Loss of classical transient receptor potential 6 channel reduces allergic airway response

Background Non-selective cation influx through canonical transient receptor potential channels (TRPCs) is thought to be an important event leading to airway inflammation. TRPC6 is highly expressed in the lung, but its role in allergic processes is still poorly understood.
Objective The purpose of this study was to evaluate the role of TRPC6 in airway hyperresponsiveness (AHR) and allergic inflammation of the lung.
Methods Methacholine-induced AHR was assessed by head-out body plethysmography of wild type (WT) and TRPC6^−/− mice. Experimental airway inflammation was induced by intraperitoneal ovalbumin (OVA) sensitization, followed by OVA aerosol challenges. Allergic inflammation and mucus production were analysed 24 h after the last allergen challenge.
Results Methacholine-induced AHR and agonist-induced contractility of tracheal rings were increased in TRPC6^−/− mice compared with WT mice, most probably due to compensatory up-regulation of TRPC3 in airway smooth muscle cells. Most interestingly, when compared with WT mice, TRPC6^−/− mice exhibited reduced allergic responses after allergen challenge as evidenced by a decrease in airway eosinophilia and blood IgE levels, as well as decreased levels of T-helper type 2 (Th2) cytokines (IL-5, IL-13) in the bronchoalveolar lavage. However, lung mucus production after allergen challenge was not altered by TRPC6 deficiency.
Conclusions TRPC6 deficiency inhibits specific allergic immune responses, pointing to an important immunological function of this cation channel in Th2 cells, eosinophils, mast cells and B cells.     

Insulin hypersensitivity in mice lacking the V1b vasopressin receptor

We have reported that [Arg⁸]-vasopressin-stimulated insulin release is blunted in islet cells isolated from V1b receptor-deficient ( V1bR ^−/−) mice. In this study, we used V1bR ^−/− mice to examine the physiological role of the V1b receptor in regulating blood glucose levels in vivo , and we found that the fasting plasma glucose, insulin and glucagon levels were lower in V1bR ^−/− mice than in wild-type ( V1bR ^+/+) mice. Next, we evaluated glucose tolerance by performing an intraperitoneal glucose tolerance test (GTT). The plasma glucose and insulin levels during the GTT were lower in V1bR ^−/− mice than in V1bR ^+/+ mice. An insulin tolerance test (ITT) revealed that, after insulin administration, plasma glucose levels were lower in V1bR ^−/− mice than in V1bR ^+/+ mice. In addition, a hyperinsulinaemic–euglycaemic clamp study showed that the glucose infusion rate was increased in V1bR ^−/− mice, indicating that insulin sensitivity was enhanced at the in vivo level in V1bR ^−/− mice. Furthermore, we found that the V1b receptor was expressed in white adipose tissue and that insulin-stimulated phosphorylation of Akt as an important signaling molecule was increased in adipocytes isolated from V1bR ^−/− mice. Thus, the blockade of the V1b receptor could result, at least in part, in enhanced insulin sensitivity by altering insulin signalling in adipocytes.     

Protein Tyrosine Phosphatase ε is a Negative Regulator of FcεRI-mediated Mast Cell Responses

Modulation of mast-cell activation may provide novel ways to control allergic diseases. Here, we show that protein tyrosine phosphatase ε (PTPε; Ptpre ) plays key regulatory roles during mast-cell activation mediated by the high-affinity IgE receptor (FcεRI). Bone marrow-derived mast cells (BMMC) from Ptpre ^−/− mice exhibited enhanced FcεRI-induced Ca²⁺ mobilization and mitogen-activated protein kinase (MAPK) (JNK and p38) activation, and showed corresponding enhancement of evoked degranulation and cytokine production, but not leukotriene production. Examination of proteins linking tyrosine kinase activation and Ca²⁺ mobilization revealed that the absence of PTPε leads to increased phosphorylation of the linker for activation of T cells and SH2 domain-containing leucocyte phosphoproteins of 76 kDa, but not Grb2-associated binder-2 (Gab2). Because Gab2 is considered to be situated downstream of Fyn kinase, we reasoned that Fyn may not be a target of PTPε. In the event, Syk but not Lyn was hyperphosphorylated in PTPε-deficient BMMC. Thus, PTPε most likely exerts its effects at the level of Syk, inhibiting downstream events including phosphorylation of SLP-76 and linker of activated T cells and mobilization of Ca²⁺. Consistent with the in vitro data, antigen- and IgE-mediated passive systemic anaphylactic reactions were augmented in Ptpre ^−/− mice. Given that the number of mast cells is unchanged in these mice, this observation most likely reflects alterations of mast cell-autonomous signalling events. These data suggest that PTPε negatively regulates FcεRI-mediated signalling pathways and thus constitutes a novel target for ameliorating allergic conditions.     

IL-12p40 is essential for the down-regulation of airway hyperresponsiveness in a mouse model of bronchial asthma with prolonged antigen exposure

Background We previously reported a mouse model of bronchial asthma showing eosinophilic inflammation, but not airway hyperresponsiveness (AHR), after prolonged antigen exposure. This model showed an increase of IL-12 in the lung.
Objective The aim of this study was to investigate the role of IL-12p40 in a murine asthma model with prolonged antigen exposures.
Methods An ovalbumin (OVA)-induced asthma model was first established in wild-type (WT) and IL-12p40-deficient (IL-12p40^−/−) mice. Both strains of mice were further exposed to either OVA (prolonged exposure group) or phosphate-buffered saline (positive control group) 3 days per week for 3 weeks. During week 4, both groups of mice were given a final challenge with OVA.
Results Prolonged antigen exposures resulted in marked suppression of airway eosinophilia in both WT and IL-12p40^−/− mice. However, AHR persisted in IL-12p40^−/− but not in WT mice. There were no significant differences of IL-5, IL-13 or IFN-γ levels in bronchoalveolar lavage fluid between WT and IL-12p40^−/− mice. The hydroxyproline content of the lung and peribronchial fibrosis were, however, significantly increased in IL-12p40^−/− mice.
Conclusion The results suggest that endogenous IL-12p40 is essential for inhibition of AHR and peribronchial fibrosis, but not eosinophilic inflammation, in a murine asthma model with prolonged antigen exposures.     

Increased responsiveness of rat colonic splanchnic afferents to 5-HT after inflammation and recovery

5-Hydroxytryptamine (5-HT) activates colonic splanchnic afferents, a mechanism by which it has been implicated in generating symptoms in postinfectious and postinflammatory states in humans. Here we compared mechanisms of colonic afferent activation by 5-HT and mechanical stimuli in normal and inflamed rat colon, and after recovery from inflammation. Colonic inflammation was induced in rats by dextran sulphate sodium. Single-fibre recordings of colonic lumbar splanchnic afferents revealed that 58% of endings responded to 5-HT (10⁻⁴ m ) in controls, 88% in acute inflammation ( P < 0.05) and 75% after 21 days recovery ( P < 0.05 versus control). Maximal responses to 5-HT were also larger, and the estimated EC₅₀ was reduced from 3.2 × 10⁻⁶ to 8 × 10⁻⁷ m in acute inflammation and recovered to 2 × 10⁻⁶ m after recovery. Responsiveness to mechanical stimulation was unaffected. 5-HT₃ receptor antagonism with alosetron reduced responses to 5-HT in controls but not during inflammation. Responses to the mast cell degranulator 48/80 mimicked those to 5-HT in inflamed tissue but not in controls, and more 5-HT-containing mast cells were seen close to calcitonin gene-related peptide-containing fibres in inflamed serosa. We conclude that colonic serosal and mesenteric endings exhibit increased sensitivity to 5-HT in inflammation, with both an increase in proportion of responders and an increase in sensitivity, which is maintained after healing of inflammation. This is associated with alterations in the roles of 5-HT₃ receptors and mast cells.     

Regulatory T cells in spontaneous autoimmune encephalomyelitis

Summary: Spontaneous experimental autoimmune encephalomyelitis (EAE) develops in 100% of mice harboring a monoclonal myelin basic protein (MBP)-specific CD4⁺αβ T-cell repertoire. Monoclonality of the αβ T-cell repertoire can be achieved by crossing MBP-specific T-cell receptor (TCR) transgenic mice with either RAG^−/− mice or TCR α^−/−/TCR β^−/− double knockout mice. Spontaneous EAE can be prevented by a single administration of purified CD4⁺ splenocytes or thymocytes obtained from wild-type syngeneic mice. The regulatory T cells (T-reg) that protect from spontaneous EAE need not express the CD25 marker, as effective protection can be attained with populations depleted of CD25⁺ T cells. Although the specificity of the regulatory T cells is important for their generation or regulatory function, T cells that protect from spontaneous EAE can have a diverse TCR α and β chain composition. T-reg cells expand poorly in vivo , and appear to be long lived. Finally, precursors for T-reg are present in fetal liver as well as in the bone marrow of aging mice. We propose that protection of healthy individuals from autoimmune diseases involves several layers of regulation, which consist of CD4⁺CD25⁺ regulatory T cells, CD4⁺CD25⁻ T-reg cells, and anti-TCR T cells, with each layer potentially operating at different stages of T-helper cell-mediated immune responses.     

Carbonic anhydrase gene expression in CA II-deficient (Car2−/−) and CA IX-deficient (Car9−/−) mice

Using real-time PCR and immunohistochemistry, we have examined the expression of carbonic anhydrase isozymes (CA) I, II, III, IV, IX, XII, XIII and XIV in the brain, kidney, stomach and colon of the wild-type, CA II-deficient ( Car2^−/− ), and CA IX deficient ( Car9^−/− ) mice. The expression of Car4, Car12, Car13 and Car14 mRNAs did not show any significant deviations between the three groups of mice, whereas both groups of CA deficient mice showed decreased expression levels of Car1 in the colon and Car3 in the kidney. The Car2 mRNA level was greatly reduced but not completely abolished in all four tissues from the Car2^−/− mice in which no CA II protein was expressed. Sequencing the Car2 cDNA isolated from C57BL6 Car2^−/− mice revealed two nucleotide differences from the wild-type C57BL6 mice. One is a silent polymorphism found in Car2 mRNA from wild-type DBA mice, which is the strain that provided the original mutagenized chromosome. The second change is a mutation that causes prematurely terminated translation at codon 155 (Gln155X). Car9 mRNA and CA IX protein expression levels were up-regulated about 2.5- and 3.6-fold, respectively, in the stomach of the Car2^−/− mice. These results suggest that the loss of function of cytosolic CA II in the stomach of Car2^−/− mice leads to up-regulation of an extracellular CA, namely CA IX, which is expressed on the cell surface of the gastric epithelium.     

Tumor necrosis factor alpha is reparative via TNFR2 [corrected] in the hippocampus and via TNFR1 [corrected] in the striatum after virus-induced encephalitis

Differentiating between injurious and reparative factors facilitates appropriate therapeutic intervention. We evaluated the role of tumor necrosis factor α (TNFα) in parenchymal brain pathology resolution following virus-induced encephalitis from a picornavirus, Theiler's murine encephalomyelitis virus (TMEV). We infected the following animals with TMEV for 7 to 270 days: B6/129 TNF^−/− mice (without TNFα expression), B6/129 TNFR1^−/− mice (without TNFα receptor 1 expression), and B6/129 TNFR2^−/− mice (without TNFα receptor 2 expression). Normal TNFα-expressing controls were TMEV-infected B6, 129/J, B6/129F1 and B6/129F2 mice. Whereas all strains developed inflammation and neuronal injury in the hippocampus and striatum 7 to 21 days postinfection (dpi), the control mice resolved the pathology by 45 to 90 dpi. However, parenchymal hippocampal and striatal injury persisted in B6/129 TNF^−/− mice following infection. Treating virus-infected mice with active recombinant mouse TNFα resulted in less hippocampal and striatal pathology, whereas TNFα-neutralizing treatment worsened pathology. T1 "black holes" appeared on MRI during early infection in the hippocampus and striatum in all mice but persisted only in TNF^−/− mice. TNFR1 mediated hippocampal pathology resolution whereas TNFR2 mediated striatal healing. These findings indicate the role of TNFα in resolution of sublethal hippocampal and striatal injury.     

Glycinergic input of widefield, displaced amacrine cells of the mouse retina

Glycine receptors (GlyRs) of displaced amacrine cells of the mouse retina were analysed using whole cell recordings and immunocytochemical staining with subunit-specific antibodies. During the recordings the cells were filled with a fluorescent tracer and 11 different morphological types could be identified. The studies were performed in wild-type mice and in mutant mice deficient in the GlyRα1 ( Glra1^spd-ot , 'oscillator' mouse), the GlyRα2 ( Glra2 ^−/−) and the GlyRα3 subunit ( Glra3 ^−/−). Based on their responses to the application of exogenous glycine in the retinas of wild-type and mutant mice, the cells were grouped into three major classes: group I cells (comprising the morphological types MA-S5, MA-S1, MA-S1/S5, A17, PA-S1, PA-S5 and WA-S1), group II cells (comprising the morphological types PA-S4, WA-S3 and WA-multi) and ON-starburst cells. For further analysis, spontaneous inhibitory postsynaptic currents (sIPSCs) were measured both in wild-type and mutant mouse retinas. Glycinergic sIPSCs and glycine induced currents of group I cells remained unaltered across wild-type and the three mutant mice (mean decay time constant of sIPSCs, τ∼25 ms). Group II cells showed glycinergic sIPSCs and glycine induced currents in wild-type, Glra1^spd-ot and Glra3 ^−/− mice (τ∼25 ms); however, glycinergic currents were absent in group II cells of Glra2 ^−/− mice. Glycine induced currents and sIPSCs recorded from ON-starburst amacrine cells did not differ significantly between wild-type and the mutant mouse retinas (τ∼50–70 ms). We propose that GlyRs of group II cells are dominated by the α2 subunit; GlyRs of ON-starburst amacrine cells appear to be dominated by the α4 subunit.     

Impaired Antibody Responses but Normal Proliferation of Specific CD4+ T Cells in Mice Lacking Complement Receptors 1 and 2

Severely impaired Ab responses are seen in animals lacking C (complement) factors C2, C3 or C4 as well as CR1/2 (C receptors 1 and 2). The molecular mechanism behind this phenomenon is not understood. One possibility is that C-containing immune complexes are endocytosed via CR2 on B cells and presented to specific CD4⁺ T cells, which would then proliferate and provide efficient help to specific B cells. In vitro , B cells can endocytose immune complexes via CR1/2 and present the Ag to T cells. Whether absence of this Ag presenting function in Cr2^−/− mice (mice lacking CR1/2) explains their low Ab response is unclear. To address this question, Cr2^−/− and wild type mice were transferred with OVA-specific T cells, obtained from the DO11.10 strain which has a transgenic TCR recognizing an OVA peptide. The animals were subsequently immunized with sheep red blood cells (SRBC) conjugated to OVA. Interestingly, proliferation of the OVA-specific T cells was normal in Cr2^−/− mice, although their Ab response to both SRBC and OVA was severely impaired. These observations suggest that the impaired Ab response in Cr2^−/− mice cannot be explained by a lack of appropriate induction of T cell help.     

γδ T cells: firefighters or fire boosters in the front lines of inflammatory responses

Summary:  Intradermal inoculation of cloned self-reactive αβ T cells into the footpads of mice induced cutaneous graft-versus-host disease (GVHD), and after recovery from GVHD, the epidermis became resistant to subsequent attempts to induce GVHD. Resistance to GVHD was not induced in the epidermis of T-cell receptor δ-deficient (TCRδ^−/−) mice that lacked γδ T cells bearing canonical Vγ5/Vδ1⁺γδTCRs, known as dendritic epidermal T cells (DETCs), and resistance was restored by reconstitution of these mutant mice with precursors of Vγ5⁺ DETCs. Pulmonary infection by Cryptococcus neoformans induced an increase of γδ T cells in the lung, and in comparison with wildtype mice, TCRδ^−/− mice eliminated C. neoformans more rapidly and synthesized more interferon-γ in the lung. In the mouse small intestine, the absence of γδ T cells is associated with a reduction in epithelial cell turnover and downregulation of the expression of major histocompatibility complex class II molecules. The protective role of γδ T cells was verified in a dextran sodium sulfate-induced inflammatory bowel disease (IBD) model, whereas in a spontaneous model of IBD, γδ T cells were involved in the exacerbation of colitis in TCRα^−/− mice. Taken together, in addition to the homeostatic regulation of epithelial tissues, γδ T cells appear to play a pivotal role in the modification of inflammatory responses induced in many organs containing epithelia.     

Ca2+ permeability of the channel pore is not essential for the δ2 glutamate receptor to regulate synaptic plasticity and motor coordination

The δ2 glutamate receptor (GluRδ2) plays a crucial role in cerebellar functions; mice with a disrupted GluR δ 2 gene ( GluR δ 2^−/− ) display impaired synapse formation and abrogated long-term depression (LTD). However, the mechanisms by which GluRδ2 functions have remained unclear. Because a GluRδ2 mutation in lurcher mice causes channel activities characterized by Ca²⁺ permeability, GluRδ2 was previously suggested to serve as a Ca²⁺-permeable channel in Purkinje cells. To test this hypothesis, we introduced a GluR δ 2 transgene, which had a mutation (Gln618Arg) in the putative channel pore, into GluR δ 2^−/− mice. Interestingly, the mutant transgene rescued the major functional and morphological abnormalities of GluR δ 2^−/− Purkinje cells, such as enhanced paired-pulse facilitation, impaired LTD at parallel fibre synapses, and sustained innervation by multiple climbing fibres. These results indicate that the conserved glutamine residue in the channel pore, which is crucial for all Ca²⁺-permeable glutamate receptors, is not essential for the function of GluRδ2.     

Differential expression of leukocyte immunoglobulin-like receptors on cord-blood-derived human mast cell progenitors and mature mast cells

The leukocyte Ig-like receptors (LILRs) comprise a family of cell-surface immunoregulatory receptors with activating and inhibitory members. The inhibitory LILRs possess cytoplasmic ITIMs that down-regulate signaling by nonreceptor tyrosine kinase cascades. The activating members have a truncated cytoplasmic domain and signal through the FcR gamma chain. We examined the expression of LILRs on human mast cells during their development in vitro. Progenitor mast cells expressed cell surface inhibitory LILRB1, -B2, -B3, and -B4 and activating LILRA1. However, although mature cord blood-derived mast cells (hMCs) had detectable mRNA encoding multiple LILRs, none were expressed on the cell surface. Culture of progenitor mast cells or hMCs with various cytokine combinations failed to retain or induce cell surface expression of the LILRs. It is interesting that hMCs expressed LILRB5 in cytoplasmic granules and upon cross-linking of the high-affinity IgE receptor, released LILRB5 into the culture medium. Our results demonstrate that LILRs are developmentally regulated in human mast cells and that LILRB5 is expressed in mast cell granules and the release of soluble LILRB5 following IgE FcR-dependent stimulation, which has potential for amplification of mast cell-dependent, inflammatory responses.     

Jejunal afferent nerve sensitivity in wild-type and TRPV1 knockout mice

The aim of this study was to investigate the contribution of the TRPV1 receptor to jejunal afferent sensitivity in the murine intestine. Multiunit activity was recorded in vitro from mesenteric afferents supplying segments of mouse jejunum taken from wild-type (WT) and TRPV1 knockout (TRPV1^−/−) animals. In WT preparations, ramp distension of the gut (up to 60 mmHg) produced biphasic changes in afferent activity so the pressure–response curve had an initial rapid increase in afferent discharge followed by a second phase of slower increase in activity. Afferent response to distension was significantly lower in TRPV1^−/− than in WT mice. Single-unit analysis revealed three functional types of afferent fibres: (1) low-threshold fibres (2) wide dynamic range fibres and (3) high-threshold fibres. There was a marked downward shift of the pressure–response curve for wide dynamic range fibres in the TRPV1^−/− mice as compared to the WT controls. The afferent response to intraluminal hydrochloric acid (20 m m ) was also attenuated in the TRPV1^−/− mice. In contrast, the response to bath application of bradykinin (1 μ m , 3 ml) was not significantly different between the two groups. The TRPV1 antagonist capsazepine (10 μ m ) significantly attenuated the nerve responses to distension, intraluminal acid and bradykinin, as well as the spontaneous discharge in WT mice. The WT jejunal afferents responded to capsaicin with rapid increases in afferent activity, whereas TRPV1^−/− afferents were not at all sensitive to capsaicin. Previous evidence indicates that TRPV1 is not mechanosensitive, so the results of the present study suggest that activation of TRPV1 may sensitize small intestinal afferent neurones.     

CD3−4−8− Thymocyte Precursors with Interleukin-2 Receptors Differentiate Phenotypically in Coculture with Thymic Stromal Cells

CD3⁻4⁻8⁻ interleukin-2 receptor positive (IL-2R⁺) thymocyte precursors from adult mice were cocultured with thymic stromal cells from syngeneic adult mice. The IL-2R⁺CD3⁻4⁻8⁻ thymocytes were obtained by positive panning of IL-2R⁺ cells followed by either sorting or negative panning of triple negative cells, and they were cocultured with primary or secondary cultures of heterogeneous thymic stromal cells. Phenotypic maturation of these precursor cells was extremely rapid. Within 2½ days significant numbers of CD4⁺8⁺ and CD3⁺4⁺8⁻ cell populations developed, the latter expressing the αβ T-cell receptor (αβ-TCR). Thus heterogeneous stromal cell cultures support the development of IL-2R⁺ precursors and with these methods it will now be possible to isolate the particular stromal cells involved at each stromal-dependent step.     

KCNQ1 is the luminal K+ recycling channel during stimulation of gastric acid secretion

Parietal cell (PC) proton secretion via H⁺/K⁺-ATPase requires apical K⁺ recycling. A variety of K⁺ channels and transporters are expressed in the PC and the molecular nature of the apical K⁺ recycling channel is under debate. This study was undertaken to delineate the exact function of KCNQ1 channels in gastric acid secretion. Acid secretory rates and electrophysiological parameters were determined in gastric mucosae of 7- to 8-day-old KCNQ1^+/+, ^+/– and ^−/− mice. Parietal cell ultrastructure, abundance and gene expression levels were quantified. Glandular structure and PC abundance, and housekeeping gene expression did not differ between the KCNQ1^−/− and ^+/+ mucosae. Microvillar secretory membranes were intact, but basal acid secretion was absent and forskolin-stimulated acid output reduced by ∼90% in KCNQ1^−/− gastric mucosa. Application of a high K⁺ concentration to the luminal membrane restored normal acid secretory rates in the KCNQ1^−/− mucosa. The study demonstrates that the KCNQ1 channel provides K⁺ to the extracellular K⁺ binding site of the H⁺/K⁺-ATPase during acid secretion, and no other gastric K⁺ channel can substitute for this function.     

Congenital tracheal malformation in cystic fibrosis transmembrane conductance regulator-deficient mice

In cystic fibrosis (CF) patients, the major alteration in pulmonary function is due to peripheral airway obstruction. In the present study, we investigated the possibility that alterations in the extrathoracic airways, particularly in the trachea that expresses high levels of CFTR (CF transmembrane conductance regulator), may contribute to respiratory dysfunction. We performed morphological analyses of the trachea and airway functional studies in adult Cftr knockout ( Cftr ^−/−) and F508del-CFTR mice and their controls. Macroscopic and histological examination of the trachea showed the presence of one to seven disrupted or incomplete cartilage rings in Cftr ^−/− mice (23/25) while only a few Cftr^+/+ mice (6/25) had one abnormal ring. Tracheal defects were mainly localized in the proximal trachea. In 14 Cftr ^−/− mice, frontal disruption of the first three to six rings below the cricoid cartilage were associated with upper tracheal constriction. Similar tracheal abnormalities were detected in adult F508del-CFTR and in newborn Cftr^−/− and F508del-CFTR mice. Tracheal and ventilatory function analyses showed in Cftr ^−/− mice a decreased contractile response of the proximal trachea and a reduced breathing rate due to an increase in the inspiratory and expiratory times. In F508del-CFTR mice, the expiratory time was longer than in controls. Therefore, these structural and functional abnormalities detected in adult and newborn CF mouse models may represent congenital malformations related to CFTR dysfunction. These results raise important questions concerning the mechanisms governing tracheal development within the context of CFTR protein dysfunction and the implication of such abnormalities in the pathogenesis of airway disease in CF.     

Transthyretin accelerates vascular Abeta deposition in a mouse model of Alzheimer's disease

Transthyretin (TTR) binds amyloid-β (Aβ) and prevents Aβ fibril formation in vitro . It was reported that the lack of neurodegeneration in a transgenic mouse model of Alzheimer's disease (AD) (Tg2576 mouse) was associated with increased TTR level in the hippocampus, and that chronic infusion of anti-TTR antibody into the hippocampus of Tg2576 mice led to increased local Aβ deposits, tau hyperphosphorylation and apoptosis. TTR is, therefore, speculated to prevent Aβ pathology in AD. However, a role for TTR in Aβ deposition is not yet known. To investigate the relationship between TTR and Aβ deposition, we generated a mouse line carrying a null mutation at the endogenous TTR locus and the human mutant amyloid precursor protein cDNA responsible for familial AD (Tg2576 /TTR ^−/− mouse) by crossing Tg2576 mice with TTR-deficient mice. We asked whether Aβ deposition was accelerated in Tg2576/ TTR ^−/− mice relative to the heterozygous mutant Tg2576 (Tg2576/ TTR ^+/−) mice. Contrary to our expectations, the degree of total and vascular Aβ burdens in the aged Tg2576/ TTR ^−/− mice was significantly reduced relative to the age-matched Tg2576/ TTR ^+/− mice. Our experiments present, for the first time, compelling evidence that TTR does not suppress but rather accelerates vascular Aβ deposition in the mouse model of AD.     

Induction of Gut Mucosal Immune Responses: Importance of Genetic Background and Th1/Th2 Cross-Regulation

The reciprocal regulation of T-helper cell (Th) subsets is widely documented in various animal models of infectious diseases. In this study IFN-γ/IL-4 double knockout (DKO) mice were used to analyse the role of Th subsets in mucosal immune responses. We found that the DKO mice had normal IgA differentiation but impaired induction of specific gut mucosal antibody responses after oral immunization using cholera toxin adjuvant. Both Th1 and Th2 responses were reduced compared with wild-type mice. Despite the absence of both IFN-γ and IL-4 in the DKO mice the overall results were similar to previous observations in IFN-γ receptor-knockout (IFN-γR^−/−) mice and did not suggest a strict cross-regulation of the two Th subsets in the gut mucosa. To further examine the role of IFN-γ in mucosal immunity we compared two different mouse strains lacking IFN-γ, i.e. IFN-γ^−/− (C57BL/6) and IFN-γR^−/− mice (129/Sv). We found that IFN-γR^−/− mice exhibited reduced mucosal antibody responses and decreased Th1 and Th2 activity after oral immunization, while IFN-γ^−/− mice had intact antibody responses and increased Th2 responses. Thus, genetic differences were found to critically affect the development of a specific gut mucosal immune response. An enhanced Th2 activity in the Peyer's patches following oral immunization was associated with an ability to mount strong intestinal IgA immunity.     

Possible mechanisms of the concentration-dependent action of substance P to induce histamine release from rat peritoneal mast cells and the effect of extracellular calcium on mast-cell activation

Rat peritoneal mast cells purified on a Percoll gradient were loaded with the fluorescent Ca²⁺ indicator fura-2 and were challenged with different concentrations of substance P (SP), and intracellular calcium concentrations ([Ca²⁺]_i) were measured by a spectrofluorometric assay. SP at 5 × 10⁻⁶ mol/1 and 10⁻⁵ mol/1 caused a significant histamine release with a significant increase in [Ca²⁺]_i in a dose-dependent manner. However, SP at 10⁻⁸-10⁻⁶ mol/1 did not induce either histamine release or increase in [Ca²⁺]_i. Extracellular calcium at 0.9 mM inhibited the histamine release with a significant reduction of [Ca²⁺]ⁱ compared with that of the cells in a nominally calcium-free condition. These results indicate that the action of SP on rat mast cells relies upon [Ca²⁺]_i to induce histamine release.