Fidgetin-Like 2: A Microtubule-Based Regulator of Wound Healing.

Wound healing is a complex process driven largely by the migration of a variety of distinct cell types from the wound margin into the wound zone. In this study, we identify the previously uncharacterized microtubule-severing enzyme, Fidgetin-like 2 (FL2), as a fundamental regulator of cell migration that can be targeted in vivo using nanoparticle-encapsulated small interfering RNA (siRNA) to promote wound closure and regeneration. In vitro, depletion of FL2 from mammalian tissue culture cells results in a more than twofold increase in the rate of cell movement, in part due to a significant increase in directional motility. Immunofluorescence analyses indicate that FL2 normally localizes to the cell edge, importantly to the leading edge of polarized cells, where it regulates the organization and dynamics of the microtubule cytoskeleton. To clinically translate these findings, we utilized a nanoparticle-based siRNA delivery platform to locally deplete FL2 in both murine full-thickness excisional and burn wounds. Topical application of FL2 siRNA nanoparticles to either wound type results in a significant enhancement in the rate and quality of wound closure both clinically and histologically relative to controls. Taken together, these results identify FL2 as a promising therapeutic target to promote the regeneration and repair of cutaneous wounds.

Management of cutaneous abscesses by dermatologists.

IMPORTANCE: There is currently no data detailing the degree to which dermatologists follow CDC/Infectious Diseases Society of America (IDSA) guidelines in the treatment of abscesses, which recommend that incision and drainage (I+D) as primary therapy for skin and soft tissue infections (SSTI). OBJECTIVE: To evaluate the management of skin abscesses by dermatologists. DESIGN, SETTING, PARTICIPANTS: A national email survey of 780 dermatologists was conducted from May-June 2012. Awareness, experience, and preparedness of respondents for abscess treatment, as well as the treatment practices in different clinical scenarios were evaluated. Response rate = 65% (n=510). ELIGIBILITY CRITERIA: board certified/eligible dermatologists practicing in US. Main practice affiliation: solo (20%), group (33%), university health system/academic (32%), multi-specialty (13%), and other (2%). Main practice setting: urban (49%), suburban (42%), and rural (9%). MAIN OUTCOME AND MEASURES: Practitioner report of: awareness of national guidelines, use of I+D in initial management of uncomplicated abscess found on face, trunk, and extremity on patients age 6 months, 3, 15, 50, and 80 years, and use of antibiotics in the initial management. RESULTS: 99% of respondents were capable of performing I+D in their practice. The IDSA recommends cultures in all patients treated with antibiotic therapy, and does not recommend antibiotics for the treatment of simple abscess. 18% of respondents reported culturing abscesses less than 50% of the time, while 91% incorporated antibiotics into initial treatment. Nearly a quarter (24%) of respondents would choose an initial antibiotic that would not cover Methicillin-resistant Staphylococcus aureus (MRSA). For facial abscesses, as the age of the patient increased from infant, respondents were more likely to incorporate I+D into their initial treatment. For abscesses on the trunk and extremities, respondents were less likely to I+D infants and toddlers, compared to adolescents, adults and the elderly. CONCLUSION: Although most dermatologists were prepared to manage uncomplicated abscesses (98%), this survey identifies gaps in clinical standards of care established by the CDC/IDSA. Identification of these practice gaps may impact physician practice and dermatology residency curricula, and may serve to improve abscess management and antibacterial stewardship in the outpatient setting.

Amphotericin B releasing nanoparticle topical treatment of Candida spp. in the setting of a burn wound.

Candida spp. infection in the context of burn wounds leads to invasive disease with a 14-70% mortality rate. Unfortunately, current administrations of AmB, an important therapeutic demonstrating minimal resistance, are only available via potentially cytotoxic IV infusions. In order to circumvent these sequelae, we investigated the efficacy of nanoparticle encapsulated AmB (AmB-np) as a topical therapeutic against Candida spp. (drug release equilibrated solubilized AmB [AmB-sol] included as control). Clinical strains demonstrated equal or enhanced killing efficacy with 72.4-91.1% growth reduction by 4 hours. AmB-nps resulted in statistically significant reduction of fungal biofilm metabolic activity ranging from 80% to 95% viability reduction (P<0.001). Using a murine full-thickness burn model, AmB-np exhibited a quicker efficiency in fungal clearance versus AmB-sol by day three, although wound healing rates were similar. These data support the concept that AmB-np can function as a topical antifungal in the setting of a burn wound. FROM THE CLINICAL EDITOR: The control of fungal infections with Candida species remains a challenge in the context of burn wounds. A nanoencapsulated topical amphotericin-B compound was studied in a murine model of full thickness burn injury, showing remarkable efficacy in controlling Candida infection. This may become a viable alternative to the potentially toxic intravenous formulations.

Evaluation of the antibiotic properties of glutathione.

Skin and soft tissue infections (SSTIs) are growing in prevalence in both the outpatient and inpatient settings and are some of the most common diseases seen by dermatologists, who are often the first point of care for these patients. Microbial resistance to antibiotics continues to rise as more virulent strains evolve, and strains predominantly found in the hospital setting are now being seen in the community. Therefore, innovative approaches to combat this trend are needed. Glutathione (GSH) is a well-described and established antioxidant. It participates in detoxification of xenobiotics, regulation of cellular growth, modulation of immune response, and maintenance of the thiol status of proteins and cellular cysteine levels. GSH is also known to have a regulatory effect on immune cells and even inherent antibacterial properties have been reported. To this end, the value of GSH as an antibiotic was evaluated by growing methicillin resistant S. aureus, E. coli, K. pneumoniae and P. aeruginosa strains isolated from human skin and soft tissue infection in the presence of GSH. At a physiologic concentration of 10 mM, GSH had no effect on bacterial growth. At concentrations above 50 mM, which created acidic conditions (pH < 4), bacterial growth was completely inhibited. When adjusted to physiologic pH, GSH exhibited a bacteriostatic effect in a concentration-dependent manner. Additionally, the cytotoxicity of GSH was evaluated in a murine cell line. GSH was relatively non-toxic to murine macrophages, even at the highest concentration tested (160 mM). These results suggest the potential utility of GSH for the prevention and/or as adjunctive treatment of infection, most significantly in disease states associated with GSH deficiency.

A nanoparticle delivery vehicle for S-nitroso-N-acetyl cysteine: sustained vascular response.

Interest in the development of nitric oxide (NO) based therapeutics has grown exponentially due to its well elucidated and established biological functions. In line with this surge, S-nitroso thiol (RSNO) therapeutics are also receiving more attention in recent years both as potential stable sources of NO as well as for their ability to serve as S-nitrosating agents; S-nitrosation of protein thiols is implicated in many physiological processes. We describe two hydrogel based RSNO containing nanoparticle platforms. In one platform the SNO groups are covalently attached to the particles (SNO-np) and the other contains S-nitroso-N-acetyl cysteine encapsulated within the particles (NAC-SNO-np). Both platforms function as vehicles for sustained activity as trans-S-nitrosating agents. NAC-SNO-np exhibited higher efficiency for generating GSNO from GSH and maintained higher levels of GSNO concentration for longer time (24 h) as compared to SNO-np as well as a previously characterized nitric oxide releasing platform, NO-np (nitric oxide releasing nanoparticles). In vivo, intravenous infusion of the NAC-SNO-np and NO-np resulted in sustained decreases in mean arterial pressure, though NAC-SNO-np induced longer vasodilatory effects as compared to the NO-np. Serum chemistries following infusion demonstrated no toxicity in both treatment groups. Together, these data suggest that the NAC-SNO-np represents a novel means to both study the biologic effects of nitrosothiols and effectively capitalize on its therapeutic potential.

The potential of nitric oxide releasing therapies as antimicrobial agents.

Nitric oxide (NO) is a short-lived, diatomic, lipophilic gas that plays an integral role in defending against pathogens. Among its many functions are involvement in immune cell signaling and in the biochemical reactions by which immune cells defend against bacteria, fungi, viruses and parasites. NO signaling directs a broad spectrum of processes, including the differentiation, proliferation, and apoptosis of immune cells. When secreted by activated immune cells, NO diffuses across cellular membranes and exacts nitrosative and oxidative damage on invading pathogens. These observations led to the development of NO delivery systems that can harness the antimicrobial properties of this evanescent gas. The innate microbicidal properties of NO, as well as the antimicrobial activity of the various NO delivery systems, are reviewed.

Nitric oxide-releasing nanoparticles accelerate wound healing in NOD-SCID mice.

Wound healing is a complex process, coordinated by various biological factors. In immunocompromised states wound healing can be interrupted as a result of decreased numbers of immune cells, impairing the production of effector molecules such as nitric oxide (NO). Therefore, topical NO-releasing platforms, such as diethylenetriamine (DETA NONOate), have been investigated to enhance wound healing. Recently, we demonstrated a nanoparticle platform that releases NO (NO-NPs) in a sustained manner, accelerating wound healing in both uninfected and infected murine wound models. Here, NO-NPs were investigated and compared to DETA NONOate in an immunocompromised wound model using non-obese, diabetic, severe combined immunodeficiency mice. NO-NP treatment accelerated wound closure as compared to controls and DETA NONOate treatment. In addition, histological assessment revealed that wounds treated with NO-NPs had less inflammation, more collagen deposition, and more blood vessel formation as compared to other groups, consistent with our previous data in immunocompetent animals. These data suggest that NO-NPs may serve as a novel wound-healing therapy in the setting of immunocompromised states associated with impaired wound healing. FROM THE CLINICAL EDITOR: Wound healing in an immunocompromised host is often incomplete and is a source of major concern in such conditions. This work demonstrates in a murine model that in these settings NO releasing nanoparticles significantly enhance wound healing.

Nitric oxide nanoparticles: pre-clinical utility as a therapeutic for intramuscular abscesses.

Nitric oxide (NO) is a critical component of host defense against invading pathogens; however, its therapeutic utility is limited due to a lack of practical delivery systems. Recently, a NO-releasing nanoparticulate platform (NO-np) was shown to have in vitro broad-spectrum antimicrobial activity and in vivo pre-clinical efficacy in a dermal abscess model. To extend these findings, both topical (TP) and intralesional (IL) NO-np administration was evaluated in a MRSA intramuscular murine abscess model and compared with vancomycin. All treatment arms accelerated abscess clearance clinically, histologically, and by microbiological assays on both days 4 and 7 following infection. However, abscesses treated with NO-np via either route demonstrated a more substantial, statistically significant decrease in bacterial survival based on colony forming unit assays and histologically revealed less inflammatory cell infiltration and preserved muscular architecture. These data suggest that the NO-np may be an effective addition to our armament for deep soft tissue infections.

Nitrosoglutathione generating nitric oxide nanoparticles as an improved strategy for combating Pseudomonas aeruginosa-infected wounds.

Pseudomonas aeruginosa is a community-acquired, nosocomial pathogen that is an important cause of human morbidity and mortality; it is intrinsically resistant to several antibiotics and is capable of developing resistance to newly developed drugs via a variety of mechanisms. P aeruginosa's ubiquity and multidrug resistance (MDR) warrants the development of innovative methods that overcome its ability to develop resistance. We have previously described a nitric oxide-releasing nanoparticle (NO-np) platform that effectively kills gram-positive and gram-negative organisms in vitro and accelerates clinical recovery in vivo in murine wound and abscess infection models. We have also demonstrated that when glutathione (GSH) is added to NO-np, the nitroso intermediate S-nitrosoglutathione (GSNO) is formed, which has greater activity against P aeruginosa and other gram-negative organisms compared with NO-np alone. In the current study, we evaluate the potential of NO-np to generate GSNO both in vitro and in vivo in a murine excisional wound model infected with an MDR clinical isolate of P aeruginosa. Whereas NO-np alone inhibited P aeruginosa growth in vitro for up to 8 hours, NO-np+GSH completely inhibited P aeruginosa growth for 24 hours. Percent survival in the NO-np+GSH-treated isolates was significantly lower than in the NO-np (36.1% vs 8.3%; P=.004). In addition, NO-np+GSH accelerated wound closure in P aeruginosa-infected wounds, and NO-np+GSH-treated wounds had significantly lower bacterial burden when compared to NO-np-treated wounds (P<.001). We conclude that GSNO is easily generated from our NO-np platform and has the potential to be used as an antimicrobial agent against MDR organisms such as P aeruginosa.