Stephen J. Wood

Pro-inflammatory chemokine CCL2 (MCP-1) promotes healing in diabetic wounds by restoring the macrophage response.

Prior studies suggest that the impaired healing seen in diabetic wounds derives from a state of persistent hyper-inflammation characterized by harmful increases in inflammatory leukocytes including macrophages. However, such studies have focused on wounds at later time points (day 10 or older), and very little attention has been given to the dynamics of macrophage responses in diabetic wounds early after injury. Given the importance of macrophages for the process of healing, we studied the dynamics of macrophage response during early and late phases of healing in diabetic wounds. Here, we report that early after injury, the diabetic wound exhibits a significant delay in macrophage infiltration. The delay in the macrophage response in diabetic wounds results from reduced Chemokine (C-C motif) ligand 2 (CCL2) expression. Importantly, one-time treatment with chemoattractant CCL2 significantly stimulated healing in diabetic wounds by restoring the macrophage response. Our data demonstrate that, rather than a hyper-inflammatory state; the early diabetic wound exhibits a paradoxical and damaging decrease in essential macrophage response. Our studies suggest that the restoration of the proper kinetics of macrophage response may be able to jumpstart subsequent healing stages. CCL2 chemokine-based therapy may be an attractive strategy to promote healing in diabetic wounds.

An ApoE-Aβ inhibition complex in Aβ fibril extension

Abstract Background: Literature reports differ dramatically in showing that apolipoprotein E either facilitates or inhibits Aβ aggregate formation in vitro . Resolution of the nature of the ApoE-Aβ interaction is critical for progress towards understanding its possible role in the modulation of Alzheimers disease. Results: Here, we show that purified ApoE-Aβ co-aggregate is a poor seed of fibril formation. We also demonstrate ApoE inhibition of Aβ fibril growth in four independent aggregation assays, arguing that the poor fibril formation observed under these conditions is real and not an analytical artifact. We also directly show ApoE binding to immobilized Aβ fibrils by surface plasmon resonance. Conclusions: The results suggest a unifying model in which ApoE binds to Aβ fibril seeds and nascent nuclei to generate stable complexes that inhibit the rapid extension of mono-component Aβ fibrils but at the same time can foster continued slow growth of mixed ApoE-Aβ aggregates. In vivo co-aggregate formation may be important in many examples of pathological protein misassembly.

Chronic Alcohol Exposure Renders Epithelial Cells Vulnerable to Bacterial Infection

Despite two centuries of reports linking alcohol consumption with enhanced susceptibility to bacterial infections and in particular gut-derived bacteria, there have been no studies or model systems to assess the impact of long-term alcohol exposure on the ability of the epithelial barrier to withstand bacterial infection. It is well established that acute alcohol exposure leads to reduction in tight and adherens junctions, which in turn leads to increases in epithelial cellular permeability to bacterial products, leading to endotoxemia and a variety of deleterious effects in both rodents and human. We hypothesized that reduced fortification at junctional structures should also reduce the epithelial barrier’s capacity to maintain its integrity in the face of bacterial challenge thus rendering epithelial cells more vulnerable to infection. In this study, we established a cell-culture based model system for long-term alcohol exposure to assess the impact of chronic alcohol exposure on the ability of Caco-2 intestinal epithelial cells to withstand infection when facing pathogenic bacteria under the intact or wounded conditions. We report that daily treatment with 0.2% ethanol for two months rendered Caco-2 cells far more susceptible to wound damage and cytotoxicity caused by most but not all bacterial pathogens tested in our studies. Consistent with acute alcohol exposure, long-term ethanol exposure also adversely impacted tight junction structures, but in contrast, it did not affect the adherens junction. Finally, alcohol-treated cells partially regained their ability to withstand infection when ethanol treatment was ceased for two weeks, indicating that alcohol’s deleterious effects on cells may be reversible.

Cell migration regulates the kinetics of cytokinesis

Cytokinesis is the final stage of cell division in which the daughter cells separate. Although a growing body of evidence suggests that cell migration-induced traction forces may be required to provide physical assistance for daughter cells to dissociate during abscission, the role of cell migration in cytokinesis has not been directly elucidated. Recently, we have demonstrated that Crk and paxillin, which are pivotal components of the cell migration machinery, localize to the midbody and are essential for the abscission. These findings provided an important link between the cell migration and cytokinesis machineries and prompted us to dissect the role of cell migration in cytokinesis. We show that cell migration controls the kinetics of cleavage furrowing, midbody extension and abscission and coordinates proper subcellular redistribution of Crk and syntaxin-2 to the midbody after ingression.

Pseudomonas aeruginosa uses T3SS to inhibit diabetic wound healing.

Diabetic foot ulcers are responsible for more hospitalizations than any other complication of diabetes. Bacterial infection is recognized as an important factor associated with impaired healing in diabetic ulcers. Pseudomonas aeruginosa is the most frequently detected Gram‐negative pathogen in diabetic ulcers. P. aeruginosa infection has been shown to impair healing in diabetic wounds in a manner that correlates with its ability to form biofilm. While the majority of infections in diabetic ulcers are biofilm associated, 33% of infections are nonbiofilm in nature. P. aeruginosa is the most prevalent Gram‐negative pathogen in all diabetic wound types, which suggests that the deleterious impact of P. aeruginosa on healing in diabetic wounds goes beyond its ability to form biofilm and likely involves other factors. The Type III Secretion System (T3SS) virulence structure is required for the pathogenesis of all P. aeruginosa clinical isolates, suggesting that it may also play a role in the inhibition of wound repair in diabetic skin ulcers. We evaluated the role of T3SS in mediating P. aeruginosa–induced tissue damage in the wounds of diabetic mice. Our data demonstrate that P. aeruginosa establishes a robust and persistent infection in diabetic wounds independent of its ability to form biofilm and causes severe wound damage in a manner that primarily depends on its T3SS.

Pseudomonas aeruginosa ExoT Induces Atypical Anoikis Apoptosis in Target Host Cells by Transforming Crk Adaptor Protein into a Cytotoxin.

Previously, we demonstrated that Pseudomonas aeruginosa ExoT induces potent apoptosis in host epithelial cells in a manner that primarily depends on its ADP-ribosyltransferase domain (ADPRT) activity. However, the mechanism underlying ExoT/ADPRT-induced apoptosis remains undetermined. We now report that ExoT/ADPRT disrupts focal adhesion sites, activates p38β and JNK, and interferes with integrin-mediated survival signaling; causing atypical anoikis. We show that ExoT/ADPRT-induced anoikis is mediated by the Crk adaptor protein. We found that Crk-/- knockout cells are significantly more resistant to ExoT-induced apoptosis, while Crk-/- cells complemented with Crk are rendered sensitive to ExoT-induced apoptosis. Moreover, a dominant negative (DN) mutant form of Crk phenocopies ExoT-induced apoptosis both kinetically and mechanistically. Crk is generally believed to be a component of focal adhesion (FA) and its role in cellular survival remains controversial in that it has been found to be either pro-survival or pro-apoptosis. Our data demonstrate that although Crk is recruited to FA sites, its function is likely not required for FA assembly or for survival per se. However, when modified by ExoT or by mutagenesis, it can be transformed into a cytotoxin that induces anoikis by disrupting FA sites and interfering with integrin survival signaling. To our knowledge, this is the first example whereby a bacterial toxin exerts its cytotoxicity by subverting the function of an innocuous host cellular protein and turning it against the host cell.

Pseudomonas aeruginosa Exotoxin T induces potent cytotoxicity against a variety of murine and human cancer cell lines.

In patients with malignancy, the major barrier to achieving complete response is emergence of resistance to current chemotherapeutic agents. One of the major mechanisms by which tumour cells become resistant to therapies is by altering cellular drug targets through mutations and/or deletions. Resistance by this mechanism is achieved more easily if the drug has limited cellular targets and/or processes. We hypothesized that as Pseudomonas aeruginosa exotoxin T (ExoT) targets six proteins that are required for cancer cell survival and proliferation, it is highly unlikely for cancer cells to develop resistance to this toxin. We assessed ExoTs cytotoxicity against multiple invasive and highly resistant tumour cell lines in order to evaluate its potential as a chemotherapeutic agent. Our data demonstrated that ExoT induced potent cytotoxicity in all tumour cell lines that we examined. Collectively, our data highlighted the potential of ExoT as a possible chemotherapeutic candidate for the treatment of cancer.

Pseudomonas aeruginosa ExoT Induces Mitochondrial Apoptosis in Target Host Cells in a Manner That Depends on Its GTPase-activating Protein (GAP) Domain Activity

Background: The GAP domain of ExoT induces apoptosis in epithelial cells, but the mechanism underlying GAP-induced apoptosis remains unknown. Results: GAP domain activates JNK1/2, causes cytochrome c release, and activates caspase-9 and caspase-3. Conclusion: GAP domain of ExoT induces intrinsic apoptosis in epithelial cells. Significance: The GAP and the ADPRT domains make ExoT into a potent cytotoxin, capable of inducing different forms of apoptosis. Pseudomonas aeruginosa is the most common cause of hospital-acquired pneumonia and a killer of immunocompromised patients. We and others have demonstrated that the type III secretion system (T3SS) effector protein ExoT plays a pivotal role in facilitating P. aeruginosa pathogenesis. ExoT possesses an N-terminal GTPase-activating protein (GAP) domain and a C-terminal ADP-ribosyltransferase (ADPRT) domain. Because it targets multiple non-overlapping cellular targets, ExoT performs several distinct virulence functions for P. aeruginosa, including induction of apoptosis in a variety of target host cells. Both the ADPRT and the GAP domain activities contribute to ExoT-induced apoptosis. The ADPRT domain of ExoT induces atypical anoikis by transforming an innocuous cellular protein, Crk, into a cytotoxin, which interferes with integrin survival signaling. However, the mechanism underlying the GAP-induced apoptosis remains unknown. In this study, we demonstrate that the GAP domain activity is both necessary and sufficient to induce mitochondrial (intrinsic) apoptosis. We show that intoxication with GAP domain results in: (i) JNK1/2 activation; (ii) substantial increases in the mitochondrial levels of activated pro-apoptotic proteins Bax and Bid, and to a lesser extent Bim; (iii) loss of mitochondrial membrane potential and cytochrome c release; and (iv) activation of initiator caspase-9 and executioner caspase-3. Further, GAP-induced apoptosis is partially mediated by JNK1/2, but it is completely dependent on caspase-9 activity. Together, the ADPRT and the GAP domains make ExoT into a highly versatile and potent cytotoxin, capable of inducing multiple forms of apoptosis in target host cells.

A novel human antimicrobial factor targets Pseudomonas aeruginosa through its type III secretion system

Pseudomonas aeruginosa is an important opportunistic bacterial pathogen. Despite its metabolic and virulence versatility, it has not been shown to infect articular joints, which are areas that are rarely infected with bacteria in general. We hypothesized that articular joints possess antimicrobial activity that limits bacterial survival in these environments. We report that cartilages secrete a novel antimicrobial factor, henceforth referred to as the cartilage-associated antimicrobial factor (CA-AMF), with potent antimicrobial activity. Importantly, CA-AMF exhibited significantly more antimicrobial activity against P. aeruginosa strains with a functional type III secretion system (T3SS). We propose that CA-AMF represents a new class of human antimicrobial factors in innate immunity, one which has evolved to selectively target pathogenic bacteria among the beneficial and commensal microflora. The T3SS is the first example, to the best of our knowledge, of a pathogen-specific molecular target in this antimicrobial defence system.

Salvage of contaminated osteochondral allografts: the effects of chlorhexidine on human articular chondrocyte viability.

Background: Because chondrocyte viability is imperative for successful osteochondral allograft transplantation, sterilization techniques must provide antimicrobial effects with minimal cartilage toxicity. Chlorhexidine gluconate (CHG) is an effective disinfectant; however, its use with human articular cartilage requires further investigation. Purpose: To determine the maximal chlorhexidine concentration that does not affect chondrocyte viability in allografts and to determine whether this concentration effectively sterilizes contaminated osteoarticular grafts. Study Design: Controlled laboratory study. Methods: Osteochondral plugs were subjected to pulse lavage with 1-L solutions of 0.002%, 0.01%, 0.05%, and 0.25% CHG and cultured for 0, 1, 2, and 7 days in media of 10% fetal bovine serum and antibiotics. Chondrocyte viability was determined via LIVE/DEAD Viability Assay. Plugs were contaminated with Staphylococcus aureus and randomized to 4 treatment groups. One group was not contaminated; the 3 others were contaminated and received no treatment, saline pulse lavage, or saline pulse lavage with 0.002% CHG. Serial dilutions were plated and colony-forming units assessed. Results: The control group and the 0.002% CHG group showed similar cell viability, ranging from 67% ± 4% to 81% ± 22% (mean ± SD) at all time points. In the 0.01% CHG group, cell viability was reduced in comparison with control by 2-fold at day 2 and remained until day 7 (P < .01). The 0.05% and 0.25% CHG groups showed a 2-fold reduction in cell viability at day 1 (P < .01). At day 7, cell viability was reduced to 15% ± 18% (4-fold decrease) for the 0.05% CHG group and 10% ± 19% (6-fold decrease) for the 0.25% CHG group (P < .01). Contaminated grafts treated with 0.002% CHG demonstrated no colony-forming units. Conclusion: Pulse lavage with 0.002% CHG does not cause significant cell death within 7 days after exposure, while CHG at concentrations >0.002% significantly decreases chondrocyte viability within 1 to 2 days after exposure and should therefore not be used for disinfection of osteochondral allograft. Pulse lavage does not affect chondrocyte viability but cannot be used in isolation to sterilize contaminated fragments. Overall, 0.002% CHG was shown to effectively decontaminate osteoarticular fragments. Clinical Relevance: This study offers a scientific protocol for sterilizing osteochondral fragments that does not adversely affect cartilage viability.