ABSTRACT
Osteoporosis is a common skeletal disorder characterized by an imbalance between bone resorption and formation, exhibiting a higher prevalence in women compared with men. While previous studies have primarily focused on genomics and genetics in osteoporosis susceptibility, there is a lack of systematic exploration of sex-specific differences in lipid levels in mouse bone marrow. Multiple reaction monitoring-based liquid chromatography-trandem mass spectrometry (LC-MS/MS) was used to quantify lipidomic profiles in bone marrow samples from three female mice and three male mice. The LC-MS/MS technique based on the multiple reaction monitoring method identified and quantified 184 lipids from 15 lipid classes. The contents of most lipids in the bone marrow cells of female mice were higher than those in male mice, including four polyunsaturated fatty acids, three phospholipids and four sphingolipids. Among all the lipid molecules, lactosylceramide (d18:0/16:0) showed the highest fold change in female mice, while its precursor lipid, glucosylceramide, was the most up-regulated in male mice. This study, focusing on bone marrow lipidomics, elucidates significant sexual dimorphism in lipid levels within bone marrow cells. It provides novel evidence supporting the higher prevalence of osteoporosis in women and enhances our understanding of the connection between sex-specific lipid levels and the risk of osteoporosis.
Subject(s)
Bone Marrow , Lipidomics , Lipids , Tandem Mass Spectrometry , Animals , Female , Male , Mice , Lipidomics/methods , Bone Marrow/metabolism , Bone Marrow/chemistry , Chromatography, Liquid/methods , Tandem Mass Spectrometry/methods , Lipids/analysis , Sex Characteristics , Mice, Inbred C57BL , Bone Marrow Cells/metabolism , Bone Marrow Cells/chemistryABSTRACT
Background: microRNA-34a (miR-34a) had been reported to have a diagnostic role in acute myeloid leukemia (AML). However, its value in the bone marrow (BM) of AML patients, in addition to its role in response to therapy is still unclear. The current study was designed to assess the diagnostic, prognostic, and predictive significance of miR-34a in the BM of AML patients. Methods: The miR-34a was assessed in BM aspirate of 82 AML patients in relation to 12 normal control subjects using qRT-PCR. The data were assessed for correlation with the relevant clinical criteria, response to therapy, disease-free survival (DFS), and overall survival (OS) rates. Results: miR-34a was significantly downregulated in AML patients [0.005 (3.3 × 10-6-1.32)], compared to the control subjects [0.108 (3.2 × 10-4-1.64), p = 0.021]. The median relative quantification (RQ) of miR-34a was 0.106 (range; 0-32.12). The specificity, sensitivity, and area under the curve (AUC) for the diagnosis of AML were (58.3%, 69.5%, 0.707, respectively, p = 0.021). patients with upregulated miR-34a showed decreased platelets count <34.5 × 109/L, and achieved early complete remission (CR, p = 0.031, p = 0.044, respectively). Similarly, patients who were refractory to therapy showed decreased miR-34a levels in comparison to those who achieved CR [0.002 (0-0.01) and 0.12 (0-32.12), respectively, p = 0.002]. Therefore, miR-34a could significantly identify patients with CR with a specificity of 75% and sensitivity of 100% at a cut-off of 0.014 (AUC = 0.927, p = 0.005). There was no considerable association between miR-34a expression and survival rates of the included AML patients. Conclusion: miR-34a could be a beneficial diagnostic biomarker for AML patients. In addition, it serves as a good indicator for response to therapy, which could possibly identify patients who are refractory to treatment with 100% sensitivity and 75% specificity.
Subject(s)
Leukemia, Myeloid, Acute , MicroRNAs , Humans , Bone Marrow/chemistry , Bone Marrow/metabolism , Leukemia, Myeloid, Acute/diagnosis , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/metabolism , Prognosis , Disease-Free SurvivalABSTRACT
Induction therapy followed by CD34+ cell mobilisation and autologous transplantation represents standard of care for multiple myeloma (MM). However, the anti-CD38 monoclonal antibodies daratumumab and isatuximab have been associated with mobilisation impairment, yet the mechanism remains unclear. In this study, we investigated the effect of three different regimens (dara-VCd, isa-KRd and VTd) on CD34+ cells using flow cytometry and transcriptomics. Decreased CD34+ cell peak concentration and yields, longer collection and delayed engraftment were reproduced after dara-VCd/isa-KRd versus VTd induction in 34 patients in total. Using flow cytometry, we detected major changes in the proportion of apheresis product and bone marrow CD34+ subsets in patients treated with regimens containing anti-CD38 therapy; however, without any decrease in CD38high B-lymphoid progenitors in both materials. RNA-seq of mobilised CD34+ cells from 21 patients showed that adhesion genes are overexpressed in CD34+ cells after dara-VCd/isa-KRd and JCAD, NRP2, MDK, ITGA3 and CLEC3B were identified as potential target genes. Finally, direct in vitro effect of isatuximab in upregulating JCAD and CLEC3B was confirmed by quantitative PCR. These findings suggest that upregulated adhesion-related interactions, rather than killing of CD34+ cells by effector mechanisms, could be leading causes of decreased mobilisation efficacy in MM patients treated with anti-CD38 therapy.
Subject(s)
Multiple Myeloma , Humans , Multiple Myeloma/therapy , Antigens, CD34/analysis , Bone Marrow/chemistry , Flow Cytometry , Hematopoietic Stem Cell Mobilization , ADP-ribosyl Cyclase 1ABSTRACT
Multiple myeloma (MM) cells from 1 out of 20 patient expressed high basal levels of membrane B-cell maturation antigen (BCMA, TNFRSF17, CD269), which was not upregulated by gamma-secretase inhibitor, suggesting a defective BCMA shedding by gamma-secretase. Genetic analyses of the patient's bone marrow DNA showed no mutations within the BCMA coding region, but rather partial deletion of PSEN1 and amplification of PSEN2, which encode alternative catalytic units of gamma-secretase. Altogether the data suggest that pt#12 MM cells express high and dysregulated BCMA with no shedding, due to genetic alterations of one or more gamma-secretase subunits.
Subject(s)
Multiple Myeloma , Humans , B-Cell Maturation Antigen , Amyloid Precursor Protein Secretases , Bone Marrow/chemistryABSTRACT
INTRODUCTION: Treatment with erythropoietin-stimulating agents (ESAs) is widely used in anemia of chronic kidney disease (CKD). Acquired ESA resistance is an important problem. The aim of this study is to examine the bone marrow findings in hemodialysis patients with ESA-resistant anemia. METHODS: The data of 210 patients with acquired ESA resistance were reviewed retrospectively. The patients were divided into groups according to having diagnosis of dysplasia and hematological disease, and survival analysis was performed. RESULTS: A total of 26 patients were included in the study. While dysplasia was present in 10 (38.5%) patients, five of them were diagnosed hematologically. When survival analysis was performed between those with and without a hematological diagnosis, a difference in survival was observed against the group with the diagnosis (24.4 vs. 72 months, p = 0.045). CONCLUSION: Unresponsiveness to ESA treatment in CKD patients or a decrease in one of the other cell lines along with hemoglobin, it would be appropriate to perform early bone marrow examination.
Subject(s)
Anemia , Erythropoietin , Hematinics , Renal Insufficiency, Chronic , Humans , Hematinics/therapeutic use , Retrospective Studies , Bone Marrow/chemistry , Bone Marrow/metabolism , Erythropoietin/therapeutic use , Anemia/drug therapy , Anemia/etiology , Renal Dialysis/adverse effects , Hemoglobins/analysis , Renal Insufficiency, Chronic/complications , Renal Insufficiency, Chronic/therapyABSTRACT
BACKGROUND: Iron overload due to the excessive use of parenteral iron in haemodialysis is now an increasingly recognised clinical issue. Before erythropoiesis-stimulating agents (ESA) were introduced, a specific feature of patients treated by dialysis and having iron overload was that iron levels in the bone marrow were paradoxically low in most of them, despite severe hepatosplenic siderosis. Whether or not this paradox persists in the actual ESA era was unknown until recently, when an autopsy study in 21 patients treated by haemodialysis revealed similarities between liver and bone marrow iron content. The aim of this study was to further explore these recent findings in a cohort of alive patients on dialysis and to analyse the determinants of iron bone marrow. METHODS: Liver iron concentration (LIC) and vertebral T2∗ (a surrogate marker of bone marrow iron) were analysed retrospectively in 152 alive patients on dialysis (38.8% female) of whom 47.4% had iron overload by quantitative magnetic resonance imaging (MRI). FINDINGS: Vertebral T2∗ differed significantly between patients classified according to liver iron content at MRI: those with mild or moderate and severe liver iron overload had increased vertebral iron content at R2∗ relaxometry MRI (mild: vertebral T2∗ = 9.9 ms (4-24.8); moderate and severe: vertebral T2∗ = 8.5 ms (4.9-22.8)) when compared to patients with normal LIC (vertebral T2∗ = 13.2 ms (6.6-30.5) (p < 0.0001 Kruskal-Wallis test)). INTERPRETATION: The paradoxical discrepancy between bone marrow and liver iron-storage compartments observed in the pre-ESA era has disappeared today, as shown by a recent autopsy study and the present study in a cohort of alive patients treated by dialysis. FUNDING: None.
Subject(s)
Hemosiderosis , Iron Overload , Humans , Female , Male , Retrospective Studies , Bone Marrow/chemistry , Renal Dialysis/adverse effects , Hemosiderosis/etiology , Hemosiderosis/pathology , Iron , Iron Overload/pathology , Liver/pathology , Magnetic Resonance Imaging/methodsABSTRACT
Low oxygen bone marrow (BM) niches (~1%-4% low O2 ) provide critical signals for hematopoietic stem/progenitor cells (HSC/HSPCs). Our presented data are the first to investigate live, sorted HSC/HSPCs in their native low O2 conditions. Transcriptional and proteomic analysis uncovered differential Ca2+ regulation that correlated with overlapping phenotypic populations consisting of robust increases of cytosolic and mitochondrial Ca2+ , ABC transporter (ABCG2) expression and sodium/hydrogen exchanger (NHE1) expression in live, HSC/HSPCs remaining in constant low O2. We identified a novel Ca2+ high population in HSPCs predominantly detected in low O2 that displayed enhanced frequency of phenotypic LSK/LSKCD150 in low O2 replating assays compared to Ca2+ low populations. Inhibition of the Ca2+ regulator NHE1 (Cariporide) resulted in attenuation of both the low O2 induced Ca2+ high population and subsequent enhanced maintenance of phenotypic LSK and LSKCD150 during low O2 replating. These data reveal multiple levels of differential Ca2+ regulation in low O2 resulting in phenotypic, signaling, and functional consequences in HSC/HSPCs.
Subject(s)
Calcium , Hematopoietic Stem Cells , Oxygen , Bone Marrow/chemistry , Bone Marrow/metabolism , Calcium/metabolism , Hematopoietic Stem Cells/metabolism , Oxygen/metabolism , Proteomics , Animals , MiceABSTRACT
BACKGROUND: The aim of the study was to improve the understanding of complex karyotype acute mixed cell leukemia containing pseudo Chediak-Higashi granules. METHODS: A case of acute mixed cell leukemia resembling AML1-ETO positive acute myeloid leukemia was reported. The results of morphological, immunophenotypic, and cytogenetic tests were analyzed by reviewing relevant literature. RESULTS: The patient was a young boy with clinical manifestations of recurrent fever. Bone marrow smear: Granulocyte system hyperplasia is obvious, visible at each stage, primitive cells account for 12%. These cells are large in volume, mostly round or class round, with abundant cell mass, stained gray blue, unbalanced development of some nuclear plasma, abnormal cytoplasmic staining, and visible "sunrise red" -like changes. Typical Auer bodies, pseudo Chadiak-Higashi granules and phagocytic erythroid substances were observed. The nuclei are irregular in shape, distorted and depressed, with fine chromatin and prominent large nucleoli. Bone marrow was extracted 3 days later, the bone marrow smear showed 65% primitive cells. The morphology of primitive cells was similar to that of 3 days ago. The results of flow cytometry showed that the primary/naive T cells in the samples possessed nuclear cells. Flow cytometry showed two groups of abnormal cells. One group accounted for 3.87% of nuclear cells and was a primitive/naive T-cell phenotype, mainly expressing: CD34+, CD7+, CD5+, CD2dim+, MPO-, CCD3 + part, CD3-, CD4-, CD8 -, CD117 -, CD13-, CD33-, HLA - DR -, CD10-, CD11b-, CD56-. The other group which accounted for 79.8% of the nuclear cells was monocyte phenotype, mainly expressing: CD34-, CD117-, CD13+ small amount, CD33+, HLA-DR-, CD11b+, CD14+, CD15+, CD36+, CD56+, CD64+, CD4+, CD85J+, CD85K + part. It matched the immunophenotype of acute mixed cell leukemia (T/MMPAL). Immunophenotypic leukemia-related fusion genes were negative, and karyotype analysis results were 45, XY, T (11; 12)(p13; Q13), -12-17, + mar [12]/90 < n > 4, idem x 2 [6]/46, XY. Combined with the above results, acute mixed cell leukemia was diagnosed. CONCLUSIONS: The flow cytometry is the gold standard in the diagnosis of acute mixed cell leukemia. The diagnosis of acute mixed cell leukemia requires the combination of clinical manifestations, cellular morphology, immunology, cytogenetics and molecular biology, and the comprehensive diagnosis efficiency is obviously better than that of morphology.
Subject(s)
Leukemia, Biphenotypic, Acute , Leukemia, Myeloid, Acute , Humans , Leukemia, Myeloid, Acute/diagnosis , Leukemia, Myeloid, Acute/genetics , HLA-DR Antigens/analysis , Bone Marrow/chemistry , Phenotype , Immunophenotyping , RUNX1 Translocation Partner 1 Protein/genetics , Oncogene Proteins, Fusion/genetics , Core Binding Factor Alpha 2 Subunit/geneticsABSTRACT
Obesity is associated with disruptions in the adaptive immune system; however, dietary fatty acids in high-fat diets (HFDs) that induce obesity have consequences that are currently unclear regarding T-cell maintenance in bone marrow (BM). C57BL/6J mice were randomly assigned to isocaloric HFDs formulated with dietary fats rich in saturated fatty acids (SFAs), monounsaturated fatty acids (MUFAs), or MUFAs supplemented with eicosapentaenoic and docosahexaenoic acids for 20 weeks, followed by an analysis of the immunophenotypic feature of lymphocytes (CD3+) T and their subsets CD4+ and CD8+ T cells in spleen and BM, identification of fatty acids in BM extracellular fluid and analysis of the correspondence between fatty acids with the frequency of T-cell subsets in BM. Splenic CD3+ T cells were reduced irrespective of HFDs. In BM, CD3+ T cells were reduced after HFD-SFAs, while CD4+ T cells were increased after HFDs enriched in MUFAs and CD8+ T cells were reduced irrespective of HFDs. In BM extracellular fluid, the content of palmitic and myristic acids increased after HFD-SFAs and that of oleic acid increased after HFDs enriched in MUFAs. There was a statistical correspondence between HFD-induced changes in fatty acids in BM extracellular fluid and HFD-induced changes in the frequency of CD3+ and CD4+ T cells in BM. These findings reveal an undervalued critical role for dietary fatty acids in the selective acquisition of T-cell subsets in BM, highlighting that oleic acid existing in the surroundings of T-cell niches during HFD-induced obesity could be instrumental in the maintenance of CD4+ T cells.
Subject(s)
Diet, High-Fat , Fatty Acids , Animals , Bone Marrow/chemistry , CD4-Positive T-Lymphocytes , CD8-Positive T-Lymphocytes , Diet, High-Fat/adverse effects , Fatty Acids/analysis , Fatty Acids, Monounsaturated , Mice , Mice, Inbred C57BL , Obesity/etiology , Oleic AcidsABSTRACT
In this study, the influence of the living conditions of red deer (Cervus elaphus) fawns (wild vs. farmed) and effect of the category of free-living animals (fawns vs. does) on the fatty acid (FA) profile of the leg bone marrow was assessed. The composition of FAs in the deer bone marrow was determined by the gas chromatography method. In all groups, oleic acid (18:1 c9) was the most abundant in deer bone marrow and comprised of approximately 37% of total FAs. The bone marrow of young wild deer was characterized by a significantly (p < 0.001) higher fat content and saturated FAs proportion, while farmed fawns contained more moisture (p < 0.005) and fat-free dry matter (p < 0.001), as well as more monounsaturated FAs cis branched-chain FAs and monounsaturated FAs trans (p < 0.001). Although no significant (p > 0.05) differences were found between fawns, in terms of partial sums of PUFA, a significantly (p < 0.001) higher level of the sum of n-3 and n-6 FAs and more favorable n-6/n-3 ratio in the bone marrow of wild fawns were determined. In general, the legs of wild fawns were better prepared for wintering than farmed ones. In turn, comparing the category-related FAs composition in the bone marrow of free-living animals, a more favorable profile was observed in the adult (does) than in the young (fawns) animals, as the bone marrow of the wild does was characterized by significantly (p < 0.001) lower percentages of saturated FAs and a higher percentage of monounsaturated FAs cis.
Subject(s)
Deer , Fatty Acids , Animals , Animals, Wild , Bone Marrow/chemistry , Ecosystem , Fatty Acids/analysisABSTRACT
Due to the considerable success of cancer immunotherapy for leukemia, the tumor immune environment has become a focus of intense research; however, there are few reports on the dynamics of the tumor immune environment in leukemia. Here, we analyzed the tumor immune environment in pediatric B cell precursor acute lymphoblastic leukemia by analyzing serial bone marrow samples from nine patients with primary and recurrent disease by mass cytometry using 39 immunophenotype markers, and transcriptome analysis. High-dimensional single-cell mass cytometry analysis elucidated a dynamic shift of T cells from naïve to effector subsets, and clarified that, during relapse, the tumor immune environment comprised a T helper 1-polarized immune profile, together with an increased number of effector regulatory T cells. These results were confirmed in a validation cohort using conventional flow cytometry. Furthermore, RNA transcriptome analysis identified the upregulation of immune-related pathways in B cell precursor acute lymphoblastic leukemia cells during relapse, suggesting interaction with the surrounding environment. In conclusion, a tumor immune environment characterized by a T helper 1-polarized immune profile, with an increased number of effector regulatory T cells, could contribute to the pathophysiology of recurrent B cell precursor acute lymphoblastic leukemia. This information could contribute to the development of effective immunotherapeutic approaches against B cell precursor acute lymphoblastic leukemia relapse.
Subject(s)
Biomarkers, Tumor/genetics , Bone Marrow/immunology , Gene Expression Profiling/methods , Neoplasm Recurrence, Local/genetics , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/genetics , Adolescent , Bone Marrow/chemistry , Child , Child, Preschool , Female , Flow Cytometry , Gene Expression Regulation, Neoplastic , Humans , Infant , Male , Neoplasm Recurrence, Local/immunology , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/immunology , Sequence Analysis, RNA , Single-Cell Analysis , Tumor Microenvironment , Up-Regulation , Young AdultABSTRACT
Conventional two-material dual-energy CT (DECT) decomposition is insufficient to model bone marrow, which contains three materials: bone minerals, red marrow (water), and yellow marrow (fat). We explore an image-domain three-material decomposition DECT technique accounting for bone minerals in a bone-water-fat phantom. Three-material decomposition fat fraction (FF3MD) exhibited stronger correlation than two-material decomposition fat fraction (FF2MD) with MRI-based fat fraction (r = 0.95 vs r = 0.69). With increasing bone minerals, correlation of FF3MD remained stable (r = 0.81-1.02), whereas correlation of FF2MD decreased (r = 0.21-0.65).
Subject(s)
Bone Marrow/anatomy & histology , Magnetic Resonance Imaging , Minerals/analysis , Radiography, Dual-Energy Scanned Projection/methods , Tomography, X-Ray Computed/methods , Bone Marrow/chemistry , Bone Marrow/diagnostic imaging , Humans , Minerals/chemistry , Phantoms, ImagingABSTRACT
BACKGROUND: The cells of the entire body, including the skeletal system, especially of young animals, may derive from the bone marrow in which they multiply. Therefore, it is important to assess whether the diet and quality of life of deer have a significant impact on the elemental composition of bone and bone marrow, which can directly affect their health and growth. The aim of this study was to determine the concentrations of macro- (Ca, calcium, P, phosphorus, Mg, magnesium, K, potassium, Na, sodium) and microelements (Li, lithium, Cr, chromium, Mn, manganese, Co, cobalt, Cu, copper, Zn, zinc, Se, selenium, Mo, molybdenum, and Sn, tin) accumulated in the bone marrow and bones of deer (Cervus elaphus). The study was carried out on 15 young stags divided into two groups: farmed and wild animals. The concentrations of macro- and microelements were analysed using the inductively coupled plasma mass spectrometry technique. This research expands our knowledge on this topic, which so far has not been extensively studied. RESULTS: The mean content of K, Na, Zn and Se in the bone marrow of farmed animals was significantly higher than in wild deer, whereas the mean content of Ca, P, Mg, K, Na and Li in the bones was higher in wild animals than in farmed individuals (p < 0.05). In addition, the mean concentration of Cr, Mn, Cu, Se and Mo in the bones of the analysed animals differed significantly (p < 0.05) and was higher in the farmed deer. The mean concentration of Se in the bone marrow of wild deer decreased with the increase of the body weight (p < 0.05). In turn, the mean content of Mn in the bone marrow and of Mo in the bones of the animals was significantly positively correlated with the animals' body weight (p < 0.05). CONCLUSIONS: The obtained results indicated different levels of micro- and macro-components in the body of farmed and wild deer, though without clear and strong variations. Generally, the higher level of macronutrients in the bones of wild deer may be related to the higher physiological importance of these minerals for life activities in the natural environment and to the limited supply of balanced food. On the other hand, the higher levels of microelements in the tissues of farmed animals may result from their significantly better nutritional status in the first year of life, achieved through appropriate nutrition as well as diet supplementation of adult females.
Subject(s)
Bone Marrow/chemistry , Bone and Bones/chemistry , Deer , Minerals/analysis , Animal Husbandry , Animals , Animals, Wild , Body Weight , Male , Metals/analysisSubject(s)
Anemia, Iron-Deficiency/diagnosis , Evidence-Based Medicine , Ferritins/blood , Practice Guidelines as Topic , Anemia, Iron-Deficiency/blood , Anemia, Iron-Deficiency/etiology , Bone Marrow/chemistry , Endoscopy, Gastrointestinal/adverse effects , Endoscopy, Gastrointestinal/methods , Female , Gastrointestinal Neoplasms/blood , Gastrointestinal Neoplasms/diagnosis , Humans , Iron/analysis , Male , Postmenopause/blood , Sensitivity and Specificity , Sex FactorsABSTRACT
BACKGROUND: Flow cytometry plays a key role in detecting bone marrow (BM) involvement in patients with diffuse large B-cell lymphoma (DLBCL). To improve its detection sensitivity, we need to explore novel markers. In this study, we detected the expression CD54 on lymphoma cells in BM specimens from DLBCL patients and clarified its diagnostic significance in BM involvement by DLBCL. METHODS: We collected BM specimens from 76 patients with DLBCL (germinal center B-cell (GCB) = 25, non-GCB = 51) and 10 control patients without lymphoma. We detected and compared the expression of CD54 on lymphoma cells and normal mature B cells by using 10-color panels. RESULTS: Normal plasma cells expressed a higher level of CD54 as compared with hematogones (p < 0.05) and normal mature B cells (p < 0.05). Among 76 patients, 23 of them (GCB = 12, non-GCB = 11) had BM involvement. Lymphoma B cells from 12 cases (GBC = 4, non-GCB = 8) expressed a higher level of CD54 compared to normal mature B cells (p < 0.05). Additionally, lymphoma cells of the non-GCB subtype frequently expressed a higher level of CD54 in comparison to the GCB subtype (p < 0.05). And the high expression of CD54 was not related to plasmacytoid differentiation. CONCLUSION: Aberrant expression of CD54 on lymphoma cells is frequently seen in patients' BM specimens involved by DLBCL, especially in the non-GCB subtype. CD54 could be used as a new marker to gate on lymphoma cells and improve the detection sensitivity of BM involvement in patients with DLBCL.
Subject(s)
Biomarkers, Tumor/analysis , Bone Marrow/chemistry , Flow Cytometry , Intercellular Adhesion Molecule-1/analysis , Lymphoma, Large B-Cell, Diffuse/chemistry , Adult , Aged , Aged, 80 and over , B-Lymphocytes/chemistry , B-Lymphocytes/metabolism , Biomarkers, Tumor/metabolism , Biopsy , Bone Marrow/metabolism , Bone Marrow/pathology , Cell Adhesion Molecules/analysis , Cell Adhesion Molecules/metabolism , Cell Differentiation , Female , Germinal Center/chemistry , Germinal Center/metabolism , Germinal Center/pathology , Humans , Immunophenotyping , Intercellular Adhesion Molecule-1/metabolism , Lymphoma, Large B-Cell, Diffuse/metabolism , Lymphoma, Large B-Cell, Diffuse/pathology , Male , Middle Aged , Plasma Cells/cytologyABSTRACT
RATIONALE: Murine syngeneic tumor models have revealed efficacious systemic antitumor responses following primary tumor in situ vaccination combined with targeted radionuclide therapy to secondary or metastatic tumors. Here we present studies on the safety and feasibility of this approach in a relevant translational companion dog model (n = 17 dogs) with advanced cancer. METHODS: The three component of the combination immuno-radiotherapy approach were employed either separately or in combination in companion dogs with advanced stage cancer. In situ vaccination was achieved through the administration of hypofractionated external beam radiotherapy and intratumoral hu14.18-IL2 fusion immunocytokine injections to the index tumor. In situ vaccination was subsequently combined with targeted radionuclide therapy using a theranostic pairing of IV 86Y-NM600 (for PET imaging and subject-specific dosimetry) and IV 90Y-NM600 (therapeutic radionuclide) prescribed to deliver an immunomodulatory 2 Gy dose to all metastatic sites in companion dogs with metastatic melanoma or osteosarcoma. In a subset of dogs, immunologic parameters preliminarily assessed. RESULTS: The components of the immuno-radiotherapy combination were well tolerated either alone or in combination, resulting in only transient low grade (1 or 2) adverse events with no dose-limiting events observed. In subject-specific dosimetry analyses, we observed 86Y-NM600 tumor:bone marrow absorbed-dose differential uptakes ≥2 in 4 of 5 dogs receiving the combination, which allowed subsequent safe delivery of at least 2 Gy 90Y-NM600 TRT to tumors. NanoString gene expression profiling and immunohistochemistry from pre- and post-treatment biopsy specimens provide evidence of tumor microenvironment immunomodulation by 90Y-NM600 TRT. CONCLUSIONS: The combination of external beam radiotherapy, intratumoral immunocytokine, and targeted radionuclide immuno-radiotherapy known to have activity against syngeneic melanoma in murine models is feasible and well tolerated in companion dogs with advanced stage, spontaneously arising melanoma or osteosarcoma and has immunomodulatory potential. Further studies evaluating the dose-dependent immunomodulatory effects of this immuno-radiotherapy combination are currently ongoing.
Subject(s)
Antibodies, Monoclonal/therapeutic use , Interleukin-2/therapeutic use , Melanoma/therapy , Osteosarcoma/therapy , Radiopharmaceuticals/therapeutic use , Animals , Antibodies, Monoclonal/adverse effects , Bone Marrow/chemistry , Bone Marrow/metabolism , Bone Marrow/pathology , Combined Modality Therapy , Dogs , Feasibility Studies , Female , Gene Expression , Interleukin-2/adverse effects , Leukocytes, Mononuclear/cytology , Leukocytes, Mononuclear/metabolism , Lymphocytes, Tumor-Infiltrating/cytology , Lymphocytes, Tumor-Infiltrating/metabolism , Male , Melanoma/immunology , Melanoma/pathology , Melanoma/veterinary , Osteosarcoma/immunology , Osteosarcoma/veterinary , Positron Emission Tomography Computed Tomography , Radiopharmaceuticals/adverse effects , Radiopharmaceuticals/chemistry , Vaccination , Yttrium Radioisotopes/chemistryABSTRACT
Isolation of pure extracellular vesicles (EVs), especially from blood, has been a major challenge in the field of EV research. The presence of lipoproteins and soluble proteins often hinders the isolation of high purity EVs upon utilization of conventional separation methods. To circumvent such problems, we designed a single-step dual size-exclusion chromatography (dSEC) column for effective isolation of highly pure EVs from bone marrow derived human plasma. With an aim to select appropriate column design parameters, we analyzed the physiochemical properties of the major substances in bone marrow derived plasma, which include EVs, lipoproteins, and soluble proteins. Based on these findings, we devised a novel dSEC column with two different types of porous beads sequentially stacked each other for efficient separation of EVs from other contaminants. The newly developed dSEC columns exhibited better performance in isolating highly pure EVs from AML plasma in comparison to conventional isolation methods.
Subject(s)
Bone Marrow/chemistry , Chromatography, Gel/methods , Equipment Design/methods , Extracellular Vesicles/chemistry , Plasma/chemistry , Apolipoproteins B/analysis , Apolipoproteins B/isolation & purification , Cholesterol, LDL/isolation & purification , Chromatography, Gel/instrumentation , Equipment Design/instrumentation , HL-60 Cells , Humans , Plasma/cytology , THP-1 Cells , Tetraspanin 30/analysis , Tetraspanin 30/isolation & purificationABSTRACT
The role of the vascularized bone marrow component as a continuous source of donor-derived hematopoietic stem cells that facilitate tolerance induction of vascularized composite allografts is not completely understood. In this study, vascularized composite tissue allograft transplantation outcomes between recipients receiving either conventional bone marrow transplantation (CBMT) or vascularized bone marrow (VBM) transplantation from Balb/c (H2d) to C57BL/6 (H2b) mice were compared. Either high- or low-dose CBMT (1.5 × 108 or 3 × 107 bone marrow cells, respectively) was applied. In addition, recipients were treated with costimulation blockade (1 mg anti-CD154 and 0.5 mg CTLA4Ig on postoperative days 0 and 2, respectively) and short-term rapamycin (3 mg/kg/day for the first posttransplant week and then every other day for another 3 weeks). Similar to high-dose conventional bone marrow transplantation, 5/6 animals in the vascularized bone marrow group demonstrated long-term allograft survival (>120 days). In contrast, significantly shorter median survival was noted in the low-dose CBMT group (~64 days). Consistently high chimerism levels were observed in the VBM transplantation group. Notably, low levels of circulating CD4+ and CD8+ T cells and a higher ratio of Treg to Teff cells were maintained in VBM transplantation and high-dose CBMT recipients (>30 days) but not in low-dose VBM transplant recipients. Donor-specific hyporesponsiveness was shown in tolerant recipients in vitro. Removal of the vascularized bone marrow component after secondary donor-specific skin transplantation did not affect either primary allograft or secondary skin graft survival.
Subject(s)
Bone Marrow Transplantation/methods , Bone Marrow/chemistry , Graft Rejection/prevention & control , Graft Survival , Immune Tolerance , Skin Transplantation/methods , Vascularized Composite Allotransplantation/methods , Animals , CD8-Positive T-Lymphocytes/immunology , Graft Rejection/immunology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Plastic Surgery Procedures , T-Lymphocytes, Regulatory/immunology , Transplantation Chimera , Transplantation, HomologousABSTRACT
BACKGROUND: Neutropenia is the most common dose-limiting side effect of cytotoxic chemotherapy in cancer-bearing dogs. Biodynamic imaging (BDI) is a functional imaging technology that measures dynamic light scattering from living, three-dimensional tissues to characterize intracellular motion within those tissues. Previous studies have associated BDI biomarkers with tumour sensitivity to chemotherapy agents in dogs with naturally occurring cancer. We hypothesized that BDI, performed ex vivo on bone marrow aspirate samples, would identify dynamic biomarkers associated with the occurrence of specific degrees of neutropenia in tumour-bearing dogs receiving doxorubicin chemotherapy. MATERIALS AND METHODS: Bone marrow aspirates were collected from 10 dogs with naturally occurring cancers prior to initiation of doxorubicin treatment. BDI was performed on bone marrow samples treated ex vivo with doxorubicin at 0.1, 1, 10 and 100 µM along with 0.1% DMSO as a control. Dogs then were treated with doxorubicin (30 mg/m2 , intravenously). Peripheral blood neutrophil counts were obtained on the day of treatment and again 7 days later. Receiver operating characteristic curves identified provisional breakpoints for BDI biomarkers that correlated with specific changes in neutrophil counts between the two time points. RESULTS: Provisional breakpoints for several BDI biomarkers were identified, specifying dogs with the largest proportionate change in neutrophils and with neutropenia that was grade 2 or higher following doxorubicin treatment. CONCLUSIONS: Biodynamic imaging of bone marrow aspirates may identify those dogs at greater risk for neutropenia following doxorubicin chemotherapy. This approach may be useful for pre-emptively modifying chemotherapy dosing in dogs to avoid unacceptable side effects.
Subject(s)
Antineoplastic Agents/adverse effects , Biomarkers, Tumor/analysis , Bone Marrow/chemistry , Dog Diseases/metabolism , Neoplasms/veterinary , Neutropenia/veterinary , Animals , Dog Diseases/chemically induced , Dogs , Neoplasms/metabolism , Neutropenia/chemically inducedABSTRACT
The aim of this study was to investigate physiological variations of the water T2 relaxation time in vertebral bone marrow with respect to age, body mass index (BMI), sex and proton density fat fraction (PDFF) based on single-voxel magnetic resonance spectroscopy (MRS) at 3 T. Multi-TE single-voxel STEAM MRS data of a single lumbar vertebra (L4 or L5) from 260 subjects (160/100 female/male, age: 0.7/37.1/77.7 years, BMI: 13.6/26.2/44.5 kg/m2 [min./median/max.]) with no history of vertebral bone marrow pathologies were retrospectively included. All data were processed using a joint series T2-constrained time domain-based water-fat model. Water T2 and PDFF data were analyzed using (a) Pearson's correlation r and (b) multiple linear regression without interactions of the independent variables. Min./median/max. water T2 and PDFF were 11.2/21.1/42.5 ms and 4.0%/36.8%/82.0%, respectively. Pearson's correlation coefficients were significant (P < .05) for water T2 versus age (r = -0.429/-0.210 female/male) and for water T2 versus PDFF (r = -0.580/-0.546 female/male) for females and males, respectively. Females showed significant higher water T2 values compared with males (P < .001). Multiple linear regression for water T2 without interactions revealed a R2 = 0.407 with PDFF (P < .001) and sex (P < .001) as significant predictors. The current study suggests that under physiological conditions vertebral bone marrow water T2 is negatively correlated with age and PDFF and shows significant differences between females and males. The observed systematic trends are of relevance for the evaluation of T2 values and T2-weighted bone marrow parameters. Further research on the exact mechanisms and drivers of the observed water T2 behavior is required.