An immune modulatory role for the fes proto-oncogene

The Fes protein tyrosine kinase is abundantly expressed in phagocytic immune cells, including tumor associated macrophages. Fes knockout mice (fes-/-) display enhanced sensitivity to LPS, and this was shown to be associated with increased NF-κB signaling and TNFα production from fes-/- macrophages. Interestingly, tumor onset in the mouse mammary tumor virus (MMTV-Neu) transgenic mouse model of breast cancer is significantly delayed in fes-/- mice, and this was associated with increased frequency of CD11b+ myeloid and CD3+ T cells in the premalignant mammary glands. Recent studies have also implicated Fes in cross-talk between MHC-I and the NF-κB and IRF-3 pathways in macrophages. Signal 3, the production of inflammatory cytokines and Type I interferons downstream of NF-κB and IRF-3 pathways in antigen presenting cells, is considered an important component of T-cell activation, after engagement of T cell receptor by MHC presented antigen (Signal 1) and co-receptors by their ligands (Signal 2). Using a lymphocytic choriomeningitis virus (LCMV) model of immune activation, I show that LPS stimulated fes-/- macrophages promote more robust activation of LCMV antigenspecific CD8+ T cells than wild type macrophages (fes+/+). Furthermore, LPS stimulated fes-/- macrophages showed increased phosphorylation of NF-B and IRF-3. I also showed that Fes colocalizes with MHC-I in dynamic vesicular structures within macrophages. These observations are consistent with a model where Fes regulates Signal 3 in antigen presenting cells through roles in cross-talk between MHC-I and the NF-kB and IRF-3 signaling pathways. This suggests that Fes plays an immune checkpoint role at the level of Signal 3, and that Fes inhibition could promote tumor immunity through increased Signal 3 driven T cell activation.

Identification of a novel population of Langerin+ dendritic cells.

Langerhans cells (LCs) are antigen-presenting cells that reside in the epidermis of the skin and traffic to lymph nodes (LNs). The general role of these cells in skin immune responses is not clear because distinct models of LC depletion resulted in opposite conclusions about their role in contact hypersensitivity (CHS) responses. While comparing these models, we discovered a novel population of LCs that resides in the dermis and does not represent migrating epidermal LCs, as previously thought. Unlike epidermal LCs, dermal Langerin(+) dendritic cells (DCs) were radiosensitive and displayed a distinct cell surface phenotype. Dermal Langerin(+) DCs migrate from the skin to the LNs after inflammation and in the steady state, and represent the majority of Langerin(+) DCs in skin draining LNs. Both epidermal and dermal Langerin(+) DCs were depleted by treatment with diphtheria toxin in Lang-DTREGFP knock-in mice. In contrast, transgenic hLang-DTA mice lack epidermal LCs, but have normal numbers of dermal Langerin(+) DCs. CHS responses were abrogated upon depletion of both epidermal and dermal LCs, but were unaffected in the absence of only epidermal LCs. This suggests that dermal LCs can mediate CHS and provides an explanation for previous differences observed in the two-model systems.

Getting to the root of neuronal signalling in plant-parasitic nematodes using RNA interference

A variety of genes expressed in preparasitic second-stage juveniles (J2) of plant-parasitic nematodes appear to be vulnerable to RNA interference (RNAi) in vitro by coupling double-stranded (ds)RNA soaking with the artificial stimulation of pharyngeal pumping. Also, there is mounting evidence that the in planta generation of nematode-specific double-stranded RNAs (dsRNAs) has real utility in the control of these pests. Although neuronally-expressed genes in Caenorhabditis elegans are commonly refractory to RNAi, we have discovered that neuronally-expressed genes in plant-parasitic nematodes are highly susceptible to RNAi and that silencing can be induced by simple soaking procedures without the need for pharyngeal stimulation. Since most front-line anthelmintics that are used for the control of nematode parasites of animals and humans act to disrupt neuromuscular coordination, we argue that intercellular signalling processes associated with neurons have much appeal as targets for transgenic plant-based control strategies for plant-parasitic nematodes. FMRFamide-like peptides (FLPs) are a large family of neuropeptides which are intimately associated with neuromuscular regulation, and our studies on flp gene function in plant-parasitic nematodes have revealed that their expression is central to coordinated locomotory activities. We propose that the high level of conservation in nervous systems across nematodes coupled with the RNAi-susceptibility of neuronally-expressed genes in plant-parasitic nematodes provides a valuable research tool which could be used to interrogate neuronal signalling processes in nematodes.

Nitric oxide synthase gene therapy enhances the toxicity of cisplatin in cancer cells

BACKGROUND AIMS: Cell-based gene therapy is an alternative to viral and non-viral gene therapy. Emerging evidence suggests that mesenchymal stem cells (MSC) are able to migrate to sites of tissue injury and have immunosuppressive properties that may be useful in targeted gene therapy for sustained specific tissue engraftment. METHODS: In this study, we injected intravenously (i.v.) 1x10(6) MSC, isolated from green fluorescent protein (GFP) transgenic rats, into Rif-1 fibrosarcoma-bearing C3H/HeN mice. The MSC had been infected using a lentiviral vector to express stably the luciferase reporter gene (MSC-GFP-luci). An in vivo imaging system (IVIS 200) and Western blotting techniques were used to detect the distribution of MSC-GFP-luci in tumor-bearing animals. RESULTS: We observed that xenogenic MSC selectively migrated to the tumor site, proliferated and expressed the exogenous gene in subcutaneous fibrosarcoma transplants. No MSC distribution was detected in other organs, such as the liver, spleen, colon and kidney. We further showed that the FGF2/FGFR pathways may play a role in the directional movement of MSC to the Rif-1 fibrosarcoma. We performed in vitro co-culture and in vivo tumor growth analysis, showing that MSC did not affect the proliferation of Rif-1 cells and fibrosarcoma growth compared with an untreated control group. Finally, we demonstrated that the xenogenic MSC stably expressing inducible nitric oxide synthase (iNOS) protein transferred by a lentivirus-based system had a significant inhibitory effect on the growth of Rif-1 tumors compared with MSC alone and the non-treatment control group. CONCLUSIONS: iNOS delivered by genetically modified iNOS-MSC showed a significant anti-tumor effect both in vitro and in vivo. MSC may be used as a target gene delivery vehicle for the treatment of fibrosarcoma and other tumors

Inhibition of Advanced Glycation and Absence of Galectin-3 Prevent Blood-Retinal Barrier Dysfunction during Short-Term Diabetes

Breakdown of the inner blood-retinal barrier (iBRB) occurs early in diabetes and is central to the development of sight-threatening diabetic macular edema (DME) as retinopathy progresses. In the current study, we examined how advanced glycation end products (AGEs) forming early in diabetes could modulate vasopermeability factor expression in the diabetic retina and alter inter-endothelial cell tight junction (TJ) integrity leading to iBRB dysfunction. We also investigated the potential for an AGE inhibitor to prevent this acute pathology and examined a role of the AGE-binding protein galectin-3 (Gal-3) in AGE-mediated cell retinal pathophysiology. Diabetes was induced in C57/BL6 wild-type (WT) mice and in Gal-3(-/-) transgenic mice. Blood glucose was monitored and AGE levels were quantified by ELISA and immunohistochemistry. The diabetic groups were subdivided, and one group was treated with the AGE-inhibitor pyridoxamine (PM) while separate groups of WT and Gal-3(-/-) mice were maintained as nondiabetic controls. iBRB integrity was assessed by Evans blue assay alongside visualisation of TJ protein complexes via occludin-1 immunolocalization in retinal flat mounts. Retinal expression levels of the vasopermeability factor VEGF were quantified using real-time RT-PCR and ELISA. WT diabetic mice showed significant AGE -immunoreactivity in the retinal microvasculature and also showed significant iBRB breakdown (P < .005). These diabetics had higher VEGF mRNA and protein expression in comparison to controls (P < .01). PM-treated diabetics had normal iBRB function and significantly reduced diabetes-mediated VEGF expression. Diabetic retinal vessels showed disrupted TJ integrity when compared to controls, while PM-treated diabetics demonstrated near-normal configuration. Gal-3(-/-) mice showed significantly less diabetes-mediated iBRB dysfunction, junctional disruption, and VEGF expression changes than their WT counterparts. The data suggests an AGE-mediated disruption of iBRB via upregulation of VEGF in the diabetic retina, possibly modulating disruption of TJ integrity, even after acute diabetes. Prevention of AGE formation or genetic deletion of Gal-3 can effectively prevent these acute diabetic retinopathy changes.

Langerhans cells are critical in the development of atopic dermatitis-like inflammation and symptoms in mice

Genetic or vitamin D3-induced overexpression of thymic stromal lymphopoietin (TSLP) by keratinocytes results in an atopic dermatitis (AD)-like inflammatory phenotype in mice echoing the discovery of high TSLP expression in epidermis from AD patients. Although skin dendritic cells (DC) are suspected to be involved in AD, direct evidence of a pathogenetic role for skin DC in TSLP-induced skin inflammation has not yet been demonstrated. In a mouse model of AD, i.e. mice treated with the low-calcemic vitamin D3 analogue, MC903, we show that epidermal Langerhans cells (LC)-depleted mice treated with MC903 do neither develop AD-like inflammation nor increased serum IgE as compared to vitamin D3 analogue-treated control mice. Accordingly, we show that, in mice treated with MC903 or in K14-TSLP transgenic mice, expression of maturation markers by LC is increased whereas maturation of dermal DC is not altered. Moreover, only LC are responsible for the polarization of naive CD4+ T cells to a Th2 phenotype, i.e. decrease in interferon-gamma and increase in interleukin (IL)-13 production by CD4+ T cells. This effect of LC on T-lymphocytes does not require OX40-L/CD134 and is mediated by a concomitant down-regulation of IL-12 and CD70. Although it was previously stated that TSLP up-regulates the production of thymus and activation-regulated chemokine (TARC)/chemokine (C-C motif) ligand 17 (CCL17) and macrophage-derived chemokine (MDC)/CCL22 by human LC in vitro, our work shows that production of these Th2- cell attracting chemokines is increased only in keratinocytes in response to TSLP overexpression. These results demonstrate that LC are required for the development of AD in mouse models of AD involving epidermal TSLP overexpression.

Foxp3+ T cells induce Perforin-Dependent Dendritic Cell Death in Tumor-Draining Lymph Nodes.

Regulatory T (Treg) cells limit the onset of effective antitumor immunity, through yet-ill-defined mechanisms. We showed the rejection of established ovalbumin (OVA)-expressing MCA101 tumors required both the adoptive transfer of OVA-specific CD8(+) T cell receptor transgenic T cells (OTI) and the neutralization of Foxp3(+) T cells. In tumor-draining lymph nodes, Foxp3(+) T cell neutralization induced a marked arrest in the migration of OTI T cells, increased numbers of dendritic cells (DCs), and enhanced OTI T cell priming. Using an in vitro cytotoxic assay and two-photon live microscopy after adoptive transfer of DCs, we demonstrated that Foxp3(+) T cells induced the death of DCs in tumor-draining lymph nodes, but not in the absence of tumor. DC death correlated with Foxp3(+) T cell-DC contacts, and it was tumor-antigen and perforin dependent. We conclude that Foxp3(+) T cell-dependent DC death in tumor-draining lymph nodes limits the onset of CD8(+) T cell responses.

NADPH oxidase-4 mediates protection against chronic load-induced stress in mouse hearts by enhancing angiogenesis

Cardiac failure occurs when the heart fails to adapt to chronic stresses. Reactive oxygen species (ROS)-dependent signaling is implicated in cardiac stress responses but the role of different ROS sources remains unclear. Here, we report that NADPH oxidase-4 (Nox4) facilitates cardiac adaptation to chronic stress. Unlike other Nox proteins, Nox4 activity is regulated mainly by its expression level which increased in cardiomyocytes during stresses such as pressure overload or hypoxia. To investigate the functional role of Nox4 during the cardiac response to stress, we generated mice with a genetic deletion of Nox4 or a cardiomyocyte-targeted overexpression of Nox4. Basal cardiac function was normal in both models but Nox4-null animals developed exaggerated contractile dysfunction, hypertrophy and cardiac dilatation during exposure to chronic overload whereas Nox4-transgenic mice were protected. Investigation of mechanisms underlying this protective effect revealed a significant Nox4-dependent preservation of myocardial capillary density after pressure overload. Nox4 enhanced stress-induced activation of cardiomyocyte Hif1 and the release of VEGF, resulting in an increased paracrine angiogenic activity. These data indicate that cardiomyocyte Nox4 is a novel inducible regulator of myocardial angiogenesis, a key determinant of cardiac adaptation to overload stress. Our results also have wider relevance to the use of non-specific antioxidant approaches in cardiac disease and may provide an explanation for the failure of such strategies in many settings.

High incidence of proviral integrations in the Hoxa locus in a new model of E2a-PBX1-induced B-cell leukemia

Relevant mouse models of E2a-PBX1-induced pre-B cell leukemia are still elusive. We now report the generation of a pre-B leukemia model using E2a-PBX1 transgenic mice, which lack mature and precursor T-cells as a result of engineered loss of CD3epsilon expression (CD3epsilon(-/-)). Using insertional mutagenesis and inverse-PCR, we show that B-cell leukemia development in the E2a-PBX1 x CD3epsilon(-/-) compound transgenic animals is significantly accelerated when compared to control littermates, and document several known and novel integrations in these tumors. Of all common integration sites, a small region of 19 kb in the Hoxa gene locus, mostly between Hoxa6 and Hoxa10, represented 18% of all integrations in the E2a-PBX1 B-cell leukemia and was targeted in 86% of these leukemias compared to 17% in control tumors. Q-PCR assessment of expression levels for most Hoxa cluster genes in these tumors revealed an unprecedented impact of the proviral integrations on Hoxa gene expression, with tumors having one to seven different Hoxa genes overexpressed at levels up to 6600-fold above control values. Together our studies set the stage for modeling E2a-PBX1-induced B-cell leukemia and shed new light on the complexity pertaining to Hox gene regulation. In addition, our results show that the Hoxa gene cluster is preferentially targeted in E2a-PBX1-induced tumors, thus suggesting functional collaboration between these oncogenes in pre-B-cell tumors.

Overexpression of cyclin D1 moderately increases ploidy in megakaryocytes

Background and Objectives. Megakalyocytes undergo a unique cell cycle by which they replicate their complete genome many times in the absence of cytokinesis, In the search for regulators of the endomitotic cell cycle, we previously produced mice transgenic for cyclin D3 to identify this cyclin as able to enhance ploidy and to increase the number of differentiated cells in the megakaryocytic lineage. Of the D-type cyclins, cyclin D3 and to a much lesser extent cyclin D1, are present in megakaryocytes undergoing endomitosis and these cyclins are, respectively, markedly and moderately upregulated following exposure to the ploidy-promoting factor, Mpl-ligand. Our objective was to explore whether cyclin D1 can mimic the effect of cyclin D3 on ploidy in megakalyocytes.

Endothelial Nox4 NADPH oxidase enhances vasodilatation and reduces blood pressure in vivo.

OBJECTIVE: Increased reactive oxygen species (ROS) production is involved in the pathophysiology of endothelial dysfunction. NADPH oxidase-4 (Nox4) is a ROS-generating enzyme expressed in the endothelium, levels of which increase in pathological settings. Recent studies indicate that it generates predominantly hydrogen peroxide (H(2)O(2)), but its role in vivo remains unclear. METHODS AND RESULTS: We generated transgenic mice with endothelium-targeted Nox4 overexpression (Tg) to study the in vivo role of Nox4. Tg demonstrated significantly greater acetylcholine- or histamine-induced vasodilatation than wild-type littermates. This resulted from increased H(2)O(2) production and H(2)O(2)-induced hyperpolarization but not altered nitric oxide bioactivity. Tg had lower systemic blood pressure than wild-type littermates, which was normalized by antioxidants. CONCLUSION: Endothelial Nox4 exerts potentially beneficial effects on vasodilator function and blood pressure that are attributable to H(2)O(2) production. These effects contrast markedly with those reported for Nox1 and Nox2, which involve superoxide-mediated inactivation of nitric oxide. Our results suggest that therapeutic strategies to modulate ROS production in vascular disease may need to separately target individual Nox isoforms.

pcDNA3.1tdTomato is superior to pDsRed2-N1 for optical fluorescence imaging in the F344/AY-27 rat model of bladder cancer

PURPOSE: Animal models are important for pre-clinical assessment of novel therapies in metastatic bladder cancer. The F344/AY-27 model involves orthotopic colonisation with AY-27 tumour cells which are syngeneic to F344 rats. One disadvantage of the model is the unknown status of colonisation between instillation and sacrifice. Non-invasive optical imaging using red fluorescence reporters could potentially detect tumours in situ and would also reduce the number of animals required for each experiment.

MATERIALS AND METHODS: AY-27 cells were stably transfected with either pDsRed2-N1 or pcDNA3.1tdTomato. The intensity and stability of fluorescence in the resultant AY-27/DsRed2-N1 and AY-27/tdTomato stable cell lines were compared using Xenogen IVIS®200 and Olympus IX51 systems.

RESULTS: AY-27/tdTomato fluorescence intensity was 60-fold brighter than AY-27/DsRed2-N1 and was sustained in AY-27/tdTomato cells following freezing and six subsequent sub-cultures. After sub-cutaneous injection, fluorescence intensity from AY-27/tdTomato cells was threefold stronger than that detected from AY-27/DsRed2-N1 cells. IVIS®200 detected fluorescence from AY-27/tdTomato and AY-27/DsRed2-N1 cells colonising resected and exteriorised bladders, respectively. However, the deep-seated position of the bladder precluded in vivo imaging. Characteristics of AY-27/tdTomato cells in vitro and in tumours colonising F344 rats resembled those of parental AY-27 cells. Tumour transformation was observed in the bladders colonised with AY-27/DsRed2-N1 cells.

CONCLUSIONS: In vivo whole-body imaging of internal red fluorescent animal tumours should use pcDNA3.1tdTomato rather than pDsRed2-N1. Optical imaging of deep-seated organs in larger animals remains a challenge which may require proteins with brighter red or far-red fluorescence and/or alternative approaches.

In vivo relevance for platelet glycoprotein Ib alpha residue Tyr276 in thrombus formation

Background: Platelet glycoprotein (GP) Ib-IX-V supports platelet adhesion on damaged vascular walls by binding to von Willebrand factor (VWF). For several decades it has been recognized that the alpha-subunit of GP (GPIb alpha) also binds thrombin but the physiological relevance, if any, of this interaction was unknown. Previous studies have shown that a sulfated tyrosine 276 (Tyr276) is essential for thrombin binding to GPIb alpha.Objectives: This study investigated the in vivo relevance of GPIb alpha residue Tyr276 in hemostasis and thrombosis.Methods: Transgenic mouse colonies expressing the normal human GPIb alpha subunit or a mutant human GPIb alpha containing a Phe substitution for Tyr276 (hTg(Y276F)) were generated. Both colonies were bred to mice devoid of murine GPIb alpha.Results: Surface-expressed GPIb alpha levels and platelet counts were similar in both colonies. hTg(Y276F) platelets were significantly impaired in binding alpha-thrombin but displayed normal binding to type I fibrillar collagen and human VWF in the presence of ristocetin. In vivo thrombus formation as a result of chemical damage (FeCl3) demonstrated that hTg(Y276F) mice have a delayed time to occlusion followed by unstable blood flow indicative of embolization. In models of laser-induced injury, thrombi developing in hTg(Y276F) animals were also less stable.Conclusions: The results demonstrate that GPIb alpha residue Tyr276 is physiologically important, supporting stable thrombus formation in vivo.

Protection against methylglyoxal-derived AGEs by regulation of glyoxalase 1 prevents retinal neuroglial and vasodegenerative pathology.

Aims/hypothesis
Methylglyoxal (MG) is an important precursor for AGEs. Normally, MG is detoxified by the glyoxalase (GLO) enzyme system (including component enzymes GLO1 and GLO2). Enhanced glycolytic metabolism in many cells during diabetes may overpower detoxification capacity and lead to AGE-related pathology. Using a transgenic rat model that overexpresses GLO1, we investigated if this enzyme can inhibit retinal AGE formation and prevent key lesions of diabetic retinopathy.
Methods
Transgenic rats were developed by overexpression of full length GLO1. Diabetes was induced in wild-type (WT) and GLO1 rats and the animals were killed after 12 or 24 weeks of hyperglycaemia. N e-(Carboxyethyl)lysine (CEL), N e-(carboxymethyl)lysine (CML) and MG-derived-hydroimidazalone-1 (MG-H1) were determined by immunohistochemistry and by ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MSMS). Müller glia dysfunction was determined by glial fibrillary acidic protein (GFAP) immunoreactivity and by spatial localisation of the potassium channel Kir4.1. Acellular capillaries were quantified in retinal flat mounts.
Results
GLO1 overexpression prevented CEL and MG-H1 accumulation in the diabetic retina when compared with WT diabetic counterparts (p?<?0.01). Diabetes-related increases in Müller glial GFAP levels and loss of Kir4.1 at the vascular end-feet were significantly prevented by GLO1 overexpression (p?<?0.05) at both 12- and 24-week time points. GLO1 diabetic animals showed fewer acellular capillaries than WT diabetic animals (p?<?0.001) at 24 weeks’ diabetes.
Conclusions/interpretation
Detoxification of MG reduces AGE adduct accumulation, which, in turn, can prevent formation of key retinal neuroglial and vascular lesions as diabetes progresses. MG-derived AGEs play an important role in diabetic retinopathy.

A quorum-quenching approach to investigate the conservation of quorum-sensing-regulated functions within the Burkholderia cepacia complex

Taxonomic studies of the past few years have shown that the Burkholderia cepacia complex, a heterogeneous group of B. cepacia-like organisms, consists of at least nine species. B. cepacia complex strains are ubiquitously distributed in nature and have been used for biocontrol, bioremediation, and plant growth promotion purposes. At the same time, B. cepacia complex strains have emerged as important opportunistic pathogens of humans, particularly those with cystic fibrosis. All B. cepacia complex species investigated thus far use quorum-sensing (QS) systems that rely on N-acylhomoserine lactone (AHL) signal molecules to express certain functions, including the production of extracellular proteases, swarming motility, biofilm formation, and pathogenicity, in a population-density-dependent manner. In this study we constructed a broad-host-range plasmid that allowed the heterologous expression of the Bacillus sp. strain 240B1 AiiA lactonase, which hydrolyzes the lactone ring of various AHL signal molecules, in all described B. cepacia complex species. We show that expression of AiiA abolished or greatly reduced the accumulation of AHL molecules in the culture supernatants of all tested B. cepacia complex strains. Phenotypic characterization of wild-type and transgenic strains revealed that protease production, swarming motility, biofilm formation, and Caenorhabditis elegans killing efficiency was regulated by AHL in the large majority of strains investigated.