bone marrow and spleen at 24 h after transplantation. Only a few HPC were detected in other organs. The seeding efficiency of homed FDCP-mix HPC to the bone marrow and spleen was approximately 1.5% and ranged between that of primary whole bone marrow cells and lineage-depleted freshly isolated bone marrow cells. Pretreatment of HPC with inhibitors of signal transduction indicated that short-term homing of multipotent HPC into haemopoietic organs is an active process requiring co-ordinated intracellular signalling through Rho family small GTPases and protein kinases. Thus, short-term homing of FDCP-mix HPC into haemopoietic organs is of low efficiency but high selectivity, and provides a system to analyse the mechanisms and manipulation of primitive HPC which saves large numbers of donor animals.

634. The Role of Ink4a-Arf Locus in Control of Hematopoietic Stem Cell Proliferation

The mechanisms that control hematopoietic stem cell (HSC) proliferation and self-renewal are incompletely understood, but are relevant for developing gene therapy strategies that target HSCs. Recent evidence shows that loss of Bmi-1, a polycomb transcriptional repressor of the Ink4a-Arf locus, results in progressive loss of HSCs in adult mice with subsequent failure of hematopoiesis. The Ink4a-Arf locus encodes two tumor suppressor proteins, p16Ink4a and p19Arf. Induced transcription of these genes results in cell cycle arrest in several model systems. Based on these facts, we have tested the hypothesis that these Ink4a-Arf gene products may play a role in limiting HSC self renewal during conditions of enhanced proliferation. We first tested whether p19Arf was induced in murine bone marrow cells that were cultured for 10–12 days in IL3, IL6, SCF, and 15% fetal calf serum, hypothesizing that the well described loss of HSCs under these conditions could be due to induction of p19Arf. Using cells from mice that had a GFP reporter gene inserted into the first exon of the p19Arf gene, we found that the p19Arf/GFP allele was significantly induced in the bulk cell population after culture, but not expressed in lineage negative, c-kit+, Sca1+ cells derived from uncultured bone marrow. Competitive repopulation studies were then performed using bone marrow from either p19Arf null mice, or from mice with a homozygous deletion that disrupt both p19Arf and p16Ink4a (Ink4a-Arf–/–). In both cases, there were no defects in repopulating activity when using either freshly isolated bone marrow cells, or cells grown in our 12 day cultures. These data indicate that the loss of HSCs with in vitro culture does not require either p19Arf or p16Ink4a. We next asked whether these genes play a role in the self-renewal of HSCs in vivo using serial bone marrow transplantation assay. Bone marrow cells from either strain or from wildtype controls were serially transplanted into lethally irradiated recipients in doses ranging from 1×105 to 1×106 cells per mouse. We are currently following the fourth group of transplant recipients. In the mice transplanted with bone marrow cells from Ink4a-Arf–/– donors, a survival advantage was noted relative to mice transplanted with strain-matched, wildtype controls. For instance, in secondary recipients receiving 1×105 bone marrow cells, 100% survival was noted in mice transplanted with Ink4a-Arf–/– cells after 10 weeks compared to 20% survival in mice receiving the same dose of wildtype cells. A similar survival advantage was also noted in groups given the lowest cell doses within the 3rd and 4th transplants. In parallel experiments using p19Arf null mice, there was no difference in survival relative to wildtype controls. Measurement of blood counts from recipients in all cohorts clearly showed that the deaths were due to hematopoietic failure. These results show that either both p16Ink4a and p19Arf can inhibit HSC self-renewal in a serial transplant setting, or that only p16Ink4a is necessary. Because animals transplanted with Ink4a-Arf–/– cells do die with hematopoietic failure, albeit later than wildtype controls, we conclude that mechanisms not associated with the Ink4a genes also play a role in limiting HSC self-renewal in vivo.

Resveratrol inhibits the release of mediators from bone marrow-derived mouse mast cells in vitro.

Resveratrol is a natural phytoalexin occurring in grapes, vines and peanuts and possesses both antitumor and antioxidation capabilities. Its chemoprotective actions are attributed partially to its anti-inflammation effect. The present study is aimed at checking the inhibitory actions of resveratrol on the release of mediators from bone marrow-derived mouse mast cells (BMMC) in vitro. Mast cells were prepared by isolating bone marrow cells from intact mice femora and culturing them for 4 weeks in the presence of IL-3 and FCS. The release reaction was triggered by IgE or calcium ionophore (A23187). Mediated by IgE, the release of histamine and tumor necrosis factor-alpha was significantly inhibited by resveratrol at a concentration of 100 microM; IgE-mediated release of leukotrienes and prostaglandin D (2) was also strongly suppressed at concentrations of 100 and 10 microM. Also, A23187-mediated release of histamine and leukotrienes release was strongly reduced by resveratrol at concentrations of 100 and 10 microM, respectively. Resveratrol exhibited its behavior without a significant cytotoxic activity against mast cells. In conclusion, resveratrol is a potent non-selective inhibitor of mediator release from activated mast cells and deserves further investigation of its biological modulations.

Isolated Bone Marrow Involvement in Diffuse Large B Cell Lymphoma: A Report of Three Cases with Review of Morphological, Immunophenotypic and Cytogenetic Findings

Diffuse large B cell lymphoma (DLBL) comprises a heterogenous entity characterized by the presence of large cells, exhibiting a mature B cell phenotype. The high proliferation rate and aggressive disease remain a therapeutic challenge, but the apparent biological diversity permits a risk-stratification model for prognostic grouping through the International Prognostic Index (IPI). Empirical to this approach is the consideration of cytogenetic data, offering an insight into the pathogenetic events which may underlie neoplastic clonal evolution and disease progression. We describe three cases of DLBL presenting with isolated marrow disease, a rare primary finding in this lymphoma. All three cases showed involvement of blood and bone marrow without evidence of splenic or lymph node involvement on imaging studies. Histological and immunophenotypic findings were similar in all three cases, outlining the phenotypic maturity of this disease. Cytogenetic analysis revealed complex karyotypes in the two cases examined. M-FISH (multicolour fluorescent in situ hybridization) performed on bone marrow from case 1 showed several cryptic translocations not evident on G-banding, including a novel translocation between 2p and 9p, and an unbalanced translocation between 14q and 11q. Cytogenetic analysis in case 2 showed abnormalities involving 7q, 9p at the site of the INK4a gene, and the bcl-2 locus, findings confirmed by M-FISH. These cases serve to highlight the biological and cytogenetic heterogenity of DLBL and emphasize the need for complementary investigations in the characterization of this entity.

IL-3 expression by myeloma cells increases both osteoclast formation and growth of myeloma cells

AbstractMacrophage inflammatory protein–1α (MIP-1α) gene expression is abnormally regulated in multiple myeloma (MM) owing to imbalanced expression of the acute myeloid leukemia–1A (AML-1A) and AML-1B transcription factors. We hypothesized that the increased expression ratios of AML-1A to AML-1B also induced abnormal expression of other hematopoietic and bone-specific genes that contribute to the poor prognosis of MM patients with high levels of MIP-1α. We found that interleukin-3 (IL-3) was also induced by the imbalanced AML-1A and AML-1B expression in myeloma. IL-3 mRNA levels were increased in CD138+ purified myeloma cells with increased AML-1A–to–AML-1B expression from MM patients, and IL-3 protein levels were significantly increased in freshly isolated bone marrow plasma from MM patients (66.4 ± 12 versus 22.1 ± 8.2 pg/mL; P = .038). IL-3 in combination with MIP-1α or receptor activator of nuclear factor–kappa B ligand (RANKL) significantly enhanced human osteoclast (OCL) formation and bone resorption compared with MIP-1α or RANKL alone. IL-3 stimulated the growth of interleukin-6 (IL-6)–dependent and IL-6–independent myeloma cells in the absence of IL-6, even though IL-3 did not induce IL-6 expression by myeloma cells. These data suggest that increased IL-3 levels in the bone marrow microenvironment of MM patients with imbalanced AML-1A and AML-1B expression can increase bone destruction and tumor cell growth.

Identification and Clinical Relevance of Receptor Activator of Nuclear Factor κB Ligand Expression of Myeloma Cells

Identification and Clinical Relevance of Receptor Activator of Nuclear Factor κB Ligand Expression of Myeloma Cells

Relapsed Acute Leukemia in Children: Oman Experience

Acute leukemia (AL) is the most common malignancy in children in Oman. It accounts for over one-third of all childhood cancers, most of which (∼75%) are acute lymphoblastic leukemia (ALL). Over a decade, a total of 128 cases of childhood acute leukemia have been diagnosed and managed at Paediatric Haematology/Oncology Unit, Sultan Qaboos University Hospital, which is the national referral center of pediatric leukemia cases. A retrospective review of case notes was used to study all children with a diagnosis of acute leukemia from January 1993 to January 2003. All the cases were diagnosed using a bone marrow aspirate with morphological and immunophenotypic classification. Over this period, 24 cases relapsed. They were classified as per BFM group as “very early,” “early,” and “late” according to the time from diagnosis to first relapse and were divided into isolated bone marrow (BM), extramedullary site, and combined relapse. Sixteen percent of ALL cases and 58.6% of acute myeloid leukemia (AML) cases so far relapsed. Most of the AML cases relapsed very early on in treatment. Eleven patients had combined relapse in BM and extramedullary site (9 in the central nervous system, 1 in the testicles, and 1 in the eye). The overall outcome of these patients is very poor, and only 6 patients out of 24 are still alive. In conclusion, the relapse rates of childhood AL are more or less similar to those of other reports but the overall outcome is very poor. A large majority of the patients in this study are either very early or early relapsers. Future studies including genetic and molecular analysis may be able to explain the difference in clinical outcome of these relapsed AL cases.

Freshly isolated bone marrow cells induce death of various carcinoma cell lines.

In some carcinomas such as digestive tract carcinomas, bone marrow infiltration by tumor cells is a frequent event but usually remains a micrometastatic disease and rarely induces overt bone lesions. The mechanisms responsible for the control of these metastases in the bone marrow remain poorly known. We show that freshly isolated bone marrow cells from human, murine and rat origin rapidly kill a wide range of syngeneic or xenogeneic carcinoma cell lines in culture. Further analysis of this cytotoxic process in the rat indicated that neither resident bone marrow macrophages nor NK cells were responsible for this cytotoxic effect that was restricted to a subpopulation of bone marrow cells expressing CD90 (Thy-1), a marker of hemopoietic precursors. The tumoricidal activity of these cells did not require long-term culture nor addition of exogenous cytokines or growth factors. A subset of CD90+ cells that rapidly differentiates into CD163(ED2)-expressing macrophages was observed to be responsible for tumor cell killing. These macrophages induced a non-apoptotic death of tumor cells, a process that required both a direct interaction with the tumor cell and nitric oxide (NO) production through the activation of inducible nitric oxide-synthase (iNOS). This ability of pluripotent hemopoietic stem cells to rapidly differentiate into macrophages capable of killing invasive tumor cells may account for the limited expansion of micrometastases of some carcinomas in the bone marrow.

Plasma cell survival is mediated by synergistic effects of cytokines and adhesion-dependent signals.

Recent results suggest that plasma cell longevity is not an intrinsic capacity, but depends on yet unknown factors produced in their environment. In this study, we show that the cytokines IL-5, IL-6, TNF-alpha, and stromal cell-derived factor-1alpha as well as signaling via CD44 support the survival of isolated bone marrow plasma cells. The cytokines IL-7 and stem cell factor, crucially important for early B cell development, do not mediate plasma cell survival, indicating that plasma cells and early B cells have different survival requirements. As shown in IL-6-deficient mice, IL-6 is required for a normal induction, but not for the maintenance of plasma cell responses in vivo, indicating that the effects of individual survival factors are redundant. Optimal survival of isolated plasma cells requires stimulation by a combination of factors acting synergistically. These results strongly support the concept that plasma cell survival depends on niches in which a combination of specific signals, including IL-5, IL-6, stromal cell-derived factor-1alpha, TNF-alpha, and ligands for CD44, provides an environment required to mediate plasma cell longevity.

Transcoronary implantation of bone marrow stromal cells ameliorates cardiac function after myocardial infarction

Objectives Bone marrow stromal cells are capable of differentiating into cardiomyogenic cells. We tested the hypothesis that transcoronary implantation of bone marrow stromal cells may regenerate infarcted myocardium and reduce cardiac dysfunction. Methods Isolated bone marrow stromal cells from the isogenic donor rats were transfected with LacZ reporter gene for cell labeling. To induce cardiomyogenic differentiation, the bone marrow stromal cells were treated with 5-azacytidine before implantation. Two weeks after left coronary ligation, these cells (1 × 10 6 in 150 μL) were infused into the briefly distally occluded ascending aorta of the recipient rats (n = 15) to simulate direct coronary infusion clinically. Control animals were infused with cell-free medium (n = 14). Cardiac function was evaluated by echocardiography at preimplantation and 4 and 8 weeks postimplantation. The hearts were then immunohistochemically studied to identify phenotypic changes of implanted bone marrow stromal cells. Results Immediately after cell infusion, the bone marrow stromal cells were trapped within coronary vessels in both infarcted and noninfarcted areas. However, after 8 weeks, most of the cells were identified in the scar and periscar tissue, expressing sarcomeric myosin heavy chain and cardiomyocyte-specific protein troponin I-C. Some bone marrow stromal cells were found to be connected to the adjacent host cardiomyocytes with gap junction. Two-way repeated-measures analysis of variance revealed significant improvement in fractional shortening and end-diastolic and end-systolic diameter of the left ventricle ( P = .0465, .002, .0004, respectively) in the bone marrow stromal cell group. Conclusions Although bone marrow stromal cells had been reported to improve cardiac function when injected directly into the myocardial scar, this study demonstrated for the first time that bone marrow stromal cells can be delivered via the coronary artery, as they are capable of targeted migration and differentiation into cardiomyocytes in the scar tissue to improve cardiac function.

Dexamethasone versus prednisone and daily oral versus weekly intravenous mercaptopurine for patients with standard-risk acute lymphoblastic leukemia: a report from the Children's Cancer Group

AbstractConventional therapy for childhood acute lymphoblastic leukemia (ALL) includes prednisone and oral 6-mercaptopurine. Prior observations suggested potential advantages for dexamethasone over prednisone and for intravenous (IV) over oral 6-mercaptopurine, which remain to be validated. We report the results of a randomized trial of more than 1000 subjects that examined the efficacy of dexamethasone and IV 6-mercaptopurine. Children with National Cancer Institute standard-risk ALL were randomly assigned in a 2 × 2 factorial design to receive dexamethasone (6 mg/m2/d) for 28 days in induction, plus taper, compared with prednisone (40 mg/m2/d). The second randomized assignment was for daily oral or weekly IV 6-mercaptopurine during consolidation. During maintenance, 5 days of the randomized steroid was given monthly, at the same dose, and all patients received daily oral 6-mercaptopurine. During delayed intensification, all patients received a dexamethasone dosage of 10 mg/m2/d for 21 days, with taper. Intrathecal (IT) methotrexate was the sole central nervous system–directed therapy. Patients randomly assigned to receive dexamethasone had a 6-year isolated central nervous system–relapse rate of 3.7% ± 0.8%, compared with 7.1% ± 1.1% for prednisone (P = .01). There was also a trend toward fewer isolated bone marrow relapses with dexamethasone. The 6-year event-free survival (EFS) was 85% ± 2% for dexamethasone and 77% ± 2% for prednisone (P = .002). EFS was similar with oral or IV 6-mercaptopurine; however, patients assigned to IV 6-mercaptopurine had decreased survival after relapse.

Fast Hematopoietic Recovery after Bone Marrow Engraftment Needs Physiological Proximity of Stromal and Stem Cells

Relatively slow hematopoietic recovery after isolated bone marrow (I-BM) engraftment is probably caused by a disrupted microenvironment of stromal and stem cells. Thus, we compared the kinetics of hematopoietic recovery of lethally irradiated rats that received I-BM versus vascularized BM (V-BM). Total body irradiated (TBI; 8 Gy) Lewis (LEW; RT1(1)) rats were either injected IV with syngeneic sex-mismatched 80 x 10(6) I-BM or transplanted with 80 x 10(6) V-BM in orthotopic hind limb grafts. Ten days later, peripheral blood (PB) and mesenteric lymph nodes (MLN) of these recipients were examined for the presence of donor-derived hematopoietic cells with a panel of monoclonal antibodies by FACS. To detect male cells in sex-mismatched female recipients, PCR was performed using male Y chromosome primers. When examined in PB and MLN, recipients transplanted with V-BM displayed significantly faster recovery of leukocytes (CD43+), monocytes (CD14+), and T cells (CD5+) in comparison with I-BM recipients. In addition, only V-BM (but not I-BM) groups contained stroma-like male-positive cells in PB and MLN. Our results suggest that V-BM transplants provided superior hematopoietic recovery in comparison to I-BM transplants. We postulated that close proximity between stromal and stem cells in V-BM is essential for efficient repopulation with progenitors of different lines of leukocytes.

Granulocyte-dendritic cell unbalance in the non-obese diabetic mice

Several investigators, including ourselves, have reported lower yield of GM-CSF bone marrow-derived dendritic cells (DC) with altered MHC class II and co-stimulatory molecules expression in the non-obese diabetic (NOD) mice. However, whether this defect was intrinsic to the DC lineage and/or related to abnormal expansion of other cell types responding to GM-CSF remained an opened issue. We performed phenotypical and morphological analysis of cells from GM-CSF-supplemented-bone marrow-cultures and of freshly isolated bone marrow and blood cells from unmanipulated prediabetic NOD mice. The results show a heretofore undescribed bias towards generation of granulocytes in NOD mice, concomitant with quantitative and qualitative alterations of the DC lineage in both the bone marrow and the blood of this mouse strain. We propose that increased generation of granulocytes in NOD mice might contribute to autoimmunity. First, high numbers of granulocytes per se might favor inflammatory environment. Second, granulocytes, by interfering with DC development, might favor unbalanced antigen presenting cell function leading to T cell autoimmunity.