Granzyme B, a novel mediator of allergic inflammation: its induction and release in blood basophils and human asthma

Histamine, leukotriene C4, IL-4, and IL-13 are major mediators of allergy and asthma. They are all formed by basophils and are released in particularly large quantities after stimulation with IL-3. Here we show that supernatants of activated mast cells or IL-3 qualitatively change the makeup of granules of human basophils by inducing de novo synthesis of granzyme B (GzmB), without induction of other granule proteins expressed by cytotoxic lymphocytes (granzyme A, perforin). This bioactivity of IL-3 is not shared by other cytokines known to regulate the function of basophils or lymphocytes. The IL-3 effect is restricted to basophil granulocytes as no constitutive or inducible expression of GzmB is detected in eosinophils or neutrophils. GzmB is induced within 6 to 24 hours, sorted into the granule compartment, and released by exocytosis upon IgE-dependent and -independent activation. In vitro, there is a close parallelism between GzmB, IL-13, and leukotriene C4 production. In vivo, granzyme B, but not the lymphoid granule marker granzyme A, is released 18 hours after allergen challenge of asthmatic patients in strong correlation with interleukin-13. Our study demonstrates an unexpected plasticity of the granule composition of mature basophils and suggests a role of granzyme B as a novel mediator of allergic diseases.

Induction of haem oxygenase-1 causes cortical non-haem iron increase in experimental pneumococcal meningitis: evidence that concomitant ferritin up-regulation prevents iron-induced oxidative damage

Desferrioxamine inhibits cortical necrosis in neonatal rats with experimental pneumococcal meningitis, suggesting that iron-induced oxidative damage might be responsible for neuronal damage. We therefore examined the spatial and temporal profile of changes in cortical iron and iron homeostatic proteins during pneumococcal meningitis. Infection was associated with a steady and global increase of non-haem iron in the cortex, particularly in neuronal cell bodies of layer II and V, and in capillary endothelial cells. The non-haem iron increase was associated with induction of haem oxygenase (HO)-1 in neurones, microglia and capillary endothelial cells, whereas HO-2 levels remained unchanged, suggesting that the non-haem iron increase might be the result of HO-1-mediated haem degradation. Indeed, treatment with the haem oxygenase inhibitor tin protoporphyrin (which completely blocked the accumulation of bilirubin detected in HO-1-positive cells) completely prevented the infection-associated non-haem iron increase. The same cells also displayed markedly increased ferritin staining, the increase of which occurred independently of HO activity. At the same time, no increase in DNA/RNA oxidation was observed in infected animals (as assessed by in situ detection of 8-hydroxy[deoxy]guanosine), strongly suggesting that ferritin up-regulation protected the brain from iron-induced oxidative damage. Thus, although pneumococcal meningitis leads to an increase of cortical non-haem iron, protective mechanisms up-regulated in parallel prevent iron-induced oxidative damage. Cortical damage does not appear to be a direct consequence of increased iron, therefore.

Induction of apoptosis and enhancement of chemosensitivity in human prostate cancer LNCaP cells using bispecific antisense oligonucleotide targeting Bcl-2 and Bcl-xL genes

OBJECTIVE: To determine whether a specifically designed bispecific (Bcl-2/Bcl-xL) antisense oligonucleotide (ASO) induces apoptosis and enhances chemosensitivity in human prostate cancer LNCaP cells, as Bcl-2 and Bcl-xL are both anti-apoptotic genes associated with treatment resistance and tumour progression in many malignancies, including prostate cancer. MATERIALS AND METHODS: Inhibition of Bcl-2 and Bcl-xL expression by the bispecific ASO was evaluated using real-time reverse transcription-polymerase chain reaction and Western blotting, while growth inhibition and induction of apoptosis were analysed by a crystal violet assay, flow cytometry and Western blotting of apoptosis-relevant proteins. The effect of combined treatment with bispecific ASO and chemotherapy or small-interference RNA (siRNA) targeting the clusterin gene was also investigated. RESULTS: Bispecific ASO reduced Bcl-2 and Bcl-xL expression in LNCaP cells in a dose-dependent manner. There was cell growth inhibition, increases in the sub-G0-G1 fraction, and cleavage of caspase-3 and poly(ADP-Ribose) polymerase proteins in LNCaP cells after bispecific ASO treatment. Interestingly, Bcl-2/Bcl-xL bispecific ASO treatment also resulted in the down-regulation of Mcl-1 and up-regulation of Bax. The sensitivity of LNCaP cells to mitoxantrone, docetaxel or paclitaxel was significantly increased, reducing the 50% inhibitory concentration by 45%, 80% or 90%, respectively. Furthermore, the apoptotic induction by Bcl-2/Bcl-xL bispecific ASO was synergistically enhanced by siRNA-mediated inhibition of clusterin, a cytoprotective chaperone that interacts with and inhibits activated Bax. CONCLUSIONS: These findings support the concept of the targeted suppression of Bcl-2 anti-apoptotic family members using multitarget inhibition strategies for prostate cancer, through the effective induction of apoptosis.

Self-assembly of sensory neurons into ganglia-like microtissues

Unraveling intra- and inter-cellular signaling networks managing cell-fate control, coordinating complex differentiation regulatory circuits and shaping tissues and organs in living systems remain major challenges in the post-genomic era. Resting on the laurels of past-century monolayer culture technologies, the cell culture community has only recently begun to appreciate the potential of three-dimensional mammalian cell culture systems to reveal the full scope of mechanisms orchestrating the tissue-like cell quorum in space and time. Capitalizing on gravity-enforced self-assembly of monodispersed primary embryonic mouse cells in hanging drops, we designed and characterized a three-dimensional cell culture model for ganglion-like structures. Within 24h, a mixture of mouse embryonic fibroblasts (MEF) and cells, derived from the dorsal root ganglion (DRG) (sensory neurons and Schwann cells) grown in hanging drops, assembled to coherent spherical microtissues characterized by a MEF feeder core and a peripheral layer of DRG-derived cells. In a time-dependent manner, sensory neurons formed a polar ganglion-like cap structure, which coordinated guided axonal outgrowth and innervation of the distal pole of the MEF feeder spheroid. Schwann cells, present in embryonic DRG isolates, tended to align along axonal structures and myelinate them in an in vivo-like manner. Whenever cultivation exceeded 10 days, DRG:MEF-based microtissues disintegrated due to an as yet unknown mechanism. Using a transgenic MEF feeder spheroid, engineered for gaseous acetaldehyde-inducible interferon-beta (ifn-beta) production by cotransduction of retro-/ lenti-viral particles, a short 6-h ifn-beta induction was sufficient to rescue the integrity of DRG:MEF spheroids and enable long-term cultivation of these microtissues. In hanging drops, such microtissues fused to higher-order macrotissue-like structures, which may pave the way for sophisticated bottom-up tissue engineering strategies. DRG:MEF-based artificial micro- and macrotissue design demonstrated accurate key morphological aspects of ganglions and exemplified the potential of self-assembled scaffold-free multicellular micro-/macrotissues to provide new insight into organogenesis.

Labor induction in preeclampsia: is misoprostol more effective than dinoprostone?

OBJECTIVE: To compare the efficacy of vaginal misoprostol versus dinoprostone for induction of labor (IOL) in patients with preeclampsia according to the WHO criteria. STUDY DESIGN: Ninety-eight patients were retrospectively analyzed. A total of 47 patients received 3 mg dinoprostone suppositories every 6 h (max. 6 mg/24 h) whereas 51 patients in the misoprostol group received either 50 mug misoprostol vaginally every 12 h, or 25 mug every 6 h (max. 100 mug/24 h). Primary outcomes were vaginal delivery within 24 and 48 h, respectively. RESULTS: The probability of delivering within 48 h was more than three-fold higher in the misoprostol than in the dinoprostone group: odds ratio (OR)=3.48; 95% confidence interval (CI) 1.24, 10.30, whereas no significant difference was observed within 24 h (P=0.34). No correlation was seen between a ripe cervix prior to IOL and delivery within 24/48 h (P=0.33 and P=1.0, respectively). More cesarean sections were performed in the dinoprostone group due to failed IOL (P=0.0009). No significant differences in adverse maternal outcome were observed between both study groups, whereas more neonates (12 vs. 6) of the dinoprostone group were admitted to the NICU (P=0.068). CONCLUSION: This study suggests that misoprostol may have some advantages compared to dinoprostone, including improved efficacy and lower cost of the drug, even in cases of preeclampsia.

Uniform vascular-endothelial-cell-specific gene expression in both embryonic and adult transgenic mice

TIE2 is a vascular endothelial-specific receptor tyrosine kinase essential for the regulation of vascular network formation and remodeling. Previously, we have shown that the 1.2-kb 5' flanking region of the TIE2 promoter is capable of directing beta-galactosidase reporter gene expression specifically into a subset of endothelial cells (ECs) of transgenic mouse embryos. However, transgene activity was restricted to early embryonic stages and not detectable in adult mice. Herein we describe the identification and characterization of an autonomous endothelial-specific enhancer in the first intron of the mouse TIE2 gene. Furthermore, combination of the TIE2 promoter with an intron fragment containing this enhancer allows it to target reporter gene expression specifically and uniformly to virtually all vascular ECs throughout embryogenesis and adulthood. To our knowledge, this is the first time that an in vivo expression system has been assembled by which heterologous genes can be targeted exclusively to the ECs of the entire vasculature. This should be a valuable tool to address the function of genes during physiological and pathological processes of vascular ECs in vivo. Furthermore, we were able to identify a short region critical for enhancer function in vivo that contains putative binding sites for Ets-like transcription factors. This should, therefore, allow us to determine the molecular mechanisms underlying the vascular-EC-specific expression of the TIE2 gene.

Induction of EROD activity by 1-phenylimidazole and beta-naphthoflavone in rainbow trout cultured hepatocytes: a comparative study

The classical pathway for induction of cytochrome P4501A (CYP1A) by xenobiotics is ligand binding to the aryl hydrocarbon receptor (AhR). However, several studies with mammalian cell systems point out a range of xenobiotics including imidazole derivatives, which are able to activate CYP1A through non-classical mechanisms. The objective of the present work is to compare induction of CYP1A (determined at the catalytic level as 7-ethoxyresorufin-O-deethylase, EROD) in rainbow trout (Oncorhynchus mykiss) hepatocytes by the prototypic AhR ligand, beta-naphthoflavone (betaNF), and by the imidazole derivative, 1-phenylimidazole (PIM). PIM was able to induce EROD activity although its potency was clearly lower than that of betaNF. In order to assess the relative importance of classical AhR ligand binding and alternative signaling pathways in CYP1A induction by PIM, co-exposure experiments with the partial AhR antagonist alpha-naphthoflavone (alphaNF) or with inhibitors of protein kinase C (staurosporine) and tyrosine kinases (genistein, herbimicine) were performed. alphaNF and herbimicin provoked a decrease of EROD induction both by betaNF and PIM, whereas staurosporine and genistein remained without effect. The overall similarities in the response of betaNF and PIM to the various inhibitors suggest that both compounds, in apparent contrast to the behaviour of some other imidazole derivatives, induce CYP1A following similar mechanisms.

Tissue-specific induction of EROD activity and CYP1A protein in Sparus aurata exposed to B(a)P and TCDD

The purpose of this study was to compare xenobiotic CYP1A induction in liver, gills, and excretory kidney of gilthead seabream, Sparus aurata. Fishes were exposed via water for 20 days to different concentrations of benzo(a)pyrene (B(a)P) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). CYP1A was measured at the enzyme activity level as 7-ethoxyresorufin-O-deethylase (EROD) activity, and at the protein level by means of ELISA. The liver displayed the highest absolute levels of EROD activity, both under non-exposed and exposed conditions. Organ- or inducer-related differences in the time course of CYP1A induction were moderate; however, the magnitude of the induction response varied between the organs and between B(a)P and TCDD. In the case of TCDD, liver, and kidney yielded a comparable induction response, whereas in the case of B(a)P, the kidney showed a substantially higher maximum induction factor than the liver. In the gills, the two xenobiotics resulted in similar maximum induction factors. In B(a)P-exposed seabream, EROD activities and CYP1A protein levels showed a good correlation in all three organs, whereas with TCDD as inducer the correlation was poor, what was mainly due to a decrease of EROD activities at the higher concentrations of TCDD, while CYP1A protein levels showed no concomitant decline. Overall, the study revealed both similarities and differences in the time-, concentration-, and inducer-dependent CYP1A responses of the three target organs, liver, kidney, and gills. Although, the findings of this study principally confirm the notion of the liver as the major metabolic organ in fish, they also provide evidence for substantial metabolic potential in gills and particularly in the kidney.

Induction of tachyzoite egress from cells infected with the protozoan Neospora caninum by nitro- and bromo-thiazolides, a class of broad-spectrum anti-parasitic drugs

Neospora caninum represents an important pathogen causing stillbirth and abortion in cattle and neuromuscular disease in dogs. Nitazoxanide (NTZ) and its deacetylated metabolite tizoxanide (TIZ) are nitro-thiazolyl-salicylamide drugs with a broad-spectrum anti-parasitic activity in vitro and in vivo. In order to generate compounds potentially applicable in food and breeding animals, the nitro group was removed, and the thiazole-moiety was modified by other functional groups. We had shown earlier that replacement of the nitro-group by a bromo-moiety did not notably affect in vitro efficacy of the drugs against N. caninum. In this study we report on the characterization of two bromo-derivatives, namely Rm4822 and its de-acetylated putative metabolite Rm4847 in relation to the nitro-compounds NTZ and TIZ. IC(50) values for proliferation inhibition were 4.23 and 4.14 microM for NTZ and TIZ, and 14.75 and 13.68 microM for Rm4822 and Rm4847, respectively. Complete inhibition (IC(99)) was achieved at 19.52 and 22.38 microM for NTZ and TIZ, and 18.21 and 17.66 microM for Rm4822 and Rm4847, respectively. However, in order to exert a true parasiticidal effect in vitro, continuous culture of infected fibroblasts in the presence of the bromo-thiazolide Rm4847 was required for a period of 3 days, while the nitro-compound TIZ required 5 days continuous drug exposure. Both thiazolides induced rapid egress of N. caninum tachyzoites from their host cells, and egress was inhibited by the cell membrane permeable Ca(2+)-chelator BAPTA-AM. Host cell entry by N. caninum tachyzoites was inhibited by Rm4847 but not by TIZ. Upon release from their host cells, TIZ-treated parasites remained associated with the fibroblast monolayer, re-invaded neighboring host cells and resumed proliferation in the absence of the drug. In contrast, Rm4847 inhibited host cell invasion and respective treated tachyzoites did not proliferate further. This demonstrated that bromo- and nitro-thiazolides exhibit differential effects against the intracellular protozoan N. caninum and bromo-thiazolides could represent a valuable alternative to the nitro-thiazolyl-salicylamide drugs.

Copper induction of lactate oxidase of Lactococcus lactis: a novel metal stress response

Lactococcus lactis IL1403, a lactic acid bacterium widely used for food fermentation, is often exposed to stress conditions. One such condition is exposure to copper, such as in cheese making in copper vats. Copper is an essential micronutrient in prokaryotes and eukaryotes but can be toxic if in excess. Thus, copper homeostatic mechanisms, consisting chiefly of copper transporters and their regulators, have evolved in all organisms to control cytoplasmic copper levels. Using proteomics to identify novel proteins involved in the response of L. lactis IL1403 to copper, cells were exposed to 200 muM copper sulfate for 45 min, followed by resolution of the cytoplasmic fraction by two-dimensional gel electrophoresis. One protein strongly induced by copper was LctO, which was shown to be a NAD-independent lactate oxidase. It catalyzed the conversion of lactate to pyruvate in vivo and in vitro. Copper, cadmium, and silver induced LctO, as shown by real-time quantitative PCR. A copper-regulatory element was identified in the 5' region of the lctO gene and shown to interact with the CopR regulator, encoded by the unlinked copRZA operon. Induction of LctO by copper represents a novel copper stress response, and we suggest that it serves in the scavenging of molecular oxygen.

Type I IFN induction in response to Listeria monocytogenes in human macrophages: evidence for a differential activation of IFN regulatory factor 3 (IRF3)

Listeria monocytogenes is a prototypic bacterium for studying innate and adaptive cellular immunity as well as host defense. Using human monocyte-derived macrophages, we report that an infection with a wild-type strain, but not a listeriolysin O-deficient strain, of the Gram-positive bacterium L. monocytogenes induces expression of IFN-beta and a bioactive type I IFN response. Investigating the activation of signaling pathways in human macrophages after infection revealed that a wild-type strain and a hemolysin-deficient strain of L. monocytogenes activated the NF-kappaB pathway and induced a comparable TNF response. p38 MAPK and activating transcription factor 2 were phosphorylated following infection with either strain, and IFN-beta gene expression induced by wild-type L. monocytogenes was reduced when p38 was inhibited. However, neither IFN regulatory factor (IRF) 3 translocation to the nucleus nor posttranslational modifications and dimerizations were observed after L. monocytogenes infection. In contrast, vesicular stomatitis virus and LPS triggered IRF3 activation and signaling. When IRF3 was knocked down using small interfering RNA, a L. monocytogenes-induced IFN-beta response remained unaffected whereas a vesicular stomatitis virus-triggered response was reduced. Evidence against the possibility that IRF7 acts in place of IRF3 is provided. Thus, we show that wild-type L. monocytogenes induced an IFN-beta response in human macrophages and propose that this response involves p38 MAPK and activating transcription factor 2. Using various stimuli, we show that IRF3 is differentially activated during type I IFN responses in human macrophages.