Objectives
Increasing numbers of reports have described hematopoietic improvement after iron chelation therapy in iron‐overloaded patients. These observations indicate that excess iron could affect ...hematopoiesis unfavorably. To investigate how excess iron affects hematopoiesis in vivo, we generated iron‐overloaded mice and examined hematopoietic parameters in these mice.
Methods
We generated iron‐overloaded mice by injecting 200 mg of iron dextran into C57BL/6J mice, and immature hematopoietic cells in the bone marrow were evaluated by flow cytometric analyses, colony‐forming assays, and bone marrow transplantation analyses. We also examined changes in molecular profiles of the hematopoietic microenvironment.
Results and Conclusions
Iron‐overloaded (IO) mice did not show significant defects in the hematopoietic data of the peripheral blood. Myeloid progenitor cells in the bone marrow were increased in IO mice, but the number and function of the erythroid progenitors and hematopoietic stem cells were not significantly affected. However, bone marrow transplantation from normal donors to IO recipients showed delayed hematopoietic reconstitution, which indicates that excess iron impacts the hematopoietic microenvironment negatively. Microarray and quantitative RT‐PCR analyses on the bone marrow stromal cells demonstrated remarkably reduced expression of CXCL12, VCAM‐1, Kit‐ligand, and IGF‐1 in the iron‐overloaded mice. In addition, erythropoietin and thrombopoietin levels were significantly suppressed, and increased oxidative stress was observed in the IO bone marrow and liver. Consequently, our findings indicate that excess iron can damage bone marrow stromal cells and other vital organs, disrupting hematopoiesis presumably by increased oxidative stress.
Abstract
A 77-year-old Japanese man with disseminated Mycobacterium avium complex (MAC) disease due to anti-interferon-gamma autoantibodies received rituximab, cyclophosphamide, doxorubicin, ...vincristine, and prednisolone (R-CHOP) chemotherapy because of non-Hodgkin lymphoma complication. The hepatobiliary nodules due to MAC resolved with R-CHOP and multidrug antimycobacterial treatment. R-CHOP could serve as an alternative adjunctive therapy for patients with anti-interferon-gamma autoantibodies.
Bone marrow mononuclear cells from 93 patients with hematological malignancies after allogeneic hematopoietic stem cell transplantation (AHSCT) were analyzed using flow cytometry (FCM). The disease ...was acute myeloblastic leukemia (50 patients), acute lymphoblastic leukemia, and others. Conditioning was myeloablative (80 patients) or reduced intensity. The stem cell source was bone marrow (75 patients), peripheral blood stem cells, or cord blood. After AHSCT, granulocyte colony-stimulating factor was given to all patients. All patients showed engraftment of the donor cells. FCM was conducted on a median of 22 days after AHSCT. The gate was set around a granulocytic region consisting of immature granulocytes. The positivity rates of CD13, CD14, CD15, CD33, CD34, CD56, and HLA-DR in the cells were 59.9 ± 27.4%, 5.8 ± 8.8%, 98.3 ± 1.9%, 92.3 ± 12.4%, 2.6 ± 5.8%, 24.3 ± 16.7%, and 9.1 ± 6.6%, respectively. The greatest value of CD56 positivity was 73.1%. On the basis of CD56 expression, cases of more than 24% CD56 positivity were assigned to the CD56-high group (39 patients), while the rest were assigned to the CD56-low/negative group. There were no significant differences between the two groups in terms of disease status, sex, donor, hematopoietic stem cells, days of FCM analysis, or peripheral blood cell counts around the days of performing FCM. These results indicate that CD56 can be expressed in normal immature granulocytes at a variety of expression levels in regenerative bone marrow. Attention should be paid when evaluating aberrant antigen expression of CD56 in granulocytes. J Clin Exp Hematop 53(3) : 247-250, 2013
TO THE EDITOR On August 8, 2003, a 23-year-old male was referred to our hospital because of an abnormal shadow in the right lung. He had no family members affected with immunodeficiency disorders. He ...was diagnosed as having pneumonia. On August 1, 2005, he was admitted again because of pneumonia. Serum IgG, IgA, and IgM levels were normal. An antihuman immunodeficiency virus test was negative. An abdominal computed tomographic scan showed multiple low-density lesions in the spleen, which led to the suspicion of fungal infection. On September 30, 2005, analysis of the peripheral blood lymphocytes showed normal T-cell subsets and a normal CD4/CD8 ratio. He developed intractable skin inflammatory lesions due to methicillin-sensitive Staphylococcus aureus. On December 14, 2010, the patient was referred to the Division of Hematology to determine a diagnosis for the repeated infections. He had low-grade fever. Peripheral blood neutrophils were morphologically normal without abnormal cytoplasmic granules. The serum CRP level was 6.33mg/dl. The patient's clinical course was strongly suggestive of disorders related to neutrophil dysfunction. Phenotypes of the peripheral blood neutrophils were CD11b+, CD13+, CD15+, CD33+, and phosphatidyl glucoside+ as well as CD10-, CD14-, CD34-, CD36-, and HLA-DR- (Fig.1).
d-Allose, the C-3 epimer of
d-glucose, is one of the rare sugars found in nature. In the present study, we have elucidated for the first time that various leukemia cell lines have different ...susceptibility to anti-proliferative activity of
d-allose, and that this difference is related to the difference in induction of thioredoxin interacting protein (TXNIP) expression. We examined 5 leukemia cell lines (MOLT-4F, IM-9, HL-60, BALL-1 and Daudi), and found that MOLT-4F (T-cell lymphoblastic leukemia) had the highest susceptibility to
d-allose, and that Daudi (Burkitt's lymphoma) had the lowest.
d-Allose significantly slowed the cell cycle progression without causing apoptosis of MOLT-4F cells. Intracellular TXNIP expression was specifically and markedly enhanced in MOLT-4F cells by
d-allose treatment, and subsequent increase of p27
kip1, a cell cycle inhibitor, was observed. On the other hand,
d-allose did not increase TXNIP and p27
kip1 levels at all in Daudi cells. These results indicate that
d-allose suppresses MOLT-4F cell proliferation possibly by the inhibition of cell cycle progression via induction of TXNIP expression.
We report on a case of severe renal failure in a 61-year-old female with multiple myeloma (MM). Two months prior to admission, the patient was diagnosed to have anemia and progressive renal failure ...associated with urinary Bence Jones protein and was referred to our hospital. A bone marrow biopsy revealed 40% plasma cells with κ light chain restriction. Thus, she was considered to have MM. A renal biopsy revealed neoplastic plasma cell infiltration within the kidney, moderate interstitial fibrosis, tubular atrophy, and punctate, electron-dense material along the peripheral capillary walls, tubular basement membrane, and in the interstitium of the kidney. This suggested that a combination of compression of the tubules and the microvasculature by the infiltrative process, and local light chain deposition-mediated tissue damage might be implicated in the development of renal failure in this patient. Despite a remission of bone marrow plasmacytosis with a bortezomib-based regimen, her renal function gradually deteriorated and a periodic hemodialysis program was finally required. Although the clinical impact of the direct kidney infiltration of neoplastic plasma cells on the longitudinal changes in renal function remains to be delineated, it is reasonable to consider that the infiltration of neoplastic plasma cells associated with local light chain depositions may result in irreversible renal injuries. Obviously, further studies and accumulation of additional experience with renal biopsy are required to better determine the precise and prognostic relationship between renal outcome and morphological alterations among MM patients with varying degrees of renal impairment.
A 77-year-old Japanese man with disseminated Mycobacterium avium complex (MAC) disease due to anti-interferon-gamma autoantibodies received rituximab, cyclophosphamide, doxorubicin, vincristine, and ...prednisolone (R-CHOP) chemotherapy because of non-Hodgkin lymphoma complication. The hepatobiliary nodules due to MAC resolved with R-CHOP and multidrug antimycobacterial treatment. R-CHOP could serve as an alternative adjunctive therapy for patients with antiinterferon- gamma autoantibodies. Keywords. anti-interferon-gamma autoantibody; cyclophosphamide; disseminated Mycobacterium avium complex diseases; R-CHOP; rituximab.
Background; Adoptive immunotherapy with chimeric antigen receptor (CAR) gene transduced CD19-CAR T cells, which are engineered to express extracellular single-chain immunoglobulin variable fragments ...to CD19, linked to cytoplasmic T cell activation domains including CD3-ζ, showed remarkable therapeutic benefits toward CD19+ B cell malignancies including acute lymphoblastic leukemia, chronic lymphoblastic leukemia and non-Hodgkin lymphoma (B-NHL). For clinical setting, the phenotype of manufactured CAR T cells is an important factor; especially less differentiated T cells are anticipated to provide a long-lasting immune reconstitution. Furthermore, in order to avoid the risk of technical error and contamination during T cell manufacturing process, a closed system needs to be established. In this study, we addressed these issues and have established a novel CD19-CAR T cell manufacturing method from a small amount of blood in a closed system for clinical trial to treat patients with B-NHL.
Methods; Peripheral blood was obtained from B-NHL patient volunteers and healthy donor volunteers who gave their written informed consents. Peripheral blood mononuclear cells (PBMCs) were isolated from 30 ml of blood using Ficoll-Paque PREMIUM density gradient centrifugation. PBMCs were stimulated in a plastic bag pre-coated with anti-CD3 monoclonal antibody (OKT3) and recombinant fibronectin fragment (RetroNectin®; RN). Following four days of stimulation, stimulated T cells were transferred into a 215 cm2 plastic bag pre-loaded with SFG-1928z retroviral vector (Brentjens et al., Clin Cancer Res. 2007) onto RN-coated substratum with low-temperature shaking (RBV-LTS method; Dodo et al., PLoS ONE, 2014). After one hour incubation, the bag was flipped over to facilitate more efficient utilization of the retroviral vector adsorbed on both top and bottom surfaces of the bag and further incubated. On Day 5, the transduction procedure was repeated, and the cells were transferred into 640 cm2 plastic bags until Day 10-14 for expansion. Closed system liquid handling was managed in all processes of manipulating T cells for stimulation, transduction, expansion and final product formulation.
Results; We have previously reported that the fold expansion of T cells under stimulation with RN together with OKT3 enhanced cell proliferation while preserving the naïve phenotype of T cells in comparison to stimulation with OKT3 alone or OKT3 and anti-CD28 monoclonal antibody co-stimulation. Although B-NHL patients’ T cells showed much lower fold expansion compared to healthy donors’ T cells, 4/7 patients’ CAR T cells reached their target dose of 1 x 106 cells/kg from 30 ml of blood on Day 10. For the other 3 patients, 70-150 ml blood was estimated to be required to reach their target dose. The delay in proliferations was marked in B-NHL patients’ T cells compared to healthy donors’ T cells by Day 5, but B-NHL patients’ T cells represented significant catch-up growth, which was superior to healthy donor T cells during Day 7-14. Gene transfer efficiency of patients’ T cells (N = 7, 17.9 ± 4.7%) was equivalent to that of healthy donors’ T cells (N = 5, 22.2 ± 5.3%), and CAR T cells showed potent anti-tumor reactivity with cytokine productions against CD19 positive Raji cells in vitro. Comparing to CD3/CD28 beads stimulation method, RN/OKT3 stimulation method showed equivalent expansion. Furthermore, RN/OKT3 stimulated T cells preserved higher proportion of CD8+/CCR7+/CD45RA+/CD62L+ naïve phenotype T cells (43.6% in healthy donor and 30.9% in patient donor) compared to CD3/CD28 beads stimulated T cells (22.8% in healthy donor and 11.7% in patient donor).
Conclusions; With our novel closed system manufacturing method utilizing RN/OKT3 stimulation combined with RBV-LTS transduction from a small amount of blood of B-NHL patients, we are able to manufacture a sufficient number of CAR T cells maintaining higher proportion of naïve phenotype, which is expected to improve the efficacy of adoptive immunotherapy. In our cell manufacturing, patients are not required to undergo leukapheresis, which is more invasive to patients. Based on our results, we employed our novel manufacturing method for the phase I/II clinical trial to treat patients with relapsed/refractory CD19+ B-NHL (clinicaltrials.gov, identifier; NCT02134262).
Chono:Takara Bio Inc.: Employment. Tahara:Takara Bio Inc.: Employment. Nukaya:Takara Bio Inc.: Employment. Mineno:Takara Bio Inc.: Membership on an entity’s Board of Directors or advisory committees. Tsukahara:Takara Bio Inc.: Research Funding. Ohmine:Takara Bio Inc.: Research Funding. Ozawa:Takara Bio Inc.: Research Funding. Takesako:Takara Bio Inc.: Membership on an entity’s Board of Directors or advisory committees.
Abstract
In the rituximab era, several large studies have suggested that full-dose rituximab plus cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP) might be the best treatment for ...patients with diffuse large B-cell lymphoma (DLBCL) aged 60 years and older. However, it remains unclear whether this is also the case for those aged 70 years and older. Previously untreated patients with DLBCL aged 70 years and older (elderly) were treated with R-70%CHOP, and patients younger than 70 years (younger) were treated with full-dose R-CHOP every 3 weeks, for a total of 6-8 cycles. Complete remission (CR) rates in elderly versus younger patients were 75 vs. 78% (p = 0.7), respectively. The 3-year overall survival, event-free survival and progression-free survival of elderly versus younger patients were 58 vs. 78% (p < 0.05), 45 vs. 70% (p < 0.05) and 64 vs. 72% (p = 0.43), respectively. Severe adverse events were more frequent in the elderly, even with the dose reduction in that age group. Three-year PFS with R-70%CHOP for patients aged 70 years and older was not significantly worse than that with full-dose R-CHOP for younger patients, suggesting that R-70% CHOP might be a reasonable choice for patients with DLBCL aged 70 years and older, especially for those with comorbidities.
Abstract 3173
Deferasirox (DFX) is an oral iron chelator that enables effective chelation by once daily administration.Since the introduction of DFX, iron chelation therapy (ICT) for transfusional ...iron overload has attracted increased attention. It is known that excess iron increases oxidative stress and affects various organs, such as the liver, heart and endocrine glands, negatively. Sufficient ICT can remove excess iron and improve organ dysfunction in iron-overloaded patients, and accumulating data has indicated that efficient ICT improves the survival of transfusion-dependent patients with myelodysplastic syndromes (MDS). Recently, we experienced a case of MDS with transfusional iron overload in which the hematopoietic data improved unexpectedly after administration of DFX without any other specific treatments (Okabe H et al. Rinsho Ketsueki, 2009). An increasing number of similar cases has been reported. This clinical observation indicates that iron overload could also affect the hematopoietic system unfavorably, via, as yet, unknown mechanisms.
We generated iron-overloaded mice to investigate how iron overload affects hematopoiesis in vivo. C57BL6 mice were injected with a total of 200 mg of iron dextran, intraperitoneally over 4 weeks. The iron-overloaded mice showed pigmented skin and hepatosplenomegaly, and histological examination showed excess iron deposition in the bone marrow, liver, spleen and heart. The serum and organ iron concentrations in these mice markedly increased. However, the iron-overloaded mice did not show any significant changes in peripheral blood counts or the proportion of immature hematopoietic cells in the bone marrow. To further examine the effects of excess iron on the biological functions of hematopoietic stem and progenitor cells (HSPCs), we performed bone marrow transplantation (BMT) assays. First, to assess the hematopoietic reconstitutional capacity of the HSPCs of iron-overloaded mice, we transplanted bone marrow cells (1×106 cells) from iron-overloaded mice or normal mice into lethally irradiated normal recipient mice along with the same number of normal competitor cells. We found no significant difference in hematopoietic reconstitution between the iron-overloaded donor cells and the normal donor cells, suggesting that the hematopoietic reconstitutional capacity of HSPCs in iron-overloaded mice is not significantly affected by iron. In contrast, when we transplanted bone marrow cells from normal mice (2×106 cells) into iron-overloaded recipients, hematopoietic recovery was significantly delayed, in particular platelet counts (at 2 weeks after BMT, normal recipients vs. iron-overloaded recipients, 63.4±9.4 vs. 18.7±4.7×104/μl, respectively, p<0.001). This indicates that excess iron disturbs the function of the bone marrow microenvironment and delays hematopoietic reconstitution. Microarray and quantitative RT-PCR analysis of non-hematopoietic bone marrow cells (CD45-/Ter119-) from the iron-overloaded mice demonstrated significant reductions in CXCL12, VCAM-1, Kit-ligand and IGF-1, which are important regulators of hematopoiesis. In addition, in the iron-overloaded mice, the serum concentration of erythropoietin and the expression level of thrombopoietin in the liver were also significantly reduced. Furthermore, increased oxidative stress levels were observed in the iron-overloaded liver and bone marrow.
We did not observe any direct effects of excessive iron on hematopoietic cells, but found significant impairment of the hematopoietic microenvironment in the bone marrow of iron-overloaded mice. These results suggest that oxidative stress induced by excess iron could disturb the hematopoiesis-supporting capacity of the bone marrow microenvironment by reducing the expression of many essential molecules.
No relevant conflicts of interest to declare.
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