Cells In Tg Mice Research Articles

1245-P: Characteristic Distribution of GRP78 in Islets and Intraislet Ductal Cells of Diabetic C414A-CRY1 Transgenic Mice

Our preceding studies demonstrated that cysteine414 (zinc-binding site of mCRY1) - alanine mutant mCRY1 transgenic mice (TG mice) exhibit diabetes characterized by the reduction of β-cell proliferation caused by senescence-associated secretory phenotype (SASP)-like characters of β-cells. Not only pancreatic intraepithelial neoplasias (PanINs) with pancreatic duct glands (PDGs) but also intra-islet ducts emerged age-dependently in TG mice (Okano S. et al., 2019). Reportedly GRP78 (also called BiP) is highly expressed in pancreatic ductal adenocarcinoma (PDAC), and in lesions of acinar-to-ductal metaplasia (ADM) which is known as precursors of PanIN. As for the islet, GRP78 has been intensively studied with regard to the unfolded protein response (UPR) in the β cells. However, the details of in vivo distribution of GRP78, particularly in endocrine cells outside β cells, are still remain elusive. In this study, we conducted staining experiments for GRP78 in the pancreatic sections of well-matured mice to explore the relation between endocrine cells and intra-islet ducts. The results showed that the expression of GRP78 was higher in PanINs and PDGs than in normal ductal cells in TG mice, suggesting that GRP78 plays important roles in the maintenance of the atypical ductal lesions. In both wild-type and TG mice, GRP78 was observed in the whole islet. The expression of GRP78 was higher in β cells than in alpha and delta cells in both mice. Also, GRP78 was highly expressed in primary intra-islet ducts. Some of the GRP78 high-expressing primary intra-islet ductal cells were co-expressed with chromogranin A, which is known as an endocrine marker. Taken together, our results suggest that the direct trans-differentiation of endocrine cells (possibly β cells) to ductal cells is involved in the generation of the intra-islet ductal cells. Disclosure S. Okano: None. A. Yasui: None. S. Kanno: None. K. Satoh: None. M. Igarashi: None. O. Nakajima: None. Funding Japan Society for the Promotion of Science (19K07498); Tohoku University

2057-P: Atypical Trefoil Factor Family 2 Expressing ß-Cells of Diabetic Mutant CRY1 Transgenic Mice with Intraislet Ducts

We have demonstrated that cysteine414 (zinc-binding site of mCRY1)-alanine mutant mCRY1 transgenic mice (Tg mice) show diabetes characterized by the reduction of β-cell proliferation and by β-cell dysfunction due to SASP (senescence-associated secretory phenotype)-like characters of β-cells. In addition, our previous studies showed that mucinous pancreatic duct glands (PDGs) occur in exocrine areas of aged Tg mice. Further, we also showed that mucin-producing intra-islet ducts develope age-dependently in Tg mice (Okano S. et al., 2019). To explore the molecular mechanisms for developing the intra-islet ducts, we conducted DNA microarray analysis using RNA from islets of aged Tg mice. The islet samples were obtained by paraffin-embedded pancreas sections by laser-capture microdissection. The result showed that the mRNA expression of Trefoil factor family 2 (TFF2), which is reportedly expressed in PDGs, was up-regulated in the islet of aged Tg mice. Immunostaining experiments with TFF2 antibodies revealed that, inside of islet of aged Tg mice, TFF2 was expressed by considerable rate in β-cells. TFF2 was located in speckle-like granules in the cytoplasm in the β-cells. In wild type controls, no TFF2 was observed in β-cells. These results suggest that the atypical β-cells have both endocrine and ductal characteristics. In α-cells, essentially no TFF2 protein expression was observed in both wild type controls and Tg mice. Taken together, our results strongly suggest that β-cells are major sources for intra-islet ductal cells in Tg mice. Our results also suggest that direct trans-differentiation of β-cells to ductal cells is involved in the generation of the intra-islet ductal cells. Disclosure S. Okano: None. A. Yasui: None. S. Kanno: None. K. Satoh: None. M. Igarashi: None. O. Nakajima: None. Funding Japan Society for the Promotion of Science (19K07498); IDAC; Tohoku University

“Cardiac Specific Adenyl Cyclase Type 8 Overexpression Induces Chronic Cardiac Exercise and Leads to a Unique Form of Cardiac Growth.”

IntroductionCardiac specific overexpression of adenylate cyclase type 8 (TGAC8) induces a robust, chronic increase in AC‐PKA‐cAMP signaling. Due elevated cardiac AC8‐ PKA‐cAMP signaling, TGAC8 mice experience a 30% increase in heart rate and a roughly 60% increase in ejection fraction1. This continuously elevated cardiac workload places a significant stress on TGAC8 mice hearts. We employed unbiased multiomics on left ventricle (LV) tissue to deduce the diverse molecular adaptions utilized by TGAC8 mice to adapt to this stress. We utilized echocardiogram to measure cardiac structure in TGAC8 and wildtype (WT) littermates, assessed changes in LV histology, and assessed key growth targets using western blotting to further study the cardiac growth phenotype of TGAC8 mice.ResultsPosterior wall thickness and interventricular septal thickening was increased by roughly 1mm and 2mm (p‐value < .05) respectively in TGAC8 vs WT. Despite wall thickening, total LVM was not significantly different, due to smaller ventricular chamber volume in TGAC8 mice. Cross‐sectional area of left ventricle cells in TG mice were significantly reduced on average by 100 um2 (p‐value < .05). Cells with an area less than 400 um2 were increased by 56% in TGAC8 mice compared to WT. Proteomic and RNA sequencing analyses reveals enrichment of transcripts and proteins involved in cell cycle regulation, cardiac growth, and cardiomyocyte development. Enrichment of standard cardiac hypertrophy markers such as ACTA2, ANP, MHY7, and BNP, however, were not observed. Western blotting results verified enrichment of regulators associated with growth signaling and cardiomyocyte proliferation, such as CITED4 and phospho‐AKT, previously not identified in our proteomics analysis.ConclusionThe cardiac growth response to chronically activated AC‐PKA‐cAMP signaling in TGAC8 mice is a key adaption to chronically elevated cardiac workload. Myocardial growth in TGAC8 mice is not mediated through classical pathological or physiological hypertrophy mechanisms, considering the average cardiac cell size is decreased in the LV of TGAC8 mice. Our findings suggest myocardial growth in TGAC8 mice is mediated through cardiac cell proliferation and maturation rather than cardiomyocyte physiological hypertrophic growth. While the various cell types that mediate proliferation remain to be studied, alterations in the activity of cardiac developmental regulators and PI3K‐AKT signaling are likely mediating this process. This unique phenotype makes TGAC8 mice a novel model for cardiac growth.Support or Funding InformationThis research was supported by the Intramural Research Program of National Institutes of Health, National Institute on Aging.Proteomic Analysis of Enriched Growth Signaling in TGACB MiceFigure 1 Multiomics Data for Myocardial Developmental Markers and Cell Cycle Regulators Gene name symbol Protein Protein RNA RNA Delta Percent Change p‐value Log2fold q‐value Neurogenic locus notch homolog protein 1 Notch 1 11.61042795 0.034634 −0.10343 0.543742 Recombining binding protein suppressor of hairless Rbpj 15.20 0.022 −0.11919 0.309147 Histone‐lysine N‐methyltransferase Smyd1 Smyd1 6.782540441 0.007642 0.107033 0.17192 T‐box transcription factor TBX5 Tbx5 15.7334461 0.039575 0.394611 0.000553 T‐box transcription factor TBX20 Mef2a 14.5453468 0.008824 0.394066 0.000553 Myocyte‐specific enhancer factor 2A Tbx20 9.838421742 0.068529 * Myocyte Enhancer Factor 2C Mef2c * −0.17984 0.038123 Homeobox protein Nkx‐2.5 Nkx2‐5 * 0.559012 0.041448 Heart‐ and neural crest derivatives‐expressed protein 2 HAND2 * 1.04129 0.000553 cyclin d1 Ccnd 1 8.20 0.325 −0.22628 0.01724 cyclin d2 Ccnd2 10.10 0.515 −0.01038 0.937869 Cyclin‐dependent kinase 2 Cdk2 18.92 0.008 0.221418 0.427878 Cyclin‐dependent kinase 6 Cdk6 31.41 0.001 0.401706 0.000553 Proliferating Cell Nuclear Antigen Pena 11.59 0.019 −0.05709 0.783869 P27 CDKN1B 12.13 0.270 0.003132 0.987798 P21 CDKN1A * 0.32033 0.934763 Meis Homeobox 1 Meis1 * −0.28486 0.018269 *Not Identified

Peripheral administration of the soluble TNF inhibitor XPro1595 modifies brain immune cell profiles, decreases beta-amyloid plaque load, and rescues impaired long-term potentiation in 5xFAD mice

Clinical and animal model studies have implicated inflammation and peripheral immune cell responses in the pathophysiology of Alzheimer's disease (AD). Peripheral immune cells including T cells circulate in the cerebrospinal fluid (CSF) of healthy adults and are found in the brains of AD patients and AD rodent models. Blocking entry of peripheral macrophages into the CNS was reported to increase amyloid burden in an AD mouse model. To assess inflammation in the 5xFAD (Tg) mouse model, we first quantified central and immune cell profiles in the deep cervical lymph nodes and spleen. In the brains of Tg mice, activated (MHCII+, CD45high, and Ly6Chigh) myeloid-derived CD11b+ immune cells are decreased while CD3+ T cells are increased as a function of age relative to non-Tg mice. These immunological changes along with evidence of increased mRNA levels for several cytokines suggest that immune regulation and trafficking patterns are altered in Tg mice. Levels of soluble Tumor Necrosis Factor (sTNF) modulate blood-brain barrier (BBB) permeability and are increased in CSF and brain parenchyma post-mortem in AD subjects and Tg mice. We report here that in vivo peripheral administration of XPro1595, a novel biologic that sequesters sTNF into inactive heterotrimers, reduced the age-dependent increase in activated immune cells in Tg mice, while decreasing the overall number of CD4+ T cells. In addition, XPro1595 treatment in vivo rescued impaired long-term potentiation (LTP) measured in brain slices in association with decreased Aβ plaques in the subiculum. Selective targeting of sTNF may modulate brain immune cell infiltration, and prevent or delay neuronal dysfunction in AD. Significance statementImmune cells and cytokines perform specialized functions inside and outside the brain to maintain optimal brain health; but the extent to which their activities change in response to neuronal dysfunction and degeneration is not well understood. Our findings indicate that neutralization of sTNF reduced the age-dependent increase in activated immune cells in Tg mice, while decreasing the overall number of CD4+ T cells. In addition, impaired long-term potentiation (LTP) was rescued by XPro1595 in association with decreased hippocampal Aβ plaques. Selective targeting of sTNF holds translational potential to modulate brain immune cell infiltration, dampen neuroinflammation, and prevent or delay neuronal dysfunction in AD.

Abstract 11081: Crucial Role of Thrombin Activatable Fibrinolysis Inhibitor in the Pathogenesis of Chronic Thromboembolic Pulmonary Hypertension

Background: Chronic thromboembolic pulmonary hypertension (CTEPH) is a fatal disease; however, its pathogenesis still remains to be elucidated. We tested our hypothesis that thrombin activatable fibrinolysis inhibitor (TAFI) plays a crucial role in the pathogenesis of CTEPH as it regulates fibrinolysis, reduces plasmin production, and prolongs clot lysis time. Methods and Results: In the lung of CTEPH patients, perivascular expression of TAFI in small pulmonary arteries (PA) was enhanced. In mice in vivo , chronic exposure to hypoxia (O 2 10%, 3 weeks) caused TAFI up-regulation in the lung. We then newly developed TAFI- overexpressing mice (Tg), in which chronic hypoxia increased right ventricular systolic pressure (RVSP) and caused RV hypertrophy and PA remodeling to a greater extent than in littermate controls (all P<0.05, n=8 each). Moreover, Tg mice (n=19) showed a significantly reduced long-term survival under hypoxia compared with controls (n=22) (63 vs. 95%, P<0.01). To evaluate the role of TAFI for inflammatory cell recruitment, we performed bone marrow reconstitution with GFP + cells, which demonstrated increased recruitment of GFP + cells in Tg mice compared with controls by 3D imaging. In experiments with pulmonary artery smooth muscle cells (Tg-PASMCs) in vitro , cell proliferation was significantly increased in Tg-PASMCs compared with controls (2.8 vs. 1.8-fold, n=8 each, P<0.01). Hypoxia (2% O 2 ) significantly increased TAFI expression and secretion from PASMCs. Furthermore, Tg-PASMCs showed significantly enhanced p38 activity and secretion of inflammatory cytokines/chemokines and growth factors compared with controls. Conditioned medium from Tg-PASMCs promoted cell proliferation compared with controls (1.3- vs. 1.1-fold, n=8 each, P<0.05). Human recombinant TAFI (100 nM) also promoted proliferation of PASMCs from CTEPH patients compared with vehicle controls (1.21- vs. 0.97-fold, n=8 each, P<0.01). Finally, plasma levels of activated TAFI were significantly increased in CTEPH patients (n=32) compared with healthy controls (n=13) (8.7-fold, P<0.01). Conclusions: These results indicate that TAFI plays a crucial role in the pathogenesis of CTEPH, suggesting that it is a novel therapeutic target for CTEPH.

P53 dysfunction precedes the activation of nuclear factor-κB during disease progression in mice expressing Tax, a human T-cell leukemia virus type 1 oncoprotein

Transgenic (Tg) mice expressing Tax, a human T-cell leukemia virus type 1 (HTLV-1) oncoprotein, develop mature T-cell leukemia/lymphoma. The leukemic cells in Tg mice expressing Tax show p53 dysfunction and nuclear factor-κB (NF-κB) activation, similar to that seen in adult T-cell leukemia/lymphoma (ATLL) cells from patients infected with HTLV-1. However, it is unclear when these effects occur in HTLV-1 carriers during the development of ATLL. Here, we examined p53 function and NF-κB activity before the onset of leukemia in Tax-expressing Tg (Tax-Tg) mice between 4 and 25 months of age. At 4-10 months of age, 71% of mice showed p53 inactivation, without evidence for NF-κB activation, even though tax expression was consistent from 4 to 25 months of age. The decline in p53 function resulted from decreased p53 accumulation after DNA damage. From 11 months of age onward, 75% of mice showed p53 dysfunction and 37.5% showed constitutive NF-κB activation with the components of p50 and RelB. An NF-κB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ), reduced NF-κB activity (i.e. p50/RelB) but did not restore p53 function. In vivo, treatment with DHMEQ until 24 months of age prevented the onset of T-cell leukemia in Tax-Tg mice. These results suggest that the Tax-induced decline in p53 function, which is independent of NF-κB activation in the early stage, might be the first stage in the onset of ATLL. NF-κB activity is involved in the later stages of ATLL onset.

Impaired response of hypoxic sensor protein HIF-1α and its downstream proteins in the spinal motor neurons of ALS model mice

We have recently reported spinal blood flow-metabolism uncoupling in an amyotrophic lateral sclerosis (ALS) animal model using Cu/Zn-superoxide dismutase 1 (SOD1)-transgenic (Tg) mice, suggesting a relative hypoxia in the spinal cord. However, the hypoxic stress sensor pathway has not been well studied in ALS. Here, we examined temporal and spatial changes of the hypoxic stress sensor proteins HIF-1α and its downstream proteins (VEGF, HO-1, and EPO) during the normoxiccourse of motor neuron (MN) degeneration in the spinal cord of these ALS model mice. We found that HIF-1α protein expression progressively increased both in the anterior large MNs and the surrounding glial cells in Tg mice from early symptomatic 14 week (W) and end stage 18W. Double immunofluorescence analysis revealed that HIF-1α, plus GFAP and Iba-1 double-positive surrounding glial cells, progressively increased from 14W to 18W, although the immunohistochemistry in large MNs did not change. Expression levels of VEGF and HO-1 also showed a progressive increase but were significant only in the surrounding glial cells at 18W. In contrast, EPO protein expression was decreased in the surrounding glial cells of Tg mice at 18W. Because HIF1-α serves as an important mediator of the hypoxic response, these findings indicate that MNs lack the neuroprotective response to hypoxic stress through the HIF-1α system, which could be an important mechanism of neurodegeneration in ALS.

FoxO1 Gain of Function in the Pancreas Causes Glucose Intolerance, Polycystic Pancreas, and Islet Hypervascularization

Genetic studies revealed that the ablation of insulin/IGF-1 signaling in the pancreas causes diabetes. FoxO1 is a downstream transcription factor of insulin/IGF-1 signaling. We previously reported that FoxO1 haploinsufficiency restored β cell mass and rescued diabetes in IRS2 knockout mice. However, it is still unclear whether FoxO1 dysregulation in the pancreas could be the cause of diabetes. To test this hypothesis, we generated transgenic mice overexpressing constitutively active FoxO1 specifically in the pancreas (TG). TG mice had impaired glucose tolerance and some of them indeed developed diabetes due to the reduction of β cell mass, which is associated with decreased Pdx1 and MafA in β cells. We also observed increased proliferation of pancreatic duct epithelial cells in TG mice and some mice developed a polycystic pancreas as they aged. Furthermore, TG mice exhibited islet hypervascularities due to increased VEGF-A expression in β cells. We found FoxO1 binds to the VEGF-A promoter and regulates VEGF-A transcription in β cells. We propose that dysregulation of FoxO1 activity in the pancreas could account for the development of diabetes and pancreatic cysts.

Morphogenetic roles of perlecan in the tooth enamel organ: An analysis of overexpression using transgenic mice

Perlecan, a heparan sulfate proteoglycan, is enriched in the intercellular space of the enamel organ. To understand the role of perlecan in tooth morphogenesis, we used a keratin 5 promoter to generate transgenic ( Tg) mice that over-express perlecan in epithelial cells, and examined their tooth germs at tissue and cellular levels. Immunohistochemistry showed that perlecan was more strongly expressed in the enamel organ cells of Tg mice than in wild-type mice. Histopathology showed wider intercellular spaces in the stellate reticulum of the Tg molars and loss of cellular polarity in the enamel organ, especially in its cervical region. Hertwig's epithelial root sheath (HERS) cells in Tg mice were irregularly aligned due to excessive deposits of perlecan along the inner, as well as on the outer sides of the HERS. Tg molars had dull-ended crowns and outward-curved tooth roots and their enamel was poorly crystallized, resulting in pronounced attrition of molar cusp areas. In Tg mice, expression of integrin β1 mRNA was remarkably higher at E18, while expression of bFGF, TGF-β1, DSPP and Shh was more elevated at P1. The overexpression of perlecan in the enamel organ resulted in irregular morphology of teeth, suggesting that the expression of perlecan regulates growth factor signaling in a stage-dependent manner during each step of the interaction between ameloblast-lineage cells and mesenchymal cells.

Over-expression of the LTC4 synthase gene in mice reproduces human aspirin-induced asthma

The pathogenesis of aspirin-induced asthma (AIA) is presumed to involve the aspirin/non-steroidal anti-inflammatory drug (NSAID)-induced abnormal metabolism of arachidonic acid, resulting in an increase in 5-lipoxygenase (5-LO) metabolites, particularly leukotriene C(4) (LTC(4) ). However, the role of LTC(4) in the development of AIA has yet to be conclusively demonstrated. The aim of this study was to evaluate the contribution of the lipid product LTC(4) secreted by the 5-LO pathway to the pathogenesis of AIA. To evaluate antigen-induced airway inflammation, the concentrations of T-helper type 2 cytokine in bronchoalveolar lavage fluid (BALF) obtained from LTC(4) synthase-transgenic (Tg) and wild-type (WT) mice after challenge with ovalbumin were measured. Subsequently, the ex vivo and in vivo effects of the NSAID sulpyrine were investigated in these Tg and WT mice by measuring the secretion of LTC(4) from sulpyrine-treated BAL cells and the levels of LTC(4) in BALF following challenge with sulpyrine. Finally, the sulpyrine-induced airway response by the administration of pranlukast, an antagonist of the cysteinyl (cs)-LT1 receptor, was analysed. The concentrations of IL-4, -5, and -13 in BALF from Tg mice were significantly higher than those in WT mice. In addition, sulpyrine augmented the secretion of LTC(4) in BALF and by BAL cells in Tg mice, but not in WT mice. Additionally, the increased airway resistance induced by sulpyrine could be reduced by treatment with pranlukast. Furthermore, the secretion of LTC(4) from mast cells, eosinophils, and macrophages was increased in the allergen-stimulated LTC(4) synthase gene Tg mice, even in the absence of sulpyrine, as well as in BAL cells after sulpyrine. The over-expression of the LTC(4) synthase in a mouse asthma model also replicates the key features of AIA. And our study supports that cys-LTs play a major role in the pathogenesis of AIA in patients with chronic asthma.

Treadmill exercise represses neuronal cell death in an aged transgenic mouse model of Alzheimer's disease

The present study was undertaken to further investigate the protective effect of treadmill exercise on the hippocampal proteins associated with neuronal cell death in an aged transgenic (Tg) mice with Alzheimer's disease (AD). To address this, Tg mouse model of AD, Tg-NSE/PS2m, which expresses human mutant PS2 in the brain, was chosen. Animals were subjected to treadmill exercise for 12 weeks from 24 months of age. The exercised mice were treadmill run at speed of 12 m/min, 60 min/day, 5 days/week on a 0% gradient for 3 months. Treadmill exercised mice improved cognitive function in water maze test. Treadmill exercised mice significantly reduced the expression of Aβ-42, Cox-2, and caspase-3 in the hippocampus. In parallel, treadmill exercised Tg mice decreased the phosphorylation levels of JNK, p38MAPK and tau (Ser404, Ser202, Thr231), and increased the phosphorylation levels of ERK, PI3K, Akt and GSK-3α/β. In addition, treadmill exercised Tg mice up-regulated the expressions of NGF, BDNF and phospho-CREB, and the expressions of SOD-1, SOD-2 and HSP-70. Treadmill exercised Tg mice up-regulated the expression of Bcl-2, and down-regulated the expressions of cytochrome c and Bax in the hippocampus. The number of TUNEL-positive cells in the hippocampus in mice was significantly decreased after treadmill exercise. Finally, serum TC, insulin, glucose, and corticosterone levels were significantly decreased in the Tg mice after treadmill exercise. As a consequence of such change, Aβ-dependent neuronal cell death in the hippocampus of Tg mice was markedly suppressed following treadmill exercise. These results strongly suggest that treadmill exercise provides a therapeutic potential to inhibit both Aβ-42 and neuronal death pathways. Therefore, treadmill exercise may be beneficial in prevention or treatment of AD.

Vasoactive intestinal peptide (VIP) up-regulates IL-17 generation by mouse CD4 T cells (100.1)

Abstract VIP produced by cholinergic and sensory nerves and by T cells affects T cell functions through the constitutively-expressed VPAC1 and inducible VPAC2 G protein-coupled receptors. Transgenic mice overexpressing VPAC2 (TG) selectively on CD4 T cells exhibit increased hypersensitivity and immunoinflammatory responses. Present studies were designed to examine the capacity of the VIP-VPAC2 axis to recruit the Th17 inflammatory subset of CD4 T cells. Splenic CD4 T cells isolated from TG and wild-type (WT) mice were incubated with adherent anti-CD3 and anti-CD28 antibodies and cytokines that initiate Th17 development, without and with IL-23 or VIP. IL-17 generation quantified by ELISA was enhanced significantly by 1μM VIP with the same time-course as for IL-23 over five days. IL-17 generation by CD4 T cells of TG mice was significantly greater than by those of WT mice with and without added VIP. Augmentation of IL-17 generation by both VIP and IL-23 was suppressed similarly by IL-27. Thus VIP may be a neuroalternative endogenous stimulus for Th17 development and recruit Th17 cells independently of IL-23 in inflammatory and autoimmune processes. (NIH grant AI029912)

CCL27 is a critical factor for the development of atopic dermatitis in the keratin-14 IL-4 transgenic mouse model

The keratin-14 IL-4 transgenic (Tg) mouse model of atopic dermatitis (AD) is characterized by skin infiltration of T cells, early up-regulation of T(h)2 cytokines and late surge of T(h)1 cytokines. In the present study, we investigated the role of CCL27, a T cell skin-homing chemokine known to be elevated in sera of human AD patients, in disease development in our animal model of AD. The results showed that the mRNA and protein levels of CCL27 in the skin and serum were significantly increased in IL-4 Tg mice. The percentage of T cells expressing CCR10 in skin draining lymph nodes of IL-4 Tg mice was increased, consistent with the findings of >80% of skin-infiltrating T cells in Tg mice expressing CCR10. Chemotaxis transmigration assay demonstrated that CCL27 promotes a greater degree of migration of T cells in diseased Tg mice. Subcutaneous injection of neutralizing anti-CCL27 to IL-4 Tg mice with early skin lesions resulted in reduced clinical progression of inflammation, accompanied with decreased T cell and mast cell infiltration in the skin, and down-regulation of inflammatory cytokines. In conclusion, CCL27 and CCR10 interaction is important for the development of skin inflammation in our AD model.

Increased Noise Level of Purkinje Cell Activities Minimizes Impact of Their Modulation during Sensorimotor Control

While firing rate is well established as a relevant parameter for encoding information exchanged between neurons, the significance of other parameters is more conjectural. Here, we show that regularity of neuronal spike activities affects sensorimotor processing in tottering mutants, which suffer from a mutation in P/Q-type voltage-gated calcium channels. While the modulation amplitude of the simple spike firing rate of their floccular Purkinje cells during optokinetic stimulation is indistinguishable from that of wild-types, the regularity of their firing is markedly disrupted. The gain and phase values of tottering's compensatory eye movements are indistinguishable from those of flocculectomized wild-types or from totterings with the flocculus treated with P/Q-type calcium channel blockers. Moreover, normal eye movements can be evoked in tottering when the flocculus is electrically stimulated with regular spike trains mimicking the firing pattern of normal simple spikes. This study demonstrates the importance of regularity of firing in Purkinje cells for neuronal information processing.

Generation and Analysis of Transgenic Mice Reveal a Role for CRE Binding Proteins in Multiple Stages of B Cell Development, Functional Maturation, Proliferation and Apoptosis.

Cyclic AMP response element binding protein-1 (CREB-1) is expressed in various stages of B cell development in the bone marrow. Antigen receptor induced DNA binding activity and activation dependent phosphorylation suggests a role for CREB-1 in early B cell development and functional maturation in-vivo. This was directly tested in transgenic mice over expressing a dominant negative Ser119-ala phosphomutant CREB-1 in bone marrow (BM). Northern blot analysis of total RNA and Western blot analysis of the protein isolated from 4 independent transgenic lines revealed expression of the transgene in the BM and peripheral B cells. Flow cytometric analysis of BM cells from transgenic (Tg) and non-transgenic (NTg) littermates revealed ~65% reduction in B220+/IgM− pro-B and pre-B population (7.56±0.89% NTg vs. 3.5±1.1% Tg mice) and B220+/IgM+ immature and mature B cells (4.4±0.98 in NTg vs 1.4± 0.19% in Tg mice). Detailed analysis of BM cells from Tg and NTg littermates by multiparameter flow cytometry revealed ~70% increase in B220+CD43+CD24+(int) pre-BI cells (39% in NTg vs 67% in Tg mice), and ~60% decrease in B220+CD43+CD24++(high) pre-BII B cells (46% in Ntg vs 20% in Tg mice), indicating a developmental block in pre-BI to pre-BII transition in mCREB-1 mice. Adoptive transfer of BM cells from Tg or NTg mice into sub-lethally irradiated RAG-2−/− mice, revealed cell intrinsic defect in Tg bone marrow B cells. RT-PCR analysis of RNA from Tg pre-BI B cells revealed increased c-jun and decreased jun-B transcripts with minimal changes in c-fos, PCNA, mb-1 and vpreB. In contrast to pre-BI cells, increased c-jun and junB transcripts were observed in pre-BII B cells. Cell cycle analysis exhibited a consistent decrease in S phase entry in pre-BII B cells from Tg compared to NTg mice at 48 hours without stimulation (41% in NTg vs 11% in Tg) or in response to IL-7 (48% in NTg vs 27% in Tg). Consistent with the BM defect, mCREB-1 Tg mice revealed ~ 40% decrease in the IgM+B220+ cells (29.3±0.96% in NTg vs. 17.7±2.9% Tg) in the spleen. This is further reflected in significant reduction in the absolute number of mature B cells in the spleen [(36±3.6 in NTg vs. 22 ±1.9% in Tg mice, p<0.01]. Interestingly when compared to NTg mice, mCREB-1 Tg mice revealed about 45% decrease in CD21dimCD23high follicular B cells with minimal change in the CD21highCD23dim marginal zone B cell (16.5±1.4x106 in NTg vs 9.5±0.92 x106 cells in Tg mice P<0.01). In contrast to the bone marrow and spleen, analysis of peritoneal cells revealed significant increase in the total cell number and B220+IgM+ population in Tg mice compared to NTg littermates [22±3% in NTg vs 33±2.6% in Tg mice (p<0.01)]. The increased cell number and the abnormal distribution reflected a 3–4 fold increase in IgMhighIgDlowMac-1Int B1 B cells. This increase is reflected in both CD5+ B-1a and CD5− B-1b B cells in the Tg mice and is attributed to resistance to apoptosis as evidenced by Annexin V/PI staining. These studies provide the first evidence for a role for CRE binding proteins in multiple stages of B cell development, functional maturation, proliferation and apoptosis. Ongoing studies are aimed to define the role of CREB-1 transcription factor in human B cell malignancies including chronic lymphocytic leukemia and acute lymphocytic leukemia.

Multiple Levels of Regulation for Self-Tolerance in Beef Insulin Transgenic Mice

To characterize the mechanism(s) of tolerance toward soluble self-antigens (Ags), beef insulin (BI) transgenic (Tg) mice were generated in which the transgene was expressed in pancreatic β-cells. Our previous data showed that: (i) Ag-specific tolerance can be induced and/or maintained in peripheral T cells in thymectomized BI-Tg mice and (ii) CD4+Th2 regulatory T cells are involved in maintaining peripheral tolerance (by anti-BI antibody response). In this paper, we have further characterized the relationship of low levels of BI expression (10−10–10−11M) in Th1/Th2 activation. In addition, we have explored intrathymic events associated with tolerance to self-Ags not expressed in the thymus and/or to circulating self-Ags. Limiting dilution analysis showed that there was a significantly higher frequency of BI-specific Th2 cells in Tg mice with a corresponding higher frequency of Th1 cells in non-Tg mice. While there was no transgene expression in the thymus (by RT–PCR), independent studies showed that BI can be processed and presented in the Tg thymus, which correlated with the Ag-specific hyporesponsiveness of mature thymocyes detectedin vitro.High-dose rIL-2 (150 U/ml) was able to restorein vitroperipheral T cell response of Tg mice to levels comparable to those of the non-Tg control. Collectively, our data suggest that: (i) there is a differential activation of BI-specific Th1/Th2 cellsin vivoin the presence of low Ag concentration; (ii) the thymus may play a role in self-tolerance to Ags whose expression in adults is restricted to the periphery; and (iii) multiple levels of regulation such as thymic selection, peripheral anergy, and active suppression may be involved in tolerance to BI in BI-Tg mice.

The eyes of transforming growth factor-sZ1 (TGF-sZ1) transgenic miceMorphology and the development of endotoxin-induced uveitis

Transforming growth factor-beta (TGFsZ) is a potent regulator of cellular growth and immune function. The authors studied ocular histology and endotoxin-induced uveitis in a TGF-sZ(1) transgenic (Tg) murine model. TGF-sZ(1) Tg mice were generated by micro injecting a gene constructed by fusing the mouse albumin enhancer/promoter and porcine TGF-sZ(1) cDNA. The eyes of Tg mice from two to 14 weeks of age were studied histologically. Tg mice, two to five weeks of age exhibited mild fragmentation of the lens fibers and retinal edema. No pathology was found from six to ten weeks of age, however, a progressive increased frequency of cataract was observed from 11 to 14 weeks of age. Plasma TGF-sZ(1) levels were much higher in Tg mice than age-matched wild type control littermates (wt). Endotoxin-induced uveitis (EIU) in six-to eight-week-old Tg and wt mice was induced by footpad injection of lipopolysaccharide (LPS). Mice were euthanized 24 hr after LPS injection, the eyes were collected for histology and serum assayed for IL-6 and TGF-sZ(1). There was a decrease in the mean numbers of infiltrating cells in Tg mice compared to wt mice. Serum IL-6 and TGF-sZ(1) were much higher in Tg mice. The authors concluded that expression of the TGF-sZ(1) transgene in the eyes may have effect on lens growth. Overexpression of TGF-sZ(1) results in little or no effect on the development of EIU.

B cells are required as APC for antigen-specific T cell proliferation but not for the differentiation or priming of those T cells.

We studied the influences of B cells on functional differentiation of T cells using SCID mice grafted with fetal thymus of C.B-17 mice (TG mice). T cells were shown to be reconstituted in TG mice without B cell development. These mice showed normal DTH response to SRBC and OVA. LN cells of these mice produced cytokines including IL-2, IL-4, IL-6 and IFN-gamma according to Con A stimulation. Thus, majority of T cell functions seem to differentiate in the absence of B cells. However, T cells of TG mice failed to proliferate in response to immunizing antigens in vitro, although they responded well to stimulation with Con A. This unresponsiveness of T cells in TG mice to these antigens was restored when antigen-primed B cells were added to the proliferation assay. Such an inability of T cells in antigen-specific proliferation was not seen in SCID mice grafted with C.B-17 fetal liver cells, in which B cells as well as T cells were efficiently reconstituted (FLT mice). T cell proliferation to immunizing antigen was also abrogated in FLT mice when B cells were depleted from lymphoid population. These results indicate that T cells can functionally differentiate and be primed in the absence of B cells, but they require B cells to proliferate in response to foreign antigens.