Number Of Marrow Cells Research Articles

Structural characterization of polysaccharides from Panax ginseng C. A. Meyer root and their triggered potential immunoregulatory and radioprotective activities

With people's increasing awareness of healthy diet, the diverse health-promoting functions of ginseng have been widely recognized. As one of the key functional components, ginseng polysaccharides have attracted increasing research interest. Here, three purified polysaccharide fractions, GPS-1a, GPS-1b, and GPS-2, were obtained from the root extract of Panax ginseng C. A. Meyer. Structurally, GPS-1a and GPS-1b were both linked in a → 6)-α-D-Glcp-(1 → pattern but composed of glucose and galactose in molar ratios of 9.76:0.24 and 9.81:0.19. In contrast, GPS-2 was composed of glucose, galactose, arabinose, rhamnose, and galacturonic acid in a molar ratio of 1.82:1.94:0.79:0.52:4.93. The main backbone consisted of →4)-α-D-GalpA-(1→, →4)-α-D-GalpA-6OMe-(1→, →3, 4)-α-D-GalpA-(1→, →3)-α-L-Rhap-(1 → linages, and its branches are composed of →5)-α-L-Araf-(1→, →4)-β-D-Galp-(1→, →2)-β-D-Glcp-(1→, α-D-GalAp-(1→. Benefitting from this structural variance, GPS-2 exhibited the most significant immunoregulatory and radioprotective efficacies. Specifically, GPS-2 promoted TLR2, NF-κB, and TRAF6 protein expression levels, thereby significantly improving macrophage phagocytosis, splenic lymphocyte proliferation, and stimulation of NO, IL-1β, IL-6, and TNF-α secretion, which activated RAW264.7 and splenic lymphocytes. The following radioprotection activity tests unveiled that GPS-2 increased the organ index, number of peripheral blood cells, cellularity of splenocytes, and bone marrow cell numbers in irradiated mice. This investigation revealed the contribution of polysaccharide structure characteristics to the bioactive expression and elucidated the potential utilization of GPS-2 as a radioprotective agent or immunomodulator.

The Expression of METTL14 in Patients with Newly Diagnosed Acute Myeloid Leukemia and Its Clinical Significance

To detect the expression of RNA methyltransferase 14(METTL14) in bone marrow of patients with newly diagnosed acute myeloid leukemia (AML), and to investigate the clinical and prognostic significance of METTL14 expression in newly diagnosed AML. Bone marrow samples were collected from 100 patients with newly diagnosed AML as observation group and 60 patients with iron deficiency anemia AML as control group. And collected the clinical data of the AML patients. Real-time quantitative PCR (qRT-PCR) was used to detect the expression level of METTL14 in AML and IDA patients. The relationship between the expression level of METTL14 and clinicopathological features, prognosis was analyzed. Kaplan-Meier curves were used to analyze the effect of METTL14 on overall survival (OS) in AML patients. Cox risk regression model was used to analyze the prognostic factors affecting in patients with AML. Compared with the control group, the expression of METTL14 was significantly increased in AML patients (P < 0.05). Compared with the METTL14 low-expression group, patients in the METTL14 high-expression group had advanced age, high bone marrow cell number, poor efficacyand poor prognosis(P < 0.05). The overall survival time of patients with the METTL14 high-expression group was significantly shorter than that of the low-expression group (P < 0.05). The high expression of METTL14 was an independent risk factor for poor prognosis in AML. METTL14 is significantly overexpressed in AML patients, and its correlated with poor clinicopathological features and poor prognosis. It can be used as a prognostic marker and potential therapeutie target for AML patients.

Impact of PARP Inhibitor and Platinum Therapy on Clonal Hematopoiesis

Poly (ADP-ribose) polymerase inhibitors (PARPi) are a promising new class of targeted therapy used in a variety of solid tumors with homologous recombination deficiencies (HRD). Data from clinical trials and observational studies have linked PARPi and platinum-based agents to therapy-related myeloid neoplasia (tMN). However, interpretation of these findings is limited by confounding variables including germline mutation status and prior therapeutic exposure. We and others have shown that the oncologic therapies known to confer an elevated risk of leukemia also promote the expansion of clonal hematopoiesis (CH) due to mutations of genes in the DNA damage response (DDR) pathway, including TP53, PPM1D and CHEK2. However, the extent to which CH is associated with targeted therapies such as PARPi is not clear. We hypothesized that exposure to PARPi confers a competitive advantage to hematopoietic stem/progenitor cells (HSPCs) containing mutations in DDR pathway genes. Moreover, we hypothesized that in patients with germline mutations in HRD genes, such as BRCA1/BRCA2genotoxic stress induced by PARPi or cytotoxic agents is increased, further enhancing the fitness of HSPCs carrying DDR gene mutations . To test these hypotheses, we performed longitudinal studies on 942 peripheral blood samples collected from 418 patients (ages 32 to 94) before and after treatment with PARPi (n = 100), carboplatin (n = 142), and untreated individuals (n = 176). Patients with lung, ovarian, uterine, prostate, and breast cancer who received PARPi or carboplatin on clinical trials or through standard of care were included. Patients were treated with a variety of PARPi including Olaparib (n=48), Talazoparib (n=33), Rucaparib (n = 13) and Niraparib (n = 6.) Of the 418 patients, 45 individuals harbored an HRD germline mutation. Samples were sequenced at a depth of 18,946x using a UMI-based panel including 9 common CH genes ( DNMT3A, TET2, ASXL1, TP53, PPM1D, SRSF2, SF3B1 and JAK2). Both carboplatin and PARPi treatment significantly increased the growth rate of CH mutations, driven almost entirely by mutations in DDR genes (Figure 1). Patients undergoing carboplatin treatment had significantly higher rates of mutational growth compared to those undergoing PARPi therapy (p-value = 5e -17). The strongest PARP-trapping member, Talazoparib, had the largest effect on the growth rate of CH mutations in DDR genes (p-value = 3e -9) but still lower compared to carboplatin. Surprisingly, DDR CH expansion was largely abrogated in patients harboring a pathogenic HRD germline variant for both carboplatin (p-value = 3e -5) and PARPi (p-value = 7e -10). To validate these findings, we developed a mouse model of TP53-mutatedCH by transplanting a limited number of Trp53 R172Hbone marrow cells along with congenic wildtype bone marrow into irradiated recipients. The percentage of Trp53 R172Hcells in the blood was measured by flow cytometry at baseline (6 weeks after transplantation) and after treatment with vehicle alone, Talazoparib or Olaparib daily, or cisplatin weekly, for 4 weeks. As expected, treatment with cisplatin resulted in a significant expansion of TP53-mutated cells (Figure 2). A non-significant trend to expansion of TP53-mutated cells also was observed after talazoparib, but not olaparib. We next modeled the effect of germline HRD mutations by generating bone marrow chimeras containing heterozygous Brca1-mutated HSPCs competed with Brca1-mutated HSPCs also carrying Trp53 R172H. Consistent with the human CH data, no expansion of TP53-mutated cells was observed after treatment with either cisplatin or Talazoparib, indeed there was a small, but significant decrease (Figure 2). Collectively, these data support the hypothesis that mutations in DDR genes provide a fitness advantage to HSPCs following PARP inhibitor and platinum therapy. However, compared to platinum therapy, PARPi shows less selective pressure on DDR CH. Importantly, these data strongly argue against our initial hypothesis that germline HRD variants enhance this fitness advantage. Indeed, our data suggest the opposite, with the fitness advantage of DDR gene-mutated HSPCs lost in cells with a heterozygous Brca1-mutations. These data suggest that PARPi therapy may have less of an impact on leukemia risk compared to carboplatin and in fact may show synergistic effects with HRD in blocking the competitive advantage of DDR CH during genotoxic stress.

A Mixture of Nucleosides and Nucleotides Increases Bone Marrow Cell and Peripheral Neutrophil Number in Mice Infected with Methicillin-Resistant Staphylococcus aureus

We studied the effects of a mixture of nucleosides and nucleotides on the peripheral neutrophil number and the proliferation of bone marrow cells in mice challenged with methicillin-resistant Staphylococcus aureus. BALB/c mice were fed a nucleotide-free 20% casein diet (control) or this diet supplemented with nucleosides and nucleotides orally (Expt. 1) or intraperitoneally (Expt. 2 and 3). On d 10, the mice were challenged intravenously with methicillin-resistant S. aureus (6.7 × 1012 colony forming units/L). In Expt. 1 and 2, numbers of total and differential counts of blood leucocytes were counted on d 0 (before), 1, 3 and 5 after the infection. In Expt. 3, 30 min before killing, bromodeoxyuridine (20 mg/kg), an analogue of thymidine, was administered intraperitoneally and its incorporation in the DNA synthetic phase of bone marrow cells was determined at 0 h (before), 3, 6 and 24 h after the infection. Mice fed the supplemented diet had higher (P < 0.05) leucocyte and neutrophil numbers on d 0 compared with the control group. The neutrophil numbers tended to be greater in the supplemented group at 1, 3 and 5 d after the infection. Intraperitoneal administration of nucleosides and nucleotides increased (P < 0.05) neutrophil numbers before and after the infection. Twenty-four h after the infection, incorporation of bromodeoxyuridine into the DNA synthetic phase of bone marrow cells in the group administered nucleosides and nucleotides was higher (P < 0.05) than in the control group. We conclude that, following methicillin-resistant S. aureus injection, intraperitoneal administration of a nucleoside-nucleotide mixture may stimulate bone marrow cell proliferation and increase the peripheral blood neutrophil numbers. Oral administration may elicit weaker effects.

Comprehensive study of health effects of plasma technology occupational environment: Exposure to high frequency and intensity noise and toxic gases

ObjectivesTo evaluate on animal models the health effects of the combined or separate exposure to main chemical and physical hazards of plasma-based material processing technology environment. Materials and methodsMale Wistar rats were exposed to actual levels of hazardous factors in plasma technology occupational environment: i.e., ozone and nitrogen oxides (O3 and NOx) in respective concentrations of 0.5 mg/m3 and 1.0 mg/m3 and high-frequency (1000–1600 Hz) of 112 dB intensity noise for 3 h/day, 5 days/week for 12 weeks, with a recovery period of 1 month. ResultsExposure to noise or its combination with chemical factors (ozone, nitrogen oxides) causes non-specific CNS changes testifying for significant excitation dominance, especially in the case of joint exposure. Histological examination of rats’ brain in experimental revealed a pronounced increase in blood filling of small vessels on the tenth day of the experiment, with subsequent intensification of vascular alterations and eventually to cerebral edema. The exposure to noise significantly reduced total thymus, bone marrow and spleen cell numbers and these was also more pronounced under the joint impact of noise and toxic gases. Thymus, but not bone marrow or spleen, mitotic activity was as well reduced under the same modes of exposure. Cytological investigation of film preparations of subcutaneous connective tissue revealed that joint exposure led to microcirculatory disorders, increased number of dark mast cells and reduced degranulation processes indicative of increased autoregulatory processes effective at microvasculature level. ConclusionsHigh-frequency and intensity noise is main stressor factor that has negative impact on CNS and immune system, morphology and functioning of hematopoietic organs (spleen, bone marrow, thymus) and connective tissue. Its negative impact is significantly potentiated by concurrent exposure to ozone and nitrogen oxide, while exposure only to these toxic gases has no significant effect on the above targets.

Quantitative Assessment of Bone Marrow Activity Using 18 F-FLT PET in Aplastic Anemia and Myelodysplastic Syndromes.

Peripheral cytopenias are typical of blood test abnormalities associated with a variety of conditions, including aplastic anemia (AA) and myelodysplastic syndromes (MDSs). We prospectively investigated the feasibility of quantitative analysis of whole-body bone marrow activity using PET with 3'-deoxy-3'- 18 F-fluorothymidine ( 18 F-FLT) in AA and MDS. Sixty-eight patients with cytopenia underwent 18 F-FLT PET/MRI scan, with simultaneous bone marrow aspiration and biopsy for hematopoiesis evaluation. SUVs were measured in the vertebrae (Th3, 6, and 9 and L3), bilateral iliac crests, and extremities. SUV and bone marrow pathology were compared between AA and MDS and analyzed in relation to severity of AA and prognosis of MDS. Of the 68 patients with cytopenia, 12 were diagnosed with AA, 27 with MDS, 12 with bone marrow neoplasia, 2 with myelofibrosis, and 15 with other conditions. Iliac 18 F-FLT SUVs were significantly correlated with bone marrow cell numbers and cell density ( r = 0.47, P < 0.001 and ρ = 0.65, P < 0.001, respectively). There was a significant positive correlation between iliac and vertebral SUVs in AA and MDS ( r = 0.65, P < 0.05 and r = 0.70, P < 0.001, respectively), and the slope of the regression line was significantly steeper in AA than in MDS ( P < 0.05). In AA patients, vertebral 18 F-FLT SUVs significantly decreased with disease progression, and in MDS patients, higher whole-body 18 F-FLT uptake was associated with shorter overall survival (hazards ratio, 3.18; 95% confidence interval, 1.07-9.47; P = 0.037). Quantitative whole-body bone marrow imaging using 18 F-FLT PET helps distinguish AA from MDS and assess the severity of AA and prognosis of MDS.

Synchrotron-based characterization of arthroprosthetic CoCrMo particles in human bone marrow

Particles released from cobalt-chromium-molybdenum (CoCrMo) alloys are considered common elicitors of chronic inflammatory adverse effects. There is a lack of data demonstrating particle numbers, size distribution and elemental composition of bone marrow resident particles which would allow for implementation of clinically relevant test strategies in bone marrow models at different degrees of exposure. The aim of this study was to investigate metal particle exposure in human periprosthetic bone marrow of three types of arthroplasty implants. Periprosthetic bone marrow sections from eight patients exposed to CoCrMo particles were analyzed via spatially resolved and synchrotron-based nanoscopic X-ray fluorescence imaging. These analyses revealed lognormal particle size distribution patterns predominantly towards the nanoscale. Analyses of particle numbers and normalization to bone marrow volume and bone marrow cell number indicated particle concentrations of up to 1 × 1011 particles/ml bone marrow or 2 × 104 particles/bone marrow cell, respectively. Analyses of elemental ratios of CoCrMo particles showed that particularly the particles’ Co content depends on particle size. The obtained data point towards Co release from arthroprosthetic particles in the course of dealloying and degradation processes of larger particles within periprosthetic bone marrow. This is the first study providing data based on metal particle analyses to be used for future in vitro and in vivo studies of possible toxic effects in human bone marrow following exposure to arthroprosthetic CoCrMo particles of different concentration, size, and elemental composition.Graphical abstract

Androgen Receptor Signaling Positively Regulates Monocytic Development.

Myeloid cells are critical cells involved in the orchestration of innate and adaptive immune responses. Most myeloid cells derive from the adult bone marrow in a process called myelopoiesis, a tightly controlled process that ensures constant production of myeloid cells. Sex differences in myeloid cell development have been observed; males exhibit greater monocytic differentiation in the bone marrow, and men have increased blood monocyte numbers when compared to women. Here we use a genetic mouse model of myeloid androgen receptor (AR) knockout (MARKO) and pharmacological inhibition of AR to investigate the role of androgen signaling in monocytic differentiation. We observe that although myeloid AR signaling does not influence total bone marrow cell numbers, it does affect the composition of the bone marrow myeloid population in both homeostatic and emergency settings. Genetic deletion of AR in myeloid cells led to reduced monocytic development in vivo. Similarly, pharmacologic inhibition of AR signaling in vitro reduced monocytic development. However, alteration in monocytic differentiation in the absence of AR signaling did not lead to reduced numbers of circulating myeloid cells, although MARKO male mice display reduced ratio of classical to non-classical monocytes in the blood, implying that blood monocyte subsets are skewed upon myeloid AR deletion. Our results suggest that the sex differences observed in monocytic differentiation are partly attributed to the positive role of the androgen–AR axis in regulating monocytic development directly at the myeloid cell level. Furthermore, we have identified a novel role for AR in regulating blood mature monocyte subset turnover. Investigating how androgen signaling affects monocytic development and monocyte subset heterogeneity will advance our understanding of sex differences in monocytic function at homeostasis and disease and can ultimately impact future therapeutic design targeting monocytes in the clinic.

VEGF/CDK2 are involved in diabetic organ regeneration

Aim/hypothesisDiabetes is a hyperglycaemic disease treated by a set of allopathic drugs and natural biomolecules along with many variety of stem cell. We aim to investigate the role of these drugs in targeting common protein molecule in diabetes and its associated disease. We also aim to investigate the organ degeneration mechanistic pathway in diabetes. MethodWe have generated diabetes using streptozotocin injection and treated them using bone marrow transplantation and curcumin administration. The organs were studied histopathologically and by immunofluorescence analysis while drugs were studied Pharmacogenomically. ResultMice injected with streptozotocin have higher glucose and lower insulin, islet number/diameter, bone marrow cell number compared to control and bone marrow transplanted and curcumin administered mice. Histopathology staining demonstrates damaged morphology of pancreas, kidney, brain and cardiac muscle. Further, upon comparison of all allopathic and ayurvedic drugs used for diabetes several protein targets have been identified by reverse pharmacophore analysis using PharmMapper. VEGF, CDK2, insulin receptor, HSp90, eNOS, Fructose1,6 bisphosphatase, neprilysin, AchE, MAPK are several common protein targets of anti-diabetic drugs. ConclusionThis article demonstrates that VEGF and CDK2 are critical marker in organ damage in diabetes as well as organ regeneration.

Hematopoietic Stem Cells

The small population of pluripotent hematopoietic stem cells (PHSCs) in the bone marrow consists of short-term reconstituting cells (STRCs) and long-term reconstituting cells (LTRCs), based on how quickly the transplanted cells can produce progeny in an irradiated recipient. They can be “purified” using a combination of cell size; density; fluorescent dye uptake; resistance to cytotoxic chemicals; and cell-surface markers including Thy 1.1 (T), Sca-1 (S), c-kit (K), lineage (L), CD38 (38), and CD34 (34). Using five-color fluorescence-activated cell sorting the long term, very primitive mouse LTRCs are L-/lo, S+, K+, 38+, 34-, and appear to mature to L-/lo, S+, K+, 38+, 34+ cells and then to L-/lo, S+, K+, 38-, 34+; thus, STRCs acquire CD34 and lose CD38 on maturation from CD34- LTRCs. CD34 has been used to isolate PHSCs for human transplantation studies; therefore, the LTRC may be lost during this procedure. Experimental transplantation studies indicate that the best reconstitution occurs when both cell populations are present, the more mature cells activating the immature cells after myeloablation, whereas the mature cells provide negative control in normal animals. Functionally the type of assay that has been most widely used for the quantitation of mouse stem cells is the in vivo repopulating assay. Different numbers of donor cells are combined with a standard number of normal bone marrow cells. The normal cells protect against the immediate effects of myeloablation and compete with the donor stem cells. The proportions of mature cells derived from the donor stem cells are determined by the detection of a donor-specific marker, such as an isoenzyme, Y-chromosome, or congenic antigen. Similarly, using limiting dilution transplant of a donor test population of cells and a standard number of stem cell—compromised serially transplanted cells, the relative contribution of the donor cells is measured as a competitive repopulating unit. Finally, the repopulating stem cell unit assay using complete myeloablation and busulfan-treated bone marrow radioprotective support cells provides comparatively rapid and sensitive detection of the very small numbers of LTRCs present in limiting dilution transplants. This procedure utilizes busulfan because it appears preferentially stem cell toxic, and it provides radioprotective support cells that are unable to compete effectively with normal donor stem cells in the population under investigation. Stem cells are selected based on their ability to produce both lymphoid and myeloid repopulation in severely ablated mice, rather than competitive ability.

Tarin stimulates granulocyte growth in bone marrow cell cultures and minimizes immunosuppression by cyclo-phosphamide in mice.

Chemotherapeutic drugs, such as cyclophosphamide, cause severe immunosuppression and patients become susceptible to infections. Based on this, the immunomodulatory potential of tarin, a lectin from Colocasia esculenta, was evaluated in bone marrow cell cultures and in cyclophosphamide-immunosuppressed mice. Tarin promoted maintenance of hematopoietic progenitors and repopulation of Gr1 cells in vitro which was supported by in vivo results. In immunosuppressed mice, tarin increased bone marrow cell numbers and altered cell profile distribution by enhancing the frequency of Gr1+ progenitors, including Ly6-CintLy6-Glo, and anticipating their proliferation/differentiation in mature cells, especially Ly6-CloLy6-Ghi. Bone marrow cells harvested from tarin-treated immunosuppressed mice proliferated in response to GM-CSF or G-CSF in vitro and, the low numbers of bone marrow cells in the G0 phase, combined with a high number cells undergoing apoptosis confirmed that tarin promoted a faster and intense proliferation/differentiation, even in the presence of CY-induced toxicity. As a result, tarin minimized leukopenia in immunosuppressed mice promoting a faster recovery of peripheral leucocytes and protected erythroid bone marrow cells from CY-cytotoxicity in a dose-dependent manner. Data suggest that tarin could be considered a potential adjuvant to decrease leukopenia and possibly ameliorate anemia, if carefully evaluated in human cancer cell lineages and in clinical trials.

Loss of the homologous recombination gene rad51 leads to Fanconi anemia-like symptoms in zebrafish

RAD51 is an indispensable homologous recombination protein, necessary for strand invasion and crossing over. It has recently been designated as a Fanconi anemia (FA) gene, following the discovery of two patients carrying dominant-negative mutations. FA is a hereditary DNA-repair disorder characterized by various congenital abnormalities, progressive bone marrow failure, and cancer predisposition. In this report, we describe a viable vertebrate model of RAD51 loss. Zebrafish rad51 loss-of-function mutants developed key features of FA, including hypocellular kidney marrow, sensitivity to cross-linking agents, and decreased size. We show that some of these symptoms stem from both decreased proliferation and increased apoptosis of embryonic hematopoietic stem and progenitor cells. Comutation of p53 was able to rescue the hematopoietic defects seen in the single mutants, but led to tumor development. We further demonstrate that prolonged inflammatory stress can exacerbate the hematological impairment, leading to an additional decrease in kidney marrow cell numbers. These findings strengthen the assignment of RAD51 as a Fanconi gene and provide more evidence for the notion that aberrant p53 signaling during embryogenesis leads to the hematological defects seen later in life in FA. Further research on this zebrafish FA model will lead to a deeper understanding of the molecular basis of bone marrow failure in FA and the cellular role of RAD51.

A PDZ-Binding Motif in DCAF7 Maintains Hematopoietic Homeostasis and Prevents Malignancy in DS-AMKL

Down syndrome-associated acute megakaryoblastic leukemia (DS-AMKL) affects 1 in 500 children with DS and associates uniformly with somatic mutations in GATA1. DS-AMKL is often treated successfully; however, treatments can cause long-term side effects, such that mechanistic insights and targeted therapies are needed.Recent studies report a recurring L340F mutation in DCAF7 in DS-AMKL, at the C-terminus of the protein. A highly conserved scaffold protein, DCAF7 is a cofactor for the chromosome 21-encoded kinase, DYRK1A, and shares 100% protein sequence homology among vertebrates. In our efforts to characterize the normal function of DCAF7 in hematopoiesis and its contribution to AMKL, we have identified an unreported PDZ-binding motif (PBM) in DCAF7, which is perturbed by the L340F mutation. Overexpression of mutant (DCAF7mut), but not wild type DCAF7 (DCAF7wt), in a mouse model of DS with a Gata1 mutation leads to rapid onset of dysplastic disease characterized by peripheral blood leukopenia and thrombocytopenia, with normal red cell numbers. Bone marrow of affected mice harbors abnormal, hyperproliferative erythroid progenitors and diminished, hyperchromatic megakaryocytes, with stark depletion of granulocytic and lymphoid lineages and widespread edema. Splenic architecture is grossly abnormal, with a complete absence of white pulp, proliferation of erythroid precursors, and significant numbers of apoptotic, hyperchromatic megakaryocytes.Conversely, targeted deletion of Dcaf7 in hematopoiesis shifts the myeloid-erythroid balance toward the myeloid lineage, with increased granulopoiesis and hyperchromatic megakaryocytes in the bone marrow and lower white blood cell numbers and dramatic thrombocytopenia in the peripheral blood. Splenic architecture is largely normal, although flow cytometry reveals defective B- and T- cell development.The bone marrow knockout phenotype of Dcaf7 is strongly reminiscent of the cellular phase of primary myelofibrosis (PMF). Combined with bone marrow overexpression phenotypes, which resemble hemophagocytic syndrome, our data suggest that DCAF7 is critically important for maintaining a healthy bone marrow environment and regulating blood cell lineage choice. In order to identify functional binding partners of the DCAF7 PDZ-binding motif (PBM) that may support these functions, we performed IP mass spectrometry using DCAF7wt or DCAF7mut as bait and found two highly conserved PDZ-domain containing scaffold proteins, PAR3 and PDLIM5, that interact robustly with DCAF7wt, but not with DCAF7mut. While both DCAF7wt and DCAF7mut are able to associate with DYRK1A, the ability of DCAF7 to bring DYRK1A activity to the PAR3 and PDLIM5 signaling hubs affects the composition of DCAF7-scaffolded transcriptional complexes and downstream transcriptional outcomes.Through RNA-sequencing and Ingenuity Pathways Analysis, overexpression of both DCAF7wt and DCAF7mut in mouse bone marrow leads to the induction of interferon response genes; however, DCAF7wt also induces a pro-apoptotic gene program, whereas DCAF7mut instead promotes survival gene expression and developmental reprogramming. Consistently, expression of DCAF7wt in bone marrow progenitors causes rapid expansion and increased initial colony forming ability, but decreased replating potential compared with vector control. Conversely, expression of DCAF7mut causes increased proliferation and colony formation accompanied by enhanced replating, indicating greater self-renewal conferred by DCAF7mu.Our data indicate DCAF7 regulates the balance of proliferation, differentiation and apoptosis in hematopoietic cells and that the PBD of DCAF7 is critically important for these functions, connecting DCAF7 to signaling hubs that translate environmental cues into appropriate transcriptional outcomes. Furthermore, DCAF7 disruption causes hematopoietic disease reminiscent of human pathology, suggesting it may have broad significance in hematopoietic malignancy. Finally, our data support a model wherein DCAF7 mutation facilitates the development of malignancy by promoting the survival and proliferation of mutant clones in the bone marrow and fundamentally altering the bone marrow microenvironment. DisclosuresNo relevant conflicts of interest to declare.

Evaluation of oxidative stress, antioxidant enzyme activities, and genetic damage in sprayers exposed to pesticides

The potential immunotoxic effect of the carbamate pesticide aminocarb on murine bone marrow cell subpopulations was evaluated by flow cytometry, C57B16 mice were exposed by gavage to sublethal doses of the pesticide and lymphocyte precursors from bone marrow population were stained with PNA lectin and a panel of monoclonal antibodies against cell surface antigens. In regard to the microenvironment-dependent maturation of B lymphocytes, the pesticide effect on the lymphoproliferative potential of bone marrow was assessed by marrow transplantation from aminocarb-exposed donor mice to normal, syngeneic, X-irradiated recipient mice. The sublethal exposure of 0.08–5.0 mg/kg body wt aminocarb to donor animals did not affect regenerating bone marrow in the recipient mice. No marked effect on bone marrow cell number was noted in pesticide-exposed animals. However, a marked shift in surface IgM density on marrow B cells was noted at 0.08 and 0.31 mg/kg body wt aminocarb. This was correlated with decreased cell frequency in G0G1 phase and increased frequency of cells in the S phase of the cell cycle. Thus, altered maturation of B lymphocytes, expressed as a shift in the density of surface IgM on mature B cells and not the lymphocyte count, was related to the direct effect of aminocarb and/or to the pesticide-related changes in bone marrow microenvironment. Overall, exposure to the carbamate pesticide aminocarb activated the cell maturation process in bone marrow.

To condition or not to condition-That is the question: The evolution ofnonmyeloablative conditions for transplantation.

In 1985, Eugene Cronkite and his colleagues published, in Experimental Hematology, data indicating that five consecutive "transfusions" of large numbers of marrow cells significantly increase the number of donor-derived cells detected by day 10 of a spleen colony-forming assay, the most primitive hematopoietic cells detectable at that time, present in the host for as long as 2months posttransfusion (Cronkite EP, Bullis JE, Brecher G. Marrow transfusions increase pluripotent stem cells in normal hosts. Exp Hematol 1985;13:802-805). These data provided the first evidence that donor hematopoietic stem cells (HSCs) may persist invivo for some time in recipients when transfused and not transplanted, that is, not subjected to treatments that deplete their marrow niches of endogenous HSCs. The limited technology available at the time prevented Dr. Cronkite from pursuing this observation into the development of nonmyeloablated transplantation procedures, and his experiment, as well as the term bone marrow transfusion, has since been long forgotten. In recent years, the scientific need to clarify HSC functions in nonstressed hosts and the clinical need to develop transplantation procedures with levels of morbidity/mortality acceptable for curing inherited hematologic disorders have inspired the search for nonmyeloablative transplantation procedures, including methods that "outcompete" endogenous host HSCs such as those pioneered by Dr. Cronkite's experiments using high transfusion doses. This review describes the technical progress made since Dr. Cronkite's insightful work, which has finally found its path to the clinic.

Use of raw Euphorbia tirucalli extract for inhibition of ascitic Ehrlich tumor.

to evaluate the effect of the Euphorbia tirucalli hydroalcoholic extract (ETHE) on the development of Ehrlich Tumor, in its ascitic form. we intraperitoneally inoculated 15 Swiss mice with 10.44 x 107 cells of Ehrlich Tumor and divided them in two groups one day after: ETHE Group (eight mice), treated with a dosage of 125 mg/kg/day of EHTE for five days; and Control Group (seven mice), treated only with 0.9% isotonic saline solution over the same period. The treatment was done by gavage. Ten days after inoculation, four mice from each group were sacrificed for quantification of tumor cell number, ascitic fluid volume and bone marrow cell number. The remaining animals were maintained to evaluate survival. The ascitic fluid volume and the tumor cell number were decreased in the ETHE group when compared with the control group, but with no statistical significance. On the other hand, survival was higher in the ETHE group, as well as the number of bone marrow cells. Treatment with ETHE after inoculation of Ehrlich Tumor decreases its development and increases survival and the bone marrow cellularity, thus reducing the myelosuppression present in the Ehrlich Tumor bearing mice.

Prostaglandin E2 Promotes the Sequential Recovery of Bone Marrow Vasculature and the Megakaryocyte Lineage Following Radiation Injury

The sinusoidal vascular niche is the site of terminal megakaryopoiesis and platelet production in the bone marrow. The association of megakaryocytes (MKs) with the sinusoidal endothelium and microenvironmental cues from the vascular niche are critical for efficient thrombopoiesis. Thrombocytopenia is an important clinical problem that can cause significant morbidity for patients receiving genotoxic treatments such as chemotherapy and radiation therapy. Following myeloablative bone marrow injury, the recovery of both hematopoietic stem and progenitor cells (HSPCs) and the MK lineage is dependent on marrow vascular recovery (Hooper et al. Cell Stem Cell 2009, Avecilla et al. Nature 2004). Accordingly, we reasoned that improving recovery of the vasculature might serve as a rational therapeutic strategy for thrombocytopenia stemming from cytotoxic treatments. We focused our efforts on the arachidonic acid derivative prostaglandin E2 (PGE2), which regulates numerous physiologic processes including microvascular endothelial cell proliferation and angiogenesis. Within the context of the bone marrow, PGE2 also has diverse roles in HSPC expansion, survival, and trafficking. Importantly, systemic administration of the long-acting analogue 16,16-dimethyl PGE2 (dmPGE2) improves hematopoietic recovery and enhances overall survival following total body irradiation (TBI) in mice (Hoggatt et al. Blood Cells 2013). Intriguingly, following sublethal TBI, dmPGE2-induced recovery of peripheral platelets precedes restoration of red blood cells and neutrophils (Porter et al. Stem Cells 2013). We hypothesized that the preferential recovery of platelets following dmPGE2 administration is mediated by accelerated vascular recovery in the marrow. We tested this in a murine model of sublethal radiation injury in which we had previously characterized MK lineage kinetics following 4Gy TBI (Niswander et al. Blood 2014). First, the kinetics of MK progenitors (MKPs), maturing MKs, and platelets were determined at days 5, 7, and 10 in mice receiving 6mg/kg dmPGE2 or vehicle at 48 and 72 hours post-4Gy TBI. Mice receiving dmPGE2 had an early decrease in both MKPs and platelets, with no change in MKs at day 5 post-TBI. However, by day 10, dmPGE2-treated mice demonstrated significantly enhanced recovery of both MKs and circulating platelets compared to controls, but no difference in the number of immunophenotypic HSPCs. Having confirmed enhanced recovery of the MK lineage with dmPGE2 treatment, we next sought to characterize vascular recovery. Marked injury and dilation of the marrow sinusoidal vasculature occurs rapidly following TBI. Immunohistochemistry with morphometric analysis revealed no change in vascular dilation at day 5 post-TBI with dmPGE2 treatment. However, mice receiving dmPGE2 exhibited a more than 25% improvement in vascular dilation that was first evident at day 7 and was sustained at day 10 post-TBI. While vascular dilation post-TBI has been posited to correlate with loss of surrounding hematopoietic cells, improvement in vascular dilation with dmPGE2 treatment is contrastingly evident in the setting of slightly fewer (day 7) or equivalent (day 10) numbers of nucleated marrow cells compared to irradiated control mice. These data suggest that dmPGE2-mediated recovery of the vasculature coincides temporally with the accelerated onset of MK lineage recovery. In addition, immunohistochemical morphometric analyses indicate that dmPGE2-treated mice had an 80% increase in maturing MKs in physical contact with the vascular endothelium, the site of platelet production, at day 10 post-TBI. Taken together, these data support the concept that dmPGE2-mediated marrow vascular recovery following radiation injury enhances the recovery of the MK lineage and accelerates the production of circulating platelets. Modulation of vascular recovery with dmPGE2 may be a promising therapeutic strategy for thrombocytopenia secondary to myeloablative bone marrow injury. DisclosuresCalvi:Fate Therapeutics: Patents & Royalties.

Aryl hydrocarbon receptor activation enhances total but not antigen-specific IgA secretion in Leishmania major-infected mice (MUC9P.748)

Abstract Secretory IgA plays a critical role in neutralizing enteric toxins and protecting against some mucosal pathogens. In mice, activation of the aryl hydrocarbon receptor (AhR) with a single dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has been shown to transiently decrease total fecal IgA levels but increase antigen-specific IgA levels after mucosal antigen challenge. The goal of this study was to determine if TCDD exposure would alter secretion of antigen-specific IgA levels after systemic pathogen challenge. Female C57Bl/6 mice were administered TCDD at 0 or 40 μg/Kg (po) one day prior to infection with Leishmania major promastigotes. Three weeks later, IgA levels were measured by ELISA. L. major-specific IgA levels in serum were readily detected but unchanged by TCDD treatment. L. major-specific lgA was undetected in the feces of any animal. In contrast, total fecal IgA levels (OD) were significantly increased by 34% in TCDD treated animals (p &amp;lt; 0.05). Total spleen and bone marrow cell numbers were reduced significantly in TCDD-treated mice. The expression of IgM heavy chain, IgJ chain, or kappa light chain in these cells (measured by RT-RT-PCR) was unchanged by TCDD treatment. In contrast, the expression of IgA heavy chain was significantly reduced by approximately 50% in TCDD-treated animals. These results suggest that AhR activation enhances bulk IgA secretion but does not enhance secretion of antigen-specific IgA following systemic pathogen challenge.

Synergy of bone marrow transplantation and curcumin ensue protective effects at early onset of diabetes in mice.

The aim of this study was to investigate the early onset effects of diabetes on pro-angiogenic signaling pathway, total number of bone marrow cells, organs (pancreas and kidney) damage and the reversal effect of diabetes by combinatorial treatment of curcumin and bone marrow transplantation in streptozotocin (STZ) induced diabetic mice. In the present study, Streptozotocin induced diabetic mice were transplanted with bone marrow cells (2 × 10(6) ) followed by the administration of curcumin (80 mg/kg bodyweight). Effect of diabetes on the different organs was studied by H&E, Western blotting and immunofluorescence using vascular endothelial growth factor (VEGF), platelet/endothelial cell adhesion molecule (PECAM), insulin, Caspase-9 and Caspase-3 antibodies. The effect of diabetes results in the reduction of the total cell number and viability of the bone marrow cells, organ degeneration and lower VEGF/PECAM expression. However, transplantation with normal bone marrow cells significantly reduced the blood glucose levels (above normal range) and initiated the organ regeneration via the VEGF/PECAM mediated manner. Curcumin treatment further reduced the blood glucose level (near normal); and accelerated the organ regeneration, enhanced VEGF/PECAM expression and decreased caspase expression level in the organs. Curcumin also had a protective role against the glucotoxicity test performed on the bone marrow cells. This study suggests that bone marrow transplantation and curcumin administration is an effective treatment in reversing the early onset effects of diabetes via the VEGF/PECAM signaling pathway.

57. Changes in profiles of brain cytokines associated with enhanced recruitment of bone marrow-derived cells into the brain in senescence-accelerated mice

Neurodegenerative diseases may be associated with altered immune-brain interaction. The senescence-accelerated mouse prone 10 (SAMP10) is a model of brain aging that undergoes an early onset cerebral neurodegeneration following immune senescence. We hypothesized that the brain-immune interaction is perturbed in SAMP10 mice. We created 4 groups of radiation chimeras by intra-bone marrow bone marrow transplantation using young and aged SAMP10 and B6 mice as recipients with 5-week-old GFP transgenic B6 mice as donors and analyzed chimeras immunohistochemically 4 months later. Donor’s marrow-derived cells of the myeloid lineage entered discrete brain regions through the attachments of choroid plexus. In chimeric mice with aged SAMP10 mice being used as recipients, larger numbers of marrow cells entered more brain regions than the other groups, especially in the diencephalon. We performed multiplex cytokine assays to determine tissue concentration of 10 cytokines in the diencephalon prepared from young and aged SAMP10 and B6 mice. Aged SAMP10 mice exhibited higher tissue concentrations of IL-6, G-CSF, CCL11, CXCL1 and CXCL10 than the other groups of chimera. Immunohistochemistry revealed that these cytokines were expressed in astrocytic processes of the attachments of choroid plexus, periventricular astrocytes, tanycytes, and hypothalamic neurons. Therefore, the enhanced recruitment of bone marrow-derived cells into the brain may be associated with region-specific changes in profiles of tissue cytokine microenvironment, which represents a manifestation of perturbed brain-immune interaction in SAMP10 mice.