S100A8 and S100A9 are both members of the S100 family and have been shown to play roles in myeloid differentiation, autophagy, apoptosis, and chemotherapy resistance. In this study we demonstrate that the BET-bromodomain inhibitor JQ1 causes rapid suppression of S100A8 and S100A9 mRNA and protein in a reversible manner. In addition, we show that JQ1 synergises with daunorubicin in causing AML cell death. Daunorubicin alone causes a dose-and time-dependent increase in S100A8 and S100A9 protein levels in AML cell lines which is overcome by cotreatment with JQ1. This suggests that JQ1 synergises with daunorubicin in causing apoptosis via suppression of S100A8 and S100A9 levels.
There is great variation in cytopenias in cirrhotic patients with same severity and hypersplenism and their causative factors are not clear. Recent studies have highlighted the role of gut microbiome in regulation of constant and emergency hematopoiesis. Broad-spectrum antibiotics can disrupt the homeostatic or adaptive microbiota in cirrhosis, leading to impaired hematopoiesis and a higher susceptibility to infections. We studied all patients with cirrhosis with cytopenia (anemia, leucopenia, and/or thrombocytopenia), admitted in the Institute of Liver & Biliary Sciences, between January 2016 and July 2017, who underwent a bone marrow examination. The effect of the different antimicrobial agents on peripheral blood counts and bone marrow cellularity was assessed. A total of 196 patients' data was analyzed for this study. Patients on antimicrobials (n = 115) had significantly lower hemoglobin (p < 0.001), total leucocyte count (p = 0.048), and platelet count (p = 0.043) compared to patients not on antimicrobials. On unadjusted analysis, significant association with thrombocytopenia existed in beta-lactams (OR = 1.56, 95% CI = 1.06-2.40), quinolones (OR = 1.66, 95% CI = 1.11-2.61), and antifungals (OR = 2.24, 95% CI = 1.96-4.34). Cephalosporins were found to be significantly associated with anemia (OR = 1.91, 95% CI = 1.07-3.41). Patients who received antimicrobials had hypocellular marrow (p < 0.001) as compared to nonrecipients of antibiotics. The adjusted analysis showed that quinolones and beta-lactam antibiotics are the drug classes having significant association with thrombocytopenia and alternative class of drug should be explored in these patients to avoid severe thrombocytopenia.
12–14 days of culturing of bone marrow (BM) cells containing various growth factors is widely used method for generating dendritic cells (DCs) from suspended cell population. Here we compared flask culture method and commercially available CD11c Positive Selection kit method. Immature BMDCs’ purity of adherent as well as suspended cell population was generated in the decreasing concentration of recombinant-murine granulocyte-macrophage colony-stimulating factor (rmGM-CSF) in nontreated tissue culture flasks. The expression of CD11c, MHCII, CD40, and CD86 was measured by flow cytometry. We found significant difference (P<0.05) between the two methods in the adherent cells population but no significant difference was observed between the suspended cell populations with respect to CD11c+ count. However, CD11c+ was significantly higher in both adhered and suspended cell population by culture method but kit method gave more CD11c+ from suspended cells population only. On the other hand, using both methods, immature DC expressed moderate level of MHC class II molecules as well as low levels of CD40 and CD86. Our findings suggest that widely used culture method gives the best results in terms of yield, viability, and purity of BMDCs from both adherent and suspended cell population whereas kit method works well for suspended cell population.
The purpose of this study was to quantify the stem cell and growth factor (GF) contents in the bone marrow aspirate concentrate (BMAC) and platelet-rich plasma (PRP) prepared from whole blood using a protocol established in our laboratory. We examined 10 patients with osteonecrosis of the femoral head who were treated by autologous BMAC transplantation at our hospital between January 2015 and June 2015. We quantified CD34+ and CD31−CD45−CD90+CD105+ cells in BMAC and PRP by flow cytometry. Additionally, we measured various GFs, that is, basic fibroblast growth factor (b-FGF), platelet-derived growth factor-BB (PDGF-BB), vascular endothelial growth factor (VEGF), transforming growth factor-β1 (TGF-β1), and bone morphogenetic protein-2 (BMP-2) in BMAC and PRP using enzyme-linked immunosorbent assays and statistical analyses. CD34+ and CD31−45−90+105+ cells accounted for approximately 1.9% and 0.03% of cells in BMAC and no cells in PRP. The concentration of b-FGF was higher in BMAC than in PRP (P < 0.001), whereas no significant differences in the levels of PDGF-BB, VEGF, TGF-β1, and BMP-2 were observed between the two types of sample. BMAC had an average of 1.9% CD34+ and 0.03% CD31−45−90+105+ cells and higher levels of b-FGF than those of PRP.
No studies have examined the transplantation of a bone marrow aspirate (BMA) containing mesenchymal stem cells (MSCs) combined with human parathyroid hormone 1-34 (hPTH1-34) administration. Therefore, we evaluated the feasibility and efficacy of autologous BMA transplantation combined with hPHT1-34 administration in a bone necrosis model. The metatarsal bones of rabbits were necrotized using liquid nitrogen, and the rabbits received a BMA transplantation or saline injection followed by hPTH1-34 (30 μg/kg) or saline administration three times per week (n = 3-4 per group). The rabbits were euthanized at 12 weeks after the initiation of treatment. No systemic adverse effects or local neoplastic lesions were observed. Importantly, the rabbits in the BMA transplantation plus hPTH1-34 group showed the highest bone volumes and histological scores of new bone. These data confirmed the feasibility of BMA transplantation combined with hPTH1-34, at least during the experimental period. The observed efficacy may be explained by a synergistic effect from the stimulation of MSC differentiation to osteoblasts with hPTH1-34-mediated suppression of apoptosis in osteoblasts. These results indicate the promising potential for BMA transplantation combined with hPTH1-34 administration in bone necrosis treatment. Longer term experiments are needed to confirm the safety of this therapeutic strategy.
Alpha-solanine (alpha-solanine) is a glycoalkaloid present in potato (Solanum tuberosum). It has been of particular interest because of its toxicity and potential teratogenic effects that include abnormalities of the central nervous system, such as exencephaly, encephalocele, and anophthalmia. Various types of cell culture have been used as experimental models to determine the effect of alpha-solanine on cell physiology. The morphological changes in the mesenchymal stem cell upon exposure to alpha-solanine have not been established. This study aimed to describe a reliable and reproducible model for assessing the structural changes induced by exposure of mouse bone marrow mesenchymal stem cells (MSCs) to different concentrations of alpha-solanine for 24h. The results demonstrate that nonlethal concentrations of alpha-solanine (26 mu M) changed the morphology of the cells, including an increase in the number of nucleoli, suggesting elevated protein synthesis, and the formation of spicules. In addition, treatment with alpha-solanine reduced the number of adherent cells and the formation of colonies in culture. Immunophenotypic characterization and staining of MSCs are proposed as a reproducible method that allows description of cells exposed to the glycoalkaloid, alpha-solanine.
Comorbidity is more common in older patients and can increase the cost of care by increasing toxicity. Using the SEER-Medicare database from 2000 to 2007, we examined the costs and life-year benefit of Auto-HSCT for MM patients over the age of 65 by evaluating the difference over time relative to comorbidity burden. One hundred ten patients had an Auto-HSCT in the early time period (2000-2003) and 160 in the late time period (2004-2007). Patients were divided by a Charlson Comorbidity Index (CCI) of 0 or greater than 1 (CCI1+). Median overall survival was 53.5 months for the late time period patients compared to 40.3 months for the early time period patients (p = 0.031). Median costs for CCI0 versus CCI1+ in the early period were, respectively, $70,900 versus $72,000 (100 d); $86,100 versus $98,300 (1 yr); and $139,200 versus $195,300 (3 yrs). Median costs for late period were, respectively, $58,400 versus $60,400 (100 d); $86,300 versus $77,700 (1 yr); and $124,400 versus $110,900 (3 yrs). Comorbidity had a significant impact on survival and cost among early time period patients but not among late time period patients. Therefore, older patients with some comorbidities can be considered for Auto-HSCT depending on clinical circumstances.
The procedure for aspiration of bone marrow from the femur of patients undergoing total knee arthroplasty (TKA) or total hip arthroplasty (THA) may vary from an OR (operating room) to OR based on the surgeon’s skill and may lead to varied extent of clotting of the marrow and this, in turn, presents difficulty in the isolation of mesenchymal stem cells (MSCs) from such clotted bone marrow. We present a simple detailed protocol for aspirating bone marrow from such patients, isolation, and characterization of MSCs from the aspirated bone marrow specimens and show that the bone marrow presented no clotting or exhibited minimal clotting. This represents an economical source and convenient source of MSCs from bone marrow for use in regenerative medicine. Also, we presented the detailed protocol and showed that the MSCs derived from such bone marrow specimens exhibited MSCs characteristics and generated micromass cartilages, the recipe for regenerative medicine for osteoarthritis. The protocols we presented can be used as standard operating procedures (SOPs) by researchers and clinicians.
Angiogenesis plays an important role in progression of tumor with vascular endothelial growth factor (VEGF) being key proangiogenic factor. It was intended to study angiogenesis in different hematological malignancies by quantifying expression of VEGF and MVD in bone marrow biopsy along with serum VEGF levels and observing its change following therapy. The study included 50 cases of hematological malignancies which were followed for one month after initial therapy along with 30 controls. All of them were subjected to immunostaining by anti-VEGF and factor VIII antibodies on bone marrow biopsy along with the measurement of serum VEGF levels. Significantly higher pretreatment VEGF scores, serum VEGF levels, and MVD were observed in cases as compared to controls (p<0.05). The highest VEGF score and serum VEGF were observed in chronic myeloid leukemia and maximum MVD in Non-Hodgkin’s Lymphoma. Significant decrease in serum VEGF levels after treatment was observed in all hematological malignancies except for AML. To conclude angiogenesis plays an important role in pathogenesis of all the hematological malignancies as reflected by increased VEGF expression and MVD in bone marrow biopsy along with increased serum VEGF level. The decrease in serum VEGF level after therapy further supports this view and also lays the importance of anti angiogenic therapy.
Background. Sickle cell anaemia (SCA) is an inherited condition whose clinical manifestations arise from the tendency of haemoglobin to polymerize and deform red blood cells into characteristic sickle shape. Allogeneic bone marrow transplantation offers a cure. The aim of this study was to determine the level of awareness, knowledge, and acceptance of this beneficial procedure in Nigeria. Materials and Methods. This multicentre cross-sectional study was conducted in 7 tertiary hospitals in Nigeria in 2015. Approval was obtained from each institution's research and ethics committee. A pretested structured questionnaire was administered to respondents aged 18 years and above and to the parents or guardians of those below 18 years of age. Results. There were 265 respondents comprising 120 males and 145 females. One hundred and seventy-one (64.5%) respondents were aware of BMT for the treatment of SCA. About 67.8% (116 of 171) of those who were aware believed SCA can be cured with BMT (p = 0.001) and 49.7% (85 of 171) of the respondents accepted BMT (p = 0.001). Conclusion. Awareness of BMT in Nigeria is low when compared with reports from developed countries. The knowledge is poor and acceptance is low. With adequate information, improved education, and psychological support, more Nigerians will embrace BMT.
Different rabbit polyclonal antilymphocyte globulins (ATGs) are used in allogeneic hematopoietic stem cell transplantation (alloHSCT) to prevent graft-versus-host disease (GvHD). We compared 2 different ATGs in alloHSCT after reduced intensity conditioning (RIC) for hematological malignancies. We reviewed 30 alloHSCT for hematologic malignancies performed between 2007 and 2010 with fludarabine and i.v. busulfan as conditioning regimen. Patients alternatingly received Thymoglobulin or ATG-F. Median followup was 3.3 (2.5-4.5) years. Adverse events appeared to occur more frequently during Thymoglobulin infusion than during ATG-F infusion but without statistical significance (P = 0.14). There were also no differences in 3-year overall survival (OS), disease-free survival (DFS), relapse incidence, and transplant related mortality (TRM) in the Thymoglobulin versus ATG-F group: 45.7% versus 46.7%, 40% versus 33.7%, 40% versus 33.3%, and 20% versus 33.3%. The same held for graft failure, rejection, infectious complications, immune reconstitution, and acute or chronic GvHD. In patients transplanted for hematologic malignancies after RIC, the use of Thymoglobulin is comparable to that of ATG-F in all the aspects evaluated in the study. However due to the small number of patients in each group we cannot exclude a possible difference that may exist.
Cigarette smoking is associated with musculoskeletal degenerative disorders, delayed fracture healing, and nonunion. Bone marrow progenitor cells (BMPCs), known to express CD105, are important in local trophic and immunomodulatory activity and central to musculoskeletal healing/regeneration. We hypothesized that smoking is associated with lower levels of BMPC. Iliac bone marrow samples were collected from individuals aged 18-65 years during the first steps of pelvic surgery, under IRB approval with informed consent. Patients with active infectious or neoplastic disease, a history of cytotoxic or radiation therapy, primary or secondary metabolic bone disease, or bone marrow dysfunction were excluded. Separation process purity and the number of BMPCs recovered were assessed with FACS. BMPC populations in self-reported smokers and nonsmokers were compared using the two-tailed t-test. 13 smokers and 13 nonsmokers of comparable age and gender were included. The average concentration of BMPCs was 3.52 x 10(5)/mL +/- 2.45 x 10(5)/mL for nonsmokers versus 1.31 x 10(5)/mL +/- 1.61 x 10(5)/mL for smokers (t = 3.2, P = 0.004). This suggests that cigarette smoking is linked to a significant decrease in the concentration of BMPCs, which may contribute to the reduced regenerative capacity of smokers, with implications for musculoskeletal maintenance and repair.
The objective of this study is to analyze the evolution of chimerism of all patients transplanted for hematologic malignancies in our unit during a 20-year period, alive without relapse at 1 year after allogeneic hematopoietic stem cell transplantation (HSCT). Chimerism was tested using short tandem repeat polymorphisms after separation into mononuclear cells and granulocytes by Ficoll density gradient centrifugation. Of 155 patients studied, 89 had full chimerism (FC), 36 mononuclear cells mixed chimerism (MNC-MC), and 30 granulocytic MC with or without mononuclear cells MC (Gran-MC). Survival was significantly better in MNC-MC than in Gran-MC patients, with FC patients being intermediate. There was more disease relapse in the Gran-MC group but not in the MNC-MC group as compared to FC. MC was stable up to 21 years in the MNC-MC group and up to 19 years in the Gran-MC group. Of MC patients alive at 10 years, MC persisted in 83% in the MNC-MC and 57% in the Gran-MC groups. In conclusion, mixed chimerism may remain stable over a very long time period. In survivors without relapse at 1 year after HSCT, determining lineage specific chimerism may be useful as outcome differs, MNC-MC being associated with better outcome than Gran-MC.
Both stem cell chemokine stromal cell-derived factor-1α (SDF-1α) and extracellular nucleotides such as adenosine triphosphate (ATP) are increased in ischemic myocardium. Since ATP has been reported to influence cell migration, we analysed the migratory response of bone marrow cells towards a combination of SDF-1 and ATP. Total nucleated cells (BM-TNCs) were isolated from bone marrow of cardiac surgery patients. Migration assays were performed in vitro. Subsequently, migrated cells were subjected to multicolor flow cytometric analysis of CD133, CD34, CD117, CD184, CD309, and CD14 expression. BM-TNCs migrated significantly towards a combination of SDF-1 and ATP. The proportions of CD34+ cells as well as subpopulations coexpressing multiple stem cell markers were selectively enhanced after migration towards SDF-1 or SDF-1 + ATP. After spontaneous migration, significantly fewer stem cells and CD184+ cells were detected. Direct incubation with SDF-1 led to a reduction of CD184+ but not stem cell marker-positive cells, while incubation with ATP significantly increased CD14+ percentage. In summary, we found that while a combination of SDF-1 and ATP elicited strong migration of BM-TNCs in vitro, only SDF-1 was responsible for selective attraction of hematopoietic stem cells. Meanwhile, spontaneous migration of stem cells was lower compared to BM-TNCs or monocytes.
Metastasis is a multistep process, which refers to the ability to leave a primary tumor through circulation toward the distant tissue and form a secondary tumor. Bone is a common site of metastasis, in which osteolytic and osteoblastic metastasis are observed. Signaling pathways, chemokines, growth factors, adhesion molecules, and cellular interactions as well as miRNAs have been known to play an important role in the development of bone metastasis. These factors provide an appropriate environment (soil) for growth and survival of metastatic tumor cells (seed) in bone marrow microenvironment. Recognition of these factors and determination of their individual roles in the development of metastasis and disruption of cellular interactions can provide important therapeutic targets for treatment of these patients, which can also be used as prognostic and diagnostic biomarkers. Thus, in this paper, we have attempted to highlight the molecular regulation of bone marrow metastasis in prostate and breast cancers.
TET2 is a methylcytosine dioxygenase that is frequently mutated in myeloid malignancies, notably myelodysplasia and acute myeloid leukemia. TET2 catalyses the conversion of 5′-methylcytosine to 5′-hydroxymethylcytosine within DNA and has been implicated in the process of genomic demethylation. However, the mechanism by which TET2 loss of function results in hematopoietic dysplasia and leukemogenesis is poorly understood. Here, we show that TET2 is expressed in undifferentiated embryonic stem cells and that its knockdown results in reduction of 5′-hydroxymethylcytosine in genomic DNA. We also present DNA methylation data from bone marrow samples obtained from patients with TET2-mutated myelodysplasia. Based on these findings, we sought to identify the role of TET2 in regulating pluripotency and differentiation. We show that overexpression of TET2 in a stably integrated transgene leads to increased alkaline phosphatase expression in differentiating ES cells and impaired differentiation in methylcellulose culture. We speculate that this effect is due to TET2-mediated expression of stem cell genes in ES cells via hydroxylation of 5′-methylcytosines at key promoter sequences within genomic DNA. This leads to relative hypomethylation of gene promoters as 5′-hydroxymethylcytosine is not a substrate for DNMT1-mediated maintenance methylation. We sought to test this hypothesis by cotransfecting the TET2 gene with methylated reporter genes. The results of these experiments are presented.
Though discovered later than osteoblastic niche, vascular niche has been regarded as an alternative indispensable niche operating regulation on hematopoietic stem cells (HSCs). As significant progresses gained on this type niche, it is gradually clear that the main work of vascular niche is undertaking to support hematopoiesis. However, compared to what have been defined in the mechanisms through which the osteoblastic niche regulates hematopoiesis, we know less in vascular niche. In this review, based on research data hitherto we will focus on component foundation and various functions of vascular niche that guarantee the normal hematopoiesis process within bone marrow microenvironments. And the possible pathways raised by various research results through which this environment undergoes its function will be discussed as well.
Molecular studies have shown that multiple myeloma is a highly genetically heterogonous disease which may manifest itself as any number of diverse subtypes each with variable clinicopathological features and outcomes. Given this genetic heterogeneity, a universal approach to treatment of myeloma is unlikely to be successful for all patients and instead we should strive for the goal of personalised therapy using rationally informed targeted strategies. Current DNA sequencing technologies allow for whole genome and exome analysis of patient myeloma samples that yield vast amounts of genetic data and provide a mutational overview of the disease. However, the clinical utility of this information currently lags far behind the sequencing technology which is increasingly being incorporated into clinical practice. This paper attempts to address this shortcoming by proposing a novel genetically based “traffic-light” risk stratification system for myeloma, termed the RAG (Red, Amber, Green) model, which represents a simplified concept of how complex genetic data may be compressed into an aggregate risk score. The model aims to incorporate all known clinically important trisomies, translocations, and mutations in myeloma and utilise these to produce a score between 1.0 and 3.0 that can be incorporated into diagnostic, prognostic, and treatment algorithms for the patient.
Autologous graft versus host disease (autoGVHD) is a rare transplant complication with significant morbidity and mortality. It has been hypothesized that patients with multiple myeloma might be predisposed to autoGVHD through dysregulation of the immune response resulting from either their disease, the immunomodulatory agents (IMiDs) used to treat it, or transplant conditioning regimen. Hematopoietic progenitor cell (HPC) products were available from 8 multiple myeloma patients with biopsy-proven autoGVHD, 16 matched multiple myeloma patients who did not develop autoGVHD, and 7 healthy research donors. The data on number of transplants prior to developing autoGVHD, mobilization regimens, exposure to proteasome inhibitors, use of IMiDs, and class I human leukocyte antigen types (HLA A and B) were collected. The HPC products were analyzed by flow cytometry for expression of CD3, CD4, CD8, CD25, CD56, and FoxP3. CD3 + cell number was significantly lower in autoGVHD patients compared to unaffected controls (P=0.047). On subset analysis of CD3 + cells, CD8 + cells (but not CD4 + cells) were found to be significantly lower in patients with autoGVHD (P=0.038). HLA-B55 expression was significantly associated with development of autoGVHD (P=0.032). Lower percentages of CD3 + and CD8 + T-cells and HLA-B55 expression may be predisposing factors for developing autoGVHD in myeloma.
Loss of heterozygosity (LOH) is a common event in malignant cells. In this work we introduce a new approach to identify patients with loss of heterozygosity in the HLA region either at first diagnosis or after HLA mismatched allogeneic HSCT. Diagnosis of LOH requires a high purity of recipient target cells. FACS is time consuming and also frequently prevented by rather nonspecific or unknown immune phenotype. The approach for recipient cell enrichment is based on HLA targeted complement-dependent cytotoxicity (CDC). Relative fluorescent quantification (RFQ) analysis of HLA intron length polymorphisms then allows analysis of HLA heterozygosity. The approach is exemplified in recent clinical cases illustrating the detection of an acquired allele loss. As illustrated in one case with DPB1, distinct HLA loci in donor and patient were sufficient for both proof of donor cell removal and evaluation of allele loss in the patient's leukemic cells. Results were confirmed using HLA-B RFQ analysis and leukemia-associated aberrant immunophenotype (LAIP) based cell sort. Both results confirmed suspected loss of HLA heterozygosity. Our approach complements or substitutes for FACS-based cell enrichment; hence it may be further developed as novel routine diagnostic tool. This allows rapid recipient cell purification and testing for loss of HLA heterozygosity before and after allogeneic HSCT in easily accessible peripheral blood samples.
