Increase In Spleen Size Research Articles

JAK2 inhibitors do not affect stem cells present in the spleens of patients with myelofibrosis

AbstractDysregulation of Janus kinase (JAK)–signal transducer and activator of transcription signaling is central to the pathogenesis of myelofibrosis (MF). JAK2 inhibitor therapy in MF patients results in a rapid reduction of the degree of splenomegaly, yet the mechanism underlying this effect remains unknown. The in vitro treatment of splenic and peripheral blood MF CD34+ cells with the JAK1/2/3 inhibitor, AZD1480, reduced the absolute number of CD34+, CD34+CD90+, and CD34+CXCR4+ cells as well as assayable hematopoietic progenitor cells (HPCs) irrespective of the JAK2 and calreticulin mutational status. Furthermore, AZD1480 treatment resulted in only a modest reduction in the proportion of HPCs that were JAK2V617F+ or had a chromosomal abnormality. To study the effect of the drug on MF stem cells (MF-SCs), splenic CD34+ cells were treated with AZD1480 and transplanted into immunodeficient mice. JAK2 inhibitor therapy did not affect the degree of human cell chimerism or the proportion of malignant donor cells. These data indicate that JAK2 inhibitor treatment affects a subpopulation of MF-HPCs, while sparing another HPC subpopulation as well as MF-SCs. This pattern of activity might account for the reduction in spleen size observed with JAK2 inhibitor therapy as well as the rapid increase in spleen size observed frequently with its discontinuation.

B cell development undergoes profound modifications and adaptations during pregnancy in mice.

Pregnancy hides an immunological riddle combining two antagonistic characteristics of immunology: the existence of a tolerance that allows the gestation of a semiallogeneic fetus and proper protection against pathogens threatening the health of the immunocompromised mother. Despite the fundamental role that B cells play in orchestrating an immune response, their behavior in the context of pregnancy has been barely investigated. Here we demonstrate that numbers of pre/pro and immature B cells were progressively diminished in the bone marrow (BM) of pregnant mice, leading to a reduced influx of B cells in blood and spleen. Correspondingly, lower levels of B cell-activating factor of the TNF family were observed in serum of pregnant mice. In contrast to immature B cells, mature B cells were accumulated in the BM during pregnancy. Accordingly, higher numbers of mature B cells were observed in the lymph nodes draining the uterus as well as in the peritoneal cavity of pregnant mice, both tissues in close contact with the fetuses. Despite an increase in spleen size, pregnant mice showed lower numbers of splenic B cells, which was mirrored by lower numbers of immature and FO B cells. However, marginal zone B cells in the spleen increased during pregnancy. Additionally, serum IgM, IgA, and IgG3 titers were elevated in pregnant mice. Collectively, our data show how the B cell compartment adapts to the presence of the semiallogeneic fetus during gravidity.

Therapeutic Tissue Regeneration by a Macrophage Colony-Stimulating Factor Fc Conjugate

Therapeutic Tissue Regeneration by a Macrophage Colony-Stimulating Factor Fc Conjugate

Correlation between platelet count and both liver fibrosis and spleen diameter in patients with schistosomiasis mansoni.

Studies have described the correlation between platelet count and the stages of fibrosis in chronic viral hepatitis, but few publications have studied this correlation in Schistosomiasis mansoni. Therefore, this study aimed to correlate platelet count with both the periportal fibrosis pattern and spleen diameter evaluated by ultrasound exam in patients with Schistosomiasis mansoni. Patients with Schistosomiasis mansoni were evaluated by abdominal ultrasound by a single examiner for the determination of periportal fibrosis pattern (Niamey classification) and spleen diameter. Platelet counts were performed in an automated cell counter. One hundred eighty-seven patients with Schistosomiasis mansoni (mean age: 50.2 years) were included in the study, 114 of whom (61%) were women. Based on the Niamey classification, the ultrasound analysis revealed that 37, 64, 64 and 22 patients exhibited patterns C, D, E and F, respectively. In these four groups, the mean number of platelets was 264, 196, 127 and 103 x 109/L and mean spleen diameter was 9.2, 11.9, 14.9 and 16.2 centimeters, respectively. A reduction in platelet count was significantly associated with both the progression of the periportal fibrosis and the increase in spleen size. Platelet count in patients with Schistosomiasis mansoni was inversely correlated with the severity of periportal fibrosis and spleen diameter.

PU.1 promotes cell cycle exit in the murine myeloid lineage associated with downregulation of E2F1

Acute myeloid leukemia (AML) is characterized by increased proliferation and reduced differentiation of myeloid lineage cells. AML is frequently associated with mutations or chromosomal rearrangements involving transcription factors. PU.1 (encoded by Sfpi1) is an E26 transformation-specific family transcription factor that is required for myeloid differentiation. Reduced PU.1 levels, caused by either mutation or repression, are associated with human AML and are sufficient to cause AML in mice. The objective of this study was to determine whether reduced PU.1 expression induces deregulation of the cell cycle in the myeloid lineage. Our results showed that immature myeloid cells expressing reduced PU.1 levels (Sfpi1(BN/BN) myeloid cells) proliferated indefinitely in cell culture and expanded invivo. Transplantation of Sfpi1(BN/BN) cells induced AML in recipient mice. Cultured Sfpi1(BN/BN) cells expressed elevated messenger RNA transcript and protein levels of E2F1, an important regulator of cell cycle entry. Restoration of PU.1 expression in Sfpi1(BN/BN) myeloid cells blocked proliferation, induced differentiation, and reduced E2F1 expression. Taken together, these data show that PU.1 controls cell cycle exit in the myeloid lineage associated with downregulation of E2F1 expression.

Abstract C9: MACC1 enhances intestinal tumorigenesis in ApcMin mice.

Abstract Colorectal cancer is one of the most frequently occurring malignancies worldwide. Although about 50% of the patients with colorectal cancer can be cured by surgery and multimodal treatment, the successful outcome of CRC patients is seriously affected by the metastatic dissemination of primary tumors. We have previously identified a novel gene termed MACC1 (metastasis-associated in colon cancer 1), which has a strong prognostic value for colon cancer metastasis, enhancing the metastatic potential of colon cancer cells both in vitro and in vivo. To further understand the role of MACC1 on colorectal carcinogenesis, we generated a transgenic mouse model with intestine-specific overexpression of MACC1 (vil-MACC1). Additionally, we crossed these animals with ApcMin mice to create vil-MACC1/ApcMin mice. Four vil-MACC1 founder lines showed MACC1 transgene mRNA and protein expression in the small intestine and colon, but not in other organs including liver, lung, and kidney. Although the villin promoter drove MACC1 expression through the whole vertical (crypt-villus) and horizontal (duodenum-colon) intestinal axes, higher levels of transgenic MACC1 were detected in the small intestine as compared with the colon, and in the villi as compared with the crypts. Histopathological analysis of the intestine of 3-months old vil-MACC1 mice showed no abnormalities as compared with age-matched wild-type littermate controls. Similar analysis is currently being performed in older animals (≥ 1 year old). In line with what we observed in the vil-MACC1 animals, the vil-MACC1/ApcMin mice displayed a similar pattern of MACC1 mRNA and protein expression throughout the whole small intestine and colon. Remarkably, transgenic overexpression of MACC1 in the intestine of vil-MACC1/ApcMin mice significantly increased the total number of adenomas, as compared with ApcMin littermate controls (P = 0.0146). Moreover, the small intestine of vil-MACC1/ApcMin mice displayed an increased number of large-sized adenomas (diameter ≥ 3 mm; P = 0.0033). Additionally, we also observed a slight increase in spleen size in vil-MACC1/ApcMin mice, as compared with ApcMin controls. Despite the increased tumor burden on vil-MACC1/ApcMin mice, we didn't observe a significant difference on survival between the two animal groups. We report here for the first time the generation of a mouse model with genetically engineered MACC1 expression. More importantly, our current findings demonstrate that MACC1 is instrumental for intestinal adenoma formation and development in vivo. These results further strengthen the importance of MACC1 for intestinal tumorigenesis and metastasis, underlying its potential as a new target for colorectal cancer treatment. Funded by the Alexander von Humboldt Foundation, the Wilhelm Sander Foundation, the Preclinical Comprehensive Cancer Center and the German Cancer Consortium Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C9. Citation Format: Clara Lemos, Cynthia Voss, Boris Jerchow, Wolfram Haider, Ulrike Stein. MACC1 enhances intestinal tumorigenesis in ApcMin mice. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C9.

Assessment of genetic correlation between bacterial cold water disease resistance and spleen index in a domesticated population of rainbow trout: identification of QTL on chromosome Omy19.

Selective breeding of animals for increased disease resistance is an effective strategy to reduce mortality in aquaculture. However, implementation of selective breeding programs is limited by an incomplete understanding of host resistance traits. We previously reported results of a rainbow trout selection program that demonstrated increased survival following challenge with Flavobacterium psychrophilum, the causative agent of bacterial cold water disease (BCWD). Mechanistic study of disease resistance identified a positive phenotypic correlation between post-challenge survival and spleen somatic-index (SI). Herein, we investigated the hypothesis of a genetic correlation between the two traits influenced by colocalizing QTL. We evaluated the inheritance and calculated the genetic correlation in five year-classes of odd- and even-year breeding lines. A total of 322 pedigreed families (n = 25,369 fish) were measured for disease resistance, and 251 families (n = 5,645 fish) were evaluated for SI. Spleen index was moderately heritable in both even-year (h2 = 0.56±0.18) and odd-year (h2 = 0.60±0.15) lines. A significant genetic correlation between SI and BCWD resistance was observed in the even-year line (rg = 0.45±0.20, P = 0.03) but not in the odd-year line (rg = 0.16±0.12, P = 0.19). Complex segregation analyses of the even-year line provided evidence of genes with major effect on SI, and a genome scan of a single family, 2008132, detected three significant QTL on chromosomes Omy19, 16 and 5, in addition to ten suggestive QTL. A separate chromosome scan for disease resistance in family 2008132 identified a significant BCWD QTL on Omy19 that was associated with time to death and percent survival. In family 2008132, Omy19 microsatellite alleles that associated with higher disease resistance also associated with increased spleen size raising the hypothesis that closely linked QTL contribute to the correlation between these traits. To our knowledge, this is the first estimation of spleen size heritability and evidence for genetic linkage with specific disease resistance in a teleost fish.

<b><i>In vivo</i> toxicological evaluation of crude sulfated polysaccharide from the green seaweed <i>Caulerpa cupressoides</i> var. <i>lycopodium</i> in Swiss mice</b> - doi: 10.4025/actascitechnol.v35i4.15699

Seaweeds are widely consumed as vegetables and medicinal products. The green seaweed Caulerpa cupressoides var. lycopodium sulfated polysaccharides (SPs) demonstrated anticoagulant (in vitro) and anti- and prothrombotic (in vivo) effects. However, their toxicity in vivo has not been fully determined. This study evaluated their toxicity in vivo in male Swiss mice. Animals (20-26 g, six group-1) received crude SP (9 mg kg-1, i.p.) or 0.9% saline (0.1 mL 10 g-1, i.p.) for 14 consecutive days, and then analyzed the wet weight of animal’s body organs-1 and biochemical/hematological parameters. Histopathological evaluation was also performed related to crude SP treatment. The results showed that crude SP did not cause toxicity and mortality. Regarding the biochemical analyses, crude SP did not lead to hepatic or renal dysfunctions, but affected (p < 0.05) the platelet count (1530.75 ± 1.05 × 103 µL-1) compared with the control (969.75 ± 0.51 × 103 µL-1) according to the hematological evaluation. Although histological changes in the liver and kidney have occurred, results suggested reversibility. The increased spleen size (p < 0.05) also had no toxicological significance based on histopathological analysis. Therefore, crude SP from C. cupressoides could represent safe pharmacological tool in future studies on immunomodulation and thrombosis in vivo.

Systemic and immunotoxicity of silver nanoparticles in an intravenous 28 days repeated dose toxicity study in rats

Because of its antibacterial activity nanosilver is one of the most commonly used nanomaterials. It is increasingly used in a variety of both medical and consumer products resulting in an increase in human exposure. However, the knowledge on the systemic toxicity of nanosilver is relatively limited. To determine the potential systemic toxicity of silver nanoparticles (Ag-NP) with different sizes (20 nm and 100 nm) a 28-days repeated dose toxicity study was performed in rats using intravenous administration. The toxic effect of the 20 nm Ag-NP was performed using the bench mark dose (BMD) approach.Treatment with a maximum dose of 6 mg/kg body weight was well tolerated by the animals. However, both for 20 nm and 100 nm Ag-NP growth retardation was observed during the treatment. A severe increase in spleen size and weight was present which was due to an increased cell number. Both T and B cell populations showed an increase in absolute cell number, whereas the relative cell numbers remained constant. At histopathological evaluation brown and black pigment indicating Ag-NP accumulation was noted in spleen, liver, and lymph nodes. Ag-NP was also detected incidentally in other organs. Clinical chemistry indicated liver damage (increased alkaline phosphatase, alanine transaminase, and aspartate transaminase) that could not be confirmed by histopathology. Hematology showed a decrease in several red blood cell parameters.The most striking toxic effect was the almost complete suppression of the natural killer (NK) cell activity in the spleen at high doses. Other immune parameters affected were: decreased interferon-γ and interleukin (IL)-10 production by concanavalin-A stimulated spleen cells, increased IL-1β and decreased IL-6, IL-10 and TNF-α production by lipopolysaccharide stimulated spleen cells, increase in serum IgM and IgE, and increase in blood neutrophilic granulocytes. For the spleen weight a critical effect dose of 0.37 mg/kg body weight (b.w.) could be established. The lowest critical effect dose (CED) for a 5% change compared to control animals was observed for thymus weight (CED05 0.01 mg/kg b.w.) and for functional immune parameters, i.e. decrease in NK cell activity (CED05 0.06 mg/kg b.w.) and LPS stimulation of spleen cells (CED05 0.04 mg/kg b.w.). These results show that for nanosilver the most sensitive parameters for potential adverse responses were effects on the immune system.

Distinct Roles for Neutrophils and Dendritic Cells in Inflammation and Autoimmunity in motheaten Mice

The motheaten mouse has long served as a paradigm for complex autoimmune and inflammatory disease. Null mutations in Ptpn6, which encodes the nonreceptor protein-tyrosine phosphatase Shp1, cause the motheaten phenotype. However, Shp1 regulates multiple signaling pathways in different hematopoietic cell types, so the cellular and molecular mechanism of autoimmunity and inflammation in the motheaten mouse has remained unclear. By using floxed Ptpn6 mice, we dissected the contribution of innate immune cells to the motheaten phenotype. Ptpn6 deletion in neutrophils resulted in cutaneous inflammation, but not autoimmunity, providing an animal model of human neutrophilic dermatoses. By contrast, dendritic cell deletion caused severe autoimmunity, without inflammation. Genetic and biochemical analysis showed that inflammation was caused by enhanced neutrophil integrin signaling through Src-family and Syk kinases, whereas autoimmunity resulted from exaggerated MyD88-dependent signaling in dendritic cells. Our data demonstrate that disruption of distinct Shp1-regulated pathways in different cell types combine to cause motheaten disease.

Update on ultrasound imaging of liver fibrosis

Update on ultrasound imaging of liver fibrosis

Caspase-8 Blocks Kinase RIPK3-Mediated Activation of the NLRP3 Inflammasome

Caspase-8 deficiency in certain cells prompts chronic inflammation. One mechanism suggested to account for this inflammation is enhanced signaling for necrotic cell death, mediated by the protein kinases RIPK1 and RIPK3 that caspase-8 can cleave. We describe an activity of caspase-8 in dendritic cells that controls the initiation of inflammation in another way. Caspase-8 deficiency in thesecells facilitated lipopolysaccharide-induced assembly and function of the NLRP3 inflammasome. This effect depended on the functions of RIPK1 and RIPK3, as well as of MLKL and PGAM5, two signaling proteins recently shown to contribute to RIPK3-mediated induction of necrosis. However, although enhancement of inflammasome assembly in the caspase-8-deficient cells shares proximal signaling events with the induction of necrosis, it occurred independently of cell death. These findings provide new insight into potentially pathological inflammatory processes to which RIPK1- and RIPK3-mediated signaling contributes.

Zeb2-Defficiency in the Adult Murine Hematopoietic Precursor Cells Leads to Differentiation Defects in Multiple Hematopoietic Lineages and a Myeloproliferative-Like Phenotype

AbstractAbstract 1199The life long replenishment of highly specialized blood cells by a small number of hematopoietic cells (HSC) requires a strict regulation between self-renewal and differentiation in the immature compartment of the bone marrow. Perturbation of this equilibrium can result in stem cell loss or hematologic malignancies. This balance is at least in part controlled by a network of transcription factors. Zeb2 is a transcriptional repressor and plays an important role during the embryonic development as a modulator of the epithelial to mesenchymal transition (EMT) as well as tumor progression and metastasis. We have previously identified the essential role of Zeb2 in murine embryonic hematopoiesis, where selective Zeb2 deficiency in the hematopoietic stem cells resulted in early lethality around day 12.5. The aim of this study was to analyze whether Zeb2 plays a specific role in the regulation of homeostasis in the adult hematopoietic system.Using the Mx1-Cre based inducible Zeb2 conditional knock out mouse model we analyzed the impact of Zeb2 loss on adult hematopoietic stem cell function. Upon the induction of Zeb2 deletion we found a significant decrease in most cell lineages of the peripheral blood, except the neutrophil granulocytes. However, the reduction of mature cells in the blood was not accompanied by reduced bone marrow cellularity, as the cellularity was similar between Zeb2Δ/Δ Mx1-Cre (Zeb2 conditional KO) mice and the control animals (Zeb2+/+Mx1-Cre). However, in the bone marrow of the Zeb2Δ/Δ Mx1-Cre animals the granulocytic lineage was dominating, whereas other lineages e.g. red blood cell precursors and B-lymphoid precursors were drastically reduced. Histological sections of the bone marrow cavity revealed megacaryocytes with abnormal morphology reflecting maturation defects and an increased production of reticular fibers in the BM of Zeb2Δ/Δ Mx1-Cre mice. In addition Zeb2Δ/Δ Mx1-Cre mice displayed a two to three fold increase in spleen size compared to control animals due to an extramedullary hematopoiesis. Analysis of the primitive hematopoietic compartment in the bone marrow and spleens revealed that Zeb2 deletion resulted in a pronounced increase in the most immature hematopoietic cells, defined as Lin-Sca1+cKit+CD48-CD150+ population, and perturbation in different lineage restricted progenitor subpopulations. No difference in cell cycling or apoptotic rate in the stem cell enriched bone marrow population (Lin-Sca1+cKit+CD48-CD150+) was detectable between the genotypes. Upon transplantation into lethally irradiated wild type recipients, Zeb2 deficient stem cells demonstrated significantly reduced ability to differentiate into multiple hematopoietic lineages indicating a niche independent effect of Zeb2 in promoting differentiation of hematopoietic stem cells. On the molecular level, gene expression analysis of hematopoietic stem and progenitor cells using microarray approach revealed increased transcripts of downstream targets of Wnt/ß-Catenin signaling, suggesting increased Wnt signaling activity in absence of Zeb2 in the hematopoietic compartment, which at least in part might be responsible for the observed phenotype.These data indicate that Zeb2 is involved in the regulation of the balance between self-renewal and differentiation at multiple stages of hematopoietic cell maturation. Furthermore the lack of Zeb2 in the hematopoietic compartment leads to a phenotype that resembles the features of human myeloproliferative disorders, especially the early stages of primary myelofibrosis with dominant granulopoiesis, production of reticular fibers in the bone marrow, and morphological abnormalities in megacaryocytes, accompanied by extramedullary hematopoiesis. Disclosures:No relevant conflicts of interest to declare.

L3MBTL1: A Polycomb Protein At the Node of Crosstalk Between the BMP4 and Hippo Signaling Pathways in Erythropoiesis.

AbstractAbstract 2296The Hippo signaling pathway, first discovered in Drosophila, is emerging as an important regulator of stem cell behavior. Upon still-unclear upstream stimuli, the hippo pathway kinase cascade phosphorylates and inhibits the function of YAP, a transcription coactivator, by inducing its cytoplasmic retention. While recent evidences indicate that inhibition of YAP affects cell fate decisions, and proliferation, in many tissues, little is known about the relevance of this pathway in hematopoiesis. However, the interaction of YAP with Smad1, identified in flies and human cells (Alarcon C. et al. Cell 2009), prevents smurf-mediated Smad1 degradation, potentially enhancing BMP signaling.Our ongoing studies have indentified crosstalk between the BMP4 and the Hippo pathways in hematopoietic cells, and in induced-pluripotent stem (iPS) cells that we differentiated towards the erythroid lineage. This crosstalk involves the chromatin-binding, Polycomb protein L3MBTL1, which clearly regulate the effects of BMP on the erythroid differentiation of hematopoietic stem/progenitor cells and on fetal globin gene expression.We find that the Lats2 kinase, a core component of the Hippo pathway, physically interacts with L3MBTL1 and that treatment with BMP4 or Erythropoietin decreases the expression of both proteins in various hematopoietic cells, including primary human cord blood-derived CD34+ cells. By altering L3MBTL1 levels in K562 cells, we were able to show that the L3MBTL1-Lats2 interaction enhances Lats-mediated phosphorylation and the cytoplasmic retention of YAP. Furthermore, L3MBTL1-depleted iPS cells have an enhanced smad-mediated transcriptional response; by analyzing the gene expression profile of these cells, we found increased expression of several BMP target genes (such as HHEX and ID genes), suggesting that L3MBTL1 negatively titrates the BMP4 signaling pathway at least in part by affecting YAP phosphorylation and localization. Gene Set Enrichment Analysis confirmed enrichment of many smad-related genes, and yet, these cells presented enhanced smad1/5/8 phosphorylation by WB analysis, indicating that BMP4 signaling is triggered by L3MBTL1 depletion. We also found that hematopoietic differentiation of L3MBTL1-KD iPS cells generates high-fetal globin gene expressing erythroid progeny, suggesting a role for the BMP4 signaling pathway and the targeting of L3MBTL1 in the treatment of hemoglobinopathies.To further evaluate the effect of BMP4 signaling on hematopoietic cells that lack L3MBTL1, we analyzed the stress erythroid response of L3MBTL1 KO mice: while no difference was observed at baseline in the null mice compared to wt littermates, the L3mbtl1 null mice had a more severe anemia, with increased leukocytosis, and thrombocytosis post-hydrazine (PHZ) or Epo. We found a significant increase in the colony-forming ability of the l3mbtl1 null spleen and bone marrow cells, compared to controls, as well as increased spleen size and an expansion of the spleen erythroid compartment. Thus, l3mbtl1 null hematopoietic stem cells are more sensitive to the PHZ-mediated cytokine storm, which includes BMP4. Interestingly, the L3mbtl1 null BM and spleen cells showed diminished expression of Lats2 and phospho-YAP, consistent with our in vitro findings.In conclusion, these investigations have shown that L3MBTL1 not only negatively titrates the BMP4 signaling pathway, but also provides a nodal point for crosstalk between the BMP4 and Hippo signaling pathways in erythropoiesis. Thus, these data provide insights into possible novel treatments for genetic red cell disorders (such as β-thalassemia) and for acquired bone marrow failure syndromes such as Epo-resistant anemia. Disclosures:Levine:Agios Pharmaceuticals: Research Funding.

Frequency and characteristic features of portal hypertensive gastropathy in patients with viral cirrhosis.

To determine the frequency and specific characteristic features of portal hypertensive gastropathy (PHG) in cirrhosis due to viral etiology. Cross-sectional descriptive study. The Aga Khan University Hospital, Karachi, from June 2006 till June 2008. Patients with hepatitis B and C cirrhosis were included who underwent screening esophago-gastroduodenoscopy (EGD) for varices. Baveno III consensus statement was used for diagnosing PHG on endoscopy and divided them into two subgroups i.e. mild and severe. Data related to platelet/spleen ratio, MELD score and Child Turcotte Pugh (CTP) score indicating severity of cirrhosis were recorded in all patients. Findings were compared by using independent sample t-test. Out of 360 patients who underwent screening EGD, 226 (62.8%) were males. Two hundred and eighty one (78%) had hepatitis C while 79 (22%) suffered from hepatitis B related cirrhosis. Three hundred patients (83.3%) had PHG while 71 (24%) had severe PHG. Higher proportion of esophageal varices (89.7%) was present among those who had PHG (p < 0.001). On univariate analysis lower platelet counts (117±55 vs. 167±90; p < 0.001), increased spleen size (14.1±2.9 cm vs. 12±2.4cm; p < 0.001) were found in PHG patients as compared to those without it. Similarly, lower platelet/spleen ratio was noted in patients with severe PHG (916±400 vs. 1477±899; p < 0.001). Furthermore, on multivariate analysis CTP score > 8 MELD score > 12 and platelets/spleen ratio < 900 were significantly associated factors with severe PHG. Frequency of PHG was 83% while severe PHG was seen in 24% cases of viral hepatic cirrhosis. MELD score > 12, CTP score ³ 8 and platelets/spleen ratio < 900 were significant factors of severe PHG.

Membrane-bound Fas ligand accelerates autoimmune disease in an induced murine model of systemic lupus erythematosus (123.3)

Abstract Lpr and gld, are loss of function mutations in the Fas death receptor and its ligand, respectively. However, in addition to its pro-apoptotic activity, FasL can also trigger the production of pro-inflammatory cytokines. The lpr and gld mutations normally result in a lymphoproliferative syndrome and exacerbate clinical disease in SLE autoimmune-prone strains of mice. Unexpectedly, the lpr and gld mutations were reported to attenuate disease progression in mice injected with pristane, an agent known to induce SLE. FasL is a transmembrane protein with a matrix metalloproteinase (MMP) cleavage site in the ectodomain. MMP cleavage inactivates membrane-bound FasL (mFasL) and releases a soluble form, sFasL, reported to have both antagonist and agonist activity. To better understand the impact of FasL cleavage, we have eliminated the cleavage site through targeted mutation, to produce the ΔCS line. ΔCS mice express more mFasL and fail to make sFasL. Pristane-injected ΔCS mice exhibit a markedly exacerbated disease profile associated with a striking change in ANA specificity, increased proteinuria and kidney pathology, a massive increase in spleen size due to infiltrating granulocytes and macrophages, a bias toward a Th2 response, and a trend towards increased numbers of Th17 T cells, compared to control mice. These results demonstrate that cleavage plays an important role in limiting the pro-inflammatory activity of FasL in the context of pristane-induced autoimmunity.

Neutropenia in Glycogen Storage Disease 1b (GSD1b)

Abstract 4791Neutropenia and recurrent infections are common manifestations of glycogen storage disease 1b (GSD1b), a disease caused by mutations in G6PT1, the glucose 6 phosphate transporter 1 gene. Patients with GSD1b have complex defects in neutrophil deployment and function. GSD1b causes recurrent infections – gingivitis, mouth ulcers, respiratory infections, abscesses and enterocolitis. Granulocyte colony-stimulating factor (G-CSF) has been used to treat these patients for more than 2 decades.We reviewed the presentations, manifestations, complications and G-CSF treatment for 54 GSD1b patients (31 adults and 23 children) at Duke University, the University of Florida and through the Severe Chronic Neutropenia International Registry at the University of Washington. Observational data were available on most patients for at least 3 years (mean = 12 years, median = 13 years, range = 1 to 23 years). Available data showed that before treatment with G-CSF, all of these patients had mild to moderate neutropenia; the mean of the median absolute neutrophil count (ANC) was 0.839 × 109/L ± 0.165 SEM (median = 0.503 × 109/L, range = 0.038 to 7.665 × 109/L). All of the patients also had recurrent infections; the most common events were mouth ulcers, otitis and skin abscesses. There were 7 patients who had enterocolitis prior to G-CSF treatment.G-CSF therapy was initiated at various ages ranging from birth to age 30 (mean = 6 years, median = 4 years). Initial daily doses of G-CSF ranged from 0.50 to 63.28 mcg/kg/day (mean = 5.86, median = 3.65) and were generally titrated upward to achieve a normal neutrophil count. The mean ANC on treatment was 2.598 × 109/L ± 0.136 SEM (median = 1.856 × 109/L, range = 0.0 to 12.645 × 109/L). G-CSF treatment was uniformly associated with an increase in blood neutrophils and reduced patterns of infection. In most patients the symptoms of enterocolitis improved within a few days to a few weeks after starting G-CSF therapy. An increase in spleen size also commonly occurred. In 630 patient-years of observation, there have been 3 deaths attributable to the following causes: 1 subject died of sepsis, 1 subject died of complications from hepatomegaly and neutropenia, and 1 subject died 6 years after a liver transplant. The subject developed AML 5 years after the liver transplant while receiving a combination of immunosuppressant drugs.Patients with GSD1b associated neutropenia respond well to G-CSF with reduced infections and symptoms of enterocolitis. Long-term complications are infrequent and G-CSF is well tolerated in most patients. Disclosures:Dale:Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Boxer:Amgen: Equity Ownership.

JAK2-V617F Expressing Stem Cells Display a Competitive Advantage At Low Limiting Dilution and Are Capable of Initiating MPN Phenotype

Abstract 615JAK2-V617F is the most common genetic alteration in myeloproliferative neoplasms (MPN). Forced expression of JAK2-V617F in animal models induced a MPN phenotype. However, MPN in these models was initiated by polyclonal expansion of multiple hematopoietic progenitor and stem cells. In contrast, patients with MPN display clonal hematopoiesis. Frequently, additional somatic mutations precede the acquisition of JAK2-V617F, suggesting that a pre-JAK2 mutation could be required for clonal expansion. Compatible with this hypothesis, in one JAK2-V617F knockin model the MPN phenotype was not transplantable and the stem cells showed signs of exhaustion. To address the question of whether JAK2-V617F alone is sufficient to initiate MPN we performed competitive repopulation assays and serial transplantations in a transgenic JAK2-V617F mouse model.Our transgenic mice express the human JAK2-V617F under the endogenous JAK2 promoter and display a polycythemia vera (PV) or essential thrombocythemia (ET) phenotype. The transgene can be activated by Cre recombinase and the donors were induced 5–6 weeks prior to transplantation. To allow monitoring of the reconstitution, a GFP expressing transgenic strain was used as the source of competitor cells. Bone marrow transplantations were performed into lethally irradiated mice in the inbred BL/6 background. A total of 2×106 bone marrow cells per recipient were transplanted with ratios of mutant (V617F) to wild type (WT) set as 1:1, 1:10 or 1:100. Results:At 1:1 and 1:10 ratios, the mice displayed a PV phenotype beginning at 4 weeks post transplantation (Hb ∼200-220 g/L and neutrophil count of 5–20×109 /L) that remained stable for ∼26 weeks. An increase in platelet counts (4-6×1012/L) followed at later time points. V617F cells rapidly outcompeted WT cells reaching >95% chimerisms in peripheral blood. The MPN phenotype was transplantable into secondary and tertiary recipients and no signs of exhaustion were observed. An increase in the frequencies of lineage-negative, Sca-1+ c-kit+ (LSK) cell numbers in bone marrow was noted (4.1% in V617F vs 1.2% for WT, p<0.05). To assess their cycling rate, LSK cells were sorted, labeled with carboxyfluorescein succinimidyl ester and transplanted into non-conditioned recipients. After 8 weeks, the number of LSK cells was 62-fold increased and V617F LSK cells outcompeted host cells (52% vs. 3% for WT, p<0.01). The number of slow-dividing (0-2 divisions) LSK cells was substantially reduced in the V617F transplanted mice (0.095% vs. 7.54% for WT, p<0.01). Thus, V617F increases the cycling of LSK cells and provides a competitive advantage.To allow detecting low numbers of donor cells in 1:100 transplantations, the GFP transgene was bred into the V617F background and a mixture of 2×104 V617F/GFP bone marrow cells plus 1.98×106 WT competitors was used. Based on the expected frequency of 3 long term hematopoietic stem cells (HSC) per 105 bone marrow cells, the recipients should receive on average 0.6 GFP-positive long term HSC. In mice transplanted with V617F/GFP and WT competitors at 1:100, we detected reconstitution defined as >0.1% GFP positive cells in 12 of 40 recipients (28%) that persisted beyond 20 weeks, whereas in mice transplanted with WT/GFP and WT competitors, only 5/40 (12%) showed reconstitution. The relative contribution in the WT group ranged from 0.1 – 3%, whereas in the V617F group we observed chimerism between 5 –100%. Four of the 12 mice that reconstituted with V617F/GFP cells developed a MPN phenotype (3PV, 1ET). Terminal workup of 3 mice (2PV and 1ET) at 29 weeks revealed that mice with erythrocytosis had enlarged spleens (∼150mg vs. 65mg for WT) and increased erythropoiesis in spleen histology. In contrast the mouse with ET phenotype showed only a slight increase in spleen size (90mg vs. 65mg for WT) and no increase in spleen erythropoiesis. Conclusions:Our results show that JAK2-V617F positive cells were able to rapidly outcompete WT cells. The MPN phenotype was transplantable into secondary and tertiary recipients. At low limiting dilution (1:100), V617F cells displayed > 2-fold higher engraftment rate (28%) than WT cells (12%) and a higher contribution to hematopoiesis (5-100% chimerism vs. 0.1–3%). Since 4/12 mice (30%) reconstituted with V617F cells also developed a MPN phenotype, our data demonstrate than JAK2-V617F as the sole genetic alteration can initiate MPN under conditions resembling monoclonal disease. Disclosures:No relevant conflicts of interest to declare.

GH, But Not GHRH, Plays a Role in the Development of Experimental Autoimmune Encephalomyelitis

GH has been suggested to influence the function of the immune system in several species. Experimental autoimmune encephalomyelitis (EAE) (an animal model for multiple sclerosis) has been reported not to occur in GH-deficient (GHD) mice. The aim of this study was to elucidate the effects of GH and GHRH replacement on development of EAE in a mouse model of isolated GHD due to removal of the GHRH gene [GHRH knockout (GHRHKO)]. We studied two groups of adult female mice: 12 GH-sufficient animals (control) and 36 GHRHKO animals. All mice were immunized with myelin oligodendrocyte glycoprotein peptide, a peptide known to induce EAE. GHRHKO mice were left untreated or were treated for 4 wk with daily sc injections of recombinant GH or of a GHRH super agonist JI-38 (JI38-GHD). Evaluation of EAE symptoms was carried out daily, and T-proliferative assay and histopathological analysis of the spinal cord were performed. GHRHKO mice were less prone to develop EAE when compared with control mice. GH (but not JI-38) restored the original susceptibility of mice to the disease, despite lack of complete serum IGF-I normalization. GH treatment was also associated with a markedly increase in spleen size and T-cell proliferation specific to myelin oligodendrocyte glycoprotein peptide. GH (but not GHRH) plays an important role in the development of EAE.

B cell activation contributes to splenomegaly in PPARγ deficient mice (153.21)

Abstract PPARγ is a nuclear transcription factor that modulates diverse biofunctions, including lipid biosynthesis, glucose metabolism and inflammation. However, the specific role of PPARγ in the immune function has not been fully explored. We have previously generated mice with PPARγ gene expression from 25% to 100% normal. We found that mice with 25% expression of PPARγ (PpargC/-) exhibited a two-fold increase in spleen size at their young age and lasted throughout the life. It has been demonstrated PPARγ deficiency enhanced bone formation and relocated hematopoiesis to the spleen. However, micro-CT analysis showed trabecular volume, number, thickness, and bone mineral density were increased in old, but not in young, PpargC/- mice. The population of hematopoietic stem cells (HSCs) and the expression level of transcription factors, crucial for HSC differentiation and self-renewal, in spleen were also increased in old PpargC/- mice. However, young PpargC/- with splenomegaly revealed neither increased bone formation nor relocation of hematopoiesis to the spleen. In vivo BrdU incorporation study demonstrated that B cells were highly proliferated in young PpargC/- mice. Therefore, further studies are needed to examine the role of PPARγ in immune system. These results suggest that splenomegaly in young PpargC/- mice was not likely caused by extramedullary hematopoiesis, but may be contributed by increased activation of B cells, implicating the beneficial role of PPARγ in immunomodulation.