Impact of fluoroquinolones and aminoglycosides on P. aeruginosa virulence factor production and cytotoxicity.

The opportunistic pathogen Pseudomonas aeruginosa is one of leading causes of disability and mortality worldwide and the world health organisation has listed it with the highest priority for the need of new antimicrobial therapies. P. aeruginosa strains responsible for the poorest clinical outcomes express either ExoS or ExoU, which are injected into target host cells via the type III secretion system (T3SS). ExoS is a bifunctional cytotoxin that promotes intracellular survival of invasive P. aeruginosa by preventing targeting of the bacteria to acidified intracellular compartments. ExoU is a phospholipase which causes destruction of host cell plasma membranes, leading to acute tissue damage and bacterial dissemination. Fluoroquinolones are usually employed as a first line of therapy as they have been shown to be more active against P. aeruginosa in vitrothan other antimicrobial classes. Their overuse over the past decade, however, has resulted in the emergence of antibiotic resistance. In certain clinical situations, aminoglycosides have been shown to be more effective then fluoroquinolones, despite their reduced potency towards P. aeruginosa in vitro. In this study, we evaluated the effects of fluoroquinolones (moxifloxacin and ciprofloxacin) and aminoglycosides (tobramycin and gentamycin) on T3SS expression and toxicity, in corneal epithelial cell infection models. We discovered that tobramycin disrupted T3SS expression and reduced both ExoS and ExoU mediated cytotoxicity, protecting infected HCE-t cells at concentrations below the minimal inhibitory concentration (MIC). The fluoroquinolones moxifloxacin and ciprofloxacin, however, up-regulated the T3SS and did not inhibit and may have increased the cytotoxic effects of ExoS and ExoU.

Aniridia-related keratopathy relevant cell signaling pathways in human fetal corneas.

We aimed to study aniridia-related keratopathy (ARK) relevant cell signaling pathways [Notch1, Wnt/ß-catenin, Sonic hedgehog (SHH) and mTOR] in normal human fetal corneas compared with normal human adult corneas and ARK corneas. We found that fetal corneas at 20 weeks of gestation (wg) and normal adult corneas showed similar staining patterns for Notch1; however 10-11 wg fetal corneas showed increased presence of Notch1. Numb and Dlk1 had an enhanced presence in the fetal corneas compared with the adult corneas. Fetal corneas showed stronger immunolabeling with antibodies against ß-catenin, Wnt5a, Wnt7a, Gli1, Hes1, p-rpS6, and mTOR when compared with the adult corneas. Gene expression of Notch1, Wnt5A, Wnt7A, ß-catenin, Hes1, mTOR, and rps6 was higher in the 9-12 wg fetal corneas compared with adult corneas. The cell signaling pathway differences found between human fetal and adult corneas were similar to those previously found in ARK corneas with the exception of Notch1. Analogous profiles of cell signaling pathway activation between human fetal corneas and ARK corneas suggests that there is a less differentiated host milieu in ARK.

Acetylcholine decreases formation of myofibroblasts and excessive extracellular matrix production in an in vitro human corneal fibrosis model.

Acetylcholine (ACh) has been reported to play various physiological roles, including wound healing in the cornea. Here, we study the role of ACh in the transition of corneal fibroblasts into myofibroblasts, and in consequence its role in the onset of fibrosis, in an in vitro human corneal fibrosis model. Primary human keratocytes were obtained from healthy corneas. Vitamin C (VitC) and transforming growth factor-ß1 (TGF-ß1) were used to induce fibrosis in corneal fibroblasts. qRT-PCR and ELISA analyses showed that gene expression and production of collagen I, collagen III, collagen V, lumican, fibronectin (FN) and alpha-smooth muscle actin (α-SMA) were reduced by ACh in quiescent keratocytes. ACh treatment furthermore decreased gene expression and production of collagen I, collagen III, collagen V, lumican, FN and α-SMA during the transition of corneal fibroblasts into myofibroblasts, after induction of fibrotic process. ACh inhibited corneal fibroblasts from developing contractile activity during the process of fibrosis, as assessed with collagen gel contraction assay. Moreover, the effect of ACh was dependent on activation of muscarinic ACh receptors. These results show that ACh has an anti-fibrotic effect in an in vitro human corneal fibrosis model, as it negatively affects the transition of corneal fibroblasts into myofibroblasts. Therefore, ACh might play a role in the onset of fibrosis in the corneal stroma.

Substance P induces fibrotic changes through activation of the RhoA/ROCK pathway in an in vitro human corneal fibrosis model.

Fibrosis is characterized by hardening, overgrowth, and development of scars in various tissues as a result of faulty reparative processes, diseases, or chronic inflammation. During the fibrotic process in the corneal stroma of the eye, the resident cells called keratocytes differentiate into myofibroblasts, specialized contractile fibroblastic cells that produce excessive amounts of disorganized extracellular matrix (ECM) and pro-fibrotic components such as alpha-smooth muscle actin (α-SMA) and fibronectin. This study aimed to elucidate the role of substance P (SP), a neuropeptide that has been shown to be involved in corneal wound healing, in ECM production and fibrotic markers expression in quiescent human keratocytes, and during the onset of fibrosis in corneal fibroblasts, in an in vitro human corneal fibrosis model. We report that SP induces keratocyte contraction and upregulates gene expression of collagens I, III, and V, and fibrotic markers: α-SMA and fibronectin, in keratocytes. Using our in vitro human corneal fibrosis model, we show that SP enhances gene expression and secretion of collagens I, III, and V, and lumican. Moreover, SP upregulates gene expression and secretion of α-SMA and fibronectin, and increases contractility of corneal fibroblasts during the onset of fibrosis. Activation of the preferred SP receptor, the neurokinin-1 receptor (NK-1R), is necessary for the SP-induced pro-fibrotic changes. In addition, SP induces the pro-fibrotic changes through activation of the RhoA/ROCK pathway. Taken together, we show that SP has a pro-fibrotic effect in both quiescent human keratocytes and during the onset of fibrosis in an in vitro human corneal fibrosis model.

Antiapoptotic Effect of Acetylcholine in Fas-Induced Apoptosis in Human Keratocytes.

PURPOSE: To investigate the possible antiapoptotic effect of acetylcholine (ACh) in Fas-mediated apoptosis of primary human keratocytes in vitro, and to explore the underlying mechanism. METHODS: Primary human keratocytes were isolated from healthy corneas. Fas ligand (FasL) was used to induce apoptosis in keratocytes. Cell death was assessed by ELISA. Activity of caspase-3, -7, -8, and -9 was measured with luminescent caspase activity assays. Expression of nuclear factor-κB (NF-κB) gene was assessed with RT-quantitative (q)PCR. Cytochrome c release apoptosis assay kit was used to extract mitochondria and cytosol. Cytochrome c release, cleavage of Bid, and expression of B-cell lymphoma 2 (Bcl-2) were determined by Western blot. RESULTS: Cell death ELISA revealed that ACh is able to reduce Fas-induced apoptosis in keratocytes. Analysis of the activity of effector caspases-3 and -7 showed that ACh, when added to Fas-treated cells, decreases the activation of both these enzymes. The activity of initiator caspases -8 and -9 also decreased when ACh was added to Fas-treated cells. This antiapoptotic effect of ACh was dependent on ACh concentration and activation of muscarinic ACh receptors. Analysis of the antiapoptotic mechanisms triggered by ACh showed that ACh downregulates expression of FasL-induced NF-κB RNA expression, upregulates expression of antiapoptotic protein Bcl-2, downregulates expression of proapoptotic protein Bad, reduces cytochrome c release, and prevents proapoptotic Bid protein cleavage. CONCLUSIONS: Acetylcholine has an antiapoptotic effect in a Fas-apoptosis model of human primary keratocytes in vitro. It is therefore possible that ACh may play a role in corneal wound healing, by modulating its initiation phase.

Loss of the DNA Damage Repair Kinase ATM Impairs Inflammasome-Dependent Anti-Bacterial Innate Immunity.

The ATM kinase is a central component of the DNA damage repair machinery and redox balance. ATM dysfunction results in the multisystem disease ataxia-telangiectasia (AT). A major cause of mortality in AT is respiratory bacterial infections. Whether ATM deficiency causes innate immune defects that might contribute to bacterial infections is not known. Here we have shown that loss of ATM impairs inflammasome-dependent anti-bacterial innate immunity. Cells from AT patients or Atm(-/-) mice exhibited diminished interleukin-1ß (IL-1ß) production in response to bacteria. In vivo, Atm(-/-) mice were more susceptible to pulmonary S. pneumoniae infection in a manner consistent with inflammasome defects. Our data indicate that such defects were due to oxidative inhibition of inflammasome complex assembly. This study reveals an unanticipated function of reactive oxygen species (ROS) in negative regulation of inflammasomes and proposes a theory for the notable susceptibility of AT patients to pulmonary bacterial infection.

Substance P Enhances Keratocyte Migration and Neutrophil Recruitment through Interleukin-8.

Keratocytes, the resident cells of the corneal stroma, are responsible for maintaining turnover of this tissue by synthesizing extracellular matrix components. When the cornea is injured, the keratocytes migrate to the wounded site and participate in the stromal wound healing. The neuropeptide substance P (SP), which is also known to be produced by non-neuronal cells, has previously been implicated in epithelial wound healing after corneal injury. Corneal scarring, which occurs in the stroma when the process of wound healing has malfunctioned, is one of the major causes of preventable blindness. This study aimed to elucidate the potential role of SP in keratocyte migration and therefore in stromal wound healing. We report that the expression and secretion of SP in human keratocytes are increased in response to injury in vitro. Moreover, SP enhances the migration of keratocytes by inducing the actin cytoskeleton reorganization and focal adhesion formation through the activation of the phosphatidylinositide 3-kinase and Ras-related C3 botulinum toxin substrate 1/Ras homolog gene family, member A pathway. Furthermore, SP stimulation leads to upregulated expression of the proinflammatory and chemotactic cytokine interleukin-8 (IL-8), which also contributes significantly to SP-enhanced keratocyte migration and is able to attract neutrophils. In addition, the preferred SP receptor, the neurokinin-1 receptor, is necessary to induce keratocyte migration and IL-8 secretion. In conclusion, we describe new mechanisms by which SP enhances migration of keratocytes and recruits neutrophils, two necessary steps in the corneal wound-healing process, which are also likely to occur in other tissue injuries.

Transforming Growth Factor Beta 1 Modulates the Functional Expression of the Neurokinin-1 Receptor in Human Keratocytes.

PURPOSE: Transforming growth factor beta 1 (TGF-ß1) is a cytokine involved in a variety of processes, such as differentiation of fibroblasts into myofibroblasts. TGF-ß1 has also been shown to delay the internalization of the neurokinin-1 receptor (NK-1 R) after its activation by its ligand, the neuropeptide substance P (SP). NK-1 R comprises two naturally occurring variants, a full-length and a truncated form, triggering different cellular responses. SP has been shown to affect important events in the cornea - such as stimulating epithelial cell proliferation - processes that are involved in corneal wound healing and thus in maintaining the transparency of the corneal stroma. An impaired signaling through NK-1 R could thus impact the visual quality. We hypothesize that TGF-ß1 modulates the expression pattern of NK-1 R in human corneal stroma cells, keratocytes. The purpose of this study was to test that hypothesis. METHODS: Cultures of primary keratocytes were set up with cells derived from healthy human corneas, obtained from donated transplantation graft leftovers, and characterized by immunocytochemistry and Western blot. Immunocytochemistry for TGF-ß receptors and NK-1 R was performed. Gene expression was assessed with real-time polymerase chain reaction (qPCR). RESULTS: Expression of TGF-ß receptors was confirmed in keratocytes in vitro. Treating the cells with TGF-ß1 significantly reduced the gene expression of NK-1 R. Furthermore, immunocytochemistry for NK-1 R demonstrated that it is specifically the expression of the full-length isotype of the receptor that is reduced after treatment with TGF-ß1, which was also confirmed with qPCR using a specific probe for the full-length receptor. CONCLUSIONS: TGF-ß1 down-regulates the gene expression of the full-length variant of NK-1 R in human keratocytes, which might impact its signaling pathway and thus explain the known delay in internalization after activation by SP seen with TGF-ß1 treatment.

Expression Profiles of Neuropeptides, Neurotransmitters, and Their Receptors in Human Keratocytes In Vitro and In Situ.

Keratocytes, the quiescent cells of the corneal stroma, play a crucial role in corneal wound healing. Neuropeptides and neurotransmitters are usually associated with neuronal signaling, but have recently been shown to be produced also by non-neuronal cells and to be involved in many cellular processes. The aim of this study was to assess the endogenous intracellular and secreted levels of the neuropeptides substance P (SP) and neurokinin A (NKA), and of the neurotransmitters acetylcholine (ACh), catecholamines (adrenaline, noradrenaline and dopamine), and glutamate, as well as the expression profiles of their receptors, in human primary keratocytes in vitro and in keratocytes of human corneal tissue sections in situ. Cultured keratocytes expressed genes encoding for SP and NKA, and for catecholamine and glutamate synthesizing enzymes, as well as genes for neuropeptide, adrenergic and ACh (muscarinic) receptors. Keratocytes in culture produced SP, NKA, catecholamines, ACh, and glutamate, and expressed neurokinin-1 and -2 receptors (NK-1R and NK-2R), dopamine receptor D2, muscarinic ACh receptors, and NDMAR1 glutamate receptor. Human corneal sections expressed SP, NKA, NK-1R, NK-2R, receptor D2, choline acetyl transferase (ChAT), M3, M4 and M5 muscarinic ACh receptors, glutamate, and NMDAR1, but not catecholamine synthesizing enzyme or the α1 and ß2 adrenoreceptors, nor M1 receptor. In addition, expression profiles assumed significant differences between keratocytes from the peripheral cornea as compared to those from the central cornea, as well as differences between keratocytes cultured under various serum concentrations. In conclusion, human keratocytes express an array of neuropeptides and neurotransmitters. The cells furthermore express receptors for neuropeptides/neurotransmitters, which suggests that they are susceptible to stimulation by these substances in the cornea, whether of neuronal or non-neuronal origin. As it has been shown that neuropeptides/neurotransmitters are involved in cell proliferation, migration, and angiogenesis, it is possible that they play a role in corneal wound healing.

Acetylcholine enhances keratocyte proliferation through muscarinic receptor activation.

Acetylcholine (ACh), a classical neurotransmitter, has been shown to be present in various non-neuronal cells, including cells of the eye, such as corneal epithelium and endothelium, and to have widespread physiological effects such as cytoskeleton reorganization, cellular proliferation, differentiation, and apoptosis. The aim of this study was to investigate the effect of ACh on corneal keratocyte proliferation, and the underlying mechanisms, in order to explore its possible effect in corneal wound healing. Primary culture of human keratocytes was established from donated corneas. Cell viability and fraction of proliferating cells were detected by MTS assay and BrdU incorporation ELISA, respectively. Expression of proliferative markers, PCNA and Ki-67, was detected by western blot and immunocytochemistry. Activation of the MAPK/Erk signaling pathway and its involvement in ACh-enhanced proliferation was determined by western blot analysis, MTS, and BrdU ELISA. We found that ACh enhanced keratocyte proliferation even at low concentrations. Stimulation of proliferation was mediated through activation of muscarinic ACh receptors (mAChRs). Western blot analysis revealed that ACh stimulation of keratocytes upregulated the expression of PCNA and Ki-67, and Ki-67 immunocytochemistry showed that ACh-treated cells were in an active phase of the cell cycle. ACh activated MAPK signaling, and this step was crucial for the ACh-enhanced proliferation, as inhibition of the MAPK pathway resulted in ACh having no proliferative effect. In conclusion, ACh enhances keratocyte proliferation and might thus play a role in proper corneal wound healing.

Adenoviral E4orf3 and E4orf6 proteins, but not E1B55K, increase killing of cancer cells by radiotherapy in vivo.

PURPOSE: Radiotherapy is widely used for treatment of many tumor types, but it can damage normal tissues. It has been proposed that cancer cells can be selectively sensitized to radiation by adenovirus replication or by using radiosensitizing transgenes. Adenoviral proteins E1B55K, E4orf3, and E4orf6 play a role in radiosensitization, by targeting the Mre11, Rad50, and NBS1 complex (MRN) and inhibiting DNA double-strand break (DSB) repair. We hypothesize that combined with irradiation, these adenoviral proteins increase cell killing through the impairment of DSB repair. METHODS AND MATERIALS: We assessed the radiosensitizing/additive potential of replication-deficient adenoviruses expressing E1B55K, E4orf3, and E4orf6 proteins. Combination treatments with low-dose external photon beam radiotherapy were studied in prostate cancer (PC-3MM2 and DU-145), breast cancer (M4A4-LM3), and head and neck cancer (UT-SCC8) cell lines. We further demonstrated radiosensitizing or additive effects in mice with PC-3MM2 tumors. RESULTS: We show enhanced cell killing with adenovirus and radiation combination treatment. Co-infection with several of the viruses did not further increase cell killing, suggesting that both E4orf6 and E4orf3 are potent in MRN inhibition. Our results show that adenoviral proteins E4orf3 and E4orf6, but not E1B55K, are effective also in vivo. Enhanced cell killing was due to inhibition of DSB repair resulting in persistent double-strand DNA damage, indicated by elevated phospho-H2AX levels at 24 h after irradiation. CONCLUSIONS: This knowledge can be applied for improving the treatment of malignant tumors, such as prostate cancer, for development of more effective combination therapies and minimizing radiation doses and reducing side effects.

Targeted chemotherapy for head and neck cancer with a chimeric oncolytic adenovirus coding for bifunctional suicide protein FCU1.

PURPOSE: Transfer of prodrug activation systems into tumors by using replication-deficient viruses has been suggested to be an effective method for achieving high local and low systemic anticancer drug concentrations. However, most current suicide gene therapy strategies are still hindered by poor efficiency of in vivo gene transfer, inefficient tumor penetration, limited bystander cell killing effect, and need of large prodrug doses. We hypothesized that local amplification provided by a replication competent platform would help overcome these limitations. EXPERIMENTAL DESIGN: We generated a transductionally and transcriptionally targeted oncolytic adenovirus Ad5/3-Delta24FCU1 expressing the fusion suicide gene FCU1. FCU1 encodes a bifunctional fusion protein that efficiently catalyzes the direct conversion of 5-FC, a relatively nontoxic antifungal agent, into the toxic metabolites 5-fluorouracil and 5-fluorouridine monophosphate, bypassing the natural resistance of certain human tumor cells to 5-fluorouracil. RESULTS: We examined the efficacy of Ad5/3-Delta24FCU1 and the replication-defective control Ad5/3-FCU1 with and without 5-FC. FCU1 expression was confirmed by Western blot, whereas enzymatic conversion levels in vitro and in vivo were determined by high-performance liquid chromatography separation. Significant antitumor effect was observed in vitro and in vivo in a murine model of head and neck squamous cell carcinoma. Although we observed a decrease in viral DNA copy number in vitro and in tumors treated with Ad5/3-Delta24FCU1 and 5-FC, suggesting an effect on virus replication, the highest antitumor effect was observed for this combination. CONCLUSIONS: It seems feasible and efficacious to combine adenovirus replication to the FCU1 prodrug activation system.

Antiangiogenic arming of an oncolytic vaccinia virus enhances antitumor efficacy in renal cell cancer models.

Oncolytic vaccinia viruses have shown compelling results in preclinical cancer models and promising preliminary safety and antitumor activity in early clinical trials. However, to facilitate systemic application it would be useful to improve tumor targeting and antitumor efficacy further. Here we report the generation of vvdd-VEGFR-1-Ig, a targeted and armed oncolytic vaccinia virus. Tumor targeting was achieved by deletion of genes for thymidine kinase and vaccinia virus growth factor, which are necessary for replication in normal but not in cancer cells. Given the high vascularization typical of kidney cancers, we armed the virus with the soluble vascular endothelial growth factor (VEGF) receptor 1 protein for an antiangiogenic effect. Systemic application of high doses of vvdd-VEGFR-1-Ig resulted in cytokine induction in an immunocompromised mouse model. Upon histopathological analysis, splenic extramedullary hematopoiesis was seen in all virus-injected mice and was more pronounced in the vvdd-VEGFR-1-Ig group. Analysis of the innate immune response after intravenous virus injection revealed high transient and dose-dependent cytokine elevations. When medium and low doses were used for intratumoral or intravenous injection, vvdd-VEGFR-1-Ig exhibited a stronger antitumor effect than the unarmed control. Furthermore, expression of VEGFR-1-Ig was confirmed, and a concurrent antiangiogenic effect was seen. In an immunocompetent model, systemic vvdd-VEGFR-1-Ig exhibited superior antitumor efficacy compared to the unarmed control virus. In conclusion, the targeted and armed vvdd-VEGFR-1-Ig has promising anticancer activity in renal cell cancer models. Extramedullary hematopoiesis may be a sensitive indicator of vaccinia virus effects in mice.