Bone Marrow Transplantation Studies Research Articles

Role of the CD39/CD73 Purinergic Pathway in Modulating Arterial Thrombosis in Mice

Circulating blood cells and endothelial cells express ectonucleoside triphosphate diphosphohydrolase-1 (CD39) and ecto-5'-nucleotidase (CD73). CD39 hydrolyzes extracellular ATP or ADP to AMP. CD73 hydrolyzes AMP to adenosine. The goal of this study was to examine the interplay between CD39 and CD73 cascade in arterial thrombosis. To determine how CD73 activity influences in vivo thrombosis, the time to ferric chloride-induced arterial thrombosis was measured in CD73-null mice. In response to 5% FeCl3, but not to 10% FeCl3, there was a significant decrease in the time to thrombosis in CD73-null mice compared with wild-type mice. In mice overexpressing CD39, ablation of CD73 did not inhibit the prolongation in the time to thrombosis conveyed by CD39 overexpression. However, the CD73 inhibitor α-β-methylene-ADP nullified the prolongation in the time to thrombosis in human CD39 transgenic (hC39-Tg)/CD73-null mice. To determine whether hematopoietic-derived cells or endothelial cell CD39 activity regulates in vivo arterial thrombus, bone marrow transplant studies were conducted. FeCl3-induced arterial thrombosis in chimeric mice revealed a significant prolongation in the time to thrombosis in hCD39-Tg reconstituted wild-type mice, but not on wild-type reconstituted hCD39-Tg mice. Monocyte depletion with clodronate-loaded liposomes normalized the time to thrombosis in hCD39-Tg mice compared with hCD39-Tg mice treated with control liposomes, demonstrating that increased CD39 expression on monocytes protects against thrombosis. These data demonstrate that ablation of CD73 minimally effects in vivo thrombosis, but increased CD39 expression on hematopoietic-derived cells, especially monocytes, attenuates in vivo arterial thrombosis.

A flexible cure rate model with dependent censoring and a known cure threshold.

We propose a flexible cure rate model that accommodates different censoring distributions for the cured and uncured groups and also allows for some individuals to be observed as cured when their survival time exceeds a known threshold. We model the survival times for the uncured group using an accelerated failure time model with errors distributed according to the seminonparametric distribution, potentially truncated at a known threshold. We suggest a straightforward extension of the usual expectation-maximization algorithm approach for obtaining estimates in cure rate models to accommodate the cure threshold and dependent censoring. We additionally suggest a likelihood ratio test for testing for the presence of dependent censoring in the proposed cure rate model. We show through numerical studies that our model has desirable properties and leads to approximately unbiased parameter estimates in a variety of scenarios. To demonstrate how our method performs in practice, we analyze data from a bone marrow transplantation study and a liver transplant study. Copyright © 2016 John Wiley & Sons, Ltd.

Abstract 633: Macrophage SR-BI Suppresses Atherosclerosis by Modulation of Autophagy via VPS34/Belclin-1 Pathway

Background: Autophagy modulates vascular cell lipid metabolism, lipid droplet turnover, foam cell formation, cell survival and death, and inflammation. Scavenger receptor class B type I (SR-BI) deficiency causes impaired lysosome function in macrophages and erythrocytes. Methods and Results: Bone marrow transplantation studies were performed in ApoE and LDLR deficient mice to examine the effects of hematopoietic SR-BI deletion on atherosclerotic lesion autophagy. In addition, in vitro studies compared WT versus SR-BI -/- macrophages. Under conditions of cholesterol induced stress, the mRNA and protein levels of critical autophagy players including ATG5, ATG6/Belcin-1, ATG7 and LC3II were decreased by 37.8% to 84.6% (P<0.05 to 0.01) in SR-B1 -/- macrophages and atherosclerotic aortic tissue compared to controls. Electron microscopic analysis showed that SR-BI -/- versus WT macrophages had 80% fewer (P<0.05) autophagsomes in response to cholesterol enrichment. Macrophage SR-BI deficiency led to 1.8-fold (P<0.05) more lipid deposition and 2.5-fold more (P<0.01) apoptosis in response to oxidized LDL. Furthermore, hematopoietic SR-BI deletion caused 2.3 fold (P<0.05) more cell death in aortic atherosclerotic lesions compared to the WT control. Pharmacologic activation of autophagy did not reduce the levels of lipid droplets or cell apoptosis in SR-BI null macrophages vs WT control. WT peritoneal macrophages were used to examine SR-BI subcellular distribution and its interaction with VPS34/Beclin-1. In response to induction of autophagy, macrophage SR-BI was expressed in lysosomes and co-localized with LC3-II. Furthermore, we found that SR-BI directly interacted with the VPS34/Beclin-1 complex. Conclusions: SR-BI deficiency leads to defective autophagy and accelerates macrophage foam cell formation and apoptosis in experimental mouse atherosclerotic lesions. Macrophage SR-BI regulates expression of critical autophagy players and directly modulates autophagy via the VPS34/Beclin-1 pathway, identifying novel targets for the treatment of atherosclerosis.

The role of the orphan nuclear receptor NR4A1 in erythro-myelopoiesis.

Abstract Studies of hematopoietic stem cells (HSCs) have advanced our understanding of the intracellular signals and microenvironmental interactions that influence HSC fate. Our lab is interested in the role of orphan nuclear receptor NR4A1, an immediate response gene sensitive to external stimuli, in HSC development. NR4A1 regulates the development of specific, mature hematopoietic cell lineages from both the innate and adaptive immune system including patrolling monocytes, a mature myeloid cell subset. More recently, we have shown that NR4A1 expression also identifies a subpopulation of myeloid-biased long-term HSCs in the bone marrow. Given that NR4A1 directs both immature and differentiated cell types, we investigated its role in myeloid cell maturation. Within the bone marrow and spleen progenitor cell compartments, we find that NR4A1 is exclusively expressed by myeloid progenitors and not by more restricted megakaryocyte, erythroid, or common monocyte progenitors. Nr4a1−/−mice exhibit skewed myeloid progenitor cell populations, exhibiting a 2-fold increase in CD105+CD150− erythroid progenitors and cKit+Ter119+CD71+ pro-erythroblasts in the spleen but not in the bone marrow. In vitro cultures of Nr4a1−/−splenic myeloid progenitors also show that NR4A1 restricts the frequency of erythroid cells, yet bone marrow transplant studies show similar erythroid progenitor reconstitution from both WT and Nr4a1−/− donors. Taken together, our data reveal a new role for NR4A1 as both a direct and indirect modulator of erythropoiesis.

Combined Id1 and Id3 Deletion Leads to Severe Erythropoietic Disturbances.

The Inhibitor of DNA Binding (Id) proteins play a crucial role in regulating hematopoiesis and are known to interact with E proteins and the bHLH family of transcription factors. Current efforts seek to elucidate the individual roles of Id members in regulating hematopoietic development and specification. However, the nature of their functional redundancies remains elusive since ablation of multiple Id genes is embryonically lethal. We developed a model to test this compensation in the adult. We report that global Id3 ablation with Tie2Cre-mediated conditional ablation of Id1 in both hematopoietic and endothelial cells (Id cDKO) extends viability to 1 year but leads to multi-lineage hematopoietic defects including the emergence of anemia associated with defective erythroid development, a novel phenotype unreported in prior single Id knockout studies. We observe decreased cell counts in the bone marrow and splenomegaly to dimensions beyond what is seen in single Id knockout models. Transcriptional dysregulation of hematopoietic regulators observed in bone marrow cells is also magnified in the spleen. E47 protein levels were elevated in Id cDKO bone marrow cell isolates, but decreased in the erythroid lineage. Chromatin immunoprecipitation (ChIP) studies reveal increased occupancy of E47 and GATA1 at the promoter regions of β-globin and E2A. Bone marrow transplantation studies highlight the importance of intrinsic Id signals in maintaining hematopoietic homeostasis while revealing a strong extrinsic influence in the development of anemia. Together, these findings demonstrate that loss of Id compensation leads to dysregulation of the hematopoietic transcriptional network and multiple defects in erythropoietic development in adult mice.

The adaptive immune system restrains Alzheimer’s disease pathogenesis by modulating microglial function

The innate immune system is strongly implicated in the pathogenesis of Alzheimer's disease (AD). In contrast, the role of adaptive immunity in AD remains largely unknown. However, numerous clinical trials are testing vaccination strategies for AD, suggesting that T and B cells play a pivotal role in this disease. To test the hypothesis that adaptive immunity influences AD pathogenesis, we generated an immune-deficient AD mouse model that lacks T, B, and natural killer (NK) cells. The resulting "Rag-5xfAD" mice exhibit a greater than twofold increase in β-amyloid (Aβ) pathology. Gene expression analysis of the brain implicates altered innate and adaptive immune pathways, including changes in cytokine/chemokine signaling and decreased Ig-mediated processes. Neuroinflammation is also greatly exacerbated in Rag-5xfAD mice as indicated by a shift in microglial phenotype, increased cytokine production, and reduced phagocytic capacity. In contrast, immune-intact 5xfAD mice exhibit elevated levels of nonamyloid reactive IgGs in association with microglia, and treatment of Rag-5xfAD mice or microglial cells with preimmune IgG enhances Aβ clearance. Last, we performed bone marrow transplantation studies in Rag-5xfAD mice, revealing that replacement of these missing adaptive immune populations can dramatically reduce AD pathology. Taken together, these data strongly suggest that adaptive immune cell populations play an important role in restraining AD pathology. In contrast, depletion of B cells and their appropriate activation by T cells leads to a loss of adaptive-innate immunity cross talk and accelerated disease progression.

Histological Study of Bone Marrow and Umbilical Cord Stromal Cell Transplantation in Regenerating Rat Peripheral Nerve.

Bone marrow and umbilical cord stromal cells are multipotential stem cells that have the ability to produce growth factors that play an important role in survival and generation of axons. The goal of this study was to evaluate the effects of the two different mesenchymal stem cells on peripheral nerve regeneration. In this experimental study, a 10 mm segment of the left sciatic nerve of male Wistar rats (250-300 g) was removed with a silicone tube interposed into this nerve gap. Bone marrow stromal cells (BMSCs) and human umbilical cord stromal cells (HUCSCs) were respectively obtained from rat and human. The cells were sepa- rately cultured and transplanted into the nerve gap. The sciatic nerve regeneration was evaluated by immunohistochemistry, and light and electron microscopy. Moreover, histo- morphology of the gastrocnemius muscle was observed. The nerve regeneration in the BMSCs and HUCSCs groups that had received the stem cells was significantly more favorable than the control group. In addition, the BM- SCs group was significantly more favorable than the HUCSCs group (P<0.05). The results of this study suggest that both homograft BMSCs and het- erograft HUCSCs may have the potential to regenerate peripheral nerve injury and transplantation of BMSCs may be more effective than HUCSCs in rat.

Abstract 18138: Endothelial PHD2 Loss Causes Pulmonary Arterial Hypertension via HIF-2 (Best of Basic Science Abstract)

Background: Key mediators of cellular adaption to hypoxia are hypoxia-inducible factor (HIF)-1 and -2, which are regulated by the prolyl hydroxylases (PHDs). Although the HIF pathway has been shown to be involved in the pathogenesis of pulmonary arterial hypertension (PAH), little is known about the cell type-specific functions of the PHD/HIF axis in this context. Here, we used a genetic approach to specifically dissect the contribution of the endothelial PHD/HIF axis in PAH. Methods: Endothelial cell specific HIF activation was achieved by crossing Vecadherin (Cdh5)-Cre transgenics to Phd2 floxed mice (ePHD2), while the contribution of each HIF isoform was assessed by generating double mutants lacking PHD2 and HIF-2 (ePHD2HIF2) or PHD2 and HIF-1 (PHD2HIF1). Right ventricular systolic pressure (RVSP) was directly measured via insertion of a 1.4F Mikro-tip catheter transducer into a surgically exposed right internal jugular vein. Results: Mice deficient for endothelial PHD2 showed significant activation of HIF-signaling as shown by immunoblot analysis of lung tissue for HIF-1 and HIF-2. These mice developed spontaneous PAH (ePHD2: 54.3±6.9 vs Cre-: 24.8±2.2 mmHg, n=5-6, P=0.005) which was associated with right ventricular hypertrophy (RVH) (Fulton Index: 0.52 in ePHD2 vs 0.28 in Cre-, P=0.0004) and increased mortality. The PAH phenotype was maintained in ePHD2HIF1 mutants but was reversed in ePHD2HIF2 mutants. To assess the contribution of endothelial HIF-2 in hypoxia induced PAH, endothelial HIF-2 single mutants or Cre- littermates were exposed to normobaric hypoxia (10% O2) for 4 weeks. In contrast to controls, eHIF2 mutants were completely protected from development of PAH and RVH. Bone marrow transplantation studies showed no contribution from hematopoietic HIF-2 in hypoxia induced PAH. Conclusions: Our data establish that endothelial PHD2 loss results in PAH through a HIF-2 dependent mechanism. Our findings identify the PHD2/HIF2 axis as a potential target for PAH therapies.

Macrophage SR-BI mediates efferocytosis via Src/PI3K/Rac1 signaling and reduces atherosclerotic lesion necrosis

Macrophage apoptosis and efferocytosis are key determinants of atherosclerotic plaque inflammation and necrosis. Bone marrow transplantation studies in ApoE- and LDLR-deficient mice revealed that hematopoietic scavenger receptor class B type I (SR-BI) deficiency results in severely defective efferocytosis in mouse atherosclerotic lesions, resulting in a 17-fold higher ratio of free to macrophage-associated dead cells in lesions containing SR-BI−/− cells, 5-fold more necrosis, 65.2% less lesional collagen content, nearly 7-fold higher dead cell accumulation, and 2-fold larger lesion area. Hematopoietic SR-BI deletion elicited a maladaptive inflammatory response [higher interleukin (IL)-1β, IL-6, and TNF-α lower IL-10 and transforming growth factor β]. Efferocytosis of apoptotic thymocytes was reduced by 64% in SR-BI−/− versus WT macrophages, both in vitro and in vivo. In response to apoptotic cells, macrophage SR-BI bound with phosphatidylserine and induced Src phosphorylation and cell membrane recruitment, which led to downstream activation of phosphoinositide 3-kinase (PI3K) and Ras-related C3 botulinum toxin substrate 1 (Rac1) for engulfment and clearance of apoptotic cells, as inhibition of Src decreased PI3K, Rac1-GTP, and efferocytosis in WT cells. Pharmacological inhibition of Rac1 reduced macrophage efferocytosis in a SR-BI-dependent fashion, and activation of Rac1 corrected the defective efferocytosis in SR-BI−/− macrophages. Thus, deficiency of macrophage SR-BI promotes defective efferocytosis signaling via the Src/PI3K/Rac1 pathway, resulting in increased plaque size, necrosis, and inflammation.

Granulocyte-colony stimulating factor as a treatment for diabetic neuropathy in rat

Effective treatment of diabetic neuropathy (DN) remains unsolved. We serendipitously observed dramatic relief of pain in several patients with painful DN receiving granulocyte-colony stimulating factor (G-CSF). The aim of this study was to determine if G-CSF could treat DN in an animal model and to ascertain its mechanism of action. In a rodent model of DN, G-CSF dramatically recovered nerve function, retarded histological nerve changes and increased the expression of neurotrophic factors within nerve. A sex-mismatched bone marrow transplantation (BMT) study revealed that G-CSF treatment increased the abundance of bone marrow (BM)-derived cells in nerves damaged by DN. However, we did not observe evidence of transdifferentiation or cell fusion of BM-derived cells. The beneficial effects of G-CSF were dependent on the integrity of BM. In conclusion, G-CSF produced a therapeutic effect in a rodent model of DN, which was attributed, at least in part, to the actions of BM-derived cells.

Autologous hematopoietic stem cell transplantation in elderly patients (≥ 70 years) with non-Hodgkin's lymphoma: A French Society of Bone Marrow Transplantation and Cellular Therapy retrospective study.

Limited data is available on the feasibility of high-dose chemotherapy followed by autologous hematopoietic stem cell transplantation (AHSCT) in elderly patients over 70 years of age with non-Hodgkin's lymphoma (NHL). In the setting of the Société Française de Greffe de Moelle et de Thérapie Cellulaire (SFGM-TC) group, we retrospectively analyzed 81 consecutive patients with NHL over 70 years of age who received AHSCT. The median age at AHSCT was 72.3 years [70-80]. Patients' were diagnosed with diffuse large B-cell lymphoma (n=40), follicular lymphoma (n=16), mantle cell lymphoma (n=15), T-cell lymphoma (n=5), and other (n=5). Hematopoietic Cell Transplantation Comorbidity Index (HCT-CI) was 0 in 73% of patients. Main conditionings were BEAM (Carmustine-Etoposide-Cytarabine-Melphalan, n=61) and melphalan alone (n=14). Median delays to reach 0.5×10⁹/L neutrophils and 20 × 10(9)/L platelets were of 12 [9-76] days and 12 [0-143] days, respectively. One hundred day and one year cumulative incidence of NRM was 5.4% and 8.5%, respectively. The main cause of death remains relapse. In conclusion, this study revealed that AHSCT seemed to be acceptable in patients over 70 years of age with NHL. Patient age is not a limiting factor if clinical condition is adequate.

The proportional odds cumulative incidence model for competing risks.

We suggest an estimator for the proportional odds cumulative incidence model for competing risks data. The key advantage of this model is that the regression parameters have the simple and useful odds ratio interpretation. The model has been considered by many authors, but it is rarely used in practice due to the lack of reliable estimation procedures. We suggest such procedures and show that their performance improve considerably on existing methods. We also suggest a goodness-of-fit test for the proportional odds assumption. We derive the large sample properties and provide estimators of the asymptotic variance. The method is illustrated by an application in a bone marrow transplant study and the finite-sample properties are assessed by simulations.

Abstract 327: Liver X Receptors (LXR) Modulate the Negative Effects on Endothelial Progenitors Cells after Feeding a High Cholesterol Diet

The liver X receptors (LXRα/β) belong to the nuclear receptor superfamily of ligand activated transcription factors. LXRs are endogenously activated by oxysterols; and one of their primary functions is to facilitate cholesterol efflux out of cells, such as macrophages, via the ABCA1 &amp; ABCG1 transporters. Bone marrow (BM) transplant studies have shown that LXRs are important for preventing the formation of atherosclerotic plaques. Loss of LXRs in the monocyte/macrophage population of the BM was thought to be the primary mediator of the increased atherosclerosis in these studies. However, the BM is comprised of a variety of cell types that are differentiated from hematopoietic stem cells (HSCs). Like numerous other vascular complications, endothelial defects are central to the pathogenesis of the early stages of atherosclerosis. Endothelial progenitor cells (EPCs), derived from HSCs in the BM, contribute to vascular health primarily through the secretion of factors that maintain and repair the endothelium of the damaged vasculature. We hypothesized that LXRs also modulate the negative effects of cholesterol on EPCs, in addition to its effects in macrophages. To study the effects of dietary cholesterol on EPCs and the roles of LXRs in modulating these effects, WT and LXRα/β-/- mice were fed a high-fat/high-cholesterol (HF/HC) diet starting at 8-9 weeks of age for a total of 12 weeks. Complete blood counts showed that in the LXRα/β-/- but not the WT mice fed the HF/HC diet, there was a significant expansion of select myeloid populations (neutrophils, monocytes, platelets). Furthermore, EPCs derived from the HF/HC fed LXRα/β-/- mice had a higher expression of the endothelial markers CD144 and VEGFR2 compared to WT mice and LXRα/β-/- mice on chow diet, suggesting enhanced differentiation to a more endothelial-like cell in the absence of LXRs. Notably, the paracrine reparative actions of EPCs have been shown in populations of cells with low expression of endothelial markers. Taken together, these results suggest that LXRs modulate the effects of HF/HC diet on the differentiation of EPCs to endothelial cells and thus, may provide another cell type within the BM that would benefit from LXR activation in modulating vascular disease.[[Unable to Display Character: &amp;#8195;]]

Abstract 589: Macrophage SR-BI Binds GST-Pi and Suppresses Cell Apoptosis in Atherosclerosis

Background: Oxidative stress accelerates atherosclerosis by promoting cell death. We hypothesize that macrophage scavenger receptor class B I (SR-BI) inhibits apoptosis by reducing oxidative stress in atherosclerosis. Methods and Results: Macrophage SR-BI deficiency led to 2.1-, 1.9-, and 2.7-fold more apoptosis after treatment with OxLDL, H2O2, or isolevugladin-phosphatidylethanolamine (IsoLG-PE) versus WT cells (P&lt;0.05), respectively. To examine the impact of SR-BI deficiency in atherosclerosis, bone marrow (BM) transplantation studies were performed using apoE-/- and LDLr-/- recipient mice fed a Western diet for 8 or 16 weeks, respectively. Apoptosis as measured by active Caspase 3 was increased 2.6-fold in atherosclerotic lesions of LDLr-/- recipients of SR-BI-/- versus WT BM (P&lt;0.01). Consistent with SR-BI reducing oxidative stress, the lesions of ApoE-/- mice reconstituted with SR-BI-/-ApoE-/- versus ApoE-/- BM showed 5.1-fold higher immunostaining for 4-HNE (P&lt;0.05) and 70.4% lower GSH/GSSG ratio as determined by an enzymatic assay of aortic tissue lysates (P&lt;0.01). Similarly, OxLDL treatment resulted in a 64.5% decrease in GSH/GSSG ratio in SR-BI-/- versus WT macrophages. ER stress was significantly increased in SR-BI-/- versus WT macrophages treated with OxLDL as evaluated by CHOP and BiP (P&lt;0.05). Similarly, ER stress was increased in atherosclerotic lesions of ApoE-/- recipients of SR-BI-/-ApoE-/- versus ApoE-/- BM. Importantly, an antibody-based proteomic assay revealed that macrophage SR-BI interacts with glutathione S-transferase pi (GST-pi). SR-BI deficiency caused a 48.7% reduction of GST-pi expression in atherosclerotic lesions (P&lt;0.05) and a 51.3% decrease in macrophage GST activity (P&lt;0.05). Forced expression of GST-pi rescued OxLDL-induced apoptosis in SR-BI-/- macrophages. A lipid-protein overlap assay showed SR-BI binds IsoLG-PE, but not native PE, suggesting that SR-BI recognizes oxidized lipid for clearance via a GST-pi detoxification pathway. Conclusion: Macrophage SR-BI interacts with GST-pi and suppresses oxidative stress-induced apoptosis in atherosclerosis. This novel finding of SR-BI GST-pi interaction provides a new target for prevention and treatment of atherosclerosis.

Abstract 31: Functional Impact of Inflammatory Cell-Originating HDL-miR-223 Communication in vivo

Atherosclerosis is associated with endothelial dysfunction and inflammation, and high-density lipoproteins (HDL) play critical roles in both. HDL transport a wide-variety of proteins, nucleic acids, and small molecules, which likely confer many of HDL’s alternative functions, including its anti-inflammatory and anti-atherogenic properties. Using in vitro studies, we demonstrated that macrophages export miR-223 to HDL and subsequently, HDL delivers functional miR-223 to human coronary artery endothelial cells (HCAEC). Moreover, intracellular adhesion molecule 1 (ICAM-1), a miR-223 target, was found to be down-regulated by HDL-miR-223 at a specific target site within ICAM-1’s 3’ untranslated region. These in vitro findings support the potential of a HDL-miRNA communication pathway that may contribute to atherogenesis. To validate HDL-miRNA communication in vivo, bone-marrow transplant (BMT) studies were completed with Mir223-/- and C57B/6J (WT) mice. To demonstrate that functional HDL-miR-223 is delivered to recipient cells/tissues in vivo, BM from WT mice was transplanted into irradiated Mir223-/- mice. Strikingly, we found that HDL-miR-223 communication was restored after BMT, as evidenced by significantly increased miR-223 levels on HDL and in the liver, white adipose, aortic endothelium, and skeletal muscle. Conversely, HDL-miR-223 intercellular communication was depleted by transplanting BM from Mir223-/- into WT mice, and resulted in significant loss of miR-223 on HDL and in many tissues. In both BMT studies, miR-223 changes led to altered expression of many target genes in multiple tissues. Most importantly, these studies identified a novel miR-223 target gene which we experimentally validated, vesicle transport through interaction with t-SNAREs homolog 1A (VTI1A), which was decreased and increased in the aortic endothelium from each BMT study, respectively. VTI1A has been previously characterized as a SNARE protein that contributes to cytokine secretion and membrane lipid dynamics. As such, HDL likely mediates an inflammatory cell-originating miR-223 communication pathway that suppresses inflammation and cytokine secretion in the aortic endothelium, thus antagonizing atherosclerosis.

Differences in Hematopoietic Stem Cells Contribute to Sexually Dimorphic Inflammatory Responses to High Fat Diet-induced Obesity

Women of reproductive age are protected from metabolic disease relative to postmenopausal women and men. Most preclinical rodent studies are skewed toward the use of male mice to study obesity-induced metabolic dysfunction because of a similar protection observed in female mice. How sex differences in obesity-induced inflammatory responses contribute to these observations is unknown. We have compared and contrasted the effects of high fat diet-induced obesity on glucose metabolism and leukocyte activation in multiple depots in male and female C57Bl/6 mice. With both short term and long term high fat diet, male mice demonstrated increased weight gain and CD11c(+) adipose tissue macrophage content compared with female mice despite similar degrees of adipocyte hypertrophy. Competitive bone marrow transplant studies demonstrated that obesity induced a preferential contribution of male hematopoietic cells to circulating leukocytes and adipose tissue macrophages compared with female cells independent of the sex of the recipient. Sex differences in macrophage and hematopoietic cell in vitro activation in response to obesogenic cues were observed to explain these results. In summary, this report demonstrates that male and female leukocytes and hematopoietic stem cells have cell-autonomous differences in their response to obesity that contribute to an amplified response in males compared with females.

Extrahepatic sources of factor VIII potentially contribute to the coagulation cascade correcting the bleeding phenotype of mice with hemophilia A.

A large fraction of factor VIII in blood originates from liver sinusoidal endothelial cells although extrahepatic sources also contribute to plasma factor VIII levels. Identification of cell-types other than endothelial cells with the capacity to synthesize and release factor VIII will be helpful for therapeutic approaches in hemophilia A. Recent cell therapy and bone marrow transplantation studies indicated that Küpffer cells, monocytes and mesenchymal stromal cells could synthesize factor VIII in sufficient amount to ameliorate the bleeding phenotype in hemophilic mice. To further establish the role of blood cells in expressing factor VIII, we studied various types of mouse and human hematopoietic cells. We identified factor VIII in cells isolated from peripheral and cord blood, as well as bone marrow. Co-staining for cell type-specific markers verified that factor VIII was expressed in monocytes, macrophages and megakaryocytes. We additionally verified that factor VIII was expressed in liver sinusoidal endothelial cells and endothelial cells elsewhere, e.g., in the spleen, lungs and kidneys. Factor VIII was well expressed in sinusoidal endothelial cells and Küpffer cells isolated from human liver, whereas by comparison isolated human hepatocytes expressed factor VIII at very low levels. After transplantation of CD34(+) human cord blood cells into NOD/SCIDγNull-hemophilia A mice, fluorescence activated cell sorting of peripheral blood showed >40% donor cells engrafted in the majority of mice. In these animals, plasma factor VIII activity 12 weeks after cell transplantation was up to 5% and nine of 12 mice survived after a tail clip-assay. In conclusion, hematopoietic cells, in addition to endothelial cells, express and secrete factor VIII: this information should offer further opportunities for understanding mechanisms of factor VIII synthesis and replenishment.

Mutation of SH2B3 (LNK), a genome-wide association study candidate for hypertension, attenuates Dahl salt-sensitive hypertension via inflammatory modulation.

Human genome-wide association studies have linked SH2B adaptor protein 3 (SH2B3, LNK) to hypertension and renal disease, although little experimental investigation has been performed to verify a role for SH2B3 in these pathologies. SH2B3, a member of the SH2B adaptor protein family, is an intracellular adaptor protein that functions as a negative regulator in many signaling pathways, including inflammatory signaling processes. To explore a mechanistic link between SH2B3 and hypertension, we targeted the SH2B3 gene for mutation on the Dahl salt-sensitive (SS) rat genetic background with zinc-finger nucleases. The resulting mutation was a 6-bp, in-frame deletion within a highly conserved region of the Src homology 2 (SH2) domain of SH2B3. This mutation significantly attenuated Dahl SS hypertension and renal disease. Also, infiltration of leukocytes into the kidneys, a key mediator of Dahl SS pathology, was significantly blunted in the Sh2b3(em1Mcwi) mutant rats. To determine whether this was because of differences in immune signaling, bone marrow transplant studies were performed in which Dahl SS and Sh2b3(em1Mcwi) mutants underwent total body irradiation and were then transplanted with Dahl SS or Sh2b3(em1Mcwi) mutant bone marrow. Rats that received Sh2b3(em1Mcwi) mutant bone marrow had a significant reduction in mean arterial pressure and kidney injury when placed on a high salt diet (4% NaCl). These data further support a role for the immune system as a modulator of disease severity in the pathogenesis of hypertension and provide insight into inflammatory mechanisms at play in human hypertension and renal disease.

Competing risks data analysis under the accelerated failure time model with missing cause of failure

Competing risks data with missing cause of failure are analyzed under the accelerated failure time model which is a popular semiparametric linear model in survival analysis. The missing mechanism is assumed to be missing at random. The inverse probability weighted and double robust techniques are used to modify the rank-based estimating functions for competing risks data with complete observations on cause of failure. Proper optimization technique is utilized to obtain the desired estimators. The proposed algorithm overcomes the difficulty in solving the rank estimating equations with discontinuous estimating functions. The asymptotic properties of the proposed estimators are established. To implement the related inferences, a nonparametric bootstrap approach as well as a score test is developed. Simulation studies are carried out to assess the finite sample performance of the proposed method and validate the theoretical findings. The new estimating procedure is illustrated with the data from a bone marrow transplant study.

Depletion of Bone Marrow CCSP-Expressing Cells Delays Airway Regeneration

The contribution of bone marrow cells (BMC) in lung repair is controversial. We previously reported a subpopulation of BMC that express Clara cell secretory protein (CCSP). To determine the contribution of endogenous CCSP(+) BMC to airway regeneration, we performed bone marrow transplantation studies using the CCtk mouse, which expresses a thymidine kinase suicide gene under regulation of the CCSP promoter. Mice were transplanted with wild-type or CCtk BMC and treated with ganciclovir to eliminate CCSP(+) cells. After airway injury using naphthalene, mice depleted of CCSP(+) BMC had more inflammatory cells in lung and decreased levels of oxygen in arterial blood. They also had reduced expression of airway epithelial genes and less Clara cells compared to control mice that had intact CCSP(+) BMC and bone marrow derived CCSP(+) cells in the airways. After naphthalene injury, administration of CCSP reproduced the beneficial effect of CCSP(+) BMC by improving recovery of airway epithelium, reducing lung inflammation and increasing oxygen in arterial blood from mice depleted of CCSP(+) BMC. Our data demonstrate that ablation of CCSP(+) BMC delays airway recovery and suggests the beneficial effect of CCSP(+) BMC in lung recovery is in part due to production of CCSP itself.