Missing C-terminal filaggrin expression, NFkappaB activation and hyperproliferation identify the dog as a putative model to study epidermal dysfunction in atopic dermatitis

Filaggrin loss-of-function mutations resulting in C-terminal protein truncations are strong predisposing factors in human atopic dermatitis (AD). To assess the possibility of similar truncations in canine AD, an exclusion strategy was designed on 16 control and 18 AD dogs of various breeds. Comparative immunofluorescence microscopy was performed with an antibody raised against the canine filaggrin C-terminus and a commercial N-terminal antibody. Concurrent with human AD-like features such as generalized NFKB activation and hyperproliferation, four distinctive filaggrin expression patterns were identified in non-lesional skin. It was found that 10/18 AD dogs exhibited an identical pattern for both antibodies with comparable (category I, 3/18) or reduced (category II, 7/18) expression to that of controls. In contrast, 4/18 dogs displayed aberrant large vesicles revealed by the C-terminal but not the N-terminal antibody (category III), while 4/18 showed a control-like N-terminal expression but lacked the C-terminal protein (category IV). The missing C-terminal filaggrin in category IV strongly points towards loss-of function mutations in 4/18 (22%) of all AD dogs analysed.

Glutathione peroxidase overexpression causes aberrant ERK activation in neonatal mouse cortex after hypoxic preconditioning

Preconditioning of neonatal mice with nonlethal hypoxia (HPC) protects the brain from hypoxic-ischemic (HI) injury. Overexpression of human glutathione peroxidase 1 (GPx1), which normally protects the developing murine brain from HI injury, reverses HPC protection, suggesting that a certain threshold of hydrogen peroxide concentration is required for activation of HPC signaling.

Disparate activation of the coronary sinus and inferior left atrium during atrial tachycardia after persistent atrial fibrillation ablation: prevalence, pitfalls, and impact on mapping

Persistent atrial fibrillation (AF) ablation may lead to partial disconnection of the coronary sinus (CS). As a result, disparate activation sequences of the local CS versus contiguous left atrium (LA) may be observed during atrial tachycardia (AT). We aimed to evaluate the prevalence of this phenomenon and its impact on activation mapping.

Topical application of 17β-estradiol (E2) improves skin flap survival through activation of endothelial nitric oxide synthase in rats

This study investigates the influence of 17β-estradiol (E2) on nitric oxide (NO) production in endothelial cell cultures and the effect of topical E2 on the survival of skin flap transplants in a rat model. Human umbilical vein endothelial cells were treated with three different E2 concentrations and nitrite (NO2) concentrations, as well as endothelial nitric oxide synthase (eNOS) protein expressions were analyzed. In vivo, random-pattern skin flaps were raised in female Wistar rats 14 days following ovariectomy and treated with placebo ointment (group 1), E2 as gel (group 2), and E2 via plaster (group 3). Flap perfusion, survival, and NO2 levels were measured on postoperative day 7. In vitro, E2 treatment increased NO2 concentration in cell supernatant and eNOS expression in cell lysates (p < 0.05). In vivo, E2 treated (gel and plaster groups) demonstrated significantly increased skin flap survival compared to the placebo group (p < 0.05). E2 plaster-treated animals exhibited higher NO2 blood levels than placebo (p < 0.05) paralleling the in vitro observations. E2 increases NO production in endothelial cells via eNOS activation. Topical E2 application can significantly increase survival of ischemically challenged skin flaps in a rat model and may augment wound healing in other ischemic situations via activation of NO production.

Theileria-induced constitutive IKK activation is independent of functional Hsp90

The intracellular parasite Theileria induces uncontrolled proliferation and host cell transformation. Parasite-induced transformation is accompanied by constitutive activation of IkappaB kinase (IKK), resulting in permanently high levels of activated nuclear factor (NF)-kappaB. IKK activation pathways normally require heat shock protein 90 (Hsp90), a chaperone that regulates the stability and activity of signalling molecules and can be blocked by the benzoquinone ansamycin compound geldanamycin (GA). In Theileria-transformed cells, IkappaBalpha and p65 phosphorylation, NF-kappaB nuclear translocation and DNA binding activity are largely resistant to GA and also NF-kappaB-dependent reporter gene expression is only partly affected. Our findings indicate that parasite-induced IKK activity does not require functional Hsp90.

Mouse lung CYP1A1 catalyzes the metabolic activation of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)

PhIP carcinogenesis is initiated by N(2)-hydroxylation, mediated by several cytochromes P450, including CYP1A1. However, the role of CYP1A1 in PhIP metabolic activation in vivo is unclear. In this study, Cyp1a1-null and wild-type (WT) mice were used to investigate the potential role of CYP1A1 in PhIP metabolic activation in vivo. PhIP N(2)-hydroxylation was actively catalyzed by lung homogenates of WT mice, at a rate of 14.9 +/- 5.0 pmol/min/g tissue, but < 1 pmol/min/g tissue in stomach and small intestine, and almost undetectable in mammary gland and colon. PhIP N(2)-hydroxylation catalyzed by lung homogenates of Cyp1a1-null mice was approximately 10-fold lower than that of WT mice. In contrast, PhIP N(2)-hydroxylation activity in lung homogenates of Cyp1a2-null versus WT mice was not decreased. Pretreatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) increased lung Cyp1a1 mRNA and lung homogenate PhIP N(2)-hydroxylase activity approximately 50-fold in WT mice, where the activity was substantially inhibited (70%) by monoclonal antibodies against CYP1A1. In vivo, 30 min after oral treatment with PhIP, PhIP levels in lung were similar to those in liver. After a single dose of 0.1 mg/kg [(14)C]PhIP, lung PhIP-DNA adduct levels in Cyp1a1-null mice, but not in Cyp1a2-null mice, were significantly lower (P=0.0028) than in WT mice. These results reveal that mouse lung has basal and inducible PhIP N(2)-hydroxylase activity predominantly catalyzed by CYP1A1. Because of the high inducibility of human CYP1A1, especially in cigarette smokers, the role of lung CYP1A1 in PhIP carcinogenesis should be considered.

Function of liver activation-regulated chemokine/CC chemokine ligand 20 is differently affected by cathepsin B and cathepsin D processing

Chemokine processing by proteases is emerging as an important regulatory mechanism of leukocyte functions and possibly also of cancer progression. We screened a large panel of chemokines for degradation by cathepsins B and D, two proteases involved in tumor progression. Among the few substrates processed by both proteases, we focused on CCL20, the unique chemokine ligand of CCR6 that is expressed on immature dendritic cells and subtypes of memory lymphocytes. Analysis of the cleavage sites demonstrate that cathepsin B specifically cleaves off four C-terminally located amino acids and generates a CCL20(1-66) isoform with full functional activity. By contrast, cathepsin D totally inactivates the chemotactic potency of CCL20 by generating CCL20(1-55), CCL20(1-52), and a 12-aa C-terminal peptide CCL20(59-70). Proteolytic cleavage of CCL20 occurs also with chemokine bound to glycosaminoglycans. In addition, we characterized human melanoma cells as a novel CCL20 source and as cathepsin producers. CCL20 production was up-regulated by IL-1alpha and TNF-alpha in all cell lines tested, and in human metastatic melanoma cells. Whereas cathepsin D is secreted in the extracellular milieu, cathepsin B activity is confined to cytosol and cellular membranes. Our studies suggest that CCL20 processing in the extracellular environment of melanoma cells is exclusively mediated by cathepsin D. Thus, we propose a model where cathepsin D inactivates CCL20 and possibly prevents the establishment of an effective antitumoral immune response in melanomas.

Intraperitoneal Echinococcus multilocularis infection in C57BL/6 mice affects CD40 and B7 costimulator expression on peritoneal macrophages and impairs peritoneal T cell activation

One of the most important immunopathological consequence of intraperitoneal alveolar echinococcosis (AE) in the mouse is suppression of T cell-mediated immune responses. We investigated whether and how intraperitoneal macrophages (MØs) are, respectively, implicated as antigen-presenting cells (APCs). In a first step we showed that peritoneal MØs from infected mice (AE-MØs) exhibited a reduced ability to present a conventional antigen (chicken ovalbumin, C-Ova) to specific responder lymph node T cells. In a subsequent step, AE-MØs as well as naïve MØs (positive control) proved their ability to uptake and process C-Ova fluorescein isthiocyanate (FITC). Furthermore, in comparison with naïve MØs, the surface expression of Ia molecules was up-regulated on AE-MØs at the early stage of infection, suggesting that AE-MØs provide the first signal via the antigen-Ia complex. To study the accessory activity of MØs, AE-MØs obtained at the early and late stages of infection were found to decrease Con A-induced proliferation of peritoneal naïve T cells as well as of AE-sensitized peritoneal T cells, in contrast to stimulation with naïve MØs. The status of accessory molecules was assessed by analysing the expression level of costimulatory molecules on AE-MØs, with naïve MØs as controls. It was found that B7-1 (CD80) and B7-2 (CD86) expression remained unchanged, whereas CD40 was down-regulated and CD54 (= ICAM-1) was slightly up-regulated. In a leucocyte reaction of AE-MØs with naïve or AE-T cells, both types of T cells increased their proliferative response when CD28 - the ligand of B7 receptors - was exposed to anti-CD28 in cultures. Conversely to naïve MØs, pulsing of AE-MØs with agonistic anti-CD40 did not even partially restore their costimulatory activity and failed to increase naïve or AE-T cell proliferation. Neutralizing anti-B7-1, in combination with anti-B7-2, reduced naïve and AE-T cell proliferation, whereas anti-CD40 treatment of naïve MØs increased their proliferative response to Con A. These results point at the key role of B7 receptors as accessory molecules and the necessity of the integrity of CD40-expression by naïve MØs to improve their accessory activity. Taken together, the obstructed presenting-activity of AE-MØs appeared to trigger an unresponsiveness of T cells, contributing to the suppression of their clonal expansion during the chronic phase of AE-infection.

Human monocytoid cells as a model to study Toll-like receptor-mediated activation

THP-1 2A9, a subclone of the monocytoid cell line THP-1 and known to be exquisitely sensitive to LPS, was tested for TNF production following triggering by excess doses of TLR ligands. TLR2, TLR4 and TLR5 agonists, but neither TLR3 nor TLR9 agonists, induced TNF production. When used at lower concentrations, priming by calcitriol strongly influenced the sensitivity of cells to LPS and different TLR2 triggers (lipoteichoic acid (LTA), trispalmitoyl-cysteyl-seryl-lysyl-lysyl-lysyl-lysine (Pam3Cys) and peptidoglycan (PGN)). Priming by calcitriol failed to modulate TLR2 and TLR4 mRNA and cell surface expression of these receptors. TNF signals elicited by TLR2 agonists were blocked by the TLR-specific antibody 2392. CD14-specific antibodies showed variable effects. CD14-specific antibodies inhibited TNF induction by LTA. High concentrations partially inhibited TNF induction by Pam3Cys. The same antibodies failed to inhibit TNF induction by PGN. Thus, THP-1 2A9 cells respond by TNF production to some, but not all TLR agonists, and the wide variety of putative TLR2 agonists interact to variable degrees also with other cell-surface-expressed binding sites such as CD14. THP-1 2A9 cells might provide a model by which to investigate in more detail the interaction of pathogen-associated molecular patterns and monocytoid cell-surface-expressed pattern recognition receptors.

TRAIL receptor-mediated JNK activation and Bim phosphorylation critically regulate Fas-mediated liver damage and lethality

TNF-related apoptosis-inducing ligand (TRAIL) is a member of the TNF family with potent apoptosis-inducing properties in tumor cells. In particular, TRAIL strongly synergizes with conventional chemotherapeutic drugs to induce tumor cell death. Thus, TRAIL has been proposed as a promising future cancer therapy. Little, however, is known regarding what the role of TRAIL is in normal untransformed cells and whether therapeutic administration of TRAIL, alone or in combination with other apoptotic triggers, may cause tissue damage. In this study, we investigated the role of TRAIL in Fas-induced (CD95/Apo-1-induced) hepatocyte apoptosis and liver damage. While TRAIL alone failed to induce apoptosis in isolated murine hepatocytes, it strongly amplified Fas-induced cell death. Importantly, endogenous TRAIL was found to critically regulate anti-Fas antibody-induced hepatocyte apoptosis, liver damage, and associated lethality in vivo. TRAIL enhanced anti-Fas-induced hepatocyte apoptosis through the activation of JNK and its downstream substrate, the proapoptotic Bcl-2 homolog Bim. Consistently, TRAIL- and Bim-deficient mice and wild-type mice treated with a JNK inhibitor were protected against anti-Fas-induced liver damage. We conclude that TRAIL and Bim are important response modifiers of hepatocyte apoptosis and identify liver damage and lethality as a possible risk of TRAIL-based tumor therapy.

Effect of monoterpenes on the formation and activation of osteoclasts in vitro

Monoterpenes, present in aromatic plants, are known to inhibit bone resorption in vivo. In this in vitro study, they inhibited the activation of osteoclasts only at high concentrations but inhibited the formation at much lower concentrations. Therefore, monoterpenes may act in vivo directly on osteoclastogenesis. INTRODUCTION: Monoterpenes are the major components of essential oils, which are formed in many plants. Typically, they are found in herbs and certain fruits. When fed to rats, they inhibit bone resorption by an unknown mechanism. In this study, their effect on the activity and formation of osteoclasts in vitro was studied. MATERIALS AND METHODS: The effect of monoterpenes on the development of osteoclasts was studied in co-cultures of bone marrow cells and osteoblasts and in cultures of spleen cells grown with colony stimulating factor (CSF)-1 and RANKL. In cultures of primary osteoblasts, alkaline phosphatase activity and levels of mRNA encoding RANKL and osteoprotegerin (OPG) mRNA (RT-PCR), and in osteoblast and spleen cell cultures, lactate dehydrogenase activity, a measure of toxicity, were determined. The activity of isolated rat osteoclasts was determined by counting the osteoclasts with actin rings using histofluorometry. RESULTS: The monoterpenes inhibited the formation of osteoclasts more strongly in co-cultures (> or = 1 microM) than in cultures of spleen cells (> or = 10 microM). They had a minor effect on osteoblasts. Toxic effects were not observed. The inhibition of the formation of osteoclasts was not reversed by the addition of farnesol and geranylgeraniol, excluding an effect of the monoterpenes through the mevalonate pathway. A high concentration of 1 mM was required to inhibit the activation of osteoclasts. This effect, shown for menthol and borneol, was reversible. CONCLUSIONS: The results suggest that the monoterpenes inhibit bone resorption in vivo through a direct effect on the formation of osteoclasts acting mainly on the hemopoietic cells.

Antagonistic and synergistic effects of glucocorticoids and IL-7 on CD4+ T cell activation

Glucocorticoids (GCs) are steroidal compounds widely used to treat chronic and acute inflammatory diseases. In particular, GCs at pharmacological doses induce apoptosis of activated and naïve T cells, inhibit their proliferation and block pro-inflammatory cytokine secretion. At physiological concentrations, the effect of these steroids on T cell immunity are not yet fully understood, and various studies reported paradoxical roles exerted by GCs on T cell immunity. Here, we show that GCs surprisingly induce proliferation of activated CD4(+) T cells in the presence of IL-7, a cytokine secreted in the thymus and at mucosal sites. Increased proliferation is dependent on a GC-mediated survival of mitotic cells. Moreover, we observe a downmodulation of Th1 cytokine secretion in cells treated with GCs, an outcome which is not affected by the presence of IL-7. GCs exert thus a positive role in the presence of IL-7 by enhancing proliferation of CD4(+) T cells and simultaneously a negative role by suppressing pro-inflammatory cytokine production.

Chronic excessive erythrocytosis induces endothelial activation and damage in mouse brain

Excessive erythrocytosis results in severely increased blood viscosity, which may have significant detrimental effects on endothelial cells and, ultimately, function of the vascular endothelium. Because blood-brain barrier stability is crucial for normal physiological function, we used our previously characterized erythropoietin-overexpressing transgenic (tg6) mouse line (which has a hematocrit of 0.8-0.9) to investigate the effect of excessive erythrocytosis on vessel number, structure, and integrity in vivo. These mice have abnormally high levels of nitric oxide (NO), a potent proinflammatory molecule, suggesting altered vascular permeability and function. In this study, we observed that brain vessel density of tg6 mice was significantly reduced (16%) and vessel diameter was significantly increased (15%) compared with wild-type mice. Although no significant increases in vascular permeability under normoxic or acute hypoxic conditions (8% O2 for 4 h) were detected, electron-microscopic analysis revealed altered morphological characteristics of the tg6 endothelium. Tg6 brain vascular endothelial cells appeared to be activated, with increased luminal protrusions reminiscent of ongoing inflammatory processes. Consistent with this observation, we detected increased levels of intercellular adhesion molecule-1 and von Willebrand factor, markers of endothelial activation and damage, in brain tissue. We propose that chronic excessive erythrocytosis and sustained high hematocrit cause endothelial damage, which may, ultimately, increase susceptibility to vascular disease.

Motion standstill leads to activation of inferior parietal lobe

Previous studies on motion perception revealed motion-processing brain areas sensitive to changes in luminance and texture (low-level) and changes in salience (high-level). The present functional magnetic resonance imaging (fMRI) study focused on motion standstill. This phenomenon, occurring at fast presentation frequencies of visual moving objects that are perceived as static, has not been previously explored by neuroimaging techniques. Thirteen subjects were investigated while perceiving apparent motion at 4 Hz, at 30 Hz (motion standstill), isoluminant static and flickering stimuli, fixation cross, and blank screen, presented randomly and balanced for rapid event-related fMRI design. Blood oxygenation level-dependent (BOLD) signal in the occipito-temporal brain region MT/V5 increased during apparent motion perception. Here we could demonstrate that brain areas like the posterior part of the right inferior parietal lobule (IPL) demonstrated higher BOLD-signal during motion standstill. These findings suggest that the activation of higher-order motion areas is elicited by apparent motion at high presentation rates (motion standstill). We interpret this observation as a manifestation of an orienting reaction in IPL towards stimulus motion that might be detected but not resolved by other motion-processing areas (i.e., MT/V5).