TG Cells Research Articles

Aberrant glycosylation and localization of polycystin-1 cause polycystic kidney in an AQP11 knockout model.

We previously reported that disruption of the aquaporin-11 (AQP11) gene in mice resulted in cystogenesis in the kidney. In this study, we aimed to clarify the mechanism of cystogenesis in AQP11(-/-) mice. To enable the analyses of AQP11 at the protein level in vivo, AQP11 BAC transgenic mice (Tg(AQP11)) that express 3×HA-tagged AQP11 protein were generated. This AQP11 localized to the endoplasmic reticulum (ER) of proximal tubule cells in Tg(AQP11) mice and rescued renal cystogenesis in AQP11(-/-) mice. Therefore, we hypothesized that the absence of AQP11 in the ER could result in impaired quality control and aberrant trafficking of polycystin-1 (PC-1) and polycystin-2 (PC-2). Compared with kidneys of wild-type mice, AQP11(-/-) kidneys exhibited increased protein expression levels of PC-1 and decreased protein expression levels of PC-2. Moreover, PC-1 isolated from AQP11(-/-) mice displayed an altered electrophoretic mobility caused by impaired N-glycosylation processing, and density gradient centrifugation of kidney homogenate and in vivo protein biotinylation revealed impaired membrane trafficking of PC-1 in these mice. Finally, we showed that the Pkd1(+/-) background increased the severity of cystogenesis in AQP11(-/-) mouse kidneys, indicating that PC-1 is involved in the mechanism of cystogenesis in AQP11(-/-) mice. Additionally, the primary cilia of proximal tubules were elongated in AQP11(-/-) mice. Taken together, these data show that impaired glycosylation processing and aberrant membrane trafficking of PC-1 in AQP11(-/-) mice could be a key mechanism of cystogenesis in AQP11(-/-) mice.

Neonatal infraorbital nerve crush-induced CNS synaptic plasticity and functional recovery

Infraorbital nerve (ION) transection in neonatal rats leads to disruption of whisker-specific neural patterns (barrelettes), conversion of functional synapses into silent synapses, and reactive gliosis in the brain stem trigeminal principal nucleus (PrV). Here we tested the hypothesis that neonatal peripheral nerve crush injuries permit better functional recovery of associated central nervous system (CNS) synaptic circuitry compared with nerve transection. We developed an in vitro whisker pad-trigeminal ganglion (TG)-brain stem preparation in neonatal rats and tested functional recovery in the PrV following ION crush. Intracellular recordings revealed that 68% of TG cells innervate the whisker pad. We used the proportion of whisker pad-innervating TG cells as an index of ION function. The ION function was blocked by ∼64%, immediately after mechanical crush, then it recovered beginning after 3 days postinjury and was complete by 7 days. We used this reversible nerve-injury model to study peripheral nerve injury-induced CNS synaptic plasticity. In the PrV, the incidence of silent synapses increased to ∼3.5 times of control value by 2-3 days postinjury and decreased to control levels by 5-7 days postinjury. Peripheral nerve injury-induced reaction of astrocytes and microglia in the PrV was also reversible. Neonatal ION crush disrupted barrelette formation, and functional recovery was not accompanied by de novo barrelette formation, most likely due to occurrence of recovery postcritical period (P3) for pattern formation. Our results suggest that nerve crush is more permissive for successful regeneration and reconnection (collectively referred to as "recovery" here) of the sensory inputs between the periphery and the brain stem.

The role of lymphotoxin signaling in the development of autoimmune pancreatitis and associated secondary extra-pancreatic pathologies

The pathogenic mechanisms of autoimmune pancreatitis (AIP), an increasingly recognized, immune-mediated form of chronic pancreatitis, have so far remained elusive. Treatment options for AIP are currently limited and disease relapse is frequent. Still, AIP can be characterized by specific clinical and histologic features. It has turned out that as described in other autoimmune diseases the generation of tertiary lymphoid organs is also a hallmark of patients with AIP. We have recently demonstrated that pancreata derived from human AIP patients display overexpression of lymphotoxin (LT) α and β and LTβR-target genes expressed by immune cells but also by irradiation resistant cells of the pancreas (e.g. acinar cells). Expression of LT α and β on acinar cells in murine pancreata Tg(Ela1-Lta,b) mice led to chronic pancreatitis and sufficed to reproduce key features of human AIP including the development of autoimmunity and AIP associated secondary extra pancreatic pathologies. Here, we review how aberrant and ectopic expression of LT α and β can induce inflammation and autoimmune diseases in general and how this knowledge might specifically lead to an alternative treatment for patients suffering from autoimmune pancreatitis.

Spontaneous Loss of Tolerance of Autoreactive B Cells in Act1-Deficient Rheumatoid Factor Transgenic Mice

Self-reactive B cells in BALB/c AM14 transgenic (Tg) rheumatoid factor mice are not subject to central or peripheral tolerization. Instead, they remain at a stage of "clonal ignorance"; that is, they do not proliferate and differentiate into Ab-forming cells. However, the immunoregulatory mechanisms that prevent autoantibody production in these mice remain unclear. In this study, we show that crossing AM14 Tg mice to a mouse strain deficient in Act1, a molecule involved in the regulation of BAFF-R and CD40-signaling in B cells, results in spontaneous activation of AM14 Tg B cells and production of AM14-specific Abs. Three- to 5-mo-old AM14 Tg Act1(-/-) mice showed significant expansion of AM14 Tg B cells, including a 2- to 3-fold increase in the spleen and cervical lymph nodes compared with AM14 Tg Act1(+/+) mice. Furthermore, in the presence of endogenous self-Ag (IgH(a) congenic background), AM14 Tg Act1(-/-) B cells were spontaneously activated and differentiated into Ab-forming cells. In contrast with previous studies using AM14 Tg MLR.Fas(lpr) mice, we found that a significant number of AM14 Tg cells AM14 Tg Act1(-/-) mice displayed phenotypic characteristics of germinal center B cells. Anti-CD40L treatment significantly limited the expansion and activation of AM14 Tg Act1(-/-) B cells, suggesting that CD40L-mediated signals are required for the retention of these cells. Our results support the important role of Act1 in the regulation of self-reactive B cells and reveal how Act1 functions to prevent the production of autoantibodies.

The magnitude of acute hepatitis is more limited by regulation of cytokine signalling than apoptosis of liver-infiltrating effector CD8 T cells (P4174)

Abstract Acute hepatitis is often mediated by CD8 T cells that kill target cells or secrete hepatotoxic cytokines. However, the parameters regulating CD8 T cell-mediated acute hepatitis are not well understood. We used the well-characterized Met-Kb Tg mouse model of acute hepatitis, in which hepatitis is mediated by lymph node activated TCR Tg CD8 recognizing their cognate antigen in the liver. To investigate whether cytokine regulation or apoptosis of CD8 T cells were critical in limiting acute hepatocellular injury, adoptive transfer experiments were performed using TCR Tg T cells deficient for either suppressor of cytokine signaling (SOCS-1) or the pro-apoptotic molecule Bim. Although there was a substantial accumulation of Bim-/- Tg cells, the outcome of hepatitis remained unchanged, suggesting that effector T cell survival was not a critical parameter in limiting liver damage. In contrast, SOCS-1-/- Tg T cells induced a heightened severity of hepatitis than their wt counterparts. SOCS-1-/- Tg cells isolated from the liver displayed upregulated IL-2Rα, required for the high affinity IL-2 receptor, and was associated with higher levels of IFN-γ expression, CTL activity, and proliferation rates, consistent with enhanced effector function. These data support a critical role for cytokines in CTL function, and demonstrate that the propensity of CD8 T cells to mediate acute hepatitis is determined by the quality rather than the number of CTLs infiltrating the liver.

SHP-1 deficiency does not restore the CD8+ T cell response to an LCMV viral variant (P6079)

Abstract Viruses employ various mechanisms to evade recognition by host immune effectors and establish persistence in the host. We have previously shown that CD8+ T cells responding to a viral variant of lymphocytic choriomeningitis virus (LCMV) have high SHP-1 activity. Src homology region 2 domain-containing phosphatase-1 (SHP-1) is a non-receptor phosphatase found in hematopoietic cells and is thought to play a primarily negative regulatory role in many signaling pathways. SHP-1 exerts important negative regulation on proximal TCR signaling by blocking early T cell activation. Because of its role in dampening TCR signaling, we hypothesized that removal of SHP-1 in T cells would restore the response to the viral variant. Using CD4 Cre Fl SHP-1 mice, we evaluated the generation, activation and functionality of CD8+ T cells generated in response to the viral escape mutant. Loss of SHP-1 did not greatly affect response of the P14 TCR Tg cells line assessed in vitro. In vivo analysis of the response to the viral escape mutant did not demonstrate increased T cell numbers or functionality (IFNg secretion) in response to the viral escape mutant or the control chronic viral LCMV infection. Polyclonal T cell responses to the viral challenges were unaffected by SHP-1 deficient. These observations suggest that removal of a negative regulator may not have the expected therapeutic benefit of an enhanced T cell response.

EpCAM Is an Endoderm-Specific Wnt Derepressor that Licenses Hepatic Development

Mechanisms underlying cell-type-specific response to morphogens or signaling molecules during embryonic development are poorly understood. To learn how response to the liver-inductive Wnt2bb signal is achieved, we identify an endoderm-enriched, single transmembrane protein, epithelial-cell-adhesion-molecule (EpCAM), as an endoderm-specific Wnt derepressor in zebrafish. hi2151/epcam mutants exhibit defective liver development similar to prt/wnt2bb mutants. EpCAM directly binds to Kremen1 and disrupts the Kremen1-Dickkopf2 (Dkk2) interaction, which prevents Kremen1-Dkk2-mediated removal of Lipoprotein-receptor-related protein 6 (Lrp6) from the cell surface. These data lead to a model in which EpCAM derepresses Lrp6 and cooperates with Wnt ligand to activate Wnt signaling through stabilizing membrane Lrp6 and allowing Lrp6 clustering into active signalosomes. Thus, EpCAM cell autonomously licenses and cooperatively activates Wnt2bb signaling in endodermal cells. Our results identify EpCAM as the key molecule and its functional mechanism to confer endodermal cells the competence to respond to the liver-inductive Wnt2bb signal.

Expression of dehydrin gene from Arctic Cerastium arcticum increases abiotic stress tolerance and enhances the fermentation capacity of a genetically engineered Saccharomyces cerevisiae laboratory strain

We investigated Arctic plants to determine if they have a specific mechanism enabling them to adapt to extreme environments because they are subject to such conditions throughout their life cycles. Among the cell defense systems of the Arctic mouse-ear chickweed Cerastium arcticum, we identified a stress-responsive dehydrin gene CaDHN that belongs to the SK5 subclass and contains conserved regions with one S segment at the N-terminus and five K segments from the N-terminus to the C-terminus. To investigate the molecular properties of CaDHN, the yeast Saccharomyces was transformed with CaDHN. CaDHN-expressing transgenic yeast (TG) cells recovered more rapidly from challenge with exogenous stimuli, including oxidants (hydrogen peroxide, menadione, and tert-butyl hydroperoxide), high salinity, freezing and thawing, and metal (Zn(2+)), than wild-type (WT) cells. TG cells were sensitive to copper, cobalt, and sodium dodecyl sulfate. In addition, the cell survival of TG cells was higher than that of WT cells when cells at the mid-log and stationary stages were exposed to increased ethanol concentrations. There was a significant difference in cultures that have an ethanol content >16 %. During glucose-based batch fermentation at generally used (30 °C) and low (18 °C) temperatures, TG cells produced a higher alcohol concentration through improved cell survival. Specifically, the final alcohol concentrations were 13.3 and 13.2 % in TG cells during fermentation at 30 and 18 °C, respectively, whereas they were 10.2 and 9.4 %, respectively, in WT cells under the same fermentation conditions. An in vitro assay revealed that purified CaDHN acted as a reactive oxygen species scavenger by neutralizing H2O2 and a chaperone by preventing high temperature-mediated catalase inactivation. Taken together, our results show that CaDHN expression in transgenic yeast confers tolerance to various abiotic stresses by improving redox homeostasis and enhances fermentation capacity, especially at low temperatures (18 °C).

A Dual Role of Evi-1 During Developmental Hematopoiesis

AbstractAbstract 765The Evi-1 locus was originally identified as a common site of retroviral integration in murine myeloid tumors. Several reports associate Evi-1 expression with aggressiveness in myeloid leukemia. More recent studies suggest involvement of Evi-1 in developmental hematopoiesis. Here, we analyzed zebrafish embryo and human induced pluripotent stem (iPS) cells to understand how Evi-1 modulates early blood development. We found that the zebrafish homologue evi-1 co-localizes with the hematopoietic markers scl, gata1 and gata2 during embryogenesis, indicating a role in primitive hematopoiesis. To analyse the functional role of evi-1, we performed in vivo loss-of-function studies by injecting control and inhibitory morpholino oligonucleotides (MO) suppressing evi-1 pre-mRNA splicing in zebrafish zygotes. To control for off-target effects, three separate MO were designed and injected. N = 100 zebrafish embryos were analyzed per experiment in each group. In situ hybridization analyses performed in evi-1 morphants versus control fish revealed strongly impaired formation of myeloid embryonic cells as shown by reduced pu.1, mpo and l-plastin expression and suppressed numbers of DsRed positive lyz-expressing cells in Tg(lyz:DsRed) fish. Interestingly, scl expression was not affected, suggesting that evi-1 acts at later stages, regulating specification and/or survival/proliferation of pu.1 expressing progenitor cells. Consistent with this notion, no changes were observed in primitive erythroid progenitor cells as monitored by gata1 and hbae3 expression and flow cytometry analyses of GFP-positive globin-expressing cells in Tg(globin:eGFP) fish. Furthermore, runx1+/cmyb+ cells were strongly reduced, suggesting a separate role of evi-1 in definitive hematopoiesis. Supporting this hypothesis, evi-1 morphants displayed profound suppression of ikzf1+ lymphoid progenitors and rag1+ T-lymphocytes, which was not due to impaired thymus development, since foxn1 expression remained unaltered in evi-1 morphants. Previous reports in adult murine hematopoietic cells suggest that Evi-1 affects HSC proliferation through regulation of Gata2. Indeed, we could show that co-injection of gata2 mRNA was able to rescue the impaired myeloid as well as the HSC phenotype in evi-1 morphants. However, data generated by TUNEL assays indicate that evi-1 also confers apoptosis resistance to developing AGM-HSC. Whether this effect is also mediated by gata2 is currently being explored. We further investigated the effect of EVI-1 during differentiation of hematopoietic cells from human iPS cells. Confirming our results collected in zebrafish embryo, inhibition of EVI-1 strongly reduced the numbers of CD34+ and CD45+ blood progenitor cells and the generation of myeloid colonies in colony forming assays, while leaving SCL expression unaltered. Taken together, our data suggest that Evi-1 plays conserved roles in zebrafish and human hematopoietic development, regulating embryonic myelopoiesis and HSC formation while leaving primitive erythropoiesis unaltered. The precise molecular basis of evi-1 mediated effects on these separate phases of developmental hematopoiesis is subject of ongoing investigations. Disclosures:No relevant conflicts of interest to declare.

Genetic and Pharmacologic Inhibition of Complement Impairs Endothelial Cell Function and Ablates Ovarian Cancer Neovascularization

Complement activation plays a critical role in controlling inflammatory responses. To assess the role of complement during ovarian cancer progression, we crossed two strains of mice with genetic complement deficiencies with transgenic mice that develop epithelial ovarian cancer (TgMISIIR-TAg). TgMISIIR-TAg mice fully or partially deficient for complement factor 3 (C3) (Tg+C3KO and Tg+C3HET, respectively) or fully deficient for complement factor C5a receptor (C5aR) (Tg+C5aRKO) develop either no ovarian tumors or tumors that were small and poorly vascularized compared to wild-type littermates (Tg+C3WT, Tg+C5aRWT). The percentage of tumor infiltrating immune cells in Tg+C3HET tumors compared to Tg+C3WT controls was either similar (macrophages, B cells, myeloid-derived suppressor cells), elevated (effector T cells), or decreased (regulatory T cells). Regardless of these ratios, cytokine production by immune cells taken from Tg+C3HET tumors was reduced on stimulation compared to Tg+C3WT controls. Interestingly, CD31+ endothelial cell (EC) function in angiogenesis was significantly impaired in both C3KO and C5aRKO mice. Further, using the C5aR antagonist PMX53, tube formation of ECs was shown to be C5a-dependent, possibly through interactions with the VEGF165 but not VEGF121 isoform. Finally, the mouse VEGF164 transcript was underexpressed in C3KO livers compare to C3WT livers. Thus, we conclude that complement inhibition blocks tumor outgrowth by altering EC function and VEGF165 expression.

The Lewis GFP Transgenic Rat Strain is a Useful Cell Donor for Neural Transplantation

Stem cell transplantation is a promising therapeutic approach in neurodegenerative diseases. Studying graft survival and development has important implications for the further development of experimental and clinical transplantation protocols. Cellular elements in neural transplants are sometimes difficult to identify. The existing labeling methods cannot reliably provide stably labeled cells that can be detected in long-term experiments. Transgenic (tg) Lewis rats ubiquitously expressing green fluorescent protein (GFP) provide an ideal donor source. The aim of this project was to investigate the potential of GFP-tg Lewis rats to serve as donor tissue for neural stem cell transplantation. Ventral mesencephalon (VM) GFP-tg E14.5-derived cells were compared to wild-type (wt) in vitro and in vivo. Firstly, cells from GFP and non-GFP VM tissue were compared with regard to their proliferation and response towards 6-OHDA-toxicity in culture. Secondly, 6-OHDA-lesioned hemiparkinsonian Sprague-Dawley/Crl:CD(SD) rats received intrastriatal grafts derived from VM of E14.5 GFP-tg rats. Due to the fact that donor and recipient belong to two different rat strains, we focused on graft survival in correlation with immunosuppression and graft GFP and tyrosine hydroxylase (TH) expression. In summary, in vitro tg cells exhibited 98% GFP expression and did not differ from wt cells in any of the measured parameters. In vivo, all experimental groups showed a significant compensation in rotation behavior after transplantation. Furthermore, there was no difference on rotation behavior or graft morphology and survival pattern as well as GFP expression between immunosuppressed and nonimmunosuppressed animals. The GFP-positive population of the graft was composed of 13.3% GFAP-positive, 56.1% NeuN-positive, and 1.9% TH-positive cells. Analysis of graft subpopulations manifested that 70.6% of GFAP-positive, 86.9% of NeuN-positive, and 80.1% of TH-positive cells coexpressed GFP. In conclusion, our data show that the Lewis GFP-tg rats serve as an excellent cell source for studying primary neural precursor cells in the transplantation paradigm.

B06 Genotoxic stress in fibroblasts and mesenchymal stem cells isolated from miniature pigs transgenic for n-terminal part of mutated human huntingtin

Background Huntington9s disease (HD) is a dominant inherited disorder caused by mutation in huntingtin (htt) protein that causes massive neural cell death leading to neurodegeneration. Mutated huntingtin (mt htt) induces a cascade of events that stimulates DNA breaks and the activation of DNA damage response pathway. This genotoxic stress is followed by activation of the MDC1 and H2AX proteins, in the early stages of HD pathogenesis. Expression of htt occurs in cells of neuronal and non-neuronal origin. Aims The aim of our research is to study genetoxic stress of mt htt on fibroblasts and mesenchymal stem cells (MSCs) of our miniature pig model for HD. Methods Currently, we have three generations of transgenic (tg) minipigs for N-terminal part of the human mt htt (548 aa, 124 Q). Each litter presents wild type (wt) and tg siblings at an approximate ratio 1:1 with the same genetic background. DNA damage was examined on fibroblasts and MSCs isolated from tg and wt minipigs using confocal microscopy, immunocytochemistry, Western blot analysis and the dynamic of tg and wt cell proliferation was tested by life cell imaging. Results The significant increase of MDC1 and γH2AX proteins, indicating DNA damage, was proved by immunocytochemistry and also Western blot analysis. Proliferation tests could not clearly establish the genotoxic stress of tg fibroblasts and MSCs. Conclusions We will continue in our studies to observe changes in genotoxic stress during the lifespan of our minipigs. HD tg miniature pig represents a perspective large animal model for the research of molecular mechanisms of mt htt.

The distribution of low-threshold TTX-resistant Na+ currents in rat trigeminal ganglion cells

The distribution of low-threshold tetrodotoxin-resistant (TTX-r) Na+ current and its co-expression with high-threshold TTX-r Na+ current were studied in randomly selected acutely dissociated rat trigeminal ganglion (non-identified TG cells) and TG cells serving the temporomandibular joint (TMJ-TG cells). Conditions previously shown to enhance NaV1.9 channel-mediated currents (holding potential (HP) −80mV, 130-mM fluoride internally) were employed to amplify the low-threshold Na+ current. Under these conditions, detectable low-threshold Na+ current was exhibited by 16 out of 21 non-identified TG cells (average, 1810±358pA), and by nine of 14 TMJ-TG cells (average, 959±525pA). The low-threshold Na+ current began to activate around −55mV and was inactivated by holding TG cells at −60mV and delivering 40-ms test potentials (TPs) to 0mV. The inactivation was long lasting, recovering only 8±3% over a 5-min period after the HP was returned to −80mV. Following low-threshold Na+ current inactivation, high-threshold TTX-r Na+ current, evoked from HP −60mV, was observed. High-threshold Na+ current amplitude averaged 16,592±3913pA for TPs to 0mV, was first detectable at an average TP of −34±1.3mV, and was ½ activated at −7.1±2.3mV. In TG cells expressing prominent low-threshold Na+ currents, changing the external solution to one containing 0mM Na+ reduced the amount of current required to hold the cells at −80mV through −50mV, the peak effect being observed at HP −60mV. TG cells recorded from with a more physiological pipette solution containing chloride instead of fluoride exhibited small low-threshold Na+ currents, which were greatly increased upon superfusion of the TG cells with the adenylyl cyclase (AC) activator forskolin. These data suggest two hypotheses: (1) low- and high-threshold NaV1.9 and NaV1.8 channels, respectively, are frequently co-expressed in TG neurons serving the TMJ and other structures, and (2), NaV1.9 channel-mediated currents are small under physiological conditions, but may be enhanced by inflammatory mediators that increase AC activity, and may mediate an inward leak that depolarizes TG neurons, increasing their excitability.

βAr-Stimulation Causes EADs but not DADs in Pre-Failure CAMKIIδC Transgenic Myocytes

Ca2+-Calmodulin dependent protein kinase II (CaMKII) is a nodal regulator of Ca2+-handling and electrophysiology in the ventricular myocyte, and transgenic mice expressing CaMKIIδc (CaMKIIδc TG) exhibit cellular afterdepolarizations and triggered arrhythmias. Here we studied myocytes isolated from these mice to determine: (1) how CaMKII-hyperactivity promotes electrophysiologic instability early in progression to HF, and (2) how βAR stimulation exacerbates this instability. METHODS: Myocytes were isolated, prior to overt HF, from cardiac-specific CaMKIIδc TG mice (TG, n = 18), and WT littermates (n = 16). Steady-state action potentials, Ca2+-handling, and electrophysiologic instability were assessed in whole-cell current clamp (1 Hz pacing), with simultaneous Ca2+ epifluorescence, and with and without βAR stimulation (100 nM Isoproterenol, Iso). RESULTS: EADs, but not DADs, were observed with Iso, and transgenic cells (8/18 cells, 19% of cycles) were more susceptible to EADs than WT (1/16 cells, 2% cycles; p < 0.05). Prior to EAD appearance, TG cells that later exhibited EADs (TG-EAD) also exhibited greater Iso-induced AP prolongation than cells that remained stable (52.3 ± 15.6 ms vs. 21.5 ± 3.9 ms; p < 0.05). TG-EAD cells also exhibited blunted baseline Ca2+ transient amplitude (CaT; 42 ± 9 nM vs. 123 ± 12 nM; p < 0.05), but a larger relative change in CaT with Iso (4.4 ± 0.47 fold vs. 2.8 ± 0.46 fold; p < 0.05). EADs could be abolished by caffeine, indicating a requirement for SR calcium release. CONCLUSIONS: Prior to overt HF, superimposing βAR stimulation upon CaMKIIδc overexpression elicits EADs without DADs. This EAD etiology requires SR calcium release, and given the larger relative effect of Iso on CaT in TG-EAD cells, it is likely that βAR stimulation combines withCaMKII overexpression to exceed the range of calcium-handling permissive of stable electrophysiology.

Abstract P293: Endogenous Lats2 Plays an Important Role in Mediating Myocardial Injury Caused by Ischemia/Reperfusion Through Inhibition of FoxO3

Lats2 is a tumor suppressor and a serine/threonine kinase, acting downstream of mammalian sterile 20 like kinase1 (Mst1), which stimulates apoptosis and inhibits hypertrophy in cardiomyocytes (CM). We investigated the role of Lats2 in mediating myocardial injury after ischemia/reperfusion (IR). Phosphorylation of YAP, an in vivo substrate of Lats2, was increased after 45 minutes ischemia followed by 24 hours reperfusion in control mouse hearts compared with sham, but not in dominant negative (DN) Lats2 transgenic mouse (Tg) hearts, suggesting that Lats2 is activated by IR. The size of myocardial infarction (MI)/area at risk was significantly smaller in Tg mice than in NTg mice (19% and 49%, p&lt;0.01). And there were fewer TUNEL positive cells in Tg than in NTg mice (0.04% and 0.11%, p&lt;0.05). Following 30 min of global ischemia and 60 min of reperfusion in Langendorff perfused heart preparations, left ventricular (LV) systolic pressure (100 vs 71mmHg, p&lt;0.05) and LV developed pressure (79 vs 47 mmHg, p&lt;0.05) were significantly greater in Tg than in NTg mice, indicating that suppression of Lats2 induces better functional recovery after IR. Oxidative stress, as evaluated by 8-OHdG staining, was attenuated in Tg mice. In cultured CMs, DN-Lats2 significantly decreased H 2 O 2 -induced cell death. Overexpression of Lats2 significantly downregulated (51% and 75%, p&lt;0.05), whereas that of DN-Last2 upregulated (100 and 70%, p&lt;0.05), MnSOD and catalase, suggesting that Lats2 negatively regulates expression of antioxidants. Reporter gene assays showed that overexpression of Lats2 significantly inhibits (−70%), whereas knocking down Lats2 by sh-Lats2 increases (+60%), FoxO3-mediated transcriptional activity. Overexpression of Lats2 in CMs inhibited FoxO3 expression, whereas that of DN-Lats2 significantly inhibited FoxO3 downregulation after IR in vivo, suggesting that Lats2 negatively regulates FoxO3 protein expression, which may lead to the downregulation of MnSOD and catalase. Taken together, these results suggest that endogenous Lats2 plays an important role in mediating myocardial injury in response to IR, In part through downregulation of FoxO3 and consequent downregulation of antioxidants and increased oxidative stress in the heart.

A repertoire-independent and cell-intrinsic defect in murine GVHD induction by effector memory T cells

AbstractEffector memory T cells (TEM) do not cause graft-versus-host disease (GVHD), though why this is has not been elucidated. To compare the fates of alloreactive naive (TN) or memory (TM) T cells, we developed a model of GVHD in which donor T cells express a transgene-encoded TCR specific for an antigenic peptide that is ubiquitously expressed in the recipient. Small numbers of naive TCR transgenic (Tg) T cells induced a robust syndrome of GVHD in transplanted recipients. We then used an established method to convert TCR Tg cells to TM and tested these for GVHD induction. This allowed us to control for the potentially different frequencies of alloreactive T cells among TN and TM, and to track fates of alloreactive T cells after transplantation. TEM caused minimal, transient GVHD whereas central memory T cells (TCM) caused potent GVHD. Surprisingly, TEM were not inert: they, engrafted, homed to target tissues, and proliferated extensively, but they produced less IFN-γ and their expansion in target tissues was limited at later time points, and local proliferation was reduced. Thus, cell-intrinsic properties independent of repertoire explain the impairment of TEM, which can initiate but cannot sustain expansion and tissue damage.

Zebrafish Growth Factor Independence Transcription Factors Establish a New Paradigm for Regulation of Primitive and Definitive Hematopoietic Lineages,

Abstract 3379The Growth Factor Independence (Gfi) zinc finger transcription factors play essential roles in hematopoiesis, differentially activating and repressing transcriptional programs required for hematopoietic lineage specification. In mammals, Gfi1 regulates hematopoietic stem cell (HSC) and lymphoid populations, while Gfi1b is required for megakaryocyte and erythroid development (van der Meer, et al. 2010 Leukemia 11:1834–43). In zebrafish, gfi1.1 plays an essential role in primitive hematopoiesis, preserving primitive HSC populations and regulating the erythroid-myeloid balance (Wei, et al. 2008 Cell Res. 6:677–85). However, little is known about the role of gfi1.1 in definitive hematopoiesis or about the role of additional hematopoietic gfi family members in zebrafish. Here, we report the isolation and characterization of an additional zebrafish gfi family transcription factor, gfi1.2b. We compare and contrast gfi1.1 and gfi1.2b, showing that they are highly expressed in the intermediate cell mass (ICM) and aorta-gonad-mesonephros (AGM), the respective sites of primitive and definitive hematopoiesis in zebrafish. Using antisense morpholino oligos (MO), whole mount in situ hybridization (WISH) and fluorescent activated cell sorting (FACS) of transgenic reporter fish, we demonstrate that gfi1.1 and gfi1.2b have distinct, essential roles in preserving primitive and definitive HSC populations, respectively. Loss of gfi1.1 specifically silences expression of scl and gata-1, markers of primitive HSC and erythroid progenitors. Conversely, loss of gfi1.2b silences expression of Tg(cd41:eGFPlo) cells, indicating an essential role for gfi1.2b in preserving definitive hematopoietic progenitors (Ma, et al. 2011 Blood 118:289–297). Consistent with the discrete roles of gfi1.1 and gfi1.2b in primitive and definitive lineages, knockdown of gfi1.2b silences lymphocyte rag-1 expression in the developing thymus, while knockdown of gfi1.1 has no effect on the thymic lymphocyte population. gfi1.1 and gfi1.2b have overlapping roles in erythropoiesis, as loss of either gfi factor reduces erythrocyte populations, while loss of both gfi paralogs results in a more profound silencing of erythrocytes. We further demonstrate that loss of gata-1 reduces gfi1.1 expression and silences gfi1.2b, suggesting that gata-1 plays an essential role in regulating the transcription of both genes. Together, these studies demonstrate that gfi1.1 and gfi1.2b have distinct and overlapping roles in zebrafish hematopoiesis and establish a new paradigm for the regulation of primitive and definitive hematopoietic lineages by gfi transcription factors. Disclosures:No relevant conflicts of interest to declare.

Halofuginone Has Anti-Proliferative Effects in Acute Promyelocytic Leukemia by Modulating the Transforming Growth Factor Beta Signaling Pathway

Promyelocytic leukemia-retinoic acid receptor alpha (PML-RARα) expression in acute promyelocytic leukemia (APL) impairs transforming growth factor beta (TGFβ) signaling, leading to cell growth advantage. Halofuginone (HF), a low-molecular-weight alkaloid that modulates TGFβ signaling, was used to treat APL cell lines and non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice subjected to transplantation with leukemic cells from human chorionic gonadotrophin-PML-RARα transgenic mice (TG). Cell cycle analysis using incorporated bromodeoxyuridine and 7-amino-actinomycin D showed that, in NB4 and NB4-R2 APL cell lines, HF inhibited cellular proliferation (P<0.001) and induced apoptosis (P = 0.002) after a 24-hour incubation. Addition of TGFβ revealed that NB4 cells were resistant to its growth-suppressive effects and that HF induced these effects in the presence or absence of the cytokine. Cell growth inhibition was associated with up-regulation of TGFβ target genes involved in cell cycle regulation (TGFB, TGFBRI, SMAD3, p15, and p21) and down-regulation of MYC. Additionally, TGFβ protein levels were decreased in leukemic TG animals and HF in vivo could restore TGFβ values to normal. To test the in vivo anti-leukemic activity of HF, we transplanted NOD/SCID mice with TG leukemic cells and treated them with HF for 21 days. HF induced partial hematological remission in the peripheral blood, bone marrow, and spleen. Together, these results suggest that HF has anti-proliferative and anti-leukemic effects by reversing the TGFβ blockade in APL. Since loss of the TGFβ response in leukemic cells may be an important second oncogenic hit, modulation of TGFβ signaling may be of therapeutic interest.

Inhibition of Thrombin Receptor Signaling on α-Smooth Muscle Actin + CD34 + Progenitors Leads to Repair After Murine Immune Vascular Injury

The goal of this study was to use mice expressing human tissue factor pathway inhibitor (TFPI) on α-smooth muscle actin (α-SMA)(+) cells as recipients of allogeneic aortas to gain insights into the cellular mechanisms of intimal hyperplasia (IH). BALB/c aortas (H-2(d)) transplanted into α-TFPI-transgenic (Tg) mice (H-2(b)) regenerated a quiescent endothelium in contrast to progressive IH seen in C57BL/6 wild-type (WT) mice even though both developed aggressive anti-H-2(d) alloresponses, indicating similar vascular injuries. Adoptively transferred Tg CD34(+) (but not CD34(-)) cells inhibited IH in WT recipients, indicating the phenotype of α-TFPI-Tg mice was due to these cells. Compared with syngeneic controls, endogenous CD34(+) cells were mobilized in significant numbers after allogeneic transplantation, the majority showing sustained expression of tissue factor and protease-activated receptor-1 (PAR-1). In WT, most were CD45(+) myeloid progenitors coexpressing CD31, vascular endothelial growth factor receptor-2 and E-selectin; 10% of these cells coexpressed α-SMA and were recruited to the neointima. In contrast, the α-SMA(+) human TFPI(+) CD34(+) cells recruited in Tg recipients were from a CD45(-) lineage. WT CD34(+) cells incubated with a PAR-1 antagonist or taken from PAR-1-deficient mice inhibited IH as Tg cells did. Specific inhibition of thrombin generation or PAR-1 signaling on α-SMA(+) CD34(+) cells inhibits IH and promotes regenerative repair despite ongoing immune-mediated damage.

Homeostatic responses by surviving cortical pyramidal cells in neurodegenerative tauopathy

Cortical neuron death is prevalent by 9 months in rTg(tau(P301L))4510 tau mutant mice (TG) and surviving pyramidal cells exhibit dendritic regression and spine loss. We used whole-cell patch-clamp recordings to investigate the impact of these marked structural changes on spontaneous excitatory and inhibitory postsynaptic currents (sEPSCs and sIPSCs) of layer 3 pyramidal cells in frontal cortical slices from behaviorally characterized TG and non-transgenic (NT) mice at this age. Frontal lobe function of TG mice was intact following a short delay interval but impaired following a long delay interval in an object recognition test, and cortical atrophy and cell loss were pronounced. Surviving TG cells had significantly reduced dendritic diameters, total spine density, and mushroom spines, yet sEPSCs were increased and sIPSCs were unchanged in frequency. Thus, despite significant regressive structural changes, synaptic responses were not reduced in TG cells, indicating that homeostatic compensatory mechanisms occur during progressive tauopathy. Consistent with this idea, surviving TG cells were more intrinsically excitable than NT cells, and exhibited sprouting of filopodia and axonal boutons. Moreover, the neuropil in TG mice showed an increased density of asymmetric synapses, although their mean size was reduced. Taken together, these data indicate that during progressive tauopathy, cortical pyramidal cells compensate for loss of afferent input by increased excitability and establishment of new synapses. These compensatory homeostatic mechanisms may play an important role in slowing the progression of neuronal network dysfunction during neurodegenerative tauopathies.