Interferon-α antagonizes IL-3-mediated immunoregulatory functions of human basophils

Human basophils are major inflammatory cells in maintaining chronic allergic asthma. It has been published that interferon-α (IFN-α) improves clinical symptoms of asthma patients. In contrast, IL-3 exacerbates airway inflammation by inducing IL-4, IL-8 and IL-13 secretion from human basophils thus regulating their immunoregulatory functions. Furthermore, IL-3 exceptionally promotes survival of basophils. Here, we assessed cellular response of human basophils treated with IFN-α alone or in combination with IL-3. Our data show that IFN-α enhances apoptosis in purified human blood basophils compared to spontaneous apoptosis of controls or IFN-γ treated cells. Furthermore, we demonstrate that both IFN-α and FasL enhance apoptosis in human basophils with similar efficiency in a rather additive than synergistic way. IFN-α inhibits IL-3-induced survival to a minor degree. Particularly however, it suppresses IL-3-induced de-novo production of IL-8 and IL-13 up to 80%. In contrast, the production of IL-4 is not affected. Analyses of signaling pathways reveal that IFN-α promotes prolonged phosphorylation of STAT1/STAT2. By using a pan-JAK inhibitor the phosphorylation of STAT1/STAT2 is inhibited and most importantly the pro-apoptotic effect of IFN-α is abolished. Although the phosphorylation of p38-MAPK in IFN-α-treated cells is comparable to non-treated cells, inhibition of p-p38 activity abrogates IFN-α-enhanced apoptosis as well. We conclude that IFN-α-enhanced apoptosis is tightly regulated by the cooperation of JAK/STAT and p38-MAPK pathways. Our study identifies IFN-α as a novel inhibitor of IL-3-induced IL-8 and IL-13 production of human basophils. Taken together our study may explain the improved clinical symptoms of asthma patients treated with IFN-α.

A mouse model of HIES reveals pro- and anti-inflammatory functions of STAT3.

Mutations of STAT3 underlie the autosomal dominant form of hyperimmunoglobulin E syndrome (HIES). STAT3 has critical roles in immune cells and thus, hematopoietic stem cell transplantation (HSCT), might be a reasonable therapeutic strategy in this disease. However, STAT3 also has critical functions in nonhematopoietic cells and dissecting the protean roles of STAT3 is limited by the lethality associated with germline deletion of Stat3. Thus, predicting the efficacy of HSCT for HIES is difficult. To begin to dissect the importance of STAT3 in hematopoietic and nonhematopoietic cells as it relates to HIES, we generated a mouse model of this disease. We found that these transgenic mice recapitulate multiple aspects of HIES, including elevated serum IgE and failure to generate Th17 cells. We found that these mice were susceptible to bacterial infection that was partially corrected by HSCT using wild-type bone marrow, emphasizing the role played by the epithelium in the pathophysiology of HIES.

Multiple functions of gingival and mucoperiosteal fibroblasts in oral wound healing and repair

Fibroblasts are cells of mesenchymal origin. They are responsible for the production of most extracellular matrix in connective tissues and are essential for wound healing and repair. In recent years, it has become clear that fibroblasts from different tissues have various distinct traits. Moreover, wounds in the oral cavity heal under very special environmental conditions compared with skin wounds. Here, we reviewed the current literature on the various interconnected functions of gingival and mucoperiosteal fibroblasts during the repair of oral wounds. The MEDLINE database was searched with the following terms: (gingival OR mucoperiosteal) AND fibroblast AND (wound healing OR repair). The data gathered were used to compare oral fibroblasts with fibroblasts from other tissues in terms of their regulation and function during wound healing. Specifically, we sought answers to the following questions: (i) what is the role of oral fibroblasts in the inflammatory response in acute wounds; (ii) how do growth factors control the function of oral fibroblasts during wound healing; (iii) how do oral fibroblasts produce, remodel and interact with extracellular matrix in healing wounds; (iv) how do oral fibroblasts respond to mechanical stress; and (v) how does aging affect the fetal-like responses and functions of oral fibroblasts? The current state of research indicates that oral fibroblasts possess unique characteristics and tightly controlled specific functions in wound healing and repair. This information is essential for developing new strategies to control the intraoral wound-healing processes of the individual patient.

Modeling immune functions of the mouse blood-cerebrospinal fluid barrier in vitro: primary rather than immortalized mouse choroid plexus epithelial cells are suited to study immune cell migration across this brain barrier.

BACKGROUND The blood-cerebrospinal fluid barrier (BCSFB) established by the choroid plexus (CP) epithelium has been recognized as a potential entry site of immune cells into the central nervous system during immunosurveillance and neuroinflammation. The location of the choroid plexus impedes in vivo analysis of immune cell trafficking across the BCSFB. Thus, research on cellular and molecular mechanisms of immune cell migration across the BCSFB is largely limited to in vitro models. In addition to forming contact-inhibited epithelial monolayers that express adhesion molecules, the optimal in vitro model must establish a tight permeability barrier as this influences immune cell diapedesis. METHODS We compared cell line models of the mouse BCSFB derived from the Immortomouse(®) and the ECPC4 line to primary mouse choroid plexus epithelial cell (pmCPEC) cultures for their ability to establish differentiated and tight in vitro models of the BCSFB. RESULTS We found that inducible cell line models established from the Immortomouse(®) or the ECPC4 tumor cell line did not express characteristic epithelial proteins such as cytokeratin and E-cadherin and failed to reproducibly establish contact-inhibited epithelial monolayers that formed a tight permeability barrier. In contrast, cultures of highly-purified pmCPECs expressed cytokeratin and displayed mature BCSFB characteristic junctional complexes as visualized by the junctional localization of E-cadherin, β-catenin and claudins-1, -2, -3 and -11. pmCPECs formed a tight barrier with low permeability and high electrical resistance. When grown in inverted filter cultures, pmCPECs were suitable to study T cell migration from the basolateral to the apical side of the BCSFB, thus correctly modelling in vivo migration of immune cells from the blood to the CSF. CONCLUSIONS Our study excludes inducible and tumor cell line mouse models as suitable to study immune functions of the BCSFB in vitro. Rather, we introduce here an in vitro inverted filter model of the primary mouse BCSFB suited to study the cellular and molecular mechanisms mediating immune cell migration across the BCSFB during immunosurveillance and neuroinflammation.

Increased spread and replication efficiency of Listeria monocytogenes in organotypic brain-slices is related to multilocus variable number of tandem repeat analysis (MLVA) complex.

BACKGROUND Listeria (L.) monocytogenes causes fatal infections in many species including ruminants and humans. In ruminants, rhombencephalitis is the most prevalent form of listeriosis. Using multilocus variable number tandem repeat analysis (MLVA) we recently showed that L. monocytogenes isolates from ruminant rhombencephalitis cases are distributed over three genetic complexes (designated A, B and C). However, the majority of rhombencephalitis strains and virtually all those isolated from cattle cluster in MLVA complex A, indicating that strains of this complex may have increased neurotropism and neurovirulence. The aim of this study was to investigate whether ruminant rhombencephalitis strains have an increased ability to propagate in the bovine hippocampal brain-slice model and can be discriminated from strains of other sources. For this study, forty-seven strains were selected and assayed on brain-slice cultures, a bovine macrophage cell line (BoMac) and a human colorectal adenocarcinoma cell line (Caco-2). They were isolated from ruminant rhombencephalitis cases (n = 21) and other sources including the environment, food, human neurolisteriosis cases and ruminant/human non-encephalitic infection cases (n = 26). RESULTS All but one L. monocytogenes strain replicated in brain slices, irrespectively of the source of the isolate or MLVA complex. The replication of strains from MLVA complex A was increased in hippocampal brain-slice cultures compared to complex C. Immunofluorescence revealed that microglia are the main target cells for L. monocytogenes and that strains from MLVA complex A caused larger infection foci than strains from MLVA complex C. Additionally, they caused larger plaques in BoMac cells, but not CaCo-2 cells. CONCLUSIONS Our brain slice model data shows that all L. monocytogenes strains should be considered potentially neurovirulent. Secondly, encephalitis strains cannot be conclusively discriminated from non-encephalitis strains with the bovine organotypic brain slice model. The data indicates that MLVA complex A strains are particularly adept at establishing encephalitis possibly by virtue of their higher resistance to antibacterial defense mechanisms in microglia cells, the main target of L. monocytogenes.

Everolimus is a potent inhibitor of activated hepatic stellate cell functions in vitro and in vivo , while demonstrating anti-angiogenic activities

Progression of liver fibrosis to HCC (hepatocellular carcinoma) is a very complex process which involves several pathological phenomena, including hepatic stellate cell activation, inflammation, fibrosis and angiogenesis. Therefore inhibiting multiple pathological processes using a single drug can be an effective choice to curb the progression of HCC. In the present study, we used the mTOR inhibitor everolimus to observe its effect on the in vitro activation of hepatic stellate cells and angiogenesis. The results of the present study demonstrated that everolimus treatment blocked the functions of the immortalized human activated hepatic stellate cell line LX-2 without affecting the viability and migration of primary human stellate cells. We also observed that treatment with everolimus (20 nM) inhibited collagen production by activated stellate cells, as well as cell contraction. Everolimus treatment was also able to attenuate the activation of primary stellate cells to their activated form. Angiogenesis studies showed that everolimus blocked angiogenesis in a rat aortic ring assay and inhibited the tube formation and migration of liver sinusoidal endothelial cells. Finally, everolimus treatment reduced the load of tumoral myofibroblasts in a rat model of HCC. These data suggest that everolimus targets multiple mechanisms, making it a potent blocker of the progression of HCC from liver fibrosis.

Fas death receptor signalling: roles of Bid and XIAP

Fas (also called CD95 or APO-1), a member of a subgroup of the tumour necrosis factor receptor superfamily that contain an intracellular death domain, can initiate apoptosis signalling and has a critical role in the regulation of the immune system. Fas-induced apoptosis requires recruitment and activation of the initiator caspase, caspase-8 (in humans also caspase-10), within the death-inducing signalling complex. In so-called type 1 cells, proteolytic activation of effector caspases (-3 and -7) by caspase-8 suffices for efficient apoptosis induction. In so-called type 2 cells, however, killing requires amplification of the caspase cascade. This can be achieved through caspase-8-mediated proteolytic activation of the pro-apoptotic Bcl-2 homology domain (BH)3-only protein BH3-interacting domain death agonist (Bid), which then causes mitochondrial outer membrane permeabilisation. This in turn leads to mitochondrial release of apoptogenic proteins, such as cytochrome c and, pertinent for Fas death receptor (DR)-induced apoptosis, Smac/DIABLO (second mitochondria-derived activator of caspase/direct IAP binding protein with low Pi), an antagonist of X-linked inhibitor of apoptosis (XIAP), which imposes a brake on effector caspases. In this review, written in honour of Juerg Tschopp who contributed so much to research on cell death and immunology, we discuss the functions of Bid and XIAP in the control of Fas DR-induced apoptosis signalling, and we speculate on how this knowledge could be exploited to develop novel regimes for treatment of cancer.

Fic domain-catalyzed adenylylation: insight provided by the structural analysis of the type IV secretion system effector BepA

Numerous bacterial pathogens subvert cellular functions of eukaryotic host cells by the injection of effector proteins via dedicated secretion systems. The type IV secretion system (T4SS) effector protein BepA from Bartonella henselae is composed of an N-terminal Fic domain and a C-terminal Bartonella intracellular delivery domain, the latter being responsible for T4SS-mediated translocation into host cells. A proteolysis resistant fragment (residues 10-302) that includes the Fic domain shows autoadenylylation activity and adenylyl transfer onto Hela cell extract proteins as demonstrated by autoradiography on incubation with α-[(32)P]-ATP. Its crystal structure, determined to 2.9-Å resolution by the SeMet-SAD method, exhibits the canonical Fic fold including the HPFxxGNGRxxR signature motif with several elaborations in loop regions and an additional β-rich domain at the C-terminus. On crystal soaking with ATP/Mg(2+), additional electron density indicated the presence of a PP(i) /Mg(2+) moiety, the side product of the adenylylation reaction, in the anion binding nest of the signature motif. On the basis of this information and that of the recent structure of IbpA(Fic2) in complex with the eukaryotic target protein Cdc42, we present a detailed model for the ternary complex of Fic with the two substrates, ATP/Mg(2+) and target tyrosine. The model is consistent with an in-line nucleophilic attack of the deprotonated side-chain hydroxyl group onto the α-phosphorus of the nucleotide to accomplish AMP transfer. Furthermore, a general, sequence-independent mechanism of target positioning through antiparallel β-strand interactions between enzyme and target is suggested.

Interhemispheric connections shape subjective experience of bistable motion

The right and left visual hemifields are represented in different cerebral hemispheres and are bound together by connections through the corpus callosum. Much has been learned on the functions of these connections from split-brain patients [1-4], but little is known about their contribution to conscious visual perception in healthy humans. We used diffusion tensor imaging and functional magnetic resonance imaging to investigate which callosal connections contribute to the subjective experience of a visual motion stimulus that requires interhemispheric integration. The "motion quartet" is an ambiguous version of apparent motion that leads to perceptions of either horizontal or vertical motion [5]. Interestingly, observers are more likely to perceive vertical than horizontal motion when the stimulus is presented centrally in the visual field [6]. This asymmetry has been attributed to the fact that, with central fixation, perception of horizontal motion requires integration across hemispheres whereas perception of vertical motion requires only intrahemispheric processing [7]. We are able to show that the microstructure of individually tracked callosal segments connecting motion-sensitive areas of the human MT/V5 complex (hMT/V5+; [8]) can predict the conscious perception of observers. Neither connections between primary visual cortex (V1) nor other surrounding callosal regions exhibit a similar relationship.

Effect of the Addition of a Fused Donor-Acceptor Ligand on a Ru(II) Complex: Synthesis, Characterization, and Photoinduced Electron Transfer Reactions of Ru(TTF-dppz)(2)(Aqphen) (2+)

The synthesis and the photophysical properties of the complex [Ru(TTF-dppz)(2)(Aqphen)](2+) (TTF = tetrathiafulvalene, dppz = dipyrido-[3,2-a:2',3'-c]phenazine, Aqphen = anthraquinone fused to phenanthroline via a pyrazine bridge) are described. In this molecular triad excitation into the metal ligand charge transfer bands results in the creation of a long-lived charge separated state with TTF acting as electron donor and anthraquinone as terminal acceptor. The lifetime of the charge-separated state is 400 ns in dichloromethane at room temperature. A mechanism for the charge separation involving an intermediate charge-separated state is proposed based on transient absorption spectroscopy.

Metformin inhibits human androgen production by regulating steroidogenic enzymes HSD3B2 and CYP17A1 and complex I activity of the respiratory chain

Metformin is treatment of choice for the metabolic consequences seen in polycystic ovary syndrome for its insulin-sensitizing and androgen-lowering properties. Yet, the mechanism of action remains unclear. Two potential targets for metformin regulating steroid and glucose metabolism are AMP-activated protein kinase (AMPK) signaling and the complex I of the mitochondrial respiratory chain. Androgen biosynthesis requires steroid enzymes 17α-Hydroxylase/17,20 lyase (CYP17A1) and 3β-hydroxysteroid dehydrogenase type 2 (HSD3B2), which are overexpressed in ovarian cells of polycystic ovary syndrome women. Therefore, we aimed to understand how metformin modulates androgen production using NCI-H295R cells as an established model of steroidogenesis. Similar to in vivo situation, metformin inhibited androgen production in NCI cells by decreasing HSD3B2 expression and CYP17A1 and HSD3B2 activities. The effect of metformin on androgen production was dose dependent and subject to the presence of organic cation transporters, establishing an important role of organic cation transporters for metformin's action. Metformin did not affect AMPK, ERK1/2, or atypical protein kinase C signaling. By contrast, metformin inhibited complex I of the respiratory chain in mitochondria. Similar to metformin, direct inhibition of complex I by rotenone also inhibited HSD3B2 activity. In conclusion, metformin inhibits androgen production by mechanisms targeting HSD3B2 and CYP17-lyase. This regulation involves inhibition of mitochondrial complex I but appears to be independent of AMPK signaling.