Spleen Cellularity Research Articles

Deletion of Nono, a Novel Double-Strand Break Repair Factor, Leads to Hematopoietic Stem Cell Defects

Three genes, SFPQ, NONO, and PSPC1 encode for a small family of tandem RRM domain-containing proteins with diverse functions in RNA processing, transcription, and DNA repair. Our previous work has shown that an SFPQ•NONO complex promotes a distinct sub-pathway of non-homologous end joining (NHEJ) in vitro, implicating it as a novel double-strand break (DSB) repair factor. Consistently, attenuation of NONO function in human fibroblasts leads to partial radiosensitivity, delayed resolution of DNA repair foci, and an increase in radiation-dependent chromosome aberrations. To investigate the in vivo function of this complex, we knocked out Nono, the mouse homolog of the human NONO gene. We hypothesized that a DSB repair deficiency would lead to stem cell exhaustion, sensitivity to DNA damaging agents, or both. First, we investigated hematopoietic stem cells (HSCs), which are known to be sensitive to impairment of DNA repair. Nono-deficient (gt/0) mice showed reduced bone marrow and spleen cellularity, potentially due to reduced body size. The proportion of HSCs (LSK-SLAM) in relation to total bone marrow cells was similar in Nono-deficient and wild type (WT) mice, and bone marrow cell cultures yielded similar numbers and types of colonies. However, HSCs from Nono-deficient mice showed severe impairment in competitive repopulation assays in primary and secondary recipients. HSCs from gt/0 mice also displayed significantly higher levels of ROS and proliferation (accessed via BrdU incorporation), as well as higher percentage of Ki-67 positivity when compared to HSCs from WT mice. Together, these results indicate that Nono-deficient HSCs exhibit impaired capacity for self-renewal that may be caused by excessive HSC proliferation, a primary defect in response to genotoxic stress or a combination of both. To further elucidate this phenomenon, we investigated potential differences in sensitivity to the DNA crosslinking agent mitomycin C (MMC) and radiation. As previously observed in other genomic instability models (e.g., Fanconi anemia), colony-forming-cells from Nono-deficient mice were highly sensitive to MMC and X-rays (2 Gγ). Utilizing FACS-sorted HSCs, we determined that NONO protein levels are induced after X-ray treatment, and that Nono-deficient HSCs show delayed resolution of γ-H2AX foci and increased apoptosis. We also evaluated changes in the testis, another organ frequently affected by DNA repair gene mutations. Accordingly, testes of Nono-deficient mice showed growth retardation that became apparent between 18 and 26 days of postnatal development. The co-occurrence of hematopoietic and germ cell defects is reminiscent of other DNA repair gene defects, including those seen in Fanconi anemia. Collectively, our data identify NONO as a novel and important regulator of both HSC maintenance and germ cell function. Its potential effect in other organ systems and mechanism of action are under investigation. DisclosuresNo relevant conflicts of interest to declare.

Immunotoxicity of silicon dioxide nanoparticles with different sizes and electrostatic charge.

Silicon dioxide (SiO2) nanoparticles (NPs) have been widely used in the biomedical field, such as in drug delivery and gene therapy. However, little is known about the biological effects and potential hazards of SiO2. Herein, the colloidal SiO2 NPs with two different sizes (20 nm and 100 nm) and different charges (L-arginine modified: SiO2EN20[R], SiO2EN100[R]; and negative: SiO2EN20[−], SiO2EN100[−] were orally administered (750 mg/kg/day) in female C57BL/6 mice for 14 days. Assessments of immunotoxicity include hematology profiling, reactive oxygen species generation and their antioxidant effect, stimulation assays for B- and T-lymphocytes, the activity of natural killer (NK) cells, and cytokine profiling. In vitro toxicity was also investigated in the RAW 264.7 cell line. When the cellularity of mouse spleen was evaluated, there was an overall decrease in the proliferation of B- and T-cells for all the groups fed with SiO2 NPs. Specifically, the SiO2EN20(−) NPs showed the most pronounced reduction. In addition, the nitric oxide production and NK cell activity in SiO2 NP-fed mice were significantly suppressed. Moreover, there was a decrease in the serum concentration of inflammatory cytokines such as interleukin (IL)-1β, IL-12 (p70), IL-6, tumor necrosis factor-α, and interferon-γ. To elucidate the cytotoxicity mechanism of SiO2 in vivo, an in vitro study using the RAW 264.7 cell line was performed. Both the size and charge of SiO2 using murine macrophage RAW 264.7 cells decreased cell viability dose-dependently. Collectively, our data indicate that different sized and charged SiO2 NPs would cause differential immunotoxicity. Interestingly, the small-sized and negatively charged SiO2 NPs showed the most potent in vivo immunotoxicity by way of suppressing the proliferation of lymphocytes, depressing the killing activity of NK cells, and decreasing proinflammatory cytokine production, thus leading to immunosuppression.

Haematopoiesis radioprotection in Balb/c mice by an aqueous mycelium extract from the Basidiomycete Pleurotus ostreatus mushroom

The study examined the radioprotective activity of an aqueous extract from Pleurotus ostreatus mycelium administered to Balb/c mice. Male mice were whole-body irradiated on day 0 (60Co, at 0.43 Gy/min) and divided into two groups. The extract was administered intraperitoneally to one group (100 mg/kg) on days − 10 to − 6 and − 2 to +1 with respect to the irradiation. The irradiated-control group was injected with saline solution; non-irradiated mice were used as negative controls. The radioprotective effect was evident by increases in bone marrow cellularity (5.1 × 106/femur vs. 1.1 × 106/femur in saline-control mice, p < 0.05), leucocyte counts (10.5 × 109/L vs. 4.5 × 109/L, p < 0.05), and spleen cellularity (11.2 × 107/spleen vs. 6.2 × 107/spleen, p < 0.05). The extract stimulated macrophage phagocytic activity as judged by a faster rate of carbon clearance in terms of absorbance ratios (1.62 vs. 2.01, p < 0.05). Therefore, this extract may be a candidate therapeutic agent with radioprotective activity for haematopoiesis damage, particularly to cells involved in immune function.

Early and late B cell immune responses in lethal and self-cured rodent malaria

ICR mice have heterogeneous susceptibility to lethal Plasmodium yoelii yoelii 17XL from the first days of experimental infection as evidenced by the different parasitemia levels and clinical outcomes. This mouse model has revealed specific immune responses on peripheral blood correlating with the infection fate of the animals. To search for immune-markers linked to parasitemia we examined B lymphocytes in organs of the immune system as key effectors of rodent immunity against malaria. To determine changes in immune cellularity fostered by the different prognostic parasitemia we examined B cell subsets in low (<15%) and high (>50%) parasitized mice during the first days of the infection. In the case of surviving mice, we studied the preservation of memory immune response 500 days after the primary P. yoelii challenge. Correlating with the parasitemia level, it was observed an increase in total cellularity of spleen during the first week of infection which remained after 16 months of the infection in surviving animals. B cell subsets were also modified across the different infection fates. Subpopulation as follicular B cells and B-1 cells proportions behaved differently depending on the parasitemia kinetics. In addition, peritoneal cavity cells proliferated in response to high parasitemia. More significantly, P. yoelii -specific memory B cells remained in the spleen 500 days after the primo-infection. This study demonstrates that B cell kinetics is influenced by the different parasitemia courses which are naturally developed within a same strain of untreated mice. We show that high levels of parasitemia at the beginning of infection promote an extremely fast and exacerbate response of several cell populations in spleen and peritoneal cavity that, in addition, do not follow the kinetics observed in peripheral blood. Furthermore, our results describe the longest persistence of memory B cells long time upon a single malaria infection in mice.

3,4-methylenedioxymethamphetamine (MDMA--Ecstasy) decreases neutrophil activity through the glucocorticoid pathway and impairs host resistance to Listeria monocytogenes infection in mice.

Ecstasy is the popular name of the abuse drug 3,4-methylenedioxymethamphetamine (MDMA) that decreases immunity in animals. The mechanisms that generate such alterations are still controversial. Seven independent pharmacological approaches were performed in mice to identify the possible mechanisms underlying the decrease of neutrophil activity induced by MDMA and the possible effects of MDMA on host resistance to Listeria monocytogenes. Our data showed that MDMA (10 mg kg(-1)) administration decreases NFκB expression in circulating neutrophils. Metyrapone or RU-486 administration prior to MDMA treatment abrogated MDMA effects on neutrophil activity and NFκB expression, while 6-OHDA or ICI-118,551 administration did not. As MDMA treatment increased the plasmatic levels of adrenaline and noradrenaline, propranolol pre-treatment effects were also evaluated. Propranolol suppressed both MDMA-induced increase in corticosterone serum levels and its effects on neutrophil activity. In a L. monocytogenes experimental infection context, we showed that MDMA: induced myelosuppression by decreasing granulocyte-macrophage hematopoietic progenitors (CFU-GM) in the bone marrow but increased CFU-GM in the spleen; decreased circulating leukocytes and bone marrow cellularity and increased spleen cellularity; decreased pro-inflammatory cytokine (IL-12p70, TNF, IFN-γ, IL-6) and chemokine (MCP-1) production 24 h after the infection; increased the production of pro-inflammatory cytokines and chemokines 72 h after infection and decreased IL-10 levels at all time points analyzed. It was proposed that MDMA immunosuppressive effects on neutrophil activity and host resistance to L monocytogenes rely on NFκB signaling, being mediated by HPA axis activity and corticosterone.

Acute hematopoietic stress in mice is followed by enhanced osteoclast maturation in the bone marrow microenvironment

Osteoclasts are components of hematopoietic stem cell (HSC) niches, but their role as contributors to the HSC homeostasis and release are still controversial. We aimed to investigate whether an acute blood loss of 10% of total blood content, along with the consequent intense hematopoiesis, would affect osteoclast differentiation and activity. Isolated peripheral blood, spleen, and bone marrow (BM) cells from bones of hind limbs were investigated for the presence of specific subpopulations of osteoclast precursors: B220(-)CD3(-)NK1.1(-)CD11b(-/low)CD115(+)CD117(+) cells in BM, and B220(-)CD3(-)NK1.1(-)Gr-1(-)CD11b(+)CD115(+) cells in peripheral blood and spleen as well as the receptor activator of nuclear factor κ-B(+) cycle-arrested quiescent osteoclast precursors. Expression of osteoclastogenesis-related genes CD115, receptor activator of nuclear factor κ-B, and cathepsin K, the potential of BM cells to form osteoclast-like cells in vitro, and osteoclast activity in vivo were also evaluated. We observed an increase in spleen cellularity and myelopoiesis during week 1 following blood loss, without any significant effects on BM cellularity or BM myeloid precursors, including cells with high osteoclastogenic potential. However, at 1 week postbleeding, hematopoiesis significantly promoted the expression of cathepsin K, interleukin-34, and bone morphogenetic protein-6. Quiescent osteoclast precursors increased significantly in spleen 2 days following bleeding, whereas osteoclast activity remained unchanged up to 2 weeks postbleeding. Osteoclast-dependent B-cell differentiation was affected at the pre-B stage of maturation in BM, whereas the Lin(-)Sca-1(+)c-kit(+) population expanded in BM and spleen after 2 days postbleeding. Our data demonstrate that an acute blood loss promotes differentiation and maturation of osteoclasts at 1 week but does not enhance osteoresorption at 2 weeks postbleeding. Our data also identify osteoclast differentiation as a consequent and important event in establishing HSC homeostasis following hematopoietic stress.

Oncogenic KIT-induced aggressive systemic mastocytosis requires SHP2/PTPN11 phosphatase for disease progression in mice.

Acquired mutations in KIT are driver mutations in systemic mastocytosis (SM). Here, we tested the role of SHP2/PTPN11 phosphatase in oncogenic KIT signaling using an aggressive SM mouse model. Stable knock-down (KD) of SHP2 led to impaired growth, colony formation, and increased rates of apoptosis in P815 cells. This correlated with defects in signaling to ERK/Bim, Btk, Lyn, and Stat5 pathways in P815-KD cells compared to non-targeting (NT). Retro-orbital injections of P815 NT cells in syngeneic DBA/2 mice resulted in rapid development of aggressive SM within 13-16 days characterized by splenomegaly, extramedullary hematopoiesis, and multifocal liver tumors. In contrast, mice injected with P815 SHP2 KD cells showed less disease burden, including normal spleen weight and cellularity, and significant reductions in mastocytoma cells in spleen, bone marrow, peripheral blood and liver compared to NT controls. Treatment of human mast cell leukemia HMC-1 cells or P815 cells with SHP2 inhibitor II-B08, resulted in reduced colony formation and cell viability. Combining II-B08 with multi-kinase inhibitor Dasatinib showed enhanced efficacy than either inhibitor alone in blocking cell growth pathways and cell viability. Taken together, these results identify SHP2 as a key effector of oncogenic KIT and a therapeutic target in aggressive SM.

Protein malnutrition alters spleen cell proliferation and IL-2 and IL-10 production by affecting the STAT-1 and STAT-3 balance.

Protein malnutrition (PM) is an important public health problem that affects resistance to infection by impairing a number of physiological processes. PM induces structural changes in the lymphoid organs that affect the roles of the immune and inflammatory responses in a crucial way. The activation of different transcription factors, including signal transducer and activator of transcription (STAT) family members, leads to the production of different cytokines, which are mediators essential to mounting adequate immune and inflammatory responses. In this study, malnourished animals presented anemia, leukopenia, and a severe reduction in spleen cellularity, with reduced numbers of most cell populations, as well as increased percentages of CD3(+) and CD4(+) cells. The proliferation rates were reduced, and cells were increasingly observed in the G0/G1 cell cycle phase; further, IL-2 production was reduced, while IL-10 production was increased. In spleen cells from malnourished animals, STAT-3 protein expression was increased, with a concomitant reduction in STAT-1 expression. Knowing that STAT-1 and STAT-3 are key transcription factors in both immunity and inflammatory pathways, these results infer, at least in part, a mechanistic pathway that affects the manner or intensity of the immune response in malnourished individuals, increasing susceptibility to infection.

Peroxisome proliferator-activated receptor delta (PPARδ) limits Th1 inflammation through effects on interferon (IFN)-γ transcription and lymphocyte homeostasis (IRM7P.480)

Abstract PPARδ is a nuclear receptor that functions as a transcriptional regulator of various processes including metabolic homeostasis, cell proliferation and differentiation. We previously reported that mice deficient in PPARδ (KO) exhibit more severe experimental autoimmune encephalomyelitis than wild type (WT) mice. This more severe inflammation correlated with an increased expansion of myelin-reactive IFNγ-producing cells in the spinal cord, suggesting that PPARδ may act to limit Th1 inflammatory responses. However, the mechanism of how PPARδ accomplishes this is not clear. Characterization of the spleen and thymus compartments revealed that KO mice exhibit reduced spleen and thymus cellularity as compared to WT mice. The resultant lymphopenia in KO mice associated with a higher frequency of memory CD4+ T cells in the periphery. Radiation bone marrow chimeras were generated and the defect in thymocyte development was found to map to the radio-resistant compartment, suggesting that PPARδ may operate in stromal cells to regulate thymocyte development. This was furthered confirmed by the finding that mice with T cell-restricted deficiency of PPARδ did not exhibit defective thymocyte homeostasis. Furthermore, we observed that naïve CD4+ T cell from both KO and T cell-restricted PPARδ-deficient mice had a higher propensity to produce IFNγ upon stimulation. Thus PPARδ appears to limit Th1 inflammation both through the control of lymphocyte homeostasis and IFNγ production.

Perfluorinated compounds: Emerging POPs with potential immunotoxicity

Perfluorinated compounds (PFCs) have been recognized as an important class of environmental contaminants commonly detected in blood samples of both wildlife and humans. These compounds have been in use for more than 60 years as surface treatment chemicals, polymerization aids, and surfactants. They possess a strong carbon–fluorine bond, which leads to their environmental persistence. There is evidence from both epidemiology and laboratory studies that PFCs may be immunotoxic, affecting both cell-mediated and humoral immunity. Reported effects of PFCs include decreased spleen and thymus weights and cellularity, reduced specific antibody production, reduced survival after influenza infection, and altered cytokine production. Immunosuppression is a critical effect associated with exposure to PFCs, as it has been reported to reduce antibody responses to vaccination in children. Mounting evidence suggests that immunotoxicity in experimental animals can occur at serum concentrations below, within, or just above the reported range for highly exposed humans and wildlife. Considering bioaccumulation and exposure to multiple PFCs, the risk of immunotoxicity for humans and wildlife cannot be discounted. This review will discuss current and recently published work exploring the immunomodulatory effects of PFCs in experimental animals and humans.

Stochastic Threshold Exponential (TE) Model for Hematopoietic Tissue Reconstitution Deficit after Radiation Damage.

Whole-body exposure to large radiation doses can cause severe loss of hematopoietic tissue cells and threaten life if the lost cells are not replaced in a timely manner through natural repopulation (a homeostatic mechanism). Repopulation to the baseline level N 0 is called reconstitution and a reconstitution deficit (repopulation shortfall) can occur in a dose-related and organ-specific manner. Scott et al. (2013) previously introduced a deterministic version of a threshold exponential (TE) model of tissue-reconstitution deficit at a given follow-up time that was applied to bone marrow and spleen cellularity (number of constituent cells) data obtained 6 weeks after whole-body gamma-ray exposure of female C.B-17 mice. In this paper a more realistic, stochastic version of the TE model is provided that allows radiation response to vary between different individuals. The Stochastic TE model is applied to post gamma-ray-exposure cellularity data previously reported and also to more limited X-ray cellularity data for whole-body irradiated female C.B-17 mice. Results indicate that the population average threshold for a tissue reconstitution deficit appears to be similar for bone marrow and spleen and for 320-kV-spectrum X-rays and Cs-137 gamma rays. This means that 320-kV spectrum X-rays could successfully be used in conducting such studies.

Assessment of the Toxic Potential of Carbosulfan in Rats Following Subchronic Treatment

The effect of Carbosulfan (CB), a carbamate insecticide which has been used widely in agriculture, was investigated using wistar rat. Carbosulfan was administered orally at sublethal concentration at a dose of 25 mg/kg to 12 male rats ad libitum during the tests for 30 days consecutively. The effect of CB on hematological constituent (Red Blood Corpuscules (RBC), White Blood Corpuscules (WBC), Mean Cell Volume (MCV), Mean Corpuscular Hemoglobin (MCH), Mean Corpuscular Hemoglobin Concentration (MCHC), platelet (PLT) Hemoglobin (HGB) and Hematocrit (HCT) levels) and serum damage marker enzymes (aspartate aminotransferase (AST), alanin aminotransferase (ALT), alkaline phosphatase (ALP) and lactate dehydrogenase (LDH)) of rats were investigated under laboratory conditions .Body weight, organ weight, cellularity of spleen, spleen cell subtypes, cytokine production and lymphocyte proliferation assay were also studied. CB treatments caused different effects on the hematological constituents and the serum marker enzymes of the treatment groups as compared to the controls. Results showed that CB-treated group had a significantly lower RBC, HGB, HCT, MCV, MCH and MCHC and significantly higher WBC and PLT. Also, serum marker enzymes were affected by CB. For example, ALT, ALP, creatinine and urea significantly increased in rats treated with CB. Our data showed that CB at 25 mg/kg/day could suppress cellular activity of the immune system. At this dose, CB significantly decreased the proliferation response to PHA and the production of IFN gamma and IL-4.

To Investigate the Necessity of STRA6 Upregulation in T Cells during T Cell Immune Responses

Our earlier study revealed that STRA6 (stimulated by retinoic acid gene 6) was up-regulated within 3 h of TCR stimulation. STRA6 is the high-affinity receptor for plasma retinol-binding protein (RBP) and mediates cellular vitamin A uptake. We generated STRA6 knockout (KO) mice to assess whether such up-regulation was critical for T-cell activation, differentiation and function. STRA6 KO mice under vitamin A sufficient conditions were fertile without apparent anomalies upon visual inspection. The size, cellularity and lymphocyte subpopulations of STRA6 KO thymus and spleen were comparable to those of their wild type (WT) controls. KO and WT T cells were similar in terms of TCR-stimulated proliferation in vitro and homeostatic expansion in vivo. Naive KO CD4 cells differentiated in vitro into Th1, Th2, Th17 as well as regulatory T cells in an analogous manner as their WT counterparts. In vivo experiments revealed that anti-viral immune responses to lymphocytic choriomeningitis virus in KO mice were comparable to those of WT controls. We also demonstrated that STRA6 KO and WT mice had similar glucose tolerance. Total vitamin A levels are dramatically lower in the eyes of KO mice as compared to those of WT mice, but the levels in other organs were not significantly affected after STRA6 deletion under vitamin A sufficient conditions, indicating that the eye is the mouse organ most sensitive to the loss of STRA6.Our results demonstrate that 1) in vitamin A sufficiency, the deletion of STRA6 in T cells does no affect the T-cell immune responses so-far tested, including those depend on STAT5 signaling; 2) STRA6-independent vitamin A uptake compensated the lack of STRA6 in lymphoid organs under vitamin A sufficient conditions in mice; 3) STRA6 is critical for vitamin A uptake in the eyes even in vitamin A sufficiency.

Assessment of possible immunotoxicity of the antipsychotic drug clozapine

The immunomodulatory effects of clozapine (CLZ), antipsychotic drug, were investigated in vivo using female Balb/c mice. The main aim of this study was to evaluate the immunomodulatory effects of CLZ, antipsychotic drug, following daily intraperitoneal injection to female Balb/c mice over a period of 21 days. Mice were divided into five groups, eight animals per group. Group I, served as a control group, received only the vehicle. Groups II-V received a daily intraperitoneal dose of CLZ (1, 5, 10 and 20 mg/kg, respectively) over a period of 21 days. CLZ has shown a significant decrease in the animal body weight, and it showed a significant decrease in the percentage of circulating neutrophils and lymphocytes while circulating monocytes were increased. The immunotoxicity has been also assessed by evaluating spleen cellularity, humoral immune response to a foreign antigen using sheep red blood cells and delayed-type hypersensitivity reaction. The results showed a marked suppression in these responses in CLZ-treated mice compared with the control group. Detectable changes have also been noticed in the histology of the footpad tissue and spleen. Results showed significant immunomodulatory effects of CLZ when used in Balb/c mice.

Bone Marrow Osteoblast Ablation Enhances Short-Term Hematopoietic Stem Cells Without Altering Long-Term Hematopoietic Stem Cell Populations and Accelerates Leukemia Development

Hematopoietic stem cells (HSCs) within the bone marrow (BM) microenvironment reside in close proximity to endosteal osteoblasts (OBs). Although OBs have been considered to provide a HSC niche, other studies suggest that perivascular mesenchymal cells or endothelial cells may be the primary HSC niches, and the specific role of OBs in regulation of HSCs requires further clarification. Moreover, the role of OBs in regulating leukemic stem cells (LSC) is even less well studied. To address these questions, we used a conditional OB ablation mouse model (Col2.3Δtk) in which a truncated version of the herpes simplex virus thymidine kinase (Δtk) is expressed under an OB-specific promoter. In these mice, daily intraperitoneal (IP) administration of ganciclovir (GCV) leads to production of a toxic DNA base analogue in OBs, resulting in their death. We crossed Col2.3Δtk mice with Col2.3GFP mice that specifically express GFP in OBs to facilitate assessment of OB ablation. We confirmed that 4 weeks of GCV administration resulted in ablation of endosteal OBs in this model using both immunofluorescence microscopy and flow cytometry analysis. OB ablation was associated with reduced BM cellularity (Δtk+ 3.7e7±3.0e6, Δtk- 4.8e7±3.8e6 per 4 lower extremity bones, p=0.04), but did not alter spleen (SP) cellularity (Δtk+ 5.1e7±5.3e6, Δtk- 6.3e7±7.4e6 cells per SP, p=0.19). OB ablation was also associated with significantly increased numbers of cells with long-term HSC (LTHSC) phenotype (Lin-Kit+Sca-1+Flt3-CD150+CD48-) in both the BM (Δtk+ 6490±1315, Δtk- 4236±922 per 4 lower extremity bones; p=0.03) and SP (Δtk+ 980±473, Δtk- 96±40 per SP; p=0.04). Significant increases in common myeloid progenitor (CMP) (Δtk+ 145114±43608, Δtk- 82200±26754; p=0.002) and granulocyte/monocyte progenitor (GMP) (Δtk+ 51411±17349, Δtk- 20206±9279, p=0.003, p=0.02) numbers were seen in SP of OB-ablated mice, whereas significant alterations in other hematopoietic populations in BM, SP or PB were not seen. We performed limiting-dilution competitive repopulation assays to determine the functional LTHSC potential of BM cells from OB-ablated and control mice. OB-ablated mice demonstrated a higher frequency of short-term repopulating cells compared to LTHSCs from non-ablated mice (5 weeks: Δtk+ 1 in 4,941; Δtk- 1 in 17,351 BM cells) but similar long-term engraftment (15 weeks: Δtk+ 1 in 22,853; Δtk- 1 in 23,137 BM cells). Transplantation of BM cells from primary transplant recipients into secondary recipients demonstrated similar long-term engraftment potential after second transplant. These results suggest that despite increased numbers of phenotypic LTHSCs in OB-ablated mice, the long-term repopulating and self-renewing capacity of BM cells remains unchanged in OB-ablated mice, but on the other hand there is an increase in functional short-term repopulating capacity. Next, to examine the role of OBs in regulation of Chronic Myelogenous Leukemia (CML) stem cells, we crossed the Col2.3GFPΔtk mice with an inducible transgenic BCR-ABL mouse model of CML (ScltTA-BCR/ABL). In these mice withdrawal of tetracycline results in induction of BCR-ABL expression in HSCs and development of a CML-like myeloproliferative disorder. GCV administration to achieve OB ablation was initiated one week prior to BCR-ABL induction by tetracycline withdrawal, and was continued for the duration of the experiment. CML development was monitored by checking blood counts every 2 weeks after induction and mice were followed for survival. We observed significantly accelerated development of CML in OB-ablated versus non-ablated mice, with 50% of the OB-ablated mice dying within 47 days of CML induction, whereas >50% of the non-ablated mice survived to day 73 (p=0.017). Collectively, these studies suggest that BM OBs are not essential for maintenance of long-term repopulating and self-renewing HSCs, but regulate the expansion of short-term HSCs in the BM. Our studies also indicate an important and previously unrecognized role for OBs in regulating the leukemogenicity of CML LSCs. Disclosures:No relevant conflicts of interest to declare.

SAT0067 Mice lacking FLT3L are protected from collagen-induced arthritis: Regulating the regulators

BackgroundAutoimmune diseases often result from inappropriate or unregulated activation of autoreactive T cells. Traditional approaches to treat autoimmune diseases have focused on direct inhibition of autoreactive T cells. A key...

Fish oil supplementation ameliorates leukopenia and thrombocytopenia and modulates nitric oxide and TNF‐α production during the acute phase of Trypanosoma cruzi infection

Omega‐3 polyunsaturated fatty acids (n‐3 PUFA) supplementation has been reported to reduce inflammation and in some circumstances diminish infectious disease resistance in rodents. We investigated whether supplementation with fish oil, a source of n‐3 PUFA, impaired host resistance against Trypanosoma cruzi, the causal agent of Chagas’ disease. C57BL/6 mice were supplemented (0.6% v/w) by gavage with phosphate buffer saline (PBS), corn oil (CO) or fish oil (FO) for 15 days prior to T. cruzi challenge and throughout the course of the infection. In this study, we observed an increase in spleen weight and cellularity in the infected‐FO supplemented mice. Importantly, FO supplementation in infected mice improved platelets and leukocytes counts, decreased heart parasitism, decreased nitric oxide production in plasma and cardiac tissue, and increased TNF‐α production by spleen cells stimulated with parasite antigen. These data suggest the potential of FO supplementation to attenuate the clinical course of T. cruzi infection and further studies are warranted.

Immuno-modulation effect of sulphated polysaccharide (porphyran) from Porphyra vietnamensis

Our investigation explores the immuno-efficiency of sulphated polysaccharides enriched Porphyra vietnamenis. Isolated polysaccharide fraction (17.1–25.8%) was characterized by FTIR and NMR spectroscopy which showed the presence of typical linear backbone structure called as porphyran. Oral administration of porphyran (200–500mg/kg) evoked a significant (P≤0.05) increase in weight of the thymus, spleen and lymphoid organ cellularity. The total leucocyte and lymphocyte count was increased significantly (P<0.005). The increase in the percent neutrophil adhesion to nylon fibres as well as a dose-dependent increase in antibody titre values was observed. A decreased response to DTH reaction induced by SRBC was recorded. A potential phagocytic response was seen and significant changes were observed in the formation of formazone crystals. It also prevented myelosuppression in cyclophosphamide drug treated rats. The results indicated that P. vietnamenis possesses potential immunomodulatory activity and has therapeutic potential for the prevention of autoimmune diseases.

Persistent Antigen and Germinal Center B Cells Sustain T Follicular Helper Cell Responses and Phenotype

T follicular helper (Tfh) cells provide help to B cells and are crucial for establishment of germinal center (GC) reactions, including production of high-affinity antibodies and generation of memory B cells and long-lived plasma cells. Here we report that the magnitude of the Tfh cell response was dictated by the amount of antigen and directly correlated with the magnitude of the GC B cell response. In addition, maintenance of the Tfh cell phenotype required sustained antigenic stimulation by GC B cells. In lymphopenic conditions, a strong and prolonged Tfh cell response led to bystander B cell activation, hypergammaglobulinemia, and production of poly- and self-reactive antibodies. These data demonstrate that antigen dose determines the size and duration of the Tfh cell response and GC reaction, highlight the transient nature of the Tfh cell phenotype, and suggest a link between overstimulation of Tfh cells and the development of dysregulated humoral immune responses.

Erythropoietin Induced Erythroid Precursor Pool Depletion Causes Erythropoietin Hyporesponsiveness

Abstract 5156 Background:Erythropoietin (EPO) hyporesponsiveness is demonstrated by a persistence of anemia despite high dose of recombinant human erythropoietin (rHuEPO) or requirement of large doses to maintain the target hemoglobin concentration. The purpose of this study is to demonstrate that the bone marrow depletion induced by rHuEPO treatment may another contributing factor to hyporesponsiveness. Methods:Healthy Wistar rats were given single dose (SD) or multiple doses (MD) of rHuEPO. In MD study, animals were challenged with thrice-weekly (100 IU/kg) over two weeks and the pharamcodynamic biomarkers included reticulocyte (RET) and red blood cells (RBC) counts, and hemoglobin (HGB) concentrations. In SD study, in addition to the biomarkers in the peripheral blood, the erythropoietic responses in bone marrow and spleen were also quantified using a flow cytometric immunophenotyping technique. The total marrow and spleen cellularity was quantitatively assessed using flow cytometry. A mathematical approach involving measuring RET age distribution was developed to evaluate the RET loss due to neocytolysis. The pharmacokinetics of rHuEPO was assessed by measuring serum concentrations over time using ELISA. Result:In MD study, a slow and oscillatory decline of RET response was observed before the administration of the last dose on day 11. The RET kept decreasing below the baseline and reached a nadir on day 22, followed by a slow return to the baseline on day 28. These observations demonstrated the tolerance and rebound phenomena. In SD study, the RET decreased below the baseline after day 6. A depletion of the bone marrow erythroid precursor cells was also observed. Meanwhile, neocytolysis of RET was only observed from day 3–5 after rHuEPO injection. These results suggested that the reduced levels of RET after day 6 resulted from the depletion in the erythroid precursor cells in bone marrow induced by rHuEPO treatment. Conclusion:The EPO hyporesponsiveness is of clinical importance because it is associated with an increased risk of death or cardiovascular events. The findings in this study demonstrate that EPO-induced erythroid precursor depletion may contribute to the rHuEPO hyporesponsiveness. When the erythroid precursor cells are depleted, an increase of rHuEPO dose would not show much benefit, due to the smaller number of cells present to initiate the erythropoiesis. These findings provide additional justification for reducing the doses of erythropoietin stimulating agents in anemic patients demonstrating hyporesponsiveness. Disclosures:No relevant conflicts of interest to declare.