Bone Marrow Expansion Research Articles

Genetic iron overload aggravates and pharmacological iron restriction improves MDS pathophysiology in preclinical study

Although iron overload is a common feature in myelodysplastic syndromes (MDS), it remains unclear how iron excess is detrimental for disease pathophysiology. Taking advantage of complementary approaches, we analyzed the impact of iron overload and restriction achieved through genetic activation (FPNC326S) and pharmacologic inhibition (vamifeport) of the iron exporter ferroportin in a MDS mouse model, respectively. While FPNC326S-induced iron overload did not significantly improve the late stages of erythroid maturation, vamifeport-mediated iron restriction ameliorated anemia and red blood cell maturation in MDS mice, through the reduction of oxidative stress and apoptosis in erythroid progenitors. Iron overload aggravated and restriction alleviated ROS formation, DNA damage and cell death in hematopoietic stem and progenitor cells, resulting in altered cell survival and quality. Finally, myeloid bias, indicated by expanded bone marrow myeloid progenitors and circulating immature myeloid blasts, was exacerbated by iron excess and attenuated by iron restriction. Overall, vamifeport treatment resulted in improved anemia and significant survival increment in MDS mice. Interestingly, the combined therapy with vamifeport and the erythroid maturation agent luspatercept has superior effect in improving anemia and myeloid bias as compared to single treatments, and offers additive beneficial effects in MDS. Our results prove for the first time in a preclinical model that iron plays a pathologic role in transfusion-independent MDS. This is likely aggravated by transfusional iron overload, as suggested by observations in the FPNC326SMDS model. Ultimately, the beneficial effects of pharmacologic FPN inhibition uncovers the therapeutic potential of early prevention of iron toxicity in transfusion-independent MDS.

Targeting exhausted cytotoxic T cells through CTLA-4 inhibition promotes elimination of neoplastic cells in human myelofibrosis xenografts.

Myeloproliferative neoplasms represent a group of clonal hematopoietic disorders of which myelofibrosis (MF) is the most aggressive. In the context of myeloid neoplasms, there is a growing recognition of the dysregulation of immune response and T-cell function as significant contributors to disease progression and immune evasion. We investigated cytotoxic T-cell exhaustion in MF to restore immune response against malignant cells. Increased expression of inhibitory receptors like CTLA-4 was observed on cytotoxic T cells from MF patients together with a reduced secretion of IFNɣ and TNFɑ. CTLA-4 ligands CD80 and CD86 were increased on MF granulocytes and monocytes highlighting a possible role for myeloid cells in suppressing T-cell activation in MF patients. Unlike healthy donors, the activation of cytotoxic T cells from MF patients was attenuated in the presence of myeloid cells and restored when T cells were cultured alone or treated with anti-CTLA-4. Moreover, anti-CTLA-4 treatment promoted elimination of neoplastic monocytes and granulocytes in a co-culture system with cytotoxic T cells. To test CTLA-4 inhibition in vivo, patient-derived xenografts were generated by transplanting MF CD34+ cells and by infusing homologous T cells in NSGS mice. CTLA-4 blockade reduced human myeloid chimerism and led to T-cell expansion in spleen and bone marrow. Overall, these findings shed light on T-cell dysfunction in MF and suggest that CTLA-4 blockade can boost the cytotoxic T cell-mediated immune response against tumor cells.

Macrocephaly and Finger Changes: A Narrative Review

Macrocephaly, characterized by an abnormally large head circumference, often co-occurs with distinctive finger changes, presenting a diagnostic challenge for clinicians. This review aims to provide a current synthetic overview of the main acquired and genetic etiologies associated with macrocephaly and finger changes. The genetic cause encompasses several categories of diseases, including bone marrow expansion disorders, skeletal dysplasias, ciliopathies, inherited metabolic diseases, RASopathies, and overgrowth syndromes. Furthermore, autoimmune and autoinflammatory diseases are also explored for their potential involvement in macrocephaly and finger changes. The intricate genetic mechanisms involved in the formation of cranial bones and extremities are multifaceted. An excess in growth may stem from disruptions in the intricate interplays among the genetic, epigenetic, and hormonal factors that regulate human growth. Understanding the underlying cellular and molecular mechanisms is important for elucidating the developmental pathways and biological processes that contribute to the observed clinical phenotypes. The review provides a practical approach to delineate causes of macrocephaly and finger changes, facilitate differential diagnosis and guide for the appropriate etiological framework. Early recognition contributes to timely intervention and improved outcomes for affected individuals.

Treatment of post-allogeneic hematopoietic stem cell transplant cytopenias with sequential doses of multipotent mesenchymal stromal/stem cells

Background aimsCytopenias after allogeneic stem cell transplantation (allo-SCT) are a common complication, the underlying pathogenic mechanisms of which remain incompletely understood. Multipotent mesenchymal stromal/stem cell (MSC) therapy has been successfully employed in the treatment of immune-related disorders and can aid in the restoration of the hematopoietic niche. MethodsA phase II clinical trial to assess the efficacy and safety of administering four sequential doses of ex-vivo expanded bone marrow MSCs from a third-party donor to patients with persistent severe cytopenias after allo-SCT was performed. ResultsThe overall response rate on day 90 was 75% among the 27 evaluable patients (comprising 12 complete responses, 8 partial responses, and 7 with no response). The median time to respond was 14.5 days. Responses were observed across different profiles, including single or multiple affected lineages, primary or secondary timing, and potential immune-mediated or post-infectious pathophysiology versus idiopathic origin. With a median follow-up for surviving patients of 85 months after MSC infusion, 53% of patients are alive. Notably, no adverse events related to MSC therapy were reported. ConclusionsIn summary, the sequential infusion of third-party MSCs emerges as a viable and safe therapeutic option, exhibiting potential benefits for patients experiencing cytopenias following allo-SCT.

Abstract 7469: Inflammation plays an important role in the progression of chronic myeloid leukemia

Abstract The development of Tyrosine Kinase Inhibitors (TKIs) resulted in a significant increase in the life expectancy of patients with chronic myeloid leukemia (CML). Nevertheless, TKIs still fail in a significant proportion of therapy-naïve patients, or their effectiveness is hampered by secondary resistance. It is therefore urgent to explore the underlying mechanisms in order to develop new therapeutic approaches to cure CML instead of controlling the disease with a lifelong TKI therapy. Using the ScltTA/TRE-BCR::ABL1 mouse model for chronic phase CML we show that BCR::ABL1 leads to the elevation of pro-inflammatory cytokines, known to be important in disease initiation and progression. This is supported by our data from CML patients, where we also observe higher levels of pro-inflammatory cytokines in the serum compared to healthy individuals. Furthermore, we demonstrate that BCR::ABL1 drives the expansion of bone marrow derived mast cells (BMMCs) and sensitizes them for FcεRI triggered degranulation. Interestingly, these BMMCs show differences in their lipid metabolism. Hence, sphingomyelin and lysophosphatidylcholine, both known to be involved in inflammation and cancer progression, are upregulated in BCR::ABL1-positive mast cells compared to their negative counterparts. By crossing in the Cpa3Cre mediated model for mast cell deficiency, we demonstrate for the first time that the lack of mast cells prevents the BCR::ABL1 induced upregulation of pro-inflammatory cytokines and also the development of splenomegaly. In addition, we were able to show that splenomegaly in CML patients is associated with high bone marrow mast cell counts and that an upregulation of pro-inflammatory cytokines in patient serum samples correlated with tryptase levels. In summary, our study identifies mast cells and their inflammatory cytokines as important factors in disease progression and suggests considering them as additional targets in CML therapy. Citation Format: Melanie Langhammer, Julia Schöpf, Timo Jaquet, Katharina Horn, Moritz Angel, Daniel Mohl, Simon Lagies, Corinna Spohr, Daniel Christen, Franziska Maria Uhl, Tiago Maié, Henrike Jacobi, Thorsten B. Feyerabend, Julia Huber, Marcus Panning, Cassian Sitaru, Ivan Costa, Robert Zeiser, Konrad Aumann, Heiko Becker, Till Braunschweig, Steffen Koschmieder, Bernd Kammerer, Khalid Shoumariyeh, Michael Huber, Mirle Schemionek-Reinders, Tilman Brummer, Sebastian Halbach. Inflammation plays an important role in the progression of chronic myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7469.

Impacts of cationic lipid-DNA complexes on immune cells and hematopoietic cells in vivo.

The inability to systemic administration of nanoparticles, particularly cationic nanoparticles, has been a significant barrier to their clinical translation due to toxicity concerns. Understanding the in vivo behavior of cationic lipids is crucial, given their potential impact on critical biological components such as immune cells and hematopoietic stem cells (HSC). These cells are essential for maintaining the body's homeostasis, and their interaction with cationic lipids is a key factor in determining the safety and efficacy of these nanoparticles. In this study, we focused on the cytotoxic effects of cationic lipid/DNA complexes (CLN/DNA). Significantly, we observed that the most substantial cytotoxic effects, including a marked increase in numbers of long-term hematopoietic stem cells (LT-HSC), occurred 24 h post-CLN/DNA treatment in mice. Furthermore, we found that CLN/DNA-induced HSC expansion in bone marrow (BM) led to a notable decrease in the ability to reestablish blood cell production. Our study provides crucial insights into the interaction between cationic lipids and vital cellular components of the immune and hematopoietic systems.

Bone Marrow Stromal Stem Cell Fate Decision: A Potential Mechanism For Bone Marrow Adipose Increase with Aging-related Osteoporosis.

Osteoporosis is a systemic bone disease that seriously threatens the health and quality of life in middle-aged and older adults. In this review, we describe the relationship between bone marrow adipose tissue and aging osteoporosis and mainly focus on bone marrow mesenchymal stem cell osteogenic-adipose differentiation fate with aging along with the relevant mechanisms responsible for these changes. We summarized recent advances in regulating the bone marrow mesenchymal stem cell differentiation due to aging in this review. Aging-related bone mass loss is accompanied by expanding bone marrow adipose because of an imbalance of bone marrow mesenchymal stem cell differentiation, resulting in adipogenesis. Ectopic adipocytes in the bone marrow increase with age and are a key factor responsible for the aging-related bone mass decrease. Transcription factors and classical regulating pathways are involved in this process during aging. As the global aging population increases, not only older women but also older men face a great fracture risk. Therefore, finding molecular mechanisms controlling the stimulating adipogenesis in BMSC during aging is important for providing the new cue for prevention and therapeutics for aging-related bone loss. Furthermore, upon physical examination of older people, except for the bone mineral density and bone turnover biochemical marker, the bone marrow adipose measurement should be taken into account when assessing the fracture risk and treatment plan that will be beneficial in clinical practice.

Flow cytometric analysis of CD34+ CD38- cells; cell frequency and immunophenotype based on CD45RA expression pattern.

The CD34+ CD38- population in bone marrow includes hematopoietic stem/progenitor cells. Recently, in acute myeloid leukemia, the focus has shifted to flow cytometry analysis targeting CD34+ CD38- leukemic cells due to their effectiveness in minimal/measurable residual disease detection and prognosis prediction. Nevertheless, the immunophenotype and cell frequency of these cells in the bone marrow, in the absence of leukemic cells, remains unknown. We aimed to evaluate detailed characteristics of CD34+ CD38- cells in both normal and leukemic cells by flow cytometry. We compared the cell frequency and immunophenotype of the CD34+ CD38- fraction in the following groups: patients with idiopathic thrombocytopenic purpura and malignant lymphoma as controls (n = 17), post-treatment patients without abnormal blasts (n = 35), and patients with myeloid malignancies (n = 86). The comparison was based on the presence or absence of CD45RA expression, a marker commonly used to prospectively isolate lymphoid-primed cell populations within the CD34+ CD38- fraction. The CD34+ CD38- CD45RA+ cell population exhibited a significant expansion in bone marrow without leukemic cells 1 month after cord blood transplantation and in various type of myeloid malignancies, compared to the control group (p < 0.01). Continuous CD45RA expression and notable expansion of the CD34+ CD38- CD45RA- population were exclusively observed in myelodysplastic syndrome-related diseases. The CD34+ CD38- CD45RA+ population displayed frequent expression of various markers in both leukemic and non-leukemic cells, in contrast to the CD34+ CD38- CD45RA- population. The CD34+ CD38- fraction should be carefully evaluated considering the nature of normal hematopoietic precursor cells, their cell frequency and immunophenotype, including CD45RA expression pattern, for improving the accuracy of myeloid malignancy diagnosis.

A review on thromboembolic events and neurological lesions in patients with β-thalassemia

Β-thalassemia is the severe form of genetic illnesses which decreases the hemoglobin synthesis. One of the major complications in thalassemic syndromes, including β-thalassemia major and intermedia is thromboembolic events. In addition, thromboembolic events are more common in non-transfusion-dependent thalassemia than those in well-transfusion-dependent β-thalassemia. A combination of hypercoagulable states including, abnormalities in red blood cells, and platelet, antithrombin III, protein C, and protein S, and splenectomy are involved in thrombotic events, but thromboembolic events can be prevented and treated in these patients via blood transfusion, hydroxyurea, anticoagulants, and aspirin. Moreover, recent studies have demonstrated the involvement of the brain lesion in β-thalassemia patients. The involvement of vascular events of brain in patients with β-thalassemia intermedia is 29-83%, but the rate of asymptomatic brain lesions in the healthy people is 0-11%. In addition, neurological complications which have been attributed to various factors are chronic hypoxia, iron overload, bone marrow expansion, and desferrioxamine neurotoxicity. This review evaluated thromboembolic events and neurological lesions in patients with β-thalassemia and its probable curative therapy.

P05 Osteoporosis in thalassemia; a case with multifactorial aetiology

Abstract Introduction Thalassemia patients are prone to osteoporosis due to multifactorial reasons, including anaemia, bone marrow expansion, iron overload and hypogonadism. Case description A 41-year-old British Asian male with beta thalassemia major was referred to rheumatology in 2018 for osteoporosis. In 2008, aged 27 years, whilst playing football, he developed a distal radial fracture and ulnar styloid process fracture and a DEXA scan had revealed low bone mineral density (BMD) for age. Subsequently, he was started on Ibandronic acid by his haematologist for a total of 10 years. After he was referred to rheumatology in 2018, bisphosphonates were discontinued as he had 5% improvement in BMD, a decade of bisphosphonates and no further fractures. Further assessment revealed multiple contributory factors for his osteoporosis. • Consistency low vitamin D – 9.6 nmol/L in 2009, average value of 20 • He was diagnosed with hypogonadism, poor compliance with IM testosterone injections • He had diabetes which was not optimally controlled • Evidence of iron overload with average serum ferritin of 2000 ug/L with deferasirox While these factors were optimised, it was decided to adopt a watch and wait policy with regards to specific osteoporosis treatment. Review in 2023 revealed serum testosterone level of 20.1 nmol/L (6.9-23.2) with better compliance with testosterone injections. Fructosamine levels were 310 (normal range 200-285) but it has reduced from a peak of 370. Serum ferritin levels have reduced to 776 down from a high of 5600. Normal vitamin D levels were maintained. The DEXA scan in 2023 revealed a spine T score of −2.4 which showed 10.2% increase in bone mineral density. DEXA scan in 2020 revealed T score of −3.1 in spine and −1.9 in hips. The P1NP level was 56 (19-69) which has reduced from 100 in 2021. Improvements in BMD and bone turnover markers were achieved through optimising contributory factors without any specific osteoporosis treatment. He will be monitored with periodic BMD and bone turnover markers. Discussion Anaemia and subsequent bone marrow expansion is a recognised cause for osteoporosis in thalassemia patients. While regular blood transfusions inhibit bone marrow expansion, ineffective erythropoiesis is not fully supressed. This patient received 3 units of blood every 4 weeks. Bone marrow expansion occurs mostly in trabecular bone resulting in low BMD is areas which generally have increased amount of trabecular bone such as the spine. Our patient had a lower BMD in the spine compared to the femoral neck. Iron overload directly disrupts bone formation and can result in endocrinopathies which contributes to osteoporosis. Hypogonadism is another well-recognised cause and is primarily results from iron deposition in pituitary and at times in the gonads. Hormonal replacement therapy reduces bone loss. Studies have shown that hypogonadic patients have more severe osteoporosis and predominant involvement of the femoral neck. Iron overload can cause diabetes mellitus and impaired growth hormone/ insulin growth factor-1 (IGF-1) axis which can contribute to osteoporosis. Our patient did have diabetes but had normal IGF-1 levels when checked previously. Deferoxamine which is one of iron chelating agents have been shown to reduce collagen synthesis and increases osteoblast apoptosis which might contribute to the development of osteoporosis. Vitamin D levels are well known to be low in patients with thalassemia compared to the general population. While there are no RCT’s to show that bisphosphonates reduce fracture risk, surrogate markers such as increased BMD and reduction in bone turnover markers are known to occur. Therefore, current guidance (Standards for the Clinical Care of Children and Adults with Thalassaemia in the UK) recommends bisphosphonates to be considered in patients with a low BMD for age (Z-score &amp;lt; −2.0, T-score &amp;lt; −2.5 if post-menopausal or over 50), if there are fragility fractures or falling BMD despite adequate vitamin D levels, and hormone supplementation. Key learning points Learning point Important to assess for osteoporosis in thalassaemic patients. There are multiple factors contributing to the development of osteoporosis which needs to be addressed together with endocrinologist and haematologist. Bisphosphonates are recommended though further RCT evidence is required to support this

P06.03.A IMMORTALIZED MACROPHAGES AS A TOOL TO STUDY PHAGOCYTOSIS AND POLARIZATION IN VITRO

Abstract BACKGROUND Macrophages, an important element of the tumour-microenvironment (TME), are classified based on polarization state: tumour inhibiting ‘classically-activated macrophages’ M1 and tumour promoting ‘alternatively-activated macrophages’ M2. It is wellknown that the glioblastoma (GBM) TME is dominated by immunosuppressive M2 macrophages contributing to tumor growth by promoting angiogenesis, invasion and immunosuppression. However, macrophage culture is difficult to maintain as the cells do not proliferate and expand, making co-culture studies challenging. In this work, we successfully immortalized and expanded bone marrow derived mouse macrophages (BMDMs) that can be used as a tool to study phagocytosis and polarization in co-culture with tumor cells. MATERIAL AND METHODS Primary macrophages, obtained from the bone marrow of C57/BL6 mice, were transduced with two lentiviral vectors: hTERT with antibiotic hygromycin (Hy) and Simian virus 40 (SV40) with mCherry selective markers. Viral-titers were prepared by co-transfecting HEK293T with hTERT and SV40 constructs and plasmids MDL-X (Gag/Pol):Vpx. Vpx is a virion-packaged accessory protein that induces degradation of SAMHD1, a GTP- activated dNTP hydrolase that inhibits lentiviral infection of macrophages. BMDMs transduced with hTERT were subjected to gradient Hy selection after 5 days infection and SV40.mCherry positive cells were sorted by flow cytometry. To assess phagocytotic capacity, BMDMs were incubated with apoptotic bodies of a murine GBM cell-line (GL261) which was treated with HSV-TK/ganciclovir (GCV) suicide gene therapy. Phagocytosis was measured by fluorescence microscopy and flow cytometry. Further, BMDMs were externally induced with either E. coli lipopolysaccharide (LPS) and interferon gamma (IFNγ) or interleukin 4 (IL4) to activate M1 and M2 polarization. Flow cytometry was used to detect iNOS, CD40 and CD86 markers for M1 phenotype and Arginase1 and CD206 for M2. RESULTS hTERT.MDL-X.Vpx.Hy BMDM showed no clear signs of immortalization with limited proliferation followed by cell death. In contrast, SV40.MDL-X.Vpx.mCherry BMDM exhibited proliferative expansion from single colony, efficient freeze-thaw cycles and were used for further studies. BMDMs showed successful phagocytosis of apoptotic bodies from HSV-tk/GCV treated GL261 cells with an increasing capacity upto 18 hours of application. The phagocytic capacity was higher compared to commercial cell lines such as BV2 and Raw 264.7. Immortalized BMDMs tend to have a M2 phenotype without external induction and it is possible to activate M1 phenotype by LPS and IFNγ induction. CONCLUSION Here, we successfully immortalized bone-marrow derived macrophages and showed that they can be used as a tool to study macrophage polarization and phagocytic capability in a tumor immunotherapy context.

Therapeutic effect of trace elements on multiple myeloma and mechanisms of cancer process.

In the human body, trace elements and other micronutrients play a vital role in growth, health and immune system function. The trace elements are Iron, Manganese, Copper, Iodine, Zinc, Cobalt, Fluoride, and Selenium. Estimating the serum levels of trace elements in hematologic malignancy patients can determine the severity of the tumor. Multiple myeloma (MM) is a hematopoietic malignancy and is characterized by plasma cell clonal expansion in bone marrow. Despite the advances in treatment methods, myeloma remains largely incurable. In addition to conventional medicine, treatment is moving toward less expensive noninvasive alternatives. One of the alternative treatments is the use of dietary supplements. In this review, we focused on the effect of three trace elements including iron, zinc and selenium on important mechanisms such as the immune system, oxidative and antioxidant factors and cell cycle. Using some trace minerals in combination with approved drugs can increase patients' recovery speed. Trace elements can be used as not only a preventive but also a therapeutic tool, especially in reducing inflammation in hematological cancers such as multiple myeloma. We hope that the prospect of the correct use of trace element supplements in the future could be promising for the treatment of diseases.

Idecabtagene vicleucel for relapsed and refractory multiple myeloma: post hoc 18-month follow-up of a phase 1 trial

Idecabtagene vicleucel (ide-cel) is a B-cell-maturation antigen (BCMA)-directed chimeric antigen receptor T cell therapy. We performed a post hoc analysis of a single-arm phase 1 multicenter study in relapsed/refractory multiple myeloma (CRB-401) (n = 62; median follow-up, 18.1 months). The primary endpoint was safety outcomes, and secondary endpoints included overall response rate (ORR), complete response (CR) and very good partial response (VGPR). The study met its primary endpoint with low rates of grade 3/grade 4 cytokine release syndrome (6.5%) and neurotoxicity (1.6%). ORR was 75.8%; 64.5% achieved VGPR or better and 38.7% achieved CR or stringent CR. Among exploratory endpoints, median duration of response, progression-free survival (PFS) and overall survival were 10.3, 8.8 and 34.2 months, respectively, and ide-cel expansion in blood and bone marrow correlated with clinical efficacy and postinfusion reduction of soluble BCMA. Patients with PFS ≥ 18 months had more naive and less exhausted T cells in apheresis material and improved functional T cell phenotype in the drug product compared with those with less durable responses. These results confirm ide-cel safety, tolerability and efficacy and describe T cell qualities that correlate with durable response. Clinicaltrials.gov identifier : NCT02658929.

A Prehispanic infant from Tenerife with diffuse microporotic lesions.

We describe diffuse microporotic lesions observed in most of the scattered skeletal remains belonging to a ≈ 6 months-old female (genetic sexing) prehispanic (antiquity ≈ 600 years BP) individual recovered from a small recess of a basaltic burial cave in the highlands (2300 m above sea level) of Tenerife. Although sphenoid wings were lacking, microporotic lesions were present in several bones, especially in the hard palate, basilar part of the occipital bone, outer aspect of the maxilla, and proximal half of the right humerus, accompanied by a subtle periosteal reaction. Although non-specific, bone lesions may be compatible with scurvy, possibly in the context of malnutrition, that probably also affected the mother, given the young age of the infant and her dependence on maternal feeding. Pathophysiological connections among iron deficiency, vitamin C deficiency and vitamin D deficiency are discussed. Both observational reports on paleopathological cases of diffuse microporotic lesions as well as experimental studies devoted to discern the relative and combined effects of hypoxia-mediated bone marrow expansion, protein-calorie malnutrition, ascorbate, vitamin D or iron deficiency on such lesions are needed.

Lkb1 loss in regulatory T cells leads to dysregulation of hematopoietic stem cell expansion and differentiation in bone marrow.

The tumor suppressor Lkb1 is known to regulate the expression of forkhead box P3 (Foxp3), thereby maintaining the levels of Foxp3+ regulatory T cells (Treg) that play a crucial role in self-tolerance. However, the effect of Lkb1 in Treg on hematopoietic stem cells (HSCs) in the bone marrow (BM) remains obscure. Here, we demonstrated that conditional deletion of Lkb1 in Treg causes loss of Treg in the BM, which leads to failure of HSC homeostasis and the abnormal expansion. Moreover, the loss of BM Treg results in dysregulation of other developing progenitors/stem cell populations, leading to the defective differentiation of T cells and B cells. In addition, HSC from the BM with Treg loss exhibited poor engraftment efficiency, indicating that loss of Treg leads to irreversible impairment of HSC. Collectively, these results demonstrated the essential role of Lkb1 in Treg for maintaining HSC homeostasis and differentiation in mice. These findings provide insight into the mechanisms of HSC regulation and guidance for a strategy to improve the outcomes and reduce complications of HSC transplantation.

CDX-301 prevents radiation-induced dysregulation of miRNA expression and biogenesis

Risks of radiation exposure necessitate the development ofradioprophylactic drugs. We have reported the efficacy of CDX-301, a recombinantly developed human protein form of Fms-related tyrosine kinase 3 ligand (Flt3L), as a radioprophylactic and radiomitigatory agent. Here, we performed global microRNA profiling to further understand the mechanism of action of CDX-301. We find that CDX-301 administration 24h prior to total body irradiation prevents radiation-induced dysregulation of microRNA biogenesis and expression in murine serum and spleen samples in a time- and tissue-dependent manner. Further analysis shows that activation of the HOTAIR regulatory pathway has a prominent function in radiation-induced injury responses, which is inhibited by pre-treatment with CDX-301. Moreover, CDX-301 attenuates radiation-induced dysregulation of several cellular functions such as inflammatory and immune responses. In corroboration, we also find that pre-treatment with CDX-301 restores the expression of bone marrow aplasia markers and inflammatory cytokines and growth factors, as well as the expression of genes associated with MAP kinase and TGF-β pathways that are altered by radiation. Our findings provide new insights into CDX-301-mediated molecular and cellular mechanisms and point to a possible novel radioprotective drug for the prevention of irradiation-induced injury and hematopoietic acute radiation syndrome.

Extramedullary Hematopoiesis Visualized on FDG-PET/CT in a Patient with Beta-Thalassemia.

Beta-thalassemia is an inherited blood disorder caused by reduced or absent synthesis of the beta chains of hemoglobin, resulting in decreased hemoglobin production. Symptoms depend on the type of beta-thalassemia ranging from no symptoms to severe illness. Ineffective erythropoiesis leads to a sequence of events responsible for bone marrow expansion, anemia, hemolysis, splenomegaly, increased iron absorption, and sometimes extramedullary hematopoiesis (EMH). We report an interesting case with EMH visualized on FDG-PET/CT and where FDG-PET/CT has also found the focus of a severe infection in a patient with beta-thalassemia.

Immune Phenotypes and Target Antigens of Clonally Expanded Bone Marrow T Cells in Treatment-Naïve Multiple Myeloma.

Multiple myeloma is a hematologic malignancy of monoclonal plasma cells that accumulate in the bone marrow. Despite their clinical and pathophysiologic relevance, the roles of bone marrow-infiltrating T cells in treatment-naïve patients are incompletely understood. We investigated whether clonally expanded T cells (i) were detectable in multiple myeloma bone marrow, (ii) showed characteristic immune phenotypes, and (iii) whether dominant clones recognized antigens selectively presented on multiple myeloma cells. Single-cell index sorting and T-cell receptor (TCR) αβ sequencing of bone marrow T cells from 13 treatment-naïve patients showed dominant clonal expansion within CD8+ cytolytic effector compartments, and only a minority of expanded T-cell clones expressed the classic immune-checkpoint molecules PD-1, CTLA-4, or TIM-3. To identify their molecular targets, TCRs of 68 dominant bone marrow clones from five selected patients were reexpressed and incubated with multiple myeloma and non-multiple myeloma cells from corresponding patients. Only 1 of 68 TCRs recognized antigen presented on multiple myeloma cells. This TCR was HLA-C-restricted, self-peptide-specific and could be activated by multiple myeloma cells of multiple patients. The remaining dominant T-cell clones did not recognize multiple myeloma cells and were, in part, specific for antigens associated with chronic viral infections. In conclusion, we showed that dominant bone marrow T-cell clones in treatment-naïve patients rarely recognize antigens presented on multiple myeloma cells and exhibit low expression of classic immune-checkpoint molecules. Our data provide experimental context for experiences from clinical immune-checkpoint inhibition trials and will inform future T cell-dependent therapeutic strategies.

Marrow changes and reduced proliferative capacity of mesenchymal stromal cells from patients with “no-option” critical limb ischemia; observations on feasibility of the autologous approach from a clinical trial

Background aimsApproximately 1 in 3 patients with critical limb ischemia (CLI) are not suitable for surgical or endovascular revascularization. Those “no-option” patients are at high risk of amputation and death. Autologous bone marrow mesenchymal stromal cells (MSCs) may provide a limb salvage option. In this study, bone marrow characteristics and expansion potentials of CLI-derived MSCs produced during a phase 1b clinical trial were compared with young healthy donor MSCs to determine the feasibility of an autologous approach. Cells were produced under Good Manufacturing Practice conditions and underwent appropriate release testing. MethodsFive bone marrow aspirates derived from patients with CLI were compared with six young healthy donor marrows in terms of number of colony-forming units–fibroblast (CFUF) and mononuclear cells. The mean population doubling times and final cell yields were used to evaluate expansion potential. The effect of increasing the volume of marrow on the CFUF count and final cell yield was evaluated by comparing 5 CLI-derived MSCs batches produced from a targeted 30 mL of marrow aspirate to five batches produced from a targeted 100 mL of marrow. ResultsCLI-derived marrow aspirate showed significantly lower numbers of mononuclear cells with no difference in the number of CFUFs when compared with healthy donors’ marrow aspirate. CLI-derived MSCs showed a significantly longer population doubling time and reduced final cell yield compared with young healthy donors' MSCs. The poor growth kinetics of CLI MSCs were not mitigated by increasing the bone marrow aspirate from 30 to 100 mL. ConclusionsIn addition to the previously reported karyotype abnormalities in MSCs isolated from patients with CLI, but not in cells from healthy donors, the feasibility of autologous transplantation of bone marrow MSCs for patients with no-option CLI is further limited by the increased expansion time and the reduced cell yield.

New Entity-Thalassemic Endocrine Disease: Major Beta-Thalassemia and Endocrine Involvement.

New Entity-Thalassemic Endocrine Disease: Major Beta-Thalassemia and Endocrine Involvement.