Beta adrenergic receptor antagonist can modify Pseudomonas aeruginosa biofilm formation in vitro: Implications for chronic wounds.

Non-healing wounds are a major medical challenge, affecting over 6.5 million people in the US alone, with associated healthcare costs of about $16 billion annually. They can result in prolonged hospitalizations, work loss, disability, poor quality of life, and in diabetic patients with foot ulcers, amputation of the affected limb in 25% of patients. Though chronic ulcers may arise from different underlying diseases, the unifying feature is chronic infection, driving persistent inflammation that prolongs the healing process. One of the most frequently cultured or genetically identified pathogens in skin wounds is Pseudomonas aeruginosa. This species avidly forms biofilms in the wound that impede bacterial eradication by the host's immune mechanisms and limit efficacy of systemic antibiotics. Thus, non-antibiotic approaches to limit the growth and biofilm formation of this wound pathogen would be an advance in the treatment of chronic wounds. Prior work has demonstrated that the growth of other microbial species can be modulated by catecholamine agonists and antagonists of the adrenergic receptors (ARs). Here, we demonstrate that not only can the growth of this common wound pathogen be modulated by catecholamines, but also that the beta-AR antagonists can significantly decrease their growth, and importantly, limit their ability to form biofilms. These findings suggest that beta adrenergic antagonists may have a therapeutic role in the treatment of chronic skin wounds.

Tunable hydrogels for mesenchymal stem cell delivery: Integrin-induced transcriptome alterations and hydrogel optimization for human wound healing.

Therapeutic applications for mesenchymal stem/stromal cells (MSCs) are growing; however, the successful implementation of these therapies requires the development of appropriate MSC delivery systems. Hydrogels are ideally suited to cultivate MSCs but tuning hydrogel properties to match their specific in vivo applications remains a challenge. Thus, further characterization of how hydrogel-based delivery vehicles broadly influence MSC function and fate will help lead to the next generation of more intelligently designed delivery vehicles. To date, few attempts have been made to comprehensively characterize hydrogel impact on the MSC transcriptome. Herein, we have synthesized cell-degradable hydrogels based on bio-inert poly(ethylene glycol) tethered with specific integrin-binding small molecules and have characterized their resulting effect on the MSC transcriptome when compared with 2D cultured and untethered 3D hydrogel cultured MSCs. The 3D culture systems resulted in alterations in the MSC transcriptome, as is evident by the differential expression of genes related to extracellular matrix production, glycosylation, metabolism, signal transduction, gene epigenetic regulation, and development. For example, genes important for osteogenic differentiation were upregulated in 3D hydrogel cultures, and the expression of these genes could be partially suppressed by tethering an integrin-binding RGD peptide within the hydrogel. Highlighting the utility of tunable hydrogels, when applied to ex vivo human wounds the RGD-tethered hydrogel was able to support wound re-epithelialization, possibly due to its ability to increase PDGF expression and decrease IL-6 expression. These results will aid in future hydrogel design for a broad range of applications.

A tractable, simplified ex vivo human skin model of wound infection.

The prevalence of infection in chronic wounds is well documented in the literature but not optimally studied due to the drawbacks of current methodologies. Here, we describe a tractable and simplified ex vivo human skin model of infection that addresses the critical drawbacks of high costs and limited translatability. Wounds were generated from excised abdominal skin from cosmetic procedures and cultured, inoculated with Staphylococcus aureus strain UAMS-1, or under aseptic conditions. After three days, the infected wounds exhibited biofilm formation and significantly impaired reepithelialization compared to the control. Additionally, promigratory and proreparative genes were significantly downregulated, while proinflammatory genes were significantly upregulated, demonstrating molecular characterizations of impaired healing as in chronic wounds. This model allows for a simplified and versatile tool for the study of wound infection and subsequent development of novel therapies.

Structural tuning of anisotropic mechanical properties in 3D-Printed hydrogel lattices.

We investigated the ability to tune the anisotropic mechanical properties of 3D-printed hydrogel lattices by modifying their geometry (lattice strut diameter, unit cell size, and unit cell scaling factor). Many soft tissues are anisotropic and the ability to mimic natural anisotropy would be valuable for developing tissue-surrogate "phantoms" for elasticity imaging (shear wave elastography or magnetic resonance elastography). Vintile lattices were 3D-printed in polyethylene glycol di-acrylate (PEGDA) using digital light projection printing. Two mechanical benchtop tests, dynamic shear testing and unconfined compression, were used to measure the apparent shear storage moduli (G') and apparent Young's moduli (E) of lattice samples. Increasing the unit cell size from 1.25 mm to 2.00 mm reduced the Young's and shear moduli of the lattices by 91% and 85%, respectively. Decreasing the strut diameter from 300 µm to 200 µm reduced the apparent shear moduli of the lattices by 95%. Increasing the geometric scaling ratio of the lattice unit cells from 1.00 × to 2.00 × increased mechanical anisotropy in shear (by a factor of 3.1) and in compression (by a factor of 2.9). Both simulations and experiments show that the effects of unit cell size and strut diameter are consistent with power law relationships between volume fraction and apparent elastic moduli. In particular, experimental measurements of apparent Young's moduli agree well with predictions of the theoretical Gibson-Ashby model. Thus, the anisotropic mechanical properties of a lattice can be tuned by the unit cell size, the strut diameter, and scaling factors. This approach will be valuable in designing tissue-mimicking hydrogel lattice-based composite materials for elastography phantoms and tissue engineered scaffolds.

FDG PET/CT Imaging 1 Week after a Single Dose of Pembrolizumab Predicts Treatment Response in Patients with Advanced Melanoma.

PURPOSE: Immunologic response to anti-programmed cell death protein 1 (PD-1) therapy can occur rapidly with T-cell responses detectable in as little as one week. Given that activated immune cells are FDG avid, we hypothesized that an early FDG PET/CT obtained approximately 1 week after starting pembrolizumab could be used to visualize a metabolic flare (MF), with increased tumor FDG activity due to infiltration by activated immune cells, or a metabolic response (MR), due to tumor cell death, that would predict response. PATIENTS AND METHODS: Nineteen patients with advanced melanoma scheduled to receive pembrolizumab were prospectively enrolled. FDG PET/CT imaging was performed at baseline and approximately 1 week after starting treatment. FDG PET/CT scans were evaluated for changes in maximum standardized uptake value (SUVmax) and thresholds were identified by ROC analysis; MF was defined as >70% increase in tumor SUVmax, and MR as >30% decrease in tumor SUVmax. RESULTS: An MF or MR was identified in 6 of 11 (55%) responders and 0 of 8 (0%) nonresponders, with an objective response rate (ORR) of 100% in the MF-MR group and an ORR of 38% in the stable metabolism (SM) group. An MF or MR was associated with T-cell reinvigoration in the peripheral blood and immune infiltration in the tumor. Overall survival at 3 years was 83% in the MF-MR group and 62% in the SM group. Median progression-free survival (PFS) was >38 months (median not reached) in the MF-MR group and 2.8 months (95% confidence interval, 0.3-5.2) in the SM group (P = 0.017). CONCLUSIONS: Early FDG PET/CT can identify metabolic changes in melanoma metastases that are potentially predictive of response to pembrolizumab and significantly correlated with PFS.

Design and characterization of 3-D printed hydrogel lattices with anisotropic mechanical properties.

The goal of this study was to design, fabricate, and characterize hydrogel lattice structures with consistent, controllable, anisotropic mechanical properties. Lattices, based on three unit-cell types (cubic, diamond, and vintile), were printed using stereolithography (SLA) of polyethylene glycol diacrylate (PEGDA). To create structural anisotropy in the lattices, unit cell design files were scaled by a factor of two in one direction in each layer and then printed. The mechanical properties of the scaled lattices were measured in shear and compression and compared to those of the unscaled lattices. Two apparent shear moduli of each lattice were measured by dynamic shear tests in two planes: (1) parallel and (2) perpendicular to the scaling direction, or cell symmetry axis. Three apparent Young's moduli of each lattice were measured by compression in three different directions: (1) the "build" direction or direction of added layers, (2) the scaling direction, and (3) the unscaled direction perpendicular to both scaling and build directions. For shear deformation in unscaled lattices, the apparent shear moduli were similar in the two perpendicular directions. In contrast, scaled lattices exhibit clear differences in apparent shear moduli. In compression of unscaled lattices, apparent Young's moduli were independent of direction in cubic and vintile lattices; in diamond lattices Young's moduli differed in the build direction, but were similar in the other two directions. Scaled lattices in compression exhibited additional differences in apparent Young's moduli in the scaled and unscaled directions. Notably, the effects of scaling on apparent modulus differed between each lattice type (cubic, diamond, or vintile) and deformation mode (shear or compression). Scaling of 3D-printed, hydrogel lattices may be harnessed to create tunable, structures of desired shape, stiffness, and mechanical anisotropy, in both shear and compression.

Combination product of dermal matrix, preconditioned human mesenchymal stem cells and timolol promotes wound healing in the porcine wound model.

A combination product of human mesenchymal stem/stromal cells (MSCs) embedded in an extracellular matrix scaffold and preconditioned with hypoxia and the beta-adrenergic receptor antagonist, timolol, combined with sustained timolol application post implantation, has shown promising results for improving wound healing in a diabetic mouse model. In the present study, we extend those findings to the more translatable large animal porcine wound model and show that the combined treatment promotes wound reepithelialization in these excisional wounds by 40.2% and increases the CD31 immunostaining marker of angiogenesis compared with the matrix control, while maintaining an accumulated timolol plasma concentration below the clinically safe level of 0.3 ng/mL after the 15-day course of topical application. Human GAPDH was not elevated in the day 15 wounds treated with MSC-containing device relative to wounds treated with matrix alone, indicating that the xenografted human MSCs in the treatment do not persist in these immune-competent animals after 15 days. The work demonstrates the efficacy and safety of the combined treatment for improving healing in the clinically relevant porcine wound model.

Dopamine transporter inhibition is necessary for cocaine-induced increases in dendritic spine density in the nucleus accumbens.

Repeated exposure to cocaine produces changes in the nervous system that facilitate drug-seeking behaviors. These drug-seeking behaviors have been studied with animal models, such as cocaine-induced locomotor sensitization. Cocaine is hypothesized to induce locomotor sensitization by neural changes, including an increase in the density of spines on the dendrites of neurons in the nucleus accumbens (NAC). However, how cocaine increases dendritic spine density in the NAC has been difficult to discern because cocaine inhibits the function of multiple targets, including the transporters for dopamine, serotonin, and norepinephrine. Previously, our lab created a tool that is useful for determining how inhibiting the dopamine transporter (DAT) contributes to the effects of cocaine by generating mice that express a cocaine-insensitive DAT (DAT-CI mice). In this study, we used DAT-CI mice to determine the contribution of DAT inhibition in cocaine-induced increases in dendritic spine density in the NAC. We repeatedly injected DAT-CI mice with either cocaine or saline, and measured both dendritic spine density in the NAC and locomotor activity. Unlike wild-type mice, DAT-CI mice did not show an increase in dendritic spine density in the NAC or in locomotor activity in response to repeated injections of cocaine. These data show that cocaine-induced increases in dendritic spine density in the NAC require DAT inhibition. Thus, DAT-inhibition may play a role in mediating the long-lasting neural changes associated with drug addiction.

Repurposing Ophthalmologic Timolol for Dermatologic Use: Caveats and Historical Review of Adverse Events.

Ophthalmic timolol solution is increasingly being repurposed as a topical therapeutic for a variety of dermatologic diseases, including pyogenic granulomas, infantile hemangiomas, and chronic wounds. There are no published guidelines or protocols for use in these indications in adults, and the dermatologic community may not be familiar with adverse events that have been extensively documented relating to its ophthalmic use. We review the evidence available relating to adverse events to topical timolol use to evaluate its safety in dermatologic applications and to alert clinicians to screening and monitoring that is needed when repurposing this drug for dermatologic use. The majority of serious adverse events associated with ophthalmic timolol were reported in the first 7 years of use, between 1978 and 1985, of which most common were cardiovascular and respiratory events, but also included 32 deaths. The available evidence suggests that ophthalmic timolol safety profiling may have been incomplete prior to widespread use. Recent clinical trials for dermatologic indications have focused on documenting efficacy and have not had rigorous monitoring for potential adverse events. Topical timolol may be safe and effective for the treatment of various dermatologic conditions in patients whose medical histories have been carefully reviewed for evidence of pre-existing cardiac or pulmonary disease and are monitored for potential adverse events. Despite the wide use of timolol in ophthalmologic practice, safe dermatologic repurposing requires recognition of the potential for facilitated systemic absorption though the skin and appreciation of its history of adverse events.

Beta-adrenergic antagonist for the healing of chronic diabetic foot ulcers: study protocol for a prospective, randomized, double-blinded, controlled and parallel-group study.

BACKGROUND: Diabetic foot ulcers (DFUs) are the most common cause of leg amputations and their management is extremely challenging. Despite many advances and expensive therapies, there has been little success in improving outcomes of DFUs. In prior work our laboratory has examined the effects of beta-adrenergic antagonists (ßAAs) on skin and skin-derived cells. We have shown that ßAAs enhance the rate of keratinocyte migration, promote angiogenesis, and hasten wound healing in scratch wounds in vitro, in animal wound models, and in anecdotally reported cases of chronic wounds that healed successfully after topical application of the ßAA timolol. Thus, we propose to test timolol directly on DFUs to determine if it improves healing above the current standard of care (SOC). This study will examine the efficacy and safety of topically applied beta-antagonist Timoptic-XE® (timolol maleate ophthalmic gel forming solution) in subjects with DFUs. METHODS/DESIGN: This is a phase two, randomized, double-blinded, controlled, and parallel-group clinical trial with two treatment arms, SOC plus topical Timoptic-XE® and SOC plus a non-biologically active gel (hydrogel, as placebo drug). Study subjects with a DFU will be selected from the Veterans Affairs Northern California Health Care System (VANCHCS). Study duration is up to 31 weeks, with three phases (screening phase for two weeks, active phase for up to 12 weeks, with an additional second consecutive confirmatory visit after 2 weeks, and follow-up phase comprising monthly visits for 4 months). Subjects will apply daily either the topical study drug or the placebo on the foot ulcer for 12 weeks or until healed, whichever comes first. Measurements of wound size and other data will be collected at baseline, followed by weekly visits for 12 weeks, and then a monthly follow-up period. DISCUSSION: This is a clinical translation study, moving the investigators' pre-clinical laboratory research into a translational study in which we will analyze clinical outcomes to assess for safety and estimate the efficacy of a topical beta-antagonist in healing of DFUs. The results from this trial may establish new treatment paradigms and safety profile for DFU treatment. TRIAL REGISTRATION: ClinicalTrials.gov, NCT03282981. Registered on June 14th, 2018.

Combination product of dermal matrix, human mesenchymal stem cells, and timolol promotes diabetic wound healing in mice.

Diabetic foot ulcers are a major health care concern with limited effective therapies. Mesenchymal stem cell (MSC)-based therapies are promising treatment options due to their beneficial effects of immunomodulation, angiogenesis, and other paracrine effects. We investigated whether a bioengineered scaffold device containing hypoxia-preconditioned, allogeneic human MSCs combined with the beta-adrenergic antagonist timolol could improve impaired wound healing in diabetic mice. Different iterations were tested to optimize the primary wound outcome, which was percent of wound epithelialization. MSC preconditioned in 1 µM timolol at 1% oxygen (hypoxia) seeded at a density of 2.5 × 105 cells/cm2 on Integra Matrix Wound Scaffold (MSC/T/H/S) applied to wounds and combined with daily topical timolol applications at 2.9 mM resulted in optimal wound epithelialization 65.6% (24.9% ± 13.0% with MSC/T/H/S vs 41.2% ± 20.1%, in control). Systemic absorption of timolol was below the HPLC limit of quantification, suggesting that with the 7-day treatment, accumulative steady-state timolol concentration is minimal. In the early inflammation stage of healing, the MSC/T/H/S treatment increased CCL2 expression, lowered the pro-inflammatory cytokines IL-1B and IL6 levels, decreased neutrophils by 44.8%, and shifted the macrophage ratio of M2/M1 to 1.9 in the wound, demonstrating an anti-inflammatory benefit. Importantly, expression of the endothelial marker CD31 was increased by 2.5-fold with this treatment. Overall, the combination device successfully improved wound healing and reduced the wound inflammatory response in the diabetic mouse model, suggesting that it could be translated to a therapy for patients with diabetic chronic wounds.

Co-translational protein targeting facilitates centrosomal recruitment of PCNT during centrosome maturation in vertebrates.

As microtubule-organizing centers of animal cells, centrosomes guide the formation of the bipolar spindle that segregates chromosomes during mitosis. At mitosis onset, centrosomes maximize microtubule-organizing activity by rapidly expanding the pericentriolar material (PCM). This process is in part driven by the large PCM protein pericentrin (PCNT), as its level increases at the PCM and helps recruit additional PCM components. However, the mechanism underlying the timely centrosomal enrichment of PCNT remains unclear. Here, we show that PCNT is delivered co-translationally to centrosomes during early mitosis by cytoplasmic dynein, as evidenced by centrosomal enrichment of PCNT mRNA, its translation near centrosomes, and requirement of intact polysomes for PCNT mRNA localization. Additionally, the microtubule minus-end regulator, ASPM, is also targeted co-translationally to mitotic spindle poles. Together, these findings suggest that co-translational targeting of cytoplasmic proteins to specific subcellular destinations may be a generalized protein targeting mechanism.

Tympanostomy Tube Placement in Children Using a Single-Pass Tool with Moderate Sedation.

Insertion of tympanostomy tubes is a common elective pediatric surgical procedure and is typically performed under general anesthesia. The potential to reduce general anesthetic requirements for young children has led to increased interest in alternatives for tympanostomy tube placement. A tympanostomy tube system, developed to enable tympanostomy tube placement in a single pass on conscious patients under moderate sedation, was evaluated. A prospective study on 128 children and 253 tympanostomy tube placements conducted at 4 centers in the United States demonstrated an 88.3% success rate in performing the procedure under moderate sedation with adverse events within normal rates reported in the literature. The feasibility of completing tympanostomy tube placement under moderate sedation enables avoidance of general anesthesia and provides additional choices to physicians and parents.

Drug-Induced Pancreatitis: A Rare Manifestation of Doxycycline Administration.

CONTEXT: Drug-induced pancreatitis (DIP) is rare, but as there are no systematic data on it, the true incidence is not known. Although numerous and varied drugs have been associated with DIP, the clinical evidence on doxycycline-induced pancreatitis is sparse. CASE REPORT: We present the case of a 58-year-old female who presented with complaints of nausea and severe epigastric pain. Her medications included doxycycline which she had been on for only 2 days. Computed tomography of her abdomen showed mild enlargement of body of the pancreas with peripancreatic fatty infiltration, along with lipase level suggestive of acute pancreatitis. In the absence of classical risk factors for acute pancreatitis, a diagnosis of DIP secondary to doxycycline therapy was made. Immediate withdrawal of the drug was accompanied by relief of symptoms and resolution of pancreatitis. CONCLUSION: This report implicates doxycycline as an etiological factor for acute pancreatitis. Knowledge regarding doxycycline related pancreatitis is of paramount importance in order to diagnose cases early and institute effective treatment in patients who are undergoing therapy with this drug.

Cervical vertebral actinomycosis mimicking malignancy in a paediatric patient.

Actinomyces spp are found in the flora of the oral cavity and vagina and may cause infection with abscess formation and draining sinuses. Cervicofacial manifestations of actinomycosis involve head and neck soft tissue, however, spread to the cervical spine is rare. We report a case of an 8-year-old boy, presenting with neck pain for 1 month and denying a history of trauma or procedures. Radiography revealed an ulceration of the posterior oropharyngeal mucosa with a defect extending to the C1-C2 vertebra, mimicking a neoplastic process. The patient underwent laryngoscopy and multiple biopsies were taken from the ulcer and bone, showing severe osteomyelitis and intraosseous filamentous organisms, morphologically consistent with Actinomyces spp. The boy received long-term antibiotics with response to treatment. Actinomycosis has rarely been reported in the cervical vertebrae of paediatric patients. This should be considered as a differential diagnosis for such a presentation as prompt antibiotic treatment may be lifesaving.

Pineal parenchymal tumour of intermediate differentiation: a rare differential diagnosis of pineal region tumours.

Pineal parenchymal tumours of intermediate differentiation are a rare type of pineal parenchymal tumours. As indicated by their name, these tumours fall between pineoblastoma (a malignant pineal parenchymal tumour) and pineocytoma (a benign pineal parenchymal tumour). In this article, we present a case of pineal parenchymal tumour of intermediate differentiation that was successfully treated by resection via the supracerebellar approach. We also discuss the differential consideration based on epidemiological, pathological and radiological findings.

Nonthrombotic Pulmonary Embolism: A Potential Complication of Polyacrylamide Hydrogel Cosmetic Injection.

Context. Polyacrylamide hydrogel (PAAG) has gained importance as a synthetic soft tissue filling agent. It has been commonly employed by physicians in Europe for facial contouring and soft tissue augmentation. Previously, PAAG is considered nontoxic and well tolerated with a few mild procedural complications. Case Presentation. A 26-year-old female was hospitalized for dry cough, worsening dyspnea, and chest discomfort after 3 hours of multiple PAAG injections in buttocks. The patient's condition deteriorated and rapidly advanced to acute respiratory failure. Therein, the diagnosis of nonthrombotic pulmonary embolism (NTPE) was established on standard set of investigations. She was intubated; corticosteroid and empiric antibiotic therapy was initiated resulting in improvement of her condition. Subsequently, extubation was done, and she was discharged from the hospital after an uneventful recovery. On 1-month follow-up, the patient had no previous symptoms. Conclusion. This report implicates clinicians to maintain a high index of suspicion for NTPE in patients presenting with respiratory symptoms following PAAG usage.

A Systematic Review of Acquired Uterine Arteriovenous Malformations: Pathophysiology, Diagnosis, and Transcatheter Treatment.

Objective An acquired uterine arteriovenous malformation (AVM) is a rare cause of vaginal bleeding and, although hysterectomy is the definitive therapy, transcatheter embolization (TCE) provides an alternative treatment option. This systematic review presents the indications, technique, and outcomes for transcatheter treatment of the acquired uterine AVMs. Study Design Literature databases were searched from 2003 to 2013 for eligible clinical studies, including the patient characteristics, procedural indication, results, complications, as well as descriptions on laterality and embolic agents utilized. Results A total of 40 studies were included comprising of 54 patients (average age of 33.4 years). TCE had a primary success rate with symptomatic control of 61% (31 patients) and secondary success rate of 91% after repeated embolization. When combined with medical therapy, symptom resolution was noted in 48 (85%) patients without more invasive surgical procedures. Conclusion Low-level evidence supports the role of TCE, including in the event of persistent bleeding following initial embolization, for the treatment of acquired uterine AVMs. The variety of embolic agents and laterality of approach delineate the importance of refining procedural protocols in the treatment of the acquired uterine AVM. Condensation A review on the management of patients with acquired uterine AVMs.