Bone Marrow Precursors Research Articles

Tumor-Released Products Promote Bone Marrow-Derived Macrophage Survival and Proliferation

Macrophages play a central role within the tumor microenvironment, with relevant implications for tumor progression. The modulation of their phenotype is one of the mechanisms used by tumors to escape from effective immune responses. This study was designed to analyze the influence of soluble products released by tumors, here represented by the tumor-conditioned media of two tumor cell lines (3LL from Lewis lung carcinoma and MN/MCA from fibrosarcoma), on murine macrophage differentiation and polarization in vitro. Data revealed that tumor-conditioned media stimulated macrophage differentiation but influenced the expression levels of macrophage polarization markers, cytokine production, and microRNAs of relevance for macrophage biology. Interestingly, tumor-derived soluble products supported the survival and proliferation rate of bone marrow precursor cells, an effect observed even with mature macrophages in the presence of M2 but not M1 inducers. Despite presenting low concentrations of macrophage colony-stimulating factor (M-CSF), tumor-conditioned media alone also supported the proliferation of cells to a similar extent as exogenous M-CSF. This effect was only evident in cells positive for the expression of the M-CSF receptor (CD115) and occurred preferentially within the CD16+ subset. Blocking CD115 partially reversed the effect on proliferation. These results suggest that tumors release soluble products that not only promote macrophage development from bone marrow precursors but also stimulate the proliferation of cells with specific phenotypes that could support protumoral functions.

BCL-2 Inhibitor ABT-737 Effectively Targets Leukemia-Initiating Cells with Differential Regulation of Relevant Genes Leading to Extended Survival in a NRAS/BCL-2 Mouse Model of High Risk-Myelodysplastic Syndrome.

AbastractDuring transformation, myelodysplastic syndromes (MDS) are characterized by reducing apoptosis of bone marrow (BM) precursors. Mouse models of high risk (HR)-MDS and acute myelogenous leukemia (AML) post-MDS using mutant NRAS and overexpression of human BCL-2, known to be poor prognostic indicators of the human diseases, were created. We have reported the efficacy of the BCL-2 inhibitor, ABT-737, on the AML post-MDS model; here, we report that this BCL-2 inhibitor also significantly extended survival of the HR-MDS mouse model, with reductions of BM blasts and lineage negative/Sca1+/KIT+ (LSK) cells. Secondary transplants showed increased survival in treated compared to untreated mice. Unlike the AML model, BCL-2 expression and RAS activity decreased following treatment and the RAS:BCL-2 complex remained in the plasma membrane. Exon-specific gene expression profiling (GEP) of HR-MDS mice showed 1952 differentially regulated genes upon treatment, including genes important for the regulation of stem cells, differentiation, proliferation, oxidative phosphorylation, mitochondrial function, and apoptosis; relevant in human disease. Spliceosome genes, found to be abnormal in MDS patients and downregulated in our HR-MDS model, such as Rsrc1 and Wbp4, were upregulated by the treatment, as were genes involved in epigenetic regulation, such as DNMT3A and B, upregulated upon disease progression and downregulated upon treatment.

Murine Dendritic Cells Grown in Serum-Free Culture Show Potent Therapeutic Activity when Loaded with Novel Th Epitopes in an Orthotopic Model of HER2pos Breast Cancer.

Preferred methods for generating mouse dendritic cells (DC) would encompass qualities of consistency, high yield, and potent function. Serum-free culture is also highly desirable, since this is the standard for cell-based therapies used in humans. We report here a serum-free modification of a culture method generating mature, activated DCs from bone marrow precursors. This is achieved through a two-stage culture comprised of 6-day expansion in Flt3 ligand and IL-6 followed by brief differentiation in a medium containing GM-CSF and IL-4, with subsequent activation using TLR ligands ODN1826 and LPS. The serum-free DCs achieve yields and surface phenotype including IL-12p70 secretion similar to standard serum-replete cultures, display a capacity to sensitize in vivo against both MHC class I- and Class II-restricted antigens, and exhibit some aspects of “killer DC” function against tumor cells. We used these DCs to help identify novel CD4pos Th epitopes on the rat ErbB2/HER-2 protein and demonstrated a subset of these as effective immunogens in a DC-based therapeutic model of HER-2pos breast cancer in Balb/c mice, where they induced powerful Th1-polarized immune responses. This method represents a useful way to efficiently produce large numbers of murine dendritic cells with excellent in vivo function well-suited for use in experimental vaccine studies.

CD14, A toll-like receptor co-factor, influences osteoclast differentiation and activity, but does not alter osteoblastic potential of bone-marrow preursors in mice

Purpose: Changes in subchondral bone structure occur in osteoarthritis (OA), and are visible by imaging as subchondral sclerosis on radiographs, and bone marrow lesions (BMLs) by MRI. These changes are the result of active bone remodeling that occurs in OA. BMLs in particular are correlated with pain in OA patients. We previously demonstrated that bone remodeling was significantly reduced after joint injury in mice deficient in CD14. CD14 enhances Toll-like receptor (TLR) mediated inflammation by acting as a co-receptor for multiple TLRs, increasing TLR sensitivity to damage associated molecular patterns (DAMPS) to activate proinflammatory signaling pathways. Soluble CD14, TLRs, and DAMPs (TLR ligands) are all elevated in OA patients, and sCD14 has been implicated as a biomarker of symptom and structural severity in OA. Increasing evidence suggestions CD14/TLR signaling influences bone remodeling as well, through activation of osteoclast precursor cells. Here, we investigate the potential mechanism by which CD14 influences bone remodeling through in vitro analysis of osteoclastogenic and osteoblastic potential and function. Methods: Bone marrow (BM) hematopoietic and mesenchymal precursors were isolated from long bones of 12-week-old male wild-type C57BL/6 (WT) and CD14-deficient (CD14-/-) mice. Briefly, BM was flushed from the femurs and tibia, and cells were cultured for 24 hours to separate non-adherent from adherent population. For osteoclastic differentiation, the non-adherent cells were seeded at a density of 26,000 cells per cm2 and were cultured in presence of monocyte/macrophage colony-stimulating factor (M-CSF; 35 ng/ml) and receptor activator of nuclear factor kβ ligand (RANKL; 100 ng/ml) for 9 days. Osteoblastic differentiation was accomplished by seeding the adherent BM cells at 30,000 cells per cm2 and culturing in the presence of 10 mM β-glycerophosphate ,50 μM ascorbic acid, and 100 nM Dexamethasone for up to 19 days. Osteoclasts were identified as multinucleated cells with positive TRAP staining. The resorptive ability of osteoclasts was assayed by quantifying resorption pits formed on bone slices. For osteoblast differentiation, Alizarin red was used to detect mineralized bone matrix. At the completion of each assay RNA was isolated from cultures and Real-time quantitative PCR analysis was performed to detect changes in mRNA levels of osteoclast (i.e. calcitonin receptor/CTR and cathepsin K/Ctsk) and osteoblast (i.e. osteopontin/OPN and osteocalcin/BLAP) differentiation markers. Results: Cells from CD14-/- mice demonstrated decreased percentages of TRAP positive cells and multi-nucleated cells (p value = 0.01; Fig. 1 A-D) and reduced Ctsk mRNA (p value = 0.03; Fig. 1 E&F), compared to WT controls. CD14-/- cells showed a 50% reduction in resorption pits and area compared to WT (Fig. 1 G&I). No significant differences were observed between strains in osteoblastic potential measured by mineralization and gene expression of osteoblastic markers (Fig. 2). Conclusions: CD14-/- BM precursor cells have reduced capacity to differentiate into functional osteoclasts, while osteoblastic potential is retained. This suggests that the reduced capacity of CD14-deficient osteoclastic cells may lead to the reduced bone remodeling in response to joint injury seen in the DMM model, by disrupting the balance between resorption and formation. Future experiments will determine the influence of DAMPs on osteoclast differentiation and activity, and the density of osteoclasts in SCB after DMM injury. These experiments will lead to a better understanding of the role of CD14 and TLR pathways on bone remodeling in response to joint injury and OA.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Primary Laryngeal Granulocytic Sarcoma: A Case Report.

Granulocytic sarcoma (GS) is a malignancy of the bone marrow precursor cells, often associated with acute or chronic myeloid leukemia, forming isolated masses with extramedullary spread. It rarely shows laryngeal involvement. In this case report, a case of GS accompanying acute myeloid leukemia involving larynx with mass of neck and mediastinum as two separate masses was presented.

The Effect of the MK0339 Neutrophil Elastase Inhibitor on Proliferation and Maturation of iPSC Derived from Patients with ELANE, TCIRG1 and Whim Syndrome Associated Neutropenia

Genetic neutropenias are heterogeneous group of hematological diseases characterized by impaired survival and maturation of myeloid precursors in bone marrow. G-CSF and HSCT are currently the only effective treatment options. MK-0339 is a potent, cell permeable, orally absorbed inhibitor of neutrophil elastase (NE), previously investigated in preclinical and clinical studies by Merck/DuPont as a potential anti-inflammatory drug. We have previously reported that MK-0339 increases neutrophil proliferation in cellular models of ELANE associated neutropenia (2016 [ASH Annual Meeting Abstracts] Blood. 2016;128:406; 2017 J Leuk Biol [in press]). To examine the specificity of these responses, we investigated the effects of MK-0339 on iPSCs from healthy volunteers and compared its effects on differentiation of iPSC derived from patients with ELANE, TCIRG-1 and CXCR4 associated neutropenia.We generated induced pluripotent stem cells (iPSC) from dermal fibroblasts from healthy volunteers and neutropenic patients harboring ELANE, TCIRG1 and CXCR4 mutations and developed a feeder free, reproducible and efficient protocol for myeloid differentiation.In our initial experiments, we observed that iPSC lines derived from patients with ELANE mutations (P139L and G214R) consistently grew much slower and formed much smaller colonies than the controls using both mTeSR1/matrigel and DEF-CS feeder free culture medium systems. We observed more than 3-fold growth reduction of patient derived lines compared to healthy volunteers.We next tested the effects of MK-0339 under the same culture conditions. Growth of control and ELANE patient cell lines was enhanced, and the cells appeared more robust, after addition of 1uM MK-0339. We were surprised to see a similar proliferative effect (~40% increase) with Ramos (human Burkitt's lymphoma cell line) cells cultured in the presence of 1uM MK-0339, although neither the Ramos cells nor the ELANE iPSCs had detectable levels of ELANE expression. The enhanced growth of control and patient iPSCs and the results with Ramos cells suggested that the effects of MK-0339 are not solely mediated via inhibition of NE.We next compared myeloid differentiation and CD34+ cell proliferation of iPSC from patients with ELANE, TCIRG1 and CXCR4 associated neutropenia in HPC expansion medium, using granulocytic differentiation markers and flow cytometry. Myeloid maturation was impaired and inefficient for both ELANE (P139L and G214R) patient derived cell lines. Myeloid differentiation of the TCIRG1 patient derived was also impaired. Introduction of MK-0339 restored the impaired myeloid differentiation capacity of both ELANE and TCIRG1 positive patient derived cell lines. An iPSC line derived from a WHIM (CXCR4 mutation) patient appeared to have normal maturation and proliferation. Western blot analysis revealed elevated levels of endoplasmic reticulum chaperone GRP78/BiP in ELANE and TCIRG1 mutant cell lines which was corrected by MK-0339 treatment.These studies demonstrate heretofore unreported proliferative effects of MK-0339 on cell lines not expressing neutrophil elastase. They suggest important mechanisms beyond enzyme inhibition to account for these findings. Although preliminary, we believe that this work suggests that MK-0339 may be a novel, oral therapy for both ELANE and non-ELANE associated neutropenia. DisclosuresDale:University of Washington: Employment, Research Funding; National Institutes of Health: Research Funding; WedMD/Medscape: Membership on an entity's Board of Directors or advisory committees; Wolter Kluwer: Other: Editor, Current Opinions in Hematology; Philips: Research Funding; X4Pharma: Research Funding; Shire: Other: Independent Review Board; Omeros: Other: DSMB; Coherus: Consultancy; Boheringer-Ingelheim: Consultancy; Prolong: Consultancy; Hospira: Consultancy; Genkyotex: Other: DSMB (work completed 6/2015); Genzyme (now owned by Sanofi-Aventis): Consultancy, Patents & Royalties, Research Funding; Cellerant: Membership on an entity's Board of Directors or advisory committees; Sanofi Aventis: Consultancy, Editor, Current Opinions in Hematology, Honoraria; Amgen: Consultancy, Research Funding; American College of Physicians: Other: Editor and author; GlaxoSmithKline: Equity Ownership; Johnson&Johnson: Equity Ownership.

Genome-Wide Analysis of the Regulatory Landscape in Different Pre-B-ALL Subtypes

BackgroundAcute pre-B leukemia results from a failure in cell differentiation. In normal development, cells commit to their lineage and differentiated phenotype state by the activation of transcription factors (TFs), or TF modules, in a series of decision points at which a choice is made between alternative lineage fates. Previously, we showed that the ETV6-RUNX1 gene translocation that occurs in 25% of pediatric leukemia patients silences regulatory regions containing RUNX binding sites (enhancers) at target gene loci implicated in lymphoid lineage differentiation (Teppo et al. 2016). However, the genome-wide changes in enhancer activities at binding sites of other TFs functioning in B-cell differentiation remain poorly understood. Moreover, the genome-wide identification of enhancers altered during leukemogenesis is incomplete.Material & MethodsTo measure transcripts generated both at enhancer and gene regions, global run-on sequencing (GRO-seq) technique was used to measure nascent RNAs (Heinäniemi et al. 2016) from human pre-B-ALL patient samples (N=35) and several cell lines representing different subtypes. For validation, additional samples were generated from cell line models with overexpression or silencing of leukemic TFs. To this collection of 151 nascent transcriptomes, additional 92 GRO-seq samples representing different cell lineages (Liivrand et al 2017) were used to compare the leukemic cells to primary cells and other cancers. Enhancer RNA (eRNA) levels were compared at TF binding motif centered enhancer regions. TF ChIP-seq data from leukemic cells and HSC were acquired from the NCBI Gene Expression Omnibus for validation of this approach.ResultsTo gain insight into the derailed regulatory landscape in leukemic cells, we identified eRNA regions both from normal and leukemic samples and performed statistical analysis distinguishing regulatory regions with differential activity between pre-B-ALL-subtypes or compared to normal CD34+ bone marrow precursors. Next, association of the significantly altered enhancers with gene transcripts was utilized to build regulatory maps for differentially expressed gene loci. For this purpose, we developed a transcript identification and quantification pipeline suitable for detecting altered expression at different coding and non-coding RNA loci from nascent transcriptomes (Liivrand et al 2017; unpublished). To globally evaluate altered TF activities across pre-B-ALL subtypes underlying the differences observed, we present here an unbiased approach based on eRNA quantification at TF motif-centered enhancers. The analysis of eRNA levels at binding sites of TFs with established role in leukemia was used as a benchmark: we could confirm low eRNA transcription at RUNX1 binding sites in the ETV6-RUNX1 subtype, elevated eRNA levels at HOXA9 and MEIS1 binding sites in the MLL rearranged subtype, while TCF3, BCL6 and PBX1 bound sites were most active in TCF3-PBX1 samples.ConclusionsOur results provide the first genome-wide transcriptional regulatory landscape of different pre-B-ALL subtypes. We identified novel subtype-specific active enhancers and annotated regulatory non-coding regions that can be utilized to evaluate the functional impact of genetic alterations outside gene regions. DisclosuresNo relevant conflicts of interest to declare.

Killer Immunoglobulin-like Receptors (KIR) in Low-Risk Myelodysplastic Syndrome: Genotyping and Gene Expression Evaluation

Introduction: MDS are a group of myeloid malignancies characterized by ineffective hematopoiesis, peripheral cytopenias, and 30% chance of transformation into AML. MDS pathogenesis involves, in some cases, a phenomenon of autoimmunity which can suppress normal hematopoiesis by direct cytotoxicity as well as by release of cytokines. Dysplasia-associated antigens released from dying cells might be responsible for evoking an adaptive immune response. NK cells appear activated, with high expression of perforin and granzyme, and mediate the cytotoxicity of bone marrow (BM) precursor cells in lower risk disease. Their activity is modulated by killer immunoglobulin-like receptors (KIR). Prior experience suggests that Tipifarnib, a farnesyl transferase inhibitor, may suppress abnormal NK cell function and autoimmunity that contributes to MDS. It is hypothesized that Tipifarnib could be especially of benefit in lower risk MDS patients where autoimmunity is known to play a role and immunosuppression is of clinical benefit in some cases.KIR genes are polymorphic and two broad haplotypes exist: KIR-haplotype A mainly encode for inhibitory receptors and only for one activating (KIR2DS4), whereas group B haplotype encodes more for activating KIRs (KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS5 und KIR3DS1). We hypothesized that KIR molecular profile could be correlated with response to Tipifarnib and molecular characteristics of patients with MDS. With this goal in mind, the key objective of the present study was to develop a methodology and a reportable range of KIR2DS2, KIR2DS5, KIR2DL2 and KIR2DL5 RNA expression and their ratios by RT-PCR in samples from low/INT-1 risk MDS patients.Methods: We evaluated the KIR genotyping and gene expression from DNA and RNA samples, respectively, in BM mononuclear cells of 50 lower risk MDS patients followed at CUMC from 2010 to 2015. KIR typing was performed by conventional PCR using commercial Miltenyi KIR genotyping kit for all known 15 human KIR genes. Gene expression was accessed by qRT-PCR for two activating (2DS2, and 2DS5), two inhibitory (2DL2, and 2DL5) KIR genes, and five leukocyte markers: CD56, CD4, GZMM, CD8A, and CD8B . Correlation between clinical and molecular data of the patients was sought.Results: Performance specifications (precision, limit of detection, linearity, and reportable range) were determined for all assays used in this study. All 50 MDS patients were positive for KIR2DL4, 3DL2, 3DL3, 2DP1 and 3DP1, whereas 2DS3 [7/55; 13%], 2DS5 [16/55; 29%], and 2DL5 [19/55; 35%] were less frequent (Figure 1). These results corroborate the existing literature showing similar distribution in the normal population. Patients with normal karyotype had higher frequency of 2DL5 [51% vs 33.3%], 2DS1 [46% vs 33.3%], 2DS3 [17% vs 6%], 2DS5 [37% vs 20%], and 3DS1 genes [43% vs 27%] than patients with abnormal cytogenetics, however none of these results was statistically significant. KIR2DS2 and 2DL2, and 2DS5 and 2DL5, co-occurred in the same patients, as already described in normal American population. All patients that presented KIR2DS2 [22/55; 44%], 2DL2 [22/55; 44%], 2DS5 [23/55; 46%], or 2DL5 [23/55; 46%] had their samples analyzed by qRT-PCR to the respective targets besides some leukocytes markers. Correlation between KIR copy number and MDS subtype, BM cellularity, WBC, RBC, Hg, and platelets was not observed.Survival analysis showed poor survival for the patients carrying KIR2DL5, 2DS1, and 3DS1 genes [p<0.05] (Fig. B,C, & D). 42% (21/50) of the patients were positive for all three genes, showing the same ratio already identified in the normal population (36%). Additionally, survival was different between the four haplotypes groups CenA/TelA, CenA/TelB, CenB/TelA, and CenB/TelB (P=0.0628; Fig. E) but a subset analysis of CenA-TelA and CenB-TelB haplotypes showed a significantly different survival between two groups (P= 0.0145; Fig 1E). There was no correlation between gene expression data and survival.Conclusions: We report on the methodology and its use to define a reportable range of KIR2DS2, KIR2DS5, KIR2DL2 and KIR2DL5 RNA expression and their ratios by RT-PCR in 50 lower risk MDS patients. Our findings suggest that KIR genes might be associated with some clinical features in patients, mainly, survival and cytogenetics profile. KIR expression will be correlated with clinical response to Tipifarnib as part of an ongoing clinical trial. [Display omitted] DisclosuresCoutinho:Kura Oncology: Consultancy. Ali:Onconova Therapeutics: Consultancy; Kura Oncology: Consultancy. Gualberto:Kura Oncology: Employment, Equity Ownership, Other: Chief Medical Officer, Patents & Royalties. Scholz:Kura Oncology: Employment, Equity Ownership, Patents & Royalties. Bracken:Kura Oncology: Employment. Jurcic:Forma Therapeutics: Research Funding; Celgene: Research Funding; Alexion Pharmaceuticals: Consultancy; Genentech: Research Funding; Incyte: Consultancy; Actinium Pharmaceuticals, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Astellas Pharma, Inc: Research Funding; Daiichi-Sankyo: Research Funding; Kura Oncology: Research Funding; Merck: Consultancy; Novartis: Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Consultancy, Research Funding; Syros Pharmaceuticals: Research Funding; Amgen: Consultancy. Raza:Novartis: Speakers Bureau; Genoptix: Speakers Bureau; Celgene Inc.: Research Funding; Kura Oncology: Research Funding; Janssen R&D: Research Funding; Syros Pharmaceuticals: Research Funding; Onconova Therapeutics: Research Funding, Speakers Bureau.

Screening of Mutations Related to Hereditary Anemias Using NGS-Targeted Sequencing Panel

Hereditary forms of anemia arise due to defects in erythrocyte differentiation from bone marrow precursor cells, or shortened survival of erythrocytes. Both cases result in the deficiency of mature cells in the bloodstream. Defects in the erythrocyte membrane proteins (e.g. hereditary spherocytosis and elliptocytosis/ pyropoikilocytosis) are common, and they manifest in association with morphological changes in the red blood cells. Other causes of hemolytic anemias include hemoglobinopathies (e.g. sickle cell diseases, thalassemias, etc) and enzymopathies (e.g. Glucose 6 phosphate dehydrogenase deficiency and pyruvate kinase deficiency), caused by defects in metabolic enzymes. Hereditary anemia can also be caused by erythroid lineage disorders such as congenital dyserythropoietic anemias (CDA), characterized by distinct morphologic abnormalities of marrow erythroblasts. Diseases that result in hereditary anemias are difficult to diagnose, due to the large clinical and genetic heterogeneity. The use of conventional techniques for the systematic screening of all potentially implicated genes would be impractical, costly and time-consuming. To address this issue, we have customized a rapid comprehensive next-generation sequencing-based assay that evaluates 58 genes with published disease-causing mutations for future clinical application. Of these genes, 27 were related with RBC cytoskeletal disorders and enzymopathies, 8 were involved with CDAs and 23 genes were related with hemochromatosis, sideroblastic anemia and porphyria. Our panel covers the complete coding region, splice site junctions, and some promoter regions. Exon capture probes were designed with the aid of the Sure Design program (Agilent Technologies, Inc., CA, USA). Targeted gene capture and library construction for next-generation sequencing (NGS) were performed using Haloplex kit (Agilent Technologies, Santa Clara, USA). Enriched samples were then sequenced on an Illumina HiSeq 2500 sequencer with 150 base pair, paired-end reads. Overall, the quality scores obtained were high, with an average of 91% of the bases analyzed with >Q30. This means that the probability that a given base is wrong is less than 0.1%. Sequencing reads were aligned to the human genome reference sequence and analysis of coverage and variants was completed using Sure Call software (Agilent Technologies, Inc., CA, USA). The validation included the benchmark sample NA12878 (Coriell Institute, NJ, USA) in triplicate. All variants were detected using Haloplex amplification followed by NGS were compared to the results available in the coding region sequencing databases of the NA12878 sample. The panel showed a sensitivity of 98.7% and a specificity of 99.9%. We also tested our panel in 43 patients with hereditary anemia searching for causative variants. Possible pathogenic variants were identified in 37 patients (86%) and explained the etiology of the diseases in 69.8% of the cases. Variants in the β-spectrin (SPTB gene) were the most frequent cause of hereditary spherocytosis (HS) in our sample, identified in 13 out of the 26 patients with previous diagnosis of HS (50%). Variants in the ankyrin (ANK1) were the second most frequent cause of HS, identified in 6 cases. Variants in SPTA1, SLC4A1, PKLR, CDAN1, G6PD, HFE and KIF23 genes were also identified. Most of the identified variants were novel. To conclude, next-generation sequencing provides a cost-effective and relatively rapid approach to molecular characterization of a disease. The identification of pathogenic variants is essential for genetic counselling, pre-implantation genetic diagnosis and even for future gene therapy. The panel we developed proved to be robust, with very low false positive and negative results and successfully determined the molecular causes of hereditary anemia in most cases. DisclosuresNo relevant conflicts of interest to declare.

A Rare Case of Pearson's Syndrome with Predominant Hematological Manifestations and Novel Mitochondrial Deletion

Summary- Pearson's syndrome is a very rare cause of transfusion dependent anemia in infants with presence of pancreatic exocrine deficiency and other systemic abnormalities. We present a case of Pearson syndrome in an infant with primary hematological manifestations and a novel mitochondrial deletion.Introduction- Pearson syndrome is a very rare cause of refractory transfusion dependent childhood anemia caused by mitochondrial DNA defects resulting in refractory sideroblastic anaemia, vacuolization of bone marrow precursors, exocrine pancreatic dysfunction and other metabolic defectsMaterial and Methods- A 6 month old infant presented with complaints of progressive pallor since one month, failure to thrive and fast breathing for 2 days. On examination he had severe anaemia with signs of congestive cardiac failure and hepatomegaly.Results- Peripheral smear findings revealed macrocytic anaemia with mild thrombocytopenia. Bone marrow aspirate revealed a cellular marrow with mild erythroid hyperplasia and characteristic cytoplasmic vacuolations in myeloid and occasional erythroid precursors. There were about 30% ring sideroblasts observed on perl's stain. In view of suspicion of Pearson's syndrome, baby was further investigated which revealed high serum lactate levels (35mg/dl). However, fecal fat excretion was normal. Mitochondrial DNA analysis revealed a point mutation in NADH dehydrogenase 3 (ND3 region) of mt DNA as opposed to large mtDNA deletions observed in previously reported cases of Pearson's syndrome.Discussion- Sideroblastic anemia presenting in infancy is an uncommon cause of anemia often requiring multiple transfusions. Pearson's syndrome is a rare cause of sideroblastic anaemia, it presents in infancy, carrying a grave prognosis and requires high index of suspicion. Clinical picture is highly variable due to Mitochondrial DNA heteroplasmia. Mitochondrial DNA deletion/duplication studies carry a confirmative value. Pyridoxine was not very effective and child required repeated transfusions. This case is presented for its rarity, hematological features and associated novel mitochondrial mutation. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Exercise of high intensity ameliorates hepatic inflammation and the progression of NASH

ObjectiveNon-alcoholic fatty liver disease (NAFLD) covers a wide spectrum of liver pathology ranging from simple fatty liver to non-alcoholic steatohepatitis (NASH). Notably, immune cell-driven inflammation is a key mechanism in the transition from fatty liver to the more serious NASH. Although exercise training is effective in ameliorating obesity-related diseases, the underlying mechanisms of the beneficial effects of exercise remain unclear. It is unknown whether there is an optimal modality and intensity of exercise to treat NAFLD. The objective of this study was to determine whether high-intensity interval training (HIIT) or moderate-intensity continuous training (MIT) is more effective at ameliorating the progression of NASH. MethodsWild-type mice were fed a high-fat, high-carbohydrate (HFHC) diet for 6 weeks and left sedentary (SED) or assigned to either an MIT or HIIT regimen using treadmill running for an additional 16 weeks. MIT and HIIT groups were pair-fed to ensure that energy intake was similar between the exercise cohorts. To determine changes in whole-body metabolism, we performed insulin and glucose tolerance tests, indirect calorimetry, and magnetic resonance imaging. NASH progression was determined by triglyceride accumulation, expression of inflammatory genes, and histological assessment of fibrosis. Immune cell populations in the liver were characterized by cytometry by time-of-flight mass spectrometry, and progenitor populations within the bone marrow were assessed by flow cytometry. Finally, we analyzed the transcriptional profile of the liver by bulk RNA sequencing. ResultsCompared with SED mice, both HIIT and MIT suppressed weight gain, improved whole-body metabolic parameters, and ameliorated the progression of NASH by reducing hepatic triglyceride levels, inflammation, and fibrosis. However, HIIT was superior to MIT at reducing adiposity, improving whole-body glucose tolerance, and ameliorating liver steatosis, inflammation, and fibrosis, without any changes in body weight. Improved NASH progression in HIIT mice was accompanied by a substantial decrease in the frequency of pro-inflammatory infiltrating, monocyte-derived macrophages in the liver and reduced myeloid progenitor populations in the bone marrow. Notably, an acute bout of MIT or HIIT exercise had no effect on the intrahepatic and splenic immune cell populations. In addition, bulk mRNA sequencing of the entire liver tissue showed a pattern of gene expression confirming that HIIT was more effective than MIT in improving liver inflammation and lipid biosynthesis. ConclusionsOur data suggest that exercise lessens hepatic inflammation during NASH by reducing the accumulation of hepatic monocyte-derived inflammatory macrophages and bone marrow precursor cells. Our findings also indicate that HIIT is superior to MIT in ameliorating the disease in a dietary mouse model of NASH.

B cell/stromal cell crosstalk in health, disease, and treatment: Follicular lymphoma as a paradigm.

Stromal cells organize specific anatomic compartments within bone marrow (BM) and secondary lymphoid organs where they finely regulate the behavior of mature normal B cells. In particular, lymphoid stromal cells (LSCs) form a phenotypically heterogeneous compartment including various cell subsets variably supporting B-cell survival, activation, proliferation, and differentiation. In turn, activated B cells trigger in-depth remodeling of LSC networks within lymph nodes (LN) and BM. Follicular lymphoma (FL) is one of the best paradigms of a B-cell neoplasia depending on a specific tumor microenvironment (TME), including cancer-associated fibroblasts (CAFs) emerging from the reprogramming of LN LSCs or poorly characterized local BM precursors. FL-CAFs support directly malignant B-cell growth and orchestrate FL permissive cell niche by contributing, through a bidirectional crosstalk, to the recruitment and polarization of immune TME subsets. Recent studies have highlighted a previously unexpected level of heterogeneity of both FL B cells and FL TME, underlined by FL-CAF plasticity. A better understanding of the signaling pathways, molecular mechanisms, and kinetic of stromal cell remodeling in FL would be useful to delineate new predictive markers and new therapeutic approaches in this still fatal malignancy.

The twilight zone: plasticity and mixed ontogeny of neutrophil and eosinophil granulocyte subsets.

It is now becoming clear that neutrophils and eosinophils are heterogeneous cells with potentially multiple subsets in health and disease. With greater marker coverage by multi-color flow cytometry and single-cell level sequencing of granulocyte populations, novel phenotypes of these cells began to emerge. Intriguingly, many newly described subsets blend distinctions between classical myeloid lineage phenotypes, which are especially true for tissue resident or recruited cells in contexts of inflammation and disease. This includes reports of neutrophils with features of eosinophils, monocytes and dendritic cells, and eosinophil subsets expressing neutrophil markers. Moreover, novel studies show the ability of immature neutrophils to transdifferentiate into mature cells belonging to other myeloid lineages (eosinophils, monocytes/macrophages). In this review, we summarize novel findings in this exciting research frontier and shed light on potential processes driving the plasticity and heterogeneity of granulocyte subsets. Specifically, we discuss the hematopoietic flexibility of granulocyte precursors in bone marrow and the adaptation of myeloid cells to local tissue microenvironments. The understanding of such intermediate and developmental phenotypes is very important, as it can teach us about origins of functionally distinct myeloid cells during inflammation, and explain reasons for successes and failures of biologics targeting terminally differentiated granulocytes.

Current advances in using tolerogenic dendritic cells as a therapeutic alternative in the treatment of type 1 diabetes.

Type 1 diabetes (T1D) is an autoimmune disease characterized by the destruction of insulin-producing β-cells of the pancreatic islets by autoreactive T cells, leading to high blood glucose levels and severe long-term complications. The typical treatment indicated in T1D is exogenous insulin administration, which controls glucose levels; however, it does not stop the autoimmune process. Various strategies have been implemented aimed at stopping β-cell destruction, such as cellular therapy. Dendritic cells (DCs) as an alternative in cellular therapy have gained great interest for autoimmune disease therapy due to their plasticity to acquire immunoregulatory properties both in vivo and in vitro, performing functions such as anti-inflammatory cytokine secretion and suppression of autoreactive lymphocytes, which are dependent of their tolerogenic phenotype, displayed by features such as semimature phenotype, low surface expression of stimulatory molecules to prime T cells, as well as the elevated expression of inhibitory markers. DCs may be obtained and propagated easily in optimal amounts from peripheral blood or bone marrow precursors, such as monocytes or hematopoietic stem cells, respectively; therefore, various protocols have been established for tolerogenic (tol)DCs manufacturing for therapeutic research in the treatment of T1D. In this review, we address the current advances in the use of tolDCs for T1D therapy, encompassing protocols for their manufacturing, the data obtained from preclinical studies carried out, and the status of clinical research evaluating the safety, feasibility, and effectiveness of tolDCs.

Differences in Steady-State Erythropoiesis in Different Mouse Bones and Postnatal Spleen.

Adult erythropoiesis is a highly controlled sequential differentiation of hematopoietic stem cells (HSCs) to mature red blood cells in the bone marrow (BM). The bones which contain BM are diverse in their structure, embryonic origin, and mode of ossification. This has created substantial heterogeneity in HSCs function in BM of different bones, however, it is not known if this heterogeneity influences erythropoiesis in different bones and different regions of the same bone. In this study, we examined steady state BM erythroid progenitors and precursors from different bones – the femur, tibia, pelvis, sternum, vertebrae, radius, humerus, frontal, parietal bone, and compared all to the femur. Trabecular and cortical regions of the femur were also compared for differences in erythropoiesis. In addition, mouse spleen was studied to determine at which age erythropoietic support by the spleen was lost postnatally. We report that total erythroid cells, and erythroid precursors in the femur are comparable to tibia, pelvis, humerus and sternum, but are significantly reduced in the vertebrae, radius, frontal, and parietal bones. Erythroid progenitors and multipotential progenitor numbers are comparable in all the bones except for reduced number in the parietal bone. In the femur, the epiphysis and metaphysis have significantly reduced number of erythroid precursors and progenitors, multipotential progenitors and myeloid progenitors compared to the diaphysis region. These results show that analysis of erythroid precursors from diaphysis region of the femur is representative of tibia, pelvis, humerus and sternum and have significant implications on the interpretation of the steady-state erythropoiesis finding from adult BM. Postnatal spleen supports erythroid precursors until 6 weeks of age which coincides with reduced number of red pulp macrophages. The residual erythroid progenitor support reaches the adult level by 3 months of age. In conclusion, our findings provide insights to the differences in erythropoiesis between different bones, between trabecular and cortical regions of the femur, and developmental changes in postnatal spleen erythropoiesis.

Heme catabolism by tumor-associated macrophages controls metastasis formation.

Although the pathological significance of tumor-associated macrophage (TAM) heterogeneity is still poorly understood, TAM reprogramming is viewed as a promising anticancer therapy. Here we show that a distinct subset of TAMs (F4/80hiCD115hiC3aRhiCD88hi), endowed with high rates of heme catabolism by the stress-responsive enzyme heme oxygenase-1 (HO-1), plays a critical role in shaping a prometastatic tumor microenvironment favoring immunosuppression, angiogenesis and epithelial-to-mesenchymal transition. This population originates from F4/80+HO-1+ bone marrow (BM) precursors, accumulates in the blood of tumor bearers and preferentially localizes at the invasive margin through a mechanism dependent on the activation of Nrf2 and coordinated by the NF-κB1-CSF1R-C3aR axis. Inhibition of F4/80+HO-1+ TAM recruitment or myeloid-specific deletion of HO-1 blocks metastasis formation and improves anticancer immunotherapy. Relative expression of HO-1 in peripheral monocyte subsets, as well as in tumor lesions, discriminates survival among metastatic melanoma patients. Overall, these results identify a distinct cancer-induced HO-1+ myeloid subgroup as a new antimetastatic target and prognostic blood marker.

Evidence for tmTNF reverse signaling in vivo: Implications for an arginase-1-mediated therapeutic effect of TNF inhibitors during inflammation

SummaryIn order to ascertain the significance of transmembrane tumor necrosis factor (tmTNF) reverse signaling in vivo, we generated a triple transgenic mouse model (3TG, TNFR1−/−, TNFR2−/−, and tmTNFKI/KI) in which all canonical tumor necrosis factor (TNF) signaling was abolished. In bone-marrow-derived macrophages harvested from these mice, various anti-TNF biologics induced the expression of genes characteristic of alternative macrophages and also inhibited the expression of pro-inflammatory cytokines mainly through the upregulation of arginase-1. Injections of TNF inhibitors during arthritis increased pro-resolutive markers in bone marrow precursors and joint cells leading to a decrease in arthritis score. These results demonstrate that the binding of anti-TNF biologics to tmTNF results in decreased arthritis severity. Collectively, our data provide evidence for the significance of tmTNF reverse signaling in the modulation of arthritis. They suggest a complementary interpretation of anti-TNF biologics effects in the treatment of inflammatory diseases and pave the way to studies focused on new arginase-1-dependent therapeutic targets.

Pig-a gene mutations in bone marrow granulocytes of procarbazine-treated F344 rats.

It was previously demonstrated that procarbazine (PCZ) is positive in the rat erythrocyte Pig-a gene mutation assay. However, since mammalian erythrocytes lack genomic DNA, it was necessary to analyze nucleated bone-marrow erythroid precursor cells to confirm that PCZ induces mutations in the Pig-a gene (Revollo et al., Environ Mol Mutagen, 2020). In this study, the association between Pig-a mutation and loss of GPI anchors was further strengthened and the genesis of Pig-a mutation in PCZ-dosed rats was evaluated by analyzing bone-marrow granulocytes. Erythrocytes and granulocytes both originate from myeloid progenitor cells, but granulocytes contain DNA throughout their developmental stages. F344 rats were treated with three doses of 150 mg/kg PCZ; 2 weeks later, CD48-deficient mutant phenotype bone-marrow granulocytes (BMGs [CD11b+ ]) were isolated by flow-cytometric sorting. Sequencing data showed that the CD48-deficient mutant phenotype BMGs contained mutations in the Pig-a gene while wild-type BMGs did not. PCZ-induced mutations included missense, nonsense and splice site variants; the majority of mutations were A > T, A > C, and A > G, with the mutated A on the nontranscribed DNA strand. The PCZ-induced mutational analysis in BMGs supports the association between the phenotype measured in the Pig-a assay and mutation in the Pig-a gene. Also, PCZ mutation spectra were similar in bone-marrow erythroids and BMGs, but none of the mutations detected in BMGs were the same as the erythroid precursor cell mutations from the same rats. Thus, mutations induced in the Pig-a assay appear to be induced after commitment of myeloid progenitor cells to either the granulocyte or erythroid pathway.

Effects of experimental protein-containing pneumococcal preparations on maturation of murine dendritic cells

Dendritic cells as the most active and highly specialized antigen presenting cells, play a key role in initiating immune responses. Currently, generation of medications activating dendritic cells for development of anti-infective and anticancer vaccines is of highly relevance. Preparations of microbial origin are promising to augment activity of dendritic cells, because they carry innate immune ligands for Toll-like receptors. Such preparations include experimental protein-containing pneumococcal preparations, obtained from acetone-inactivated microbial mass of the deposited S. pneumoniae 6B vaccine strain No. 296, followed by aqueous extraction and separation of 30—100 kDa fraction. Dendritic cells were obtained from bone marrow cells of CBA mice (n = 15), and cultured in complete growth medium RPMI-1640 added with recombinant GM-CSF and IL-4 (Biosource, USA). On day 6, experimental protein-containing pneumococcal preparations (50 pl/ml) were administered to the cultured immature dendritic cells. Commercial TNFa (20 ng/ml, Biosource, USA) was used as a standard maturation inducer (positive control). Immunophenotyping of dendritic cells was conducted by using flow cytometry with FITC- and PE-labeled monoclonal antibodies against cell surface receptors: CD34, CD38, CD83, CD86, CD80, CD11c, MHC II, CD14, CD282 (TLR2), CD284 (TLR4), (eBioscience, USA). Studying an effect of preparations on maturation of dendritic cells revealed that morphological characteristics of dendritic cells generated by using experimental protein-containing preparations did not differ significantly between each other as well as those induced by TNFα. The cells were characterized by large sizes, oval or irregular shape, veiled cytoplasm, eccentrically located nucleus and numerous long thin protrusions. Experimental proteincontaining preparations induced in cultured dendritic cells decrease in count of CD34+ immature and TLR2/TLR4+ cells, increased count of cells expressing markers of adhesion (CD38+), activation (MHC II+), costimulation (CD80/ CD86+) and terminal differentiation (CD83+), which may evidence about events of differentiation upon dendritic cell maturation. The 30—100 kDa fraction increased count of cells expressing adhesion molecules to a greater extent than aqueous extract that more pronouncedly stimulated rise in count of dendritic cells bearing costimulatory molecules (p 0.05). The activity of the examined proteins regarding their effect on CD83+ cells was comparable. Experimental protein-containing antigens derived from pneumococcal vaccine strain were shown to induce maturation of dendritic cells from bone marrow precursors, induce a decrease in the count of TLR2 and TLR4-expressing cells accounting for activating effect on innate immune effectors.

CLEC12A and CD33 coexpression as a preferential target for pediatric AML combinatorial immunotherapy

AbstractEmerging immunotherapies such as chimeric antigen receptor T cells have advanced the treatment of acute lymphoblastic leukemia. In contrast, long-term control of acute myeloid leukemia (AML) cannot be achieved by single lineage-specific targeting while sparing benign hematopoiesis. In addition, heterogeneity of AML warrants combinatorial targeting, and several suitable immunotargets (HAVCR2/CD33 and HAVCR2/CLEC12A) have been identified in adult AML. However, clinical and biologic characteristics of AML differ between children and the elderly. Here, we analyzed 36 bone marrow (BM) samples of pediatric AML patients and 13 age-matched healthy donors using whole RNA sequencing of sorted CD45dim and CD34+CD38−CD45dim BM populations and flow cytometry for surface expression of putative target antigens. Pediatric AML clusters apart from healthy myeloid BM precursors in principal-component analysis. Known immunotargets of adult AML, such as IL3RA, were not overexpressed in pediatric AML compared with healthy precursors by RNA sequencing. CD33 and CLEC12A were the most upregulated immunotargets on the RNA level and showed the highest surface expression on AML detected by flow cytometry. KMT2A-mutated infant AML clusters separately by RNA sequencing and overexpresses FLT3, and hence, CD33/FLT3 cotargeting is an additional specific option for this subgroup. CLEC12A and CD33/CLEC12Adouble-positive expression was absent in CD34+CD38−CD45RA−CD90+ hematopoietic stem cells (HSCs) and nonhematopoietic tissue, while CD33 and FLT3 are expressed on HSCs. In summary, we show that expression of immunotargets in pediatric AML differs from known expression profiles in adult AML. We identify CLEC12A and CD33 as preferential generic combinatorial immunotargets in pediatric AML and CD33 and FLT3 as immunotargets specific for KMT2A-mutated infant AML.