Double Tg Research Articles

Amorphous-Amorphous Phase Separation of Freeze-Concentrated Protein and Amino Acid Excipients for Lyophilized Formulations.

The objective of this study was to elucidate the mixing state of proteins and amino acid excipients concentrated in the amorphous non-ice region of frozen solutions. Thermal analysis of frozen aqueous solutions was performed in heating scans before and after a heat treatment. Frozen aqueous solutions containing a protein (e.g., recombinant human albumin, gelatin) or a polysaccharide (dextran) and an amino acid excipient (e.g., L-arginine, L-arginine hydrochloride, L-arginine monophosphate, sodium L-glutamate) at varied mass ratios showed single or double Tg' (glass transition temperature of maximally freeze-concentrated solutes). Some mixture frozen solutions rich in the polymers maintained the single Tg' of the freeze-concentrated amorphous solute-mixture phase. In contrast, amino acid-rich mixture frozen solutions revealed two Tg's that suggested transition of concentrated non-crystalline solute-mixture phase and excipient-dominant phase. Post-freeze heat treatment induced splitting of the Tg' in some intermediate mass ratio mixture solutions. The mixing state of proteins and amino acids varied depending on their structure, salt types, mass ratio, composition of co-solutes (e.g., NaCl) and thermal history. Information on the varied mixing states should be valuable for the rational use of amino acid excipients in lyophilized protein pharmaceuticals.

Co-Expression of NUP98-HOXD13 and Mutant IDH2 Triggers an Early T-Cell Precursor-like Leukemia in Mice

Background and hypothesis: Acute lymphoblastic leukemia (ALL) is the most common pediatric malignancy, comprising of B lineage ALL and T-cell lineage ALL (about 85% and 15%, respectively). In most cases, treatment regimens for ALL patients have proved efficient and prognosis is usually encouraging. However, a recently described subgroup of T-cell ALL patients have been found to have a poor prognosis when treated with conventional therapy. These higher risk T-cell leukemias have a cell of origin thought to resemble an early T-cell precursor (ETP), which retains the capability to differentiate into both myeloid or T-lineage cells, and are therefore termed ETP ALL. ETP ALL is characterized by lack of or low expression of typical T-cell surface markers, aberrant expression of myeloid and hematopoietic stem cell (HSC) surface markers and a gene expression signature resembling that of murine ETP cells. Recently, ETP ALL patients were found to have a unique mutational landscape that includes acquired mutations in genes typically associated with myeloid differentiation. The ambiguous lineage attributes of ETP ALL may explain the ineffectiveness of current therapies, stressing the need for establishing pre-clinical animal models. Here we show that co-expression of Nup98-Hoxd13 (NHD13) fusion gene and a mutant IDH2 gene (IDH2 R140Q) triggers an ETP-like leukemia in mice. Expression of NHD13 has been reported in MDS and AML patients, and NHD13 mice have been previously shown to develop MDS, AML, and T-cell ALL. Mutations in IDH1/2 genes occur in several cancers, including AML; the most common IDH mutation in AML patients is the IDH2 R140Q. However, expression of mutant IDH1/2 in mice has been reported to induce an expansion of the HSC compartment but was not sufficient for establishment of leukemia. Targeted sequencing of NHD13 mice has identified recurrent mutations in IDH1, suggesting that these two aberrations may collaborate to cause leukemia.Study Design and Methods: IDH2 R140Q mice were crossed with NHD13 mice and the progeny were monitored for survival. Classification of leukemic samples was done using flow cytometry and PCR analysis of TCRβ gene rearrangements. Global expression and mutational patterns of IDH2/NHD13 leukemias were analyzed using gene chip arrays and whole exome sequencing (WES).Results and conclusions: Transgenic IDH2/NHD13 mice showed decreased survival compared to all control groups. Detailed analysis of surface marker expression revealed that most double Tg cases (10/11 tested) developed leukemias resembling ETP (cKit+CD44+CD25-) or double negative (DN) 2 cells (cKit+CD44+CD25+). PCR analysis of the TCRβ locus revealed that unlike NHD13 T-cell leukemias, IDH2/NHD13 leukemias do not have clonal VDJ rearrangements. However, most of IDH2/NHD13 leukemias have clonal or oligoclonal DJ rearrangements of the TCRβ locus, suggesting that these leukemias emerged during an early stage in T cell development. Expression analysis revealed that the IDH2/NHD13 leukemia expression signature is enriched in genes that are upregulated in ETP or DN2 cells. In addition, we found that the IDH2/NHD13 leukemia expression signature is also enriched in genes uniquely expressed in ETP ALL patients. Finally, WES showed correspondence between genes mutated in the mouse ETP-like leukemia and genes recurrently mutated in ETP ALL patients. These results indicate that IDH2/NHD13 leukemias resemble ETP ALL in terms of gene expression, immunophenotype, and cooperative mutations.Relevance and importance: The poor prognosis of ETP ALL may be related to incomplete effectiveness of treatment strategies, emphasizing the need for novel therapies. IDH2/NHD13 mice can serve as an excellent pre-clinical model to study ETP ALL and to test potential treatments. DisclosuresAplan:NIH Office of Technology Transfer: Patents & Royalties.

B-1 B cell progenitors transiently and partially express keratin 5 during differentiation in bone marrow

BackgroundKeratin 5 (K5) is a cytoskeletal tissue-specific protein expressed in the epithelial cells of skin and esophagus and ectopic K5 expression in lymphocytes has never been reported. ObjectiveHere we demonstrate an ectopic epidermal self-protein expression in B-1 B cell by fate mapping of K5-expressing cells. MethodsK5-Cre×CAG-CAT-loxP-EGFP double Tg (K5×GFP) mice that express enhanced GFP under the control of the K5 promoter were employed. ResultsUnexpectedly, B220+GFP+ cells were found in LN, spleen, peripheral blood and peritoneal cavity. These cells were IgM+IgDlowCD23−CD43+CD19+CD93−, indicating that they were B-1 B cells. The number of B220+GFP+ cells was significantly larger in spleen than in the other tissues tested. Although GFP+ B-1 cells did not express K5 in the periphery, Lin−CD93+B220low−negCD19+ B-1 B cell progenitors expressed GFP and B220+CD93+ progenitor cells expressed K5 and MHC-class II in BM, indicating that GFP+ B-1 cells transiently expressed K5 and the progenitor cells were potential APC. GFP+ B-1 cells in the periphery continued expressing MHC class II and had exogenous antigen-presenting capacity comparable to non-follicular B cells. GFP+ B-1 cells spontaneously secreted more IgM than GFP− B-1 cells in vitro. ConclusionThese results indicate that B-1 B cells transiently and partially express K5 in BM and are potent for both natural antibody production and antigen presentation.

Development of the pancreas in medaka, Oryzias latipes, from embryo to adult.

To address conserved and unique features of fish pancreas development, we performed extensive analyses of pancreatic development in medaka embryos and adults using pdx1- and ptf1a-transgenic medaka, in situ hybridization and immunohistochemistry. The markers used in these analyses included pdx1, nkx6.1, nkx6.2, nkx2.2, Islet1, insulin, Somatostatin, glucagon, ptf1a, ela3l, trypsin, and amylase. The double transgenic (Tg) fish produced in the present study visualizes the development of endocrine (pdx1+) and exocrine (ptf1a+) parts simultaneously in living fishes. Like other vertebrates, the medaka pancreas develops as two (dorsal and ventral) buds in the anterior gut tube, which soon fuse into a single anlagen. The double Tg fish demonstrates that the differential property between the two buds is already established at the initial phase of bud development as indicated by strong pdx1 expression in the dorsal one. This Tg fish also allowed us to examine the gross morphology and the structure of adult pancreas and revealed unique characters of medaka pancreas such as broad and multiple connections with the gut tube along the anterior-posterior axis.

Interleukin-2/anti-interleukin-2 antibody complexes expand T regulatory cells and protect against allergen-induced airway hyperreactivity (HYP7P.256)

Abstract T-cells play a key role in the development and maintenance of allergic diseases and thus represent a promising target for therapeutic interventions. Evidence from SIT studies suggest that regulatory T-cells (Treg) might counterbalance the Th2-biased immune response. We here created double transgenic (tg) allergy mice expressing a human TCR specific for the major mugwort (Artemisia vulgaris) pollen allergen Art v 1 and HLA-DR1 to study the response to the aero-allergen in vivo. After allergen-specific sensitization, only double tg allergy mice, but not single tg or wt control mice showed enhanced airway-hyperreactivity (AHR) upon challenge, which was associated with airway and lung inflammation. Significantly, treatment of allergy mice with three daily i.p. injections of IL-2/anti-IL-2 mAb complexes induced a 6-fold increase of CD25+Foxp3+ Treg among CD3+CD4+T-cells in peripheral blood (17.9±6.2% versus 3.1±1.4%) compared to a 8-fold increase of CD39+ and KLRG1+ Treg subsets, respectively (17.2±7.9% versus 2.1±1.5%; 1.6±0.2% versus 0.2±0.2%). Furthermore, expansion of Treg by IL-2/anti-IL-2 mAb complexes before sensitization prevented mice from allergen-induced AHR upon challenge and significantly reduced Art v 1-specific serum IgE, when compared to sham-treated mice. Such in vivo expanded Treg might have similar effects in therapeutic settings. In summary, expansion of Treg by IL-2/anti-IL-2 mAb complexes seems to be a promising strategy to interfere with allergic diseases.

Daylight vision repair by cell transplantation.

Human daylight vision depends on cone photoreceptors and their degeneration results in visual impairment and blindness as observed in several eye diseases including age-related macular degeneration, cone-rod dystrophies, or late stage retinitis pigmentosa, with no cure available. Preclinical cell replacement approaches in mouse retina have been focusing on rod dystrophies, due to the availability of sufficient donor material from the rod-dominated mouse retina, leaving the development of treatment options for cone degenerations not well studied. Thus, an abundant and traceable source for donor cone-like photoreceptors was generated by crossing neural retina leucine zipper-deficient (Nrl(-/-) ) mice with an ubiquitous green fluorescent protein (GFP) reporter line resulting in double transgenic tg(Nrl(-/-); aGFP) mice. In Nrl(-/-) retinas, all rods are converted into cone-like photoreceptors that express CD73 allowing their enrichment by CD73-based magnetic activated cell sorting prior transplantation into the subretinal space of adult wild-type, cone-only (Nrl(-/-)), or cone photoreceptor function loss 1 (Cpfl1) mice. Donor cells correctly integrated into host retinas, acquired mature photoreceptor morphology, expressed cone-specific markers, and survived for up to 6 months, with significantly increased integration rates in the cone-only Nrl(-/-) retina. Individual retinal ganglion cell recordings demonstrated the restoration of photopic responses in cone degeneration mice following transplantation suggesting, for the first time, the feasibility of daylight vision repair by cell replacement in the adult mammalian retina.

PTPROt-mediated regulation of p53/Foxm1 suppresses leukemic phenotype in a CLL mouse model.

The gene encoding PTPROt is methylated and suppressed in Chronic Lymphocytc Leukemia. PTPROt exhibits in vitro tumor suppressor characteristics through the regulation of B-cell receptor signaling. Here, we generated transgenic (Tg) mice with B-cell specific expression of PTPROt. While lymphocyte development is normal in these mice, crossing them with TCL1 Tg mouse model of CLL results in a survival advantage compared to the TCL1 Tg mice. Gene expression profiling of splenic B-lymphocytes before detectable signs of CLL followed by Ingenuity Pathway Analysis revealed that the most prominently regulated functions in TCL1 Tg vs non-transgenic (NTg) and TCL1 Tg vs PTPROt/TCL1 double Tg are the same and also biologically relevant to this study. Further, enhanced expression of the chemokine Ccl3, the oncogenic transcription factor Foxm1 and its targets in TCL1 Tg mice were significantly suppressed in the double Tg mice suggesting a protective function of PTPROt against leukemogenesis. This study also showed that PTPROt mediated regulation of Foxm1 involves activation of p53, a transcriptional repressor of Foxm1, which is facilitated through suppression of B-cell receptor signaling. These results establish the in vivo tumor suppressive function of PTPROt, and identify p53/Foxm1 axis as a key downstream effect of PTPROt-mediated suppression of BCR signaling.

Striatum and entorhinal cortex atrophy in AD mouse models: MRI comprehensive analysis

Alzheimer's disease is experimentally modeled in transgenic (Tg) mice overexpressing mutated forms of the human amyloid precursor protein either alone or combined with mutated presenilins and tau. In the present study, we developed a systematic approach to compare double (TASTPM) and triple (APP/PS2/Tau) Tg mice by serial magnetic resonance imaging and spectroscopy analysis from 4 to 26 months of age to define homologous biomarkers between mice and humans. Hippocampal atrophy was found in Tg mice compared with WT. In APP/PS2/Tau the effect was age-dependent, whereas in TASTPM it was detectable from the first investigated time point. Importantly, both mice displayed an age-related entorhinal cortex thinning and robust striatal atrophy, the latter associated with a significant loss of synaptophysin. Hippocampal magnetic resonance spectroscopy revealed lower glutamate levels in both Tg mice and a selective myo-inositol increase in TASTPM. This noninvasive magnetic resonance imaging analysis, revealed common biomarkers between humans and mice, and could, thus, be promoted as a fully translational tool to be adopted in the preclinical investigation of therapeutic approaches.

Abstract 4602: Blocking of p14/ARF and DX2 binding by novel small chemical can improve the chemo-sensitivity of small cell lung cancer and non-small cell lung carcinoma

Abstract In recent, it has been reported that alternative splicing variant of AIMP2, AIMP2/DX2 (DX2), is frequently expressed in human lung cancer and is related with p53-mediated tumor suppression pathway. Here we show that DX2 can promote tumor progression and increase incidence, cooperatively with oncogenic K-Ras in transgenic mouse model. Moreover, it can induce small cell lung carcinoma (SCLC) as well as contribute to progression of non-small cell lung carcinoma (NSCLC). In fact, DX2 expression is elevated in human small cell lung cancer cell lines. Based on the cellular localization and responsibility of si-DX2, we revealed that DX2 is an inhibitor of p14/ARF. Elimination of DX2 can induce p14/ARF expression and DX2 Transgenic (Tg) mouse cells show the low expression of p19/ARF. Since DX2, but not its original product AIMP2, is selectively interacted with p14/ARF, we screened the specific binding inhibitor and obtained the single compound (SLC36) from about 9000 chemicals. This chemical can block the interaction of p14/ARF-DX2 and also AIMP2-DX2, but not the binding of p14/ARF-p53 or AIMP2-p53. In addition, it can induce p14/ARF expression and cell death in human lung cancer cell lines including SCLC cell lines. In our in vivo study, treatment of SLC36, combined with low dosage of adriamycin (1 mg/kg) can suppress the cancer incidence as well as progression in K-RasLA2 and K-Ras/DX2 double Tg model. These results indicate that DX2 can contribute to lung cancer progression and development, including SCLC, through inhibition of p14/ARF, and blocking of DX2-p14/ARF binding would be useful therapeutic strategy of human lung cancers including SCLC as well as NSCLC. Citation Format: Ah Young Oh, Youn-Sang Jung, Su-Jin Lee, Jung Hyun Jo, Ho Young Chun, Bum-Joon Park. Blocking of p14/ARF and DX2 binding by novel small chemical can improve the chemo-sensitivity of small cell lung cancer and non-small cell lung carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4602. doi:10.1158/1538-7445.AM2014-4602

Cerebrolysin™ efficacy in a transgenic model of tauopathy: role in regulation of mitochondrial structure.

BackgroundAlzheimer’s Disease (AD) and Fronto temporal lobar dementia (FTLD) are common causes of dementia in the aging population for which limited therapeutical options are available. These disorders are associated with Tau accumulation. We have previously shown that CerebrolysinTM (CBL), a neuropeptide mixture with neurotrophic effects, ameliorates the behavioral deficits and neuropathological alterations in amyloid precursor protein (APP) transgenic (tg) mouse model of AD by reducing hyper-phosphorylated Tau. CBL has been tested in clinical trials for AD, however it’s potential beneficial effects in FTLD are unknown. For this purpose we sought to investigate the effects of CBL in a tg model of tauopathy. Accordingly, double tg mice expressing mutant Tau under the mThy-1 promoter and GSK3β (to enhance Tau phosphorylation) were treated with CBL and evaluated neuropathologically.ResultsCompared to single Tau tg mice the Tau/GSK3β double tg model displayed elevated levels of Tau phosphorylation and neurodegeneration in the hippocampus. CBL treatment reduced the levels of Tau phosphorylation in the dentate gyrus and the degeneration of pyramidal neurons in the temporal cortex and hippocampus of the Tau/GSK3β double tg mice. Interestingly, the Tau/GSK3β double tg mice also displayed elevated levels of Dynamin-related protein-1 (Drp-1), a protein that hydrolyzes GTP and is required for mitochondrial division. Ultrastructural analysis of the mitochondria in the Tau/GSK3β double tg mice demonstrated increased numbers and fragmentation of mitochondria in comparison to non-tg mice. CBL treatment normalized levels of Drp-1 and restored mitochondrial structure.ConclusionsThese results suggest that the ability of CBL to ameliorate neurodegenerative pathology in the tauopathy model may involve reducing accumulation of hyper-phosphorylated Tau and reducing alterations in mitochondrial biogenesis associated with Tau.

Programmed cell death 1 (PD-1) regulates the effector function of CD8 T cells via PD-L1 expressed on target keratinocytes

Programmed cell death 1 (PD-1) is an inhibitory molecule expressed by activated T cells. Its ligands (PD-L1 and -L2; PD-Ls) are expressed not only by a variety of leukocytes but also by stromal cells. To assess the role of PD-1 in CD8 T cell-mediated diseases, we used PD-1-knockout (KO) OVA-specific T cell-receptor transgenic (Tg) CD8 T cells (OT-I cells) in a murine model of mucocutaneous graft-versus-host disease (GVHD). We found that mice expressing OVA on epidermal keratinocytes (K14-mOVA mice) developed markedly enhanced GVHD-like disease after transfer of PD-1-KO OT-I cells as compared to those mice transferred with wild-type OT-I cells. In addition, K14-mOVA × OT-I double Tg (DTg) mice do not develop GVHD-like disease after adoptive transfer of OT-I cells, while transfer of PD-1-KO OT-I cells caused GVHD-like disease in a Fas/Fas-L independent manner. These results suggest that PD-1/PD-Ls-interactions have stronger inhibitory effects on pathogenic CD8 T cells than does Fas/Fas-L-interactions. Keratinocytes from K14-mOVA mice with GVHD-like skin lesions express PD-L1, while those from mice without the disease do not. These findings reflect the fact that primary keratinocytes express PD-L1 when stimulated by interferon-γ in vitro. When co-cultured with K14-mOVA keratinocytes for 2 days, PD-1-KO OT-I cells exhibited enhanced proliferation and activation compared to wild-type OT-I cells. In addition, knockdown of 50% PD-L1 expression on the keratinocytes with transfection of PD-L1-siRNA enhanced OT-I cell proliferation. In aggregate, our data strongly suggest that PD-L1, expressed on activated target keratinocytes presenting autoantigens, regulates autoaggressive CD8 T cells, and inhibits the development of mucocutaneous autoimmune diseases.

APOMORPHINE THERAPY FOR NEURONAL INSULIN RESISTANCE IN ALZHEIMER'S MICE

detected by immunoprecipitation and double-labeling immunofluorescence. Results: As oligomer, PrP c, Fyn, NMDA receptor, GSK3s, and phosphorylated tau were detected in lipid rafts of doubleTg mice. As oligomer-PrP c complex accumulated in lipid rafts. According with As oligomer accumulation, fyn, phosphorylated NMDA receptor, phosphorylated tau, and GSK3s increased in lipid rafts of double Tg mice compared to that of single tau Tg mice. Conclusions: These findings suggest that As oligomer-PrP c complex may induce Fyn, and phosphorylation of both NMDA receptor and tau in lipid rafts leading to neurodegeneration.

Analysis of CNS tissue-specific Tregs and dendritic cells in T cell responses and autoimmunity (MUC7P.757)

Abstract We assessed locally generated, locally acting peripheral regulatory T cells (pTregs) in retinal immune homeostasis and ability of retinal dendritic cells (DC) to support T cell activation/expansion within retina. We used transgenic (Tg) mice expressing beta-galactosidase (bgal mice) as retinal neo-self antigen, TCR Tg mice (BG2) specific for bgal, and mice with labeled, depletable Tregs or DC, based on expression of diphtheria toxin (DTx) receptor and/or GFP controlled by FoxP3 or CD11c promoter (FDG or CDG mice). Treg and DC depletion from retina was done by DTx injection into the eye. T cells and DC were assayed by FACS of retina or blood. Retinal autoimmunity was assessed by histological analysis. After bgal (FDG/bgal mice) or IRBP (FDG mice) immunization, Treg depleted retinas showed increased incidence and severity of autoimmunity. We found local depletion of Tregs from retina sufficient to induce spontaneous autoimmunity in FDG/bgal/BG2 mice but not in single or double Tg mice. Dose of DTx used to eliminate retinal Tregs did not induce retinal autoimmunity if given systemically. DC depletion from the retina prevented Treg and Teff generation within retina after bgal injection. Retinal microglia remaining after DC depletion did not make up for the loss of DC-dependent antigen presentation. We conclude local Tregs within the retina protect against spontaneous organ-specific autoimmunity and local DC must be present within the retina for antigen presentation to T cells.

Cardiomyocyte-specific expression of CRNK, the C-terminal domain of PYK2, maintains ventricular function and slows ventricular remodeling in a mouse model of dilated cardiomyopathy

Up-regulation and activation of PYK2, a member of the FAK family of protein tyrosine kinases, is involved in the pathogenesis of left ventricular (LV) remodeling and heart failure (HF). PYK2 activation can be prevented by CRNK, the C-terminal domain of PYK2. We previously demonstrated that adenoviral-mediated CRNK gene transfer improved survival and LV function, and slowed LV remodeling in a rat model of coronary artery ligation-induced HF. We now interrogate whether cardiomyocyte-specific, transgenic CRNK expression prevents LV remodeling and HF in a mouse model of dilated cardiomyopathy (DCM) caused by constitutively active Protein Kinase Cε (caPKCε). Transgenic (TG; FVB/N background) mice were engineered to express rat CRNK under control of the α-myosin heavy chain promoter, and crossed with FVB/N mice with cardiomyocyte-specific expression of caPKCε to create double TG mice. LV structure, function, and gene expression were evaluated in all 4 groups (nonTG FVB/N; caPKCε(+/−); CRNK(+/−); and caPKCε×CRNK (PXC) double TG mice) at 1, 3, 6, 9 and 12mo of age. CRNK expression followed a Mendelian distribution, and CRNK mice developed and survived normally through 12mo. Cardiac structure, function and selected gene expression of CRNK mice were similar to nonTG littermates. CRNK had no effect on caPKCε expression and vice versa. PYK2 was up-regulated ~6-fold in caPKCε mice, who developed a non-hypertrophic, progressive DCM with reduced systolic (Contractility Index=151±5 vs. 90±4s−1) and diastolic (Tau=7.5±0.5 vs. 14.7±1.3ms) function, and LV dilatation (LV Remodeling Index (LVRI)=4.2±0.1 vs. 6.0±0.3 for FVB/N vs. caPKCε mice, respectively; P<0.05 for each at 12mo). In double TG PXC mice, CRNK expression significantly prolonged survival, improved contractile function (Contractile Index=115±8s−1; Tau=9.5±1.0ms), and reduced LV remodeling (LVRI=4.9±0.1). Cardiomyocyte-specific expression of CRNK improves contractile function and slows LV remodeling in a mouse model of DCM.

Neurofibrillary tangle formation by introducing wild-type human tau into APP transgenic mice

Senile plaques comprised of Aβ aggregates and neurofibrillary tangles (NFTs) composed of hyperphosphorylated tau filaments are the hallmarks of Alzheimer's disease (AD). A number of amyloid precursor protein (APP) transgenic (Tg) mice harboring APP mutations have been generated as animal models of AD. These mice successfully display amyloid plaque formation and subsequent tau hyperphosphorylation, but seldom induce NFT formations. We have demonstrated that the APPOSK-Tg mice, which possess the E693Δ (Osaka) mutation in APP and thereby accumulate Aβ oligomers without plaques, exhibit tau hyperphosphorylation at 8 months, but not NFT formation even at 24 months. We assumed that APP-Tg mice, including ours, failed to form NFTs because NFT formation requires human tau. To test this hypothesis, we crossbred APPOSK-Tg mice with tau-Tg mice (tau264), which express low levels of 3-repeat and 4-repeat wild-type human tau without any pathology. The resultant double Tg mice displayed tau hyperphosphorylation at 6 months and NFT formation at 18 months in the absence of tau mutations. Importantly, these NFTs contained both 3-repeat and 4-repeat human tau, similar to those in AD. Furthermore, the double Tg mice exhibited Aβ oligomer accumulation, synapse loss, and memory impairment at 6 months and neuronal loss at 18 months, all of which appeared earlier than in the parent APPOSK-Tg mice. These results suggest that Aβ and human tau synergistically interact to accelerate each other's pathology, that the presence of human tau is critical for NFT formation, and that Aβ oligomers can induce NFTs in the absence of amyloid plaques.

Identification of CD3+CD4−CD8− T Cells as Potential Regulatory Cells in an Experimental Murine Model of Graft-Versus-Host Skin Disease (GVHD)

We have developed K14-mOVA transgenic (Tg) mice that express membrane-associated ovalbumin (mOVA) under the control of a K14 promoter as well as double Tg mice by crossing them with OT-I mice that have a T cell receptor (TCR) recognizing OVA peptide. When injected with CD8+ OT-I cells, K14-mOVATg mice develop graft-vs-host disease (GvHD), whereas double Tg mice are protected. This suggests that, in double Tg mice, regulatory mechanisms may prevent infused OT-I cells from inducing GvHD. We demonstrated that, after adoptive transfer, TCRαβ+CD3+CD4-CD8-NK1.1- double negative (DN) T cells are increased in the peripheral lymphoid organs and skin of double Tg mice and exhibit a Vα2+Vβ5+TCR that is the same TCR specificity as OT-I cells. These DN T cells isolated from tolerant double Tg mice proliferated in response to OVA peptide and produced IFN-γ in the presence of IL-2. These cells could also suppress the proliferation of OT-I cells and were able to specifically kill activated OT-I cells through Fas/Fas ligand interaction. These findings suggest that DN T cells that accumulate in double Tg mice have regulatory functions and may play a role in the maintenance of peripheral tolerance in vivo.

Co‐expression of HLA‐B7 and HLA‐B27 alleles is associated with B7‐restricted immunodominant responses following influenza infection

It is recognized that host response following viral infection is characterized by immunodominance, but deciphering the different factors contributing to immunodominance has proved a challenge due to concurrent expression of multiple MHC class I alleles. To address this, we generated H2-K(-/-)/D(-/-) double-knockout transgenic mice expressing either one or two human MHC-I alleles. We hypothesized that co-expression of different allele combinations figures critically in immunodominance and examined this in influenza-infected, double Tg MHC-I mice. In A2/B7 or A2/B27 mice, using ELISpot assays with the A2-restricted matrix I.58-66, the B7-restricted NP418-426 or the B27-restricted NP383-391 influenza A (flu) epitopes, we observed the expected recognition of both peptides for both alleles. In contrast, in flu-infected B7/B27 mice, a significantly reduced level of B27/NP383-restricted CTL response was detected while there was no change in the B7/NP418-restricted CTL response. Flu-specific tetramer studies revealed a partial deletion of Vβ8.1(+) NP383/B27-restricted CD8(+) T cells, and a diminished Vβ12(+) CD8(+) T-cell expansion in B7/B27 Tg mice. Using HLA Tg chimeric mice, we confirmed these findings. These findings shed light on the immune consequences of co-dominant expression of MHC-I alleles for host immune response to pathogens.

Water's second glass transition.

The glassy states of water are of common interest as the majority of H2O in space is in the glassy state and especially because a proper description of this phenomenon is considered to be the key to our understanding why liquid water shows exceptional properties, different from all other liquids. The occurrence of water's calorimetric glass transition of low-density amorphous ice at 136 K has been discussed controversially for many years because its calorimetric signature is very feeble. Here, we report that high-density amorphous ice at ambient pressure shows a distinct calorimetric glass transitions at 116 K and present evidence that this second glass transition involves liquid-like translational mobility of water molecules. This "double Tg scenario" is related to the coexistence of two liquid phases. The calorimetric signature of the second glass transition is much less feeble, with a heat capacity increase at Tg,2 about five times as large as at Tg,1. By using broadband-dielectric spectroscopy we resolve loss peaks yielding relaxation times near 100 s at 126 K for low-density amorphous ice and at 110 K for high-density amorphous ice as signatures of these two distinct glass transitions. Temperature-dependent dielectric data and heating-rate-dependent calorimetric data allow us to construct the relaxation map for the two distinct phases of water and to extract fragility indices m = 14 for the low-density and m = 20-25 for the high-density liquid. Thus, low-density liquid is classified as the strongest of all liquids known ("superstrong"), and also high-density liquid is classified as a strong liquid.

Glutaminyl cyclase-mediated toxicity of pyroglutamate-beta amyloid induces striatal neurodegeneration

BackgroundPosttranslational modifications of beta amyloid (Aβ) have been shown to affect its biophysical and neurophysiological properties. One of these modifications is N-terminal pyroglutamate (pE) formation. Enzymatic glutaminyl cyclase (QC) activity catalyzes cyclization of truncated Aβ(3-x), generating pE3-Aβ. Compared to unmodified Aβ, pE3-Aβ is more hydrophobic and neurotoxic. In addition, it accelerates aggregation of other Aβ species. To directly investigate pE3-Aβ formation and toxicity in vivo, transgenic (tg) ETNA (E at the truncated N-terminus of Aβ) mice expressing truncated human Aβ(3–42) were generated and comprehensively characterized. To further investigate the role of QC in pE3-Aβ formation in vivo, ETNA mice were intercrossed with tg mice overexpressing human QC (hQC) to generate double tg ETNA-hQC mice.ResultsExpression of truncated Aβ(3–42) was detected mainly in the lateral striatum of ETNA mice, leading to progressive accumulation of pE3-Aβ. This ultimately resulted in astrocytosis, loss of DARPP-32 immunoreactivity, and neuronal loss at the sites of pE3-Aβ formation. Neuropathology in ETNA mice was associated with behavioral alterations. In particular, hyperactivity and impaired acoustic sensorimotor gating were detected. Double tg ETNA-hQC mice showed similar Aβ levels and expression sites, while pE3-Aβ were significantly increased, entailing increased astrocytosis and neuronal loss.ConclusionsETNA and ETNA-hQC mice represent novel mouse models for QC-mediated toxicity of truncated and pE-modified Aβ. Due to their significant striatal neurodegeneration these mice can also be used for analysis of striatal regulation of basal locomotor activity and sensorimotor gating, and possibly for DARPP-32-dependent neurophysiology and neuropathology. The spatio-temporal correlation of pE3-Aβ and neuropathology strongly argues for an important role of this Aβ species in neurodegenerative processes in these models.

Exosomal pMHC-I complex targets T cell-based vaccine to directly stimulate CTL responses leading to antitumor immunity in transgenic FVBneuN and HLA-A2/HER2 mice and eradicating trastuzumab-resistant tumor in athymic nude mice

One of the major obstacles in human epidermal growth factor receptor 2 (HER2)-specific trastuzumab antibody immunotherapy of HER2-positive breast cancer is the development of trastuzumab resistance, warranting the search for other therapeutic strategies. Using mouse models, we previously demonstrated that ovalbumin (OVA)-specific dendritic cell (DC)-released exosome (EXOOVA)-targeted CD4(+) T cell-based (OVA-TEXO) vaccine stimulates efficient cytotoxic T lymphocyte (CTL) responses via exosomal peptide/major histocompatibility complex (pMHC)-I, exosomal CD80 and endogenous IL-2 signaling; and long-term CTL memory by means of via endogenous CD40L signaling. In this study, using two-photon microscopy, we provide the first visual evidence on targeting OVA-TEXO to cognate CD8(+) T cells in vivo via exosomal pMHC-I complex. We prepared HER2/neu-specific Neu-TEXO and HER2-TEXO vaccines using adenoviral vector (AdVneu and AdVHER2)-transfected DC (DCneu and DCHER2)-released EXOs (EXOneu and EXOHER2), and assessed their stimulatory effects on HER2/neu-specific CTL responses and antitumor immunity. We demonstrate that Neu-TEXO vaccine is capable of stimulating efficient neu-specific CTL responses, leading to protective immunity against neu-expressing Tg1-1 breast cancer in all 6/6 transgenic (Tg) FVBneuN mice with neu-specific self-immune tolerance. We also demonstrate that HER2-TEXO vaccine is capable of inducing HER2-specific CTL responses and protective immunity against transgene HLA-A2(+)HER2(+) BL6-10A2/HER2 B16 melanoma in 2/8 double Tg HLA-A2/HER2 mice with HER2-specific self-immune tolerance. The remaining 6/8 mice had significantly prolonged survival. Furthermore, we demonstrate that HER2-TEXO vaccine stimulates responses of CD8(+) T cells capable of not only inducing killing activity to HLA-A2(+)HER2(+) BL6-10A2/HER2 melanoma and trastuzumab-resistant BT474A2 breast cancer cells in vitro but also eradicating 6-day palpable HER2(+) BT474A2 breast cancer (3-4mm in diameter) in athymic nude mice. Therefore, the novel T cell-based HER2-TEXO vaccine may provide a new therapeutic alternative for women with HER2(+) breast cancer, especially for trastuzumab-resistant HER2(+) breast cancer patients.