Phentolamine Significantly Enhances Macrolide Antibiotic Antibacterial Activity against MDR Gram-Negative Bacteria.

OBJECTIVES: Multidrug-resistant (MDR) Gram-negative bacterial infections have limited treatment options due to the impermeability of the outer membrane. New therapeutic strategies or agents are urgently needed, and combination therapies using existing antibiotics are a potentially effective means to treat these infections. In this study, we examined whether phentolamine can enhance the antibacterial activity of macrolide antibiotics against Gram-negative bacteria and investigated its mechanism of action. METHODS: Synergistic effects between phentolamine and macrolide antibiotics were evaluated by checkerboard and time-kill assays and in vivo using a Galleria mellonella infection model. We utilized a combination of biochemical tests (outer membrane permeability, ATP synthesis, ΔpH gradient measurements, and EtBr accumulation assays) with scanning electron microscopy to clarify the mechanism of phentolamine enhancement of macrolide antibacterial activity against Escherichia coli. RESULTS: In vitro tests of phentolamine combined with the macrolide antibiotics erythromycin, clarithromycin, and azithromycin indicated a synergistic action against E. coli test strains. The fractional concentration inhibitory indices (FICI) of 0.375 and 0.5 indicated a synergic effect that was consistent with kinetic time-kill assays. This synergy was also seen for Salmonella typhimurium, Klebsiella pneumoniae, and Actinobacter baumannii but not Pseudomonas aeruginosa. Similarly, a phentolamine/erythromycin combination displayed significant synergistic effects in vivo in the G. mellonella model. Phentolamine added singly to bacterial cells also resulted in direct outer membrane damage and was able to dissipate and uncouple membrane proton motive force from ATP synthesis that, resulted in enhanced cytoplasmic antibiotic accumulation via reduced efflux pump activity. CONCLUSIONS: Phentolamine potentiates macrolide antibiotic activity via reducing efflux pump activity and direct damage to the outer membrane leaflet of Gram-negative bacteria both in vitro and in vivo.

Unsatisfactory Upper Blepharoplasty Revision Technique: Reconstruction of the Upper Orbital Septum and its Fusion Point.

BACKGROUND: The orbital septum prevents both superficial eyelid infection and the spread of bleeding into the orbit. The fusion point between the upper eyelid orbital septum and the levator aponeurosis or the tarsal plate plays an important role in determining the width of the natural double eyelid. We observed a number of unsatisfactory upper eyelid blepharoplasty outcomes resulting from improper handling of the orbital septum-such as excessive destruction of orbital septum tissue and failure to form a firm attachment point between the orbital septum and the levator aponeurosis or palpebrae plate-during primary surgery. OBJECTIVES: The three most common types of unsatisfactory upper blepharoplasty outcomes include abnormally high double eyelid creases, multiple creases, and disappearance of creases. In the repair operation, we try to determine the remaining orbital septum tissue for reconstruction and form a firm attachment between the orbital septum and the levator aponeurosis or tarsal plate. Follow-up after surgery was performed to observe whether our technique can ensure effective and favorable long-term natural-looking upper eyelid blepharoplasty outcome. METHODS: From January 2018 to January 2020, secondary blepharoplasty involving the above-mentioned unsatisfactory double eyelid results was performed in 83 patients, including 63 patients (141 eyes) with abnormally high skin creases, 6 patients (8 eyes) with multiple creases, and 14 patients (24 eyes) with double eyelid disappearance. The outcomes were assessed 6 months to 2 years after the surgery by reviewing the photographs to evaluate the esthetic outcomes including stability of double eyelid, double fold curve, symmetry, patient satisfaction, and the incidence of complications. RESULTS: After an average follow-up of 12 months, most patients achieved a better double eyelid appearance. The esthetic outcome was graded as good in 80 patients, poor due to recurrence of double eyelid disappearance in 2, and poor because of asymmetry of the double eyelid curve in length or width in 1 patient. All patients had acceptable scars. No cases of infection or ptosis were observed. CONCLUSION: Reconstructing the orbital septum and ensuring a firm fixation with the levator aponeurosis or tarsal plate is an effective method to repair unsatisfactory upper eyelid blepharoplasty. Moreover, it is very important to protect the orbital septum and proper treatment during the initial surgery. LEVEL OF EVIDENCE IV: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Construction of a dermis-fat composite in vivo: Optimizing heterogeneous acellular dermal matrix with in vitro pretreatment.

Dermis-fat composite tissues have been widely used in plastic and reconstructive surgery and were previously constructed using hydrogel-type scaffolds. The constructs can be used for in vitro cosmetic and pharmaceutical testing but are not mechanically strong enough for in vivo applications. In this study, we used heterogeneous (porcine) acellular dermal matrix (PADM) as dermal layer scaffold. PADM was pretreated with the laser micropore technique and then precultured with rat adipose-derived stem cells (rADSCs) in vitro. rADSCs proliferated well on pretreated/unpretreated PADM, showing increased expression of genes associated with inflammatory regulation, proangiogenesis, and stemness, indicating that pretreated/unpretreated PADM both provide a beneficial microenvironment for rADSCs to exert their paracrine function. After in vitro processing, the rADSCs-polyporous PADM and PADM without pretreatments were implanted into the back of rats respectively, followed by adipose tissue transplantation. After implantation, the inflammation induced by pretreated PADM was significantly attenuated and localized compared to the unpretreated group. Moreover, the vascularization was faster, and more adipose tissue was formed in the pretreated group. Sound dermis-fat composite tissue was constructed with sufficient strength, which can potentially be used for actual repair application.

Pre-culture of adipose-derived stem cells and heterologous acellular dermal matrix: paracrine functions promote post-implantation neovascularization and attenuate inflammatory response.

Heterologous acellular dermal matrix (ADM) has good biocompatibility and sufficient strength for clinical use for the repair of defects, tissue filling, and resurfacing of deep wounds. However, ADM tissue has such a compact structure that it can easily result in delayed vascularization after implantation. Moreover, in spite of the low immunogenicity of heterologous ADM, it can still cause varying degrees of inflammation in the host. These two drawbacks limit the efficacy and scope of clinical applications for heterologous ADM. Adipose-derived stem cells (ADSCs) have multiple effects on promoting vascularization and regulating immunological responses through paracrine signaling. Pre-culturing heterologous ADM with ADSCs may address these problems; however, it is unknown if ADSCs can exert their paracrine functions within a heterologous ADM microenvironment. This study examined the effect of porcine ADM (PADM) on the paracrine function of rat ADSCs (rADSCs) and showed that the expression of genes associated with inflammatory regulation, pro-angiogenesis factors, and stemness increased when rADSCs were seeded on PADM compared to rADSCs seeded on microplates. This indicates that PADM can provide a beneficial microenvironment for ADSCs to exert their paracrine function. After pre-culture, in vivo implanted rADSC-PADM exhibited improved vascularization and mitigated inflammatory response compared to untreated PADM. This study is the first to report that ADM can provide a suitable microenvironment for ADSCs and that pre-culturing improved the ADM implantation quality in vivo. These results suggest that it could be possible to apply heterologous ADM more effectively and broadly for repair and reconstruction treatments.