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High spinal cord injuries (SCI) induce the deafferentation of phrenic motoneurons, leading to permanent diaphragm paralysis. This involves secondary injury associated with pathologic and inflammatory processes at the site of injury, and at the level of phrenic motoneurons. In the present study, we evaluated the antioxidant response in phrenic motoneurons involving the AMPK-Nrf2 signaling pathway following C2 spinal cord lateral hemi-section in rats. We showed that there is an abrupt reduction in the expression of phosphorylated AMPK and Nrf2 at one hour post-injury in phrenic motoneurons. A rebound is then observed at one day post-injury, reflecting a return to homeostasis condition. In the total spinal cord around phrenic motoneurons, the increase in phosphorylated AMPK and Nrf2 occurred at three days post-injury, showing the differential antioxidant response between phrenic motoneurons and other cell types. Taken together, our results display the implication of the AMPK-Nrf2 signaling pathway in phrenic motoneurons’ response to oxidative stress following high SCI. Harnessing this AMPK-Nrf2 signaling pathway could improve the antioxidant response and help in spinal rewiring to these deafferented phrenic motoneurons to improve diaphragm activity in patients suffering high SCI.

International audience; High spinal cord injuries (SCI) lead to permanent respiratory insufficiency, and the search for new therapeutics to restore this function is essential. To date, the most documented preclinical model for high SCI is the rat cervical C2 hemisection. However, molecular studies with this SCI model are limited due to the poor availability of genetically modified specimens. The aim of this work was to evaluate the pathophysiology of respiratory activity following a cervical C2 injury at different times post-injury in a C57BL/6 mouse model. No significant spontaneous recovery of diaphragmatic activity was observed up to 30 days post-injury in eupneic condition. However, during a respiratory challenge, i.e. mild asphyxia, a partial restoration of the injured diaphragm was observed at 7 days post-injury, corresponding to the crossed phrenic phenomenon. Interestingly, the diaphragmatic recording between 2 respiratory bursts on the injured side showed an amplitude increase between 1–7 days post-injury, reflecting a change in phrenic motoneuronal excitability. This increase in inter-burst excitability returned to pre-injured values when the crossed phrenic phenomenon started to be effective at 7 days post-injury. Taken together, these results demonstrate the ability of the mouse respiratory system to express long-lasting plasticity following a C2 cervical hemisection and genetically modified animals can be used to study the pathophysiological effects on these plasticity phenomena.

The innate immune system, including phagocytic cells, is the first line of defense against pathogens. During infection by microorganisms such as viruses, bacteria, or parasites, phagocytic cells produce an excess of oxidants, a crucial process for the clearance of pathogens. This increase in oxidants creates an imbalance between oxidants and endogenous antioxidants. Left unchecked, this acute or chronic oxidative stress can lead to apoptotic cell-death and oxidative stress-induced diseases including neurodegenerative and cardiovascular disorders, premature aging, secondary infections, and cancer. The activation of nuclear factor E2-related factor 2 (Nrf2) is an efficient antioxidant defensive mechanism used by host cells to counteract oxidative stress. The transcription factor Nrf2 has been identified as the master regulator of several hundred of genes involved in the antioxidant defense response. The review objectives were to collect recent findings on the contribution of oxidative stress to complications of infection, and to highlight the beneficial impact of antioxidants in reducing inflammation and oxidant-related tissue damage. Furthermore, a direct relationship between infection and decline in Nrf2 activity has been demonstrated. Thus, an interesting therapeutic approach in disease prevention and treatment of stress-related diseases may consist in optimizing antibiotic or antiviral therapy with a combination of Nrf2 inducer treatment.

Keratin 19 is a member of the cytokeratin family that is critical for maintenance of cellular architecture and organization, especially of epithelia. The pancreas has three distinct cell types, ductal, acinar, and islet, each with different functions. Embryologically, the pancreatic and duodenal homeobox 1 (PDX1) homeodomain protein is critical for the initiation of all pancreatic lineages; however, the later differentiation of the endocrine pancreas is uniquely dependent upon high PDX1 expression, whereas PDX1 is down-regulated in the ductal and acinar cell lineages. We find that this down-regulation may be required for normal ductal expression of cytokeratin K19. The K19 promoter-reporter gene assay demonstrates that ectopic PDX1 inhibits K19 reporter gene activity in primary pancreatic ductal cells. This is reinforced by our findings that retrovirally mediated stable transduction of PDX1 in primary pancreatic ductal cells suppresses K19 expression, and short interfering RNA to PDX1 in Min6 insulinoma cells results in the induction of normally undetectable K19. Complementary functional and biochemical approaches led to the unexpected finding that a multimeric complex of PDX1 and two members of the TALE homeodomain factor family, MEIS1a and PBX1b, regulates K19 gene transcription through a specific cis-regulatory element (-341 to -325) upstream of the K19 transcription start site. These data suggest a unifying mechanism whereby PDX1, myeloid ecotropic viral insertion site (MEIS), and pre-B-cell leukemia transcription factor 1 (PBX) may regulate ductal and acinar lineage specification during pancreatic development. Specifically, concomitant PDX1 suppression and MEIS isoform expression result in proper ductal and acinar lineage specification. Furthermore, PDX1 may inhibit the ductal differentiation program in the pancreatic endocrine compartment, particularly beta cells.

The genetic basis of pancreatic ductal adenocarcinoma, which constitutes the most common type of pancreatic malignancy, involves the sequential activation of oncogenes and inactivation of tumor suppressor genes. Among the pivotal genetic alterations are Ki-RAS oncogene activation and p53 tumor suppressor gene inactivation. We explain that the combination of these genetic events facilitates pancreatic carcinogenesis as revealed in novel three-dimensional cell (spheroid cyst) culture and in vivo subcutaneous and orthotopic xenotransplantation models. N-cadherin, a member of the classic cadherins important in the regulation of cell-cell adhesion, is induced in the presence of Ki-RAS mutation but subsequently downregulated with the acquisition of p53 mutation as revealed by gene microarrays and corroborated by reverse transcription-PCR and Western blotting. N-cadherin modulates the capacity of pancreatic ductal cells to migrate and invade, in part via complex formation with keratinocyte growth factor receptor and neural cell adhesion molecule and in part via interaction with p120-catenin. However, modulation of these complexes by Ki-RAS and p53 leads to enhanced cell migration and invasion. This preferentially induces the downstream effector AKT over mitogen-activated protein kinase to execute changes in cellular behavior. Thus, we are able to define molecules that in part are directly affected by Ki-RAS and p53 during pancreatic ductal carcinogenesis, and this provides a platform for potential new molecularly based therapeutic interventions.

Pancreatic ductal adenocarcinoma is the most common pancreatic neoplasm. There are approximately 33,000 new cases of pancreatic ductal adenocarcinoma annually in the United States with approximately the same number of deaths. Surgery represents the only opportunity for cure, but this is restricted to early stage pancreatic cancer. Pancreatic ductal adenocarcinoma evolves from a progressive cascade of cellular, morphological and architectural changes from normal ductal epithelium through preneoplastic lesions termed pancreatic intraepithelial neoplasia (PanIN). These PanIN lesions are in turn associated with somatic alterations in canonical oncogenes and tumor suppressor genes. Most notably, early PanIN lesions and almost all pancreatic ductal adenocarcinomas involve mutations in the K-ras oncogene. Thus, it is believed that activating K-ras mutations are critical for initiation of pancreatic ductal carcinogenesis. This has been proven through elegant genetically engineered mouse models in which a Cre-activated K-RasG12D allele is knocked into the endogenous K-Ras locus and crossed with mice expressing Cre recombinase in pancreatic tissue. As a result, mechanistic insights are now possible into how K-Ras contributes to pancreatic ductal carcinogenesis, what cooperating events are required, and armed with this knowledge, new therapeutic approaches can be pursued and tested.

SummaryTo define the genetic requirements for pancreatic ductal adenocarcinoma (PDA), we have targeted concomitant endogenous expression of Trp53R172H and KrasG12D to the mouse pancreas, revealing the cooperative development of invasive and widely metastatic carcinoma that recapitulates the human disease. The primary carcinomas and metastases demonstrate a high degree of genomic instability manifested by nonreciprocal translocations without obvious telomere erosion—hallmarks of human carcinomas not typically observed in mice. No mutations were discovered in other cardinal tumor suppressor gene pathways, which, together with previous results, suggests that there are distinct genetic pathways to PDA with different biological behaviors. These findings have clear implications for understanding mechanisms of disease pathogenesis, and for the development of detection and targeted treatment strategies.

A cervical spinal cord injury induces permanent paralysis, and often leads to respiratory distress. To date, no efficient therapeutics have been developed to improve/ameliorate the respiratory failure following high cervical spinal cord injury (SCI). Here we propose a murine pre-clinical model of high SCI at the cervical 2 (C2) metameric level to study diverse post-lesional respiratory neuroplasticity. The technique consists of a surgical partial injury at the C2 level, which will induce a hemiparalysis of the diaphragm due to a deafferentation of the phrenic motoneurons from the respiratory centers located in the brainstem. The contralateral side of the injury remains intact and allows the animal recovery. Unlike other SCIs which affect the locomotor function (at the thoracic and lumbar level), the respiratory function does not require animal motivation and the quantification of the deficit/recovery can be easily performed (diaphragm and phrenic nerve recordings, whole body ventilation). This pre-clinical C2 SCI model is a powerful, useful, and reliable pre-clinical model to study various respiratory and non-respiratory neuroplasticity events at different levels (molecular to physiology) and to test diverse putative therapeutic strategies which might improve the respiration in SCI patients.

Predicting the toxic potential of compounds to the ocular surface has depended on the Draize test for the past half century. Alternatives to Draize testing have recently been sought for a number of reasons. Stress gene expression has emerged as a means of quantifying cellular reaction and, thus, the toxic potential of the compound in question. This study examines the expression of the major stress response gene heme oxygenase-1 (HO-1) in a human corneal epithelial cell line (HCE-T) following challenges with a number of known ocular irritants.HCE-T was used to investigate the effect of ocular irritants on cell viability and HO-1 expression. Irritants tested included hydrogen peroxide, isopropyl alcohol, sodium hydroxide and trichloroacetic acid. HCE-T cells were grown to 80% confluency and treated with the listed irritants at a concentration range of 10-100 microM. Cell viability and northern blot analysis were performed following a 24 and 48 hr incubation period.HCE-T cells expressed HO-1 mRNA and HO activity similar to other human cell lines. Northern blot analysis demonstrated that levels of HO-1 mRNA transcripts increased regularly after exposure to the irritants in a concentration-dependent manner. Studies on the effect of various inhibitors and inducers of HO-1 on cell viability showed that inhibition of HO-1 potentiates the cytotoxic effect of ocular irritants. In contrast, pre-induction of HO-1 in HCE-T decreases the effect of various irritants on cell viability.These results are consistent with the idea that HO-1 mRNA levels may be used as an indicator of toxicity resulting from ocular irritants and that HCE-T cells respond to stress in a fashion similar to other human cell lines. This strategy for testing may be important in the development of an alternative to Draize testing. The results of this study also suggest that HO-1 may constitute a part of the protective defense mechanism against chemical injury.

Pancreatic ductal adenocarcinoma (PDA) is an extremely aggressive malignancy, which carries a dismal prognosis. Activating mutations of the Kras gene are common to the vast majority of human PDA. In addition, recent studies have demonstrated that embryonic signaling pathway such as Hedgehog and Notch are inappropriately upregulated in this disease. The role of another embryonic signaling pathway, namely the canonical Wnt cascade, is still controversial. Here, we use gene array analysis as a platform to demonstrate general activation of the canonical arm of the Wnt pathway in human PDA. Furthermore, we provide evidence for Wnt activation in mouse models of pancreatic cancer. Our results also indicate that Wnt signaling might be activated downstream of Hedgehog signaling, which is an early event in PDA evolution. Wnt inhibition blocked proliferation and induced apoptosis of cultured adenocarcinoma cells, thereby providing evidence to support the development of novel therapeutical strategies for Wnt inhibition in pancreatic adenocarcinoma.

In a recent issue of Cell Death and Discovery, we described a new mechanism by which sulforaphane decreased mycobacterial burden in an in vitro model of THP-1-derived macrophage infection by Mycobacterium abscessus.1 This sulforaphane-mediated diminution was due to the induction of a caspase-independent cell apoptosis and necessitated activation of both nuclear factor E2-related factor 2 (Nrf2) and p38 mitogen-activated protein kinases (MAPK) signaling pathways. M. abscessus is a nontuberculous mycobacterium that belongs to the group of rapid growing mycobacteria. This emerging pathogen is able to cause skin, bone and soft tissue infections, and more generally exacerbations of lung diseases.2 Its high tolerance to antibiotics, which limits patient treatment, has become a public health concern worldwide. M. abscessus has been increasingly involved in patients with cystic fibrosis and in immunosuppressed patients,3 enhancing their risk of developing chronic airway infections and leading to a fatal outcome. Bacterial infection induces an imbalance between oxidants and antioxidants, triggering an oxidative burst that is well known for its bactericidal effect. Yet, some selected mycobacteria are able to thrive in this oxidative environment. It is then unsurprising that the use of oxidant scavengers such as MnTE-2-PyP or N-acetyl-L-cysteine has been shown to decrease M. abscessus load by activating bacterial killing in phagolysosomes.4 In our study, we decreased the oxidative environment in THP-1-derived macrophages by activating Nrf2, the key transcription factor that controls the cascade of cytoprotective and antioxidant defense mechanisms, and the maintenance of the redox homeostasis. Upon oxidative stress or infection, cytoplasmic Nrf2 is released from its inhibitor Keap-1, translocates into the nucleus, and forms a cofactor complex that binds to specific antioxidant responsive elements (ARE) found in promoters of phase II antioxidant and detoxifying enzymes. Nrf2 signaling pathway has been shown to have an important role whether beneficial or detrimental, in microbial infections.5 M. abscessus infection of THP-1-derived macrophages activates Nrf2 signaling pathway, and induces the expression of heme oxygenase-1 and NADPH quinone oxidoreductase-1, two downstream targets of Nrf2. In addition, M. abscessus induced reactive oxygen species (ROS) production in infected macrophages and blocked phagosomal acidification thus allowing its intracellular proliferation. Sulforaphane, a well-established activator of Nrf2 was used to pretreat macrophages before mycobacterial infection. The results showed an inhibition of mycobacterial growth 7 days postinfection in sulforaphane pretreated macrophages compared with vehicle pretreated ones. This sulforaphane-induced mycobacterial growth inhibition was neither caused by phagosomal pH acidification nor increase in cell phagocytosis, but rather induction of cell apoptosis. Contrary to the well-known cyprotective effect of sulforaphane and Nrf2 signaling pathway,6 we demonstrated that pre-stimulation of Nrf2 signaling pathway by sulforaphane before M. abscessus infection triggered a caspase-independent cell apoptosis. Pretreatment of macrophages with sulforaphane alone had no significant effect on cell death. To our knowledge, this is the first study showing sulforaphane-induced Nrf2 triggers a caspase3/7-independent and p38 MAPK-dependent cell apoptosis in mycobacteria infected macrophages (Figure 1). Figure 1 Infection of THP-1 derived macrophages by M. abscessus elicits caspase3/7-dependent cell apoptosis. Pretreatment of macrophages with an Nrf2 activator before mycobacterial challenge initiates a caspase-independent cell apoptosis Cell death in macrophages has a critical role in the host response to mycobacterial infection. Cell apoptosis is an effective mechanism used by the innate defense system to fight bacterial infection. This energy-dependent mechanism generates apoptotic bodies that will facilitate T-dependent antigen response and induce mycobacterial killing by uninfected neighboring macrophages.7 As M. abscessus has only been recently identified as a separate species, very little is known in the dynamics between M. abscessus and macrophage cell survival/cell death. In our in vitro model, M. abscessus induced cell necrosis in untreated cells but not cell apoptosis. This phenomenon has been observed in M. tuberculosis that has the ability to inhibit cell apoptosis in favor of cell necrosis.8 Recent highlights on caspase-independent forms of apoptosis triggered by infection have been described in the literature. Necroptosis, also known as programmed necrosis, can be initiated by various agents including microbial infection, and is characterized by activation of the receptor-interacting protein (RIP) kinases RIP1 and RIP3-dependent signaling pathway. This programmed cell death is a critical antiviral process in the host innate immune defense system. Phagocytes infected with viruses that are able to inhibit the classical caspase-dependent apoptosis pathway can undergo necroptosis to facilitate viral clearance by the neighboring immune system.9, 10 Moreover, Roca and Ramakrishnan have demonstrated a tumor-necrosis factor (TNF)-induced necroptosis in a M. tuberculosis infected zebrafish model. In the early stages of mycobacteria infection, injection of an excess of TNF in infected macrophages induced mitochondrial ROS production through a RIP1/RIP3-dependent kinase pathway, process necessary for the bacterial clearance. However, ROS-mediated necroptosis rapidly led to release and propagation of M. tuberculosis in the extracellular microenvironment.11 Another caspase 3/7-independent apoptosis is the inflammation-mediated cell death known as pyroptosis. This particular programmed cell death participates in the host cell defense system against pathogens and requires activation of caspase 1, IL-1β and IL-18.12 Recently, Bai et al.13 have shown that M. tuberculosis-mediated production of IL-32γ in THP-1-derived macrophages induced caspase 1-dependent pyroptosis which help reduce intracellular M. tuberculosis burden. In conclusion, additional studies are required to specifically identify the caspase-independent form(s) of apoptosis initiated by the combination of Nrf2 activation and M. abscessus infection. Nevertheless, our findings strongly suggest that Nrf2 activators may be of great interest as future therapeutic treatments in supplement to the actual lengthy multi-drug therapies used in patients diagnosed with mycobacterial infection.

Background & Aims: The Ki-RAS oncogene is altered in pancreatic ductal neoplasms. Pancreatic ductal cells (PDCs) were purified from cytokeratin 19 (K19)- Ki-RAS G12V transgenic mice and control littermates to identify properties of Ki-Ras activation in a cell-type-specific context. Because Ki-RAS mutation has prognostic significance in patients treated with radiation, we studied the influence of Ki-RAS status on radiation survival. Methods: Pancreatic ductal fragments from mice with Ki-RAS G12V mutation or wild-type (WT)- Ki-RAS were cultured. Growth curves, electron microscopy, flow cytometry, and analysis of signaling and cell-cycle proteins were established. Farnesyltransferase inhibitor (FTI) treatment with R115777 before and after irradiation was used to determine the effect of Ki-Ras farnesylation on cell survival. Results: PDCs from WT and K19-Ki-RAS G12V mice had features of ductal cells with formation of 3-dimensional structures on collagen without differences in morphology, growth, and cell-cycle distribution. This may result from up-regulation of p16INK4 and p27 Kip1 and lack of hyperstimulation of the mitogen-activated protein kinase pathway in Ki-RAS G12V PDCs. No differences in radiation survival between Ki-RAS G12V PDCs and WT PDCs were observed. However, Ki-RAS G12V PDCs expressing mutant p53 V143A had enhanced survival compared with WT PDCs transduced with p53 V143A . R115777 treatment sensitized Ki-RAS G12V PDCs and Ki-RAS G12V /p53 V143A PDCs, but not WT PDCs. Conclusions: Novel characterization of murine WT PDCs and Ki-RAS G12V PDCs is described. Induction of cell-cycle regulators and lack of mitogen-activated protein kinase hyperstimulation likely are responsible for constraining activated Ki-RAS G12V -mediated proliferation. Because its activation was required for sensitization by an FTI, R115777 may be useful against pancreatic tumors expressing oncogenic Ki-Ras.

A frequent genetic alteration found in premalignant stages of pancreatic adenocarcinoma is K-ras oncogene point mutation. The mechanistic basis for the inability of K-ras mutation to transform pancreatic ductal cells is unclear, although cooperating events with p16 inactivation, p53 mutation, and SMAD 4 mutation are recognized to be necessary. We have generated a novel mouse model in which the cytokeratin 19 promoter, specifically active in pancreatic ductal cells but not other cell types of the pancreas, is fused to mutant K-ras. This is of direct relevance to human pancreatic cancer because premalignant lesions are found specifically in ductal cells. There is dramatic periductal lymphocytic infiltration in the pancreata of transgenic mice, predominantly CD4+ T lymphocytes, which may act as an adaptive immune response to activated ras-mediated signaling. In addition, gene array analysis reveals an induction of N-cadherin in transgenic mice pancreatic ductal cells, the significance of which relates to promotion of cell adhesion and deterrence of cell migration. Apart from these important biological considerations, there is parallel activity of the cytokeratin 19 promoter in the stem cell region of the gastric epithelium, namely in mucous neck cells. Activated K-ras in this context causes mucous neck cell hyperplasia, a precursor to gastric adenocarcinoma. There is concomitant parietal cell decrease, which is a key step toward gastric adenocarcinoma. Taken together, we have defined how mutant K-ras signaling modulates important molecular events in the initiating events of pancreatic and gastric carcinogenesis.

Heme oxygenase-1 (HO-1) is a stress protein, and its induction has been suggested to participate in defense mechanisms against agents that promote oxidative injury such as endotoxins and heme. We have shown that the inflammatory cytokines, interleukin-6 (IL-6) and heme-induced HO-1 gene expression, were suppressed by dexamethasone (Dex) in a sustained manner. We examined the mechanism by which the anti-inflammatory agent, Dex, inhibits IL-6 and heme-induced HO-1 expression in rabbit coronary endothelial cells. Endothelial cells treated with heme (10 microM) and IL-6 (25 ng/ml), increased HO-1 mRNA 15- and 60-fold, respectively. The activity of HO was increased 3-fold after such treatment. Although Dex failed to inhibit heme-mediated HO-1 mRNA and HO activity, it was able to reverse IL-6-stimulated HO activity. Several human HO-1 promoter-drive chloramphenicol acetyltransferase (CAT) constructs were examined to analyze IL-6 and Dex-mediated modulation of the HO-1 gene in endothelial cells. CAT assays revealed that the HO-1 promoter region between -180 and -1500 might contain a Dex-mediated negative regulator. Gel mobility shift assays using nuclear extracts from IL-6-treated endothelial cells showed a binding to the synthetic 21 base pairs of the HO-1 sequence that contains the putative STAT3 sequence. STAT3-specific probe inhibited nuclear binding protein to the putative HO-1-STAT3 sequence. This suggests that IL-6 induction of human HO-1 is mediated via the JAK-STAT pathway and that Dex inhibition of gene expression is carried out by activation of a transcriptional protein in competition with the STAT3 binding site.