Release the hens: a study on the complexities of guinea fowl as tick control.

Established tick control strategies often involve methods that can be damaging to existing environmental conditions or natural host ecology. To find more environmentally friendly methods, biological controls, like predators of ticks, have been suggested. There are natural predators of ticks, but most are generalists and not expected to control tick populations. Helmeted guinea fowl (Numida meleagris (L.) (Galliformes: Numididae)) have been suggested to be biological controls of ticks, and therefore, tick-borne pathogens, but their potential role as hosts for ticks complicates the relationship. A study was conducted to assess whether guinea fowl reduces the abundance of lone star ticks, Amblyomma americanum (L.) (Acari: Ixodidae), or whether they are hosts of ticks. Using mark-recapture techniques, painted lone star ticks were placed into 3 different treatments: penned, excluded, and free range. The recapture rates of painted ticks were compared. There was a significant difference between excluded and free-range treatments, but not between excluded and penned or between free range and penned. To investigate the role of guinea fowl as hosts of ticks, coop floors were examined for engorged ticks. Engorged lone star nymphs that had fed on guinea fowl were found. Lastly, ticks collected were tested to identify the potential reduction in risk of tick-borne pathogens. This study found no evidence that guinea fowl are an effective biological control of lone star ticks or tick-borne pathogens, but they are hosts of lone star nymphs. Future studies are needed to assess the complex ecology of a biological control of ticks that is also a host.

Identification of a broadly fibrogenic macrophage subset induced by type 3 inflammation.

Macrophages are central orchestrators of the tissue response to injury, with distinct macrophage activation states playing key roles in fibrosis progression and resolution. Identifying key macrophage populations found in human fibrotic tissues could lead to new treatments for fibrosis. Here, we used human liver and lung single-cell RNA sequencing datasets to identify a subset of CD9+TREM2+ macrophages that express SPP1, GPNMB, FABP5, and CD63. In both human and murine hepatic and pulmonary fibrosis, these macrophages were enriched at the outside edges of scarring and adjacent to activated mesenchymal cells. Neutrophils expressing MMP9, which participates in the activation of TGF-ß1, and the type 3 cytokines GM-CSF and IL-17A coclustered with these macrophages. In vitro, GM-CSF, IL-17A, and TGF-ß1 drive the differentiation of human monocytes into macrophages expressing scar-associated markers. Such differentiated cells could degrade collagen IV but not collagen I and promote TGF-ß1-induced collagen I deposition by activated mesenchymal cells. In murine models blocking GM-CSF, IL-17A or TGF-ß1 reduced scar-associated macrophage expansion and hepatic or pulmonary fibrosis. Our work identifies a highly specific macrophage population to which we assign a profibrotic role across species and tissues. It further provides a strategy for unbiased discovery, triage, and preclinical validation of therapeutic targets based on this fibrogenic macrophage population.

Bourbon Virus Transmission, New York, USA.

In July 2019, Bourbon virus RNA was detected in an Amblyomma americanum tick removed from a resident of Long Island, New York, USA. Tick infection and white-tailed deer (Odocoileus virginianus) serosurvey results demonstrate active transmission in New York, especially Suffolk County, emphasizing a need for surveillance anywhere A. americanum ticks are reported.

NLRP3 inflammasome activation in cigarette smoke priming for Pseudomonas aeruginosa-induced acute lung injury.

It is increasingly recognized that cigarette smoke (CS) exposure increases the incidence and severity of acute respiratory distress syndrome (ARDS) in critical ill humans and animals. However, the mechanism(s) is not well understood. This study aims to investigate mechanism underlying the priming effect of CS on Pseudomonas aeruginosa-triggered acute lung injury, by using pre-clinic animal models and genetically modified mice. We demonstrated that CS impaired P. aeruginosa-induced mitophagy flux, promoted p62 accumulation, and exacerbated P. aeruginosa-triggered mitochondrial damage and NLRP3 inflammasome activation in alveolar macrophages; an effect associated with increased acute lung injury and mortality. Pharmacological inhibition of caspase-1, a component of inflammasome, attenuated CS primed P. aeruginosa-triggered acute lung injury and improved animal survival. Global or myeloid-specific knockout of IL-1ß, a downstream component of inflammasome activation, also attenuated CS primed P. aeruginosa-triggered acute lung injury. Our results suggest that NLRP3 inflammasome activation is an important mechanism for CS primed P. aeruginosa-triggered acute lung injury. (total words: 155).

Best of Intentions: Influential Factors in Infant Feeding Intent among Marshallese Pregnant Women.

The purpose of this study is to explore the beliefs, intentions, and influences that serve as barriers and facilitators to exclusive breastfeeding intent among Marshallese pregnant women in the United States (US). The study used a descriptive qualitative design. In total, 36 Marshallese women in their third trimester of pregnancy participated. Participants described exclusive breastfeeding as the preferred method of infant feeding, from both individual and community perspectives. Exclusive breastfeeding was viewed as the healthiest for the infant, viewed as offering protection against sickness, and viewed as better for the overall development of the infant. Of the 36 participants, 28 participants (77.8%) stated that their infant feeding intentions included a hybrid of breastfeeding and formula feeding. The dominant barrier to exclusive breastfeeding was the need to work outside of the home. Unexpected barriers to exclusive breastfeeding were the desire for autonomy and a preference to exclusively breastfeed female infants more than male infants. Exclusive breastfeeding facilitators included support from the Special Supplemental Nutrition Program for Women, Infants, and Children and support and encouragement from female family/community members. This study is the first to document beliefs, intentions, and influences that serve as barriers and facilitators to exclusive breastfeeding among Marshallese pregnant women residing in the US.

Trends and Opportunities in Tick-Borne Disease Geography.

Tick-borne diseases are a growing problem in many parts of the world, and their surveillance and control touch on challenging issues in medical entomology, agricultural health, veterinary medicine, and biosecurity. Spatial approaches can be used to synthesize the data generated by integrative One Health surveillance systems, and help stakeholders, managers, and medical geographers understand the current and future distribution of risk. Here, we performed a systematic review of over 8,000 studies and identified a total of 303 scientific publications that map tick-borne diseases using data on vectors, pathogens, and hosts (including wildlife, livestock, and human cases). We find that the field is growing rapidly, with the major Ixodes-borne diseases (Lyme disease and tick-borne encephalitis in particular) giving way to monitoring efforts that encompass a broader range of threats. We find a tremendous diversity of methods used to map tick-borne disease, but also find major gaps: data on the enzootic cycle of tick-borne pathogens is severely underutilized, and mapping efforts are mostly limited to Europe and North America. We suggest that future work can readily apply available methods to track the distributions of tick-borne diseases in Africa and Asia, following a One Health approach that combines medical and veterinary surveillance for maximum impact.

Scoping review of distribution models for selected Amblyomma ticks and rickettsial group pathogens.

The rising prevalence of tick-borne diseases in humans in recent decades has called attention to the need for more information on geographic risk for public health planning. Species distribution models (SDMs) are an increasingly utilized method of constructing potential geographic ranges. There are many knowledge gaps in our understanding of risk of exposure to tick-borne pathogens, particularly for those in the rickettsial group. Here, we conducted a systematic scoping review of the SDM literature for rickettsial pathogens and tick vectors in the genus Amblyomma. Of the 174 reviewed articles, only 24 studies used SDMs to estimate the potential extent of vector and/or pathogen ranges. The majority of studies (79%) estimated only tick distributions using vector presence as a proxy for pathogen exposure. Studies were conducted at different scales and across multiple continents. Few studies undertook original data collection, and SDMs were mostly built with presence-only datasets from public database or surveillance sources. The reliance on existing data sources, using ticks as a proxy for disease risk, may simply reflect a lag in new data acquisition and a thorough understanding of the tick-pathogen ecology involved.

Exploring the Niche of Rickettsia montanensis (Rickettsiales: Rickettsiaceae) Infection of the American Dog Tick (Acari: Ixodidae), Using Multiple Species Distribution Model Approaches.

The American dog tick, Dermacentor variabilis (Say) (Acari: Ixodidae), is a vector for several human disease-causing pathogens such as tularemia, Rocky Mountain spotted fever, and the understudied spotted fever group rickettsiae (SFGR) infection caused by Rickettsia montanensis. It is important for public health planning and intervention to understand the distribution of this tick and pathogen encounter risk. Risk is often described in terms of vector distribution, but greatest risk may be concentrated where more vectors are positive for a given pathogen. When assessing species distributions, the choice of modeling framework and spatial layers used to make predictions are important. We first updated the modeled distribution of D. variabilis and R. montanensis using maximum entropy (MaxEnt), refining bioclimatic data inputs, and including soil variables. We then compared geospatial predictions from five species distribution modeling frameworks. In contrast to previous work, we additionally assessed whether the R. montanensis positive D. variabilis distribution is nested within a larger overall D. variabilis distribution, representing a fitness cost hypothesis. We found that 1) adding soil layers improved the accuracy of the MaxEnt model; 2) the predicted 'infected niche' was smaller than the overall predicted niche across all models; and 3) each model predicted different sizes of suitable niche, at different levels of probability. Importantly, the models were not directly comparable in output style, which could create confusion in interpretation when developing planning tools. The random forest (RF) model had the best measured validity and fit, suggesting it may be most appropriate to these data.

A Simple, Inexpensive Method for Mark-Recapture of Ixodid Ticks.

Mark-recapture techniques have been widely used and specialized to study organisms throughout the field of biology. To mark-recapture ticks (Ixodida), we have created a simple method to mark ticks using nail polish applied with an insect pin secured in a pencil that allows for a variety of questions to be answered. For measuring tick control efficacy, estimating population estimates, or measuring movement of ticks, this inexpensive mark-recapture method has been easily applied in the field and in the lab to provide useful data to answer a variety of questions about ticks.

New Antimicrobial Bioactivity against Multidrug-Resistant Gram-Positive Bacteria of Kinase Inhibitor IMD0354.

Multidrug-resistant pathogens pose a serious threat to human health. For decades, the antibiotic vancomycin has been a potent option when treating Gram-positive multidrug-resistant infections. Nonetheless, in recent decades, we have begun to see an increase in vancomycin-resistant bacteria. Here, we show that the nuclear factor-kappa B (NF-κB) inhibitor N-[3,5-Bis(trifluoromethyl)phenyl]-5-chloro-2-hydroxybenzamide (IMD0354) was identified as a positive hit through a Caenorhabditis elegans-methicillin-resistant Staphylococcus aureus (MRSA) infection screen. IMD0354 was a potent bacteriostatic drug capable of working at a minimal inhibitory concentration (MIC) as low as 0.06 µg/mL against various vancomycin-resistant strains. Interestingly, IMD0354 showed no hemolytic activity at concentrations as high as 16 µg/mL and is minimally toxic to C. elegans in vivo with 90% survival up to 64 µg/mL. In addition, we demonstrated that IMD0354's mechanism of action at high concentrations is membrane permeabilization. Lastly, we found that IMD0354 is able to inhibit vancomycin-resistant Staphylococcus aureus (VRSA) initial cell attachment and biofilm formation at sub-MIC levels and above. Our work highlights that the NF-κB inhibitor IMD0354 has promising potential as a lead compound and an antimicrobial therapeutic candidate capable of combating multidrug-resistant bacteria.

Mitochondrial Fission Mediated Cigarette Smoke-induced Pulmonary Endothelial Injury.

Cigarette smoke (CS) exposure increases the risk for acute respiratory distress syndrome in humans and promotes alveolar-capillary barrier permeability and acute lung injury in animal models. However, the underlying mechanisms are not well understood. Mitochondrial fusion and fission are essential for mitochondrial homeostasis in health and disease. In this study, we hypothesized that CS caused endothelial injury via an imbalance of mitochondrial fusion and fission and resultant mitochondrial oxidative stress and dysfunction. We noted that CS altered mitochondrial morphology by shortening mitochondrial networks and causing perinuclear accumulation of damaged mitochondria in primary rat lung microvascular endothelial cells. We also found that CS increased mitochondrial fission likely by decreasing Drp1-S637 and increasing FIS1, Drp1-S616 phosphorylation, mitochondrial translocation, and tetramerization and reduced mitochondrial fusion likely by decreasing Mfn2 in lung microvascular endothelial cells and mouse lungs. CS also caused aberrant mitophagy, increased mitochondrial oxidative stress, and reduced mitochondrial respiration. An inhibitor of mitochondrial fission and a mitochondria-specific antioxidant prevented CS-induced increased endothelial barrier dysfunction and apoptosis. Our data suggest that excessive mitochondrial fission and resultant oxidative stress are essential mediators of CS-induced endothelial injury and that inhibition of mitochondrial fission and mitochondria-specific antioxidants may be useful therapeutic strategies for CS-induced endothelial injury and associated pulmonary diseases.

Caenorhabditis elegans mounts a p38 MAPK pathway-mediated defence to Cutibacterium acnes infection.

Cutibacterium acnes is capable of inducing inflammation in acne and can lead to a chronic prostatic infection. The diverse pathogenicity among different strains of C. acnes has been presented, but simple appropriate animal models for the evaluation of this bacterium are lacking. In this study, the nematode Caenorhabditis elegans was used as an invertebrate infection model. We revealed that C. acnes type strain ATCC 6919 caused lethal infections to C. elegans in solid and liquid culture media (p < .0001). Compared with the strain ATCC 6919, the antibiotic-resistant strain HM-513 was more virulent, resulting in reduced survival (p < .0001). Four different C. acnes strains killed worms with a p value of less than .0001 when provided to C. elegans at 4.8 × 108 CFU/ml. The infection model was also employed to explore host defence responses. An increase in numerous immune effectors in response to C. acnes was detected. We focused on nine C-type lectins, including: clec-13, clec-17, clec-47, clec-52, clec-60, clec-61, clec-70, clec-71 and clec-227. The induced expression of these C-type lectin genes was down-regulated in mutant worms deficient in the p38 mitogen-activated protein kinase (MAPK) pathway. Meanwhile, PMK-1 (MAPK) was phosphorylated and activated at the onset of C. acnes infection. By monitoring the survival of mutant worms, we found that PMK-1, SEK-1 (MAPKK) and TIR-1 (MAPKKK) were critical in responding to C. acnes infection. C. elegans pmk-1 and tir-1 mutants exhibited higher mortality to C. acnes infection (p < .0001). In conclusion, C. elegans serves as a simple and valuable model to study C. acnes virulence and facilitates improvements in understanding of host innate immune responses.

Regulation of adipose tissue inflammation by interleukin 6.

Obesity is associated with a chronic state of low-grade inflammation and progressive tissue infiltration by immune cells and increased expression of inflammatory cytokines. It is established that interleukin 6 (IL6) regulates multiple aspects of metabolism, including glucose disposal, lipolysis, oxidative metabolism, and energy expenditure. IL6 is secreted by many tissues, but the role of individual cell types is unclear. We tested the role of specific cells using a mouse model with conditional expression of the Il6 gene. We found that IL6 derived from adipocytes increased, while IL6 derived from myeloid cells and muscle suppressed, macrophage infiltration of adipose tissue. These opposite actions were associated with a switch of IL6 signaling from a canonical mode (myeloid cells) to a noncanonical trans-signaling mode (adipocytes and muscle) with increased expression of the ADAM10/17 metalloprotease that promotes trans-signaling by the soluble IL6 receptor α. Collectively, these data demonstrate that the source of IL6 production plays a major role in the physiological regulation of metabolism.

Blockade of equilibrative nucleoside transporter 1/2 protects against Pseudomonas aeruginosa-induced acute lung injury and NLRP3 inflammasome activation.

Pseudomonas aeruginosa infections are increasingly multidrug resistant and cause healthcare-associated pneumonia, a major risk factor for acute lung injury (ALI)/acute respiratory distress syndrome (ARDS). Adenosine is a signaling nucleoside with potential opposing effects; adenosine can either protect against acute lung injury via adenosine receptors or cause lung injury via adenosine receptors or equilibrative nucleoside transporter (ENT)-dependent intracellular adenosine uptake. We hypothesized that blockade of intracellular adenosine uptake by inhibition of ENT1/2 would increase adenosine receptor signaling and protect against P. aeruginosa-induced acute lung injury. We observed that P. aeruginosa (strain: PA103) infection induced acute lung injury in C57BL/6 mice in a dose- and time-dependent manner. Using ENT1/2 pharmacological inhibitor, nitrobenzylthioinosine (NBTI), and ENT1-null mice, we demonstrated that ENT blockade elevated lung adenosine levels and significantly attenuated P. aeruginosa-induced acute lung injury, as assessed by lung wet-to-dry weight ratio, BAL protein levels, BAL inflammatory cell counts, pro-inflammatory cytokines, and pulmonary function (total lung volume, static lung compliance, tissue damping, and tissue elastance). Using both agonists and antagonists directed against adenosine receptors A2AR and A2BR, we further demonstrated that ENT1/2 blockade protected against P. aeruginosa -induced acute lung injury via activation of A2AR and A2BR. Additionally, ENT1/2 chemical inhibition and ENT1 knockout prevented P. aeruginosa-induced lung NLRP3 inflammasome activation. Finally, inhibition of inflammasome prevented P. aeruginosa-induced acute lung injury. Our results suggest that targeting ENT1/2 and NLRP3 inflammasome may be novel strategies for prevention and treatment of P. aeruginosa-induced pneumonia and subsequent ARDS.

Impact of Beta-lactam Allergy on Treatment of Outpatient Infections.

PURPOSE: The most commonly reported medication allergies in the United States involve beta-lactam antibiotics, creating an important consideration for prescribers when choosing optimal treatment of infections. Currently, few data exist on outpatient prescribing patterns in response to patients with a beta-lactam allergy. This study sought to evaluate the appropriateness of outpatient antibiotic therapy in patients with documented beta-lactam allergies within a Veterans Affairs health care system to evaluate areas of improvement in prescribing practices. METHODS: Patients receiving outpatient oral antibiotics were prospectively identified through real-time electronic alerts from June 2017 through February 2018. Prescriptions were then reviewed retrospectively to identify appropriateness of antibiotic, drug choice, dose, and duration based on current guideline recommendations. Data were compared between patients with a listed beta-lactam allergy and patients without a beta-lactam allergy to determine the impact on prescribing patterns and outcomes. Baseline characteristics were compared by using descriptive statistics. Significant risk factors for inappropriate prescribing were identified through a multivariable analysis. FINDINGS: The cohort included 1844 antibiotic prescriptions (documented beta-lactam allergy, 221; no beta-lactam allergy, 1623). Appropriate drug, dose, and duration for antibiotics prescribed in patients reporting a beta-lactam allergy versus nonallergic patients were 44.3% versus 53.0% (P = 0.02), 91.4% versus 86.2% (P = 0.03), and 75.1% versus 76.2% (P = 0.83), respectively. Patients with a reported beta-lactam allergy were 31% less likely to receive the correct drug for indication empirically (95% CI, 0.52-0.92) in the multivariable regression model when adjusted for fluoroquinolone use. In addition, patients reporting a beta-lactam allergy were 2.2 times (95% CI, 1.6-3.0) more likely to receive a fluoroquinolone antibiotic. Antibiotics were considered overall inappropriate based on at least one aspect of therapy in 79.6% of patients reporting a beta-lactam allergy and in 71% of nonallergic patients. IMPLICATIONS: Antibiotic therapy in patients with a documented beta-lactam allergy was less likely to be appropriate overall, suggesting an area of improvement for prescribing habits. Future interventions should focus on prescriber education regarding first-line and alternative treatments for patients with beta-lactam allergies to ensure that optimal treatment is being provided.

Evaluation of Antibiotic Prescribing in a Veterans Affairs Outpatient Setting: Identification of Stewardship Targets.

OBJECTIVE: To evaluate antibiotic prescribing practices for geriatric outpatients in a Veterans Affairs (VA) health care system.
DESIGN: This is a single-center, observational, prospective cohort study.
SETTING: Veterans Affairs Healthcare System.
PATIENTS: Outpatients treated with oral antibiotics between June and September 2017.
INTERVENTIONS: None.
MAIN OUTCOME MEASURE(S): Appropriate therapy was assessed based on clinical practice guidelines. Multivariable logistic regression was used to identify predictors of appropriate treatment.
RESULTS: This study yielded 1,063 prescriptions for analysis. No significant difference was observed for antibiotic indicated (60%), correct drug (50%), or correct duration (75%). Patients older than 65 years of age were more likely to receive an inappropriate dose (86% vs. 76%; P < 0.002). In the multivariable analysis, patients with chronic obstructive pulmonary disease (COPD) were more than 1.4 times likely to be treated appropriately (95% confidence interval 1.03-1.9) versus those without COPD. Older patients were not more likely to be re-treated or admitted within 30 days.
CONCLUSION: Antibiotics are often inappropriately used in the outpatient setting; but not more frequently in elderly patients. Older adults were more likely to be prescribed an antibiotic at an inappropriate dose. Opportunities exist for stewardship teams to provide value in the outpatient setting to ensure appropriate antibiotic prescribing with a focus on dosing.

Antibiotic stewardship targets in the outpatient setting.

BACKGROUND: Outpatient prescriptions comprise 60% of antibiotic use. This study prospectively identified inappropriate antibiotic use enabling a focused approach to outpatient antimicrobial stewardship. METHODS: Outpatients at the Veterans Affairs Western New York Healthcare System were identified via an electronic antibiotic alert from June 2017 to September 2017. Descriptive statistics and multivariable logistic regression identified stewardship targets. RESULTS: Of the 1,063 patients, 40% of antibiotic prescriptions were not indicated. Urinary tract infections (21%), bronchitis (20%), skin structure infections (17%), and sinusitis (10%) were common causes of inappropriate antibiotic use. Azithromycin (37%) was prescribed unnecessarily most often, followed by ciprofloxacin (16%), amoxicillin/clavulanate (13%), and cephalexin (12%). The correct drug was chosen in 52%, dose in 81%, and duration in 75% of patients. When the antibiotic was indicated, the correct drug was 2.9 times more likely to be prescribed and 2 times more likely to have the correct duration and receive care in the emergency room. DISCUSSION: Focusing on 4 drugs; amoxicillin/clavulanate, azithromycin, ciprofloxacin, and cephalexin accounted for 80% of unnecessary drug use. This study provides a guide to concentrate efforts during implementation of an outpatient stewardship program. CONCLUSIONS: Poor antibiotic prescribing was found in the outpatient setting. This study identifies areas for improvement via stewardship.

Dynamics of two pathogens in a single tick population.

A mathematical model for a two-pathogen, one-tick, one-host system is presented and explored. The model system is based on the dynamics of Amblyomma americanum, Rickettsia parkeri, and Rickettsia amblyommatis. The goal of this model is to determine how long an invading pathogen, R. parkeri, persists within a tick population, A. americanum, in which a resident pathogen, R. amblyommatis, is already established. The numerical simulations of the model demonstrate the parameter ranges that allow for coexistence of the two pathogens. Sensitivity analysis highlights the importance of vector-borne, tick-to-host, transmission rates on the invasion reproductive number and persistence of the pathogens over time. The model is then applied to a case study based on a reclaimed swampland field site in south-eastern Virginia using field and laboratory data. The results pinpoint the thresholds required for persistence of both pathogens in the local tick population. However, R. parkeri, is not predicted to persist beyond 3 years. Understanding the persistence and coexistence of tick-borne pathogens will allow public health officials increased insight into tick-borne disease dynamics.

TickBot: a novel robotic device for controlling tick populations in the natural environment.

A semi-autonomous 4-wheeled robot (TickBot) was fitted with a denim cloth treated with an acaricide (permethrin™) and tested for its ability to control ticks in a tick-infested natural environment in Portsmouth, Virginia. The robot's sensors detect a magnetic field signal from a guide wire encased in 80m polyethylene tubing, enabling the robot to follow the trails, open areas and other terrain where the tubing was located. To attract ticks to the treated area, CO2 was distributed through the same tubing, fitted with evenly spaced pores and flow control valves, which permitted uniform CO2 distribution. Tests were done to determine the optimum frequency for TickBot to traverse the wire-guided treatment site as well as the duration of operation that could be accomplished on a single battery charge. Prior to treatment, dragging was done to determine the natural abundance of ticks in the test site. Controls were done without CO2 and without permethrin. TickBot proved highly effective in reducing the overall tick densities to nearly zero with the treatment that included both carbon dioxide pretreatment and the permethrin treated cloth. Following a 60min traverse of the treatment areas, adult tick numbers, almost entirely Amblyomma americanum, was reduced to zero within 1h and remained at or near zero for 24h. Treatments without CO2 also showed reduction of ticks to near zero within 1h, but the populations were no different than the control sections at 4h. This study demonstrates the efficacy of TickBot as a tick control device to significantly reduce the risk of tick bites and disease transmission to humans and companion animals visiting a previously tick-infested natural environment. Continued deployment of TickBot for additional days or weeks can assure a relatively tick-safe environment for enjoyment by the public.

Targeting Cx43 and N-cadherin, which are abnormally upregulated in venous leg ulcers, influences migration, adhesion and activation of Rho GTPases.

BACKGROUND: Venous leg ulcers can be very hard to heal and represent a significant medical need with no effective therapeutic treatment currently available. PRINCIPAL FINDINGS: In wound edge biopsies from human venous leg ulcers we found a striking upregulation of dermal N-cadherin, Zonula Occludens-1 and the gap junction protein Connexin43 (Cx43) compared to intact skin, and in stark contrast to the down-regulation of Cx43 expression seen in acute, healing wounds. We targeted the expression of these proteins in 3T3 fibroblasts to evaluate their role in venous leg ulcers healing. Knockdown of Cx43 and N-cadherin, but not Zonula Occludens-1, accelerated cell migration in a scratch wound-healing assay. Reducing Cx43 increased Golgi reorientation, whilst decreasing cell adhesion and proliferation. Furthermore, Connexin43 and N-cadherin knockdown led to profound effects on fibroblast cytoskeletal dynamics after scratch-wounding. The cells exhibited longer lamelipodial protrusions lacking the F-actin belt seen at the leading edge in wounded control cells. This phenotype was accompanied by augmented activation of Rac-1 and RhoA GTPases, as revealed by Förster Resonance Energy Transfer and pull down experiments. CONCLUSIONS: Cx43 and N-cadherin are potential therapeutic targets in the promotion of healing of venous leg ulcers, by acting at least in part through distinct contributions of cell adhesion, migration, proliferation and cytoskeletal dynamics.