PCTR1 Enhances Repair and Bacterial Clearance in Skin Wounds

Tissue injury elicits an inflammatory response that facilitates host defense. Resolution of inflammation promotes the transition to tissue repair and is governed, in part, by specialized pro-resolving mediators (SPM). The complete structures of a novel series of cysteinyl-SPM (cys-SPM) were recently elucidated, and proved to stimulate tissue regeneration in planaria and resolve acute inflammation in mice. Their functions in mammalian tissue repair are of interest. Here, nine structurally distinct cys-SPM were screened and PCTR1 uniquely enhanced human keratinocyte migration with efficacy similar to epidermal growth factor. In skin wounds of mice, PCTR1 accelerated closure. Wound infection increased PCTR1 that coincided with decreased bacterial burden. Addition of PCTR1 reduced wound bacteria levels and decreased inflammatory monocytes/macrophages, which was coupled with increased expression of genes involved in host defense and tissue repair. These results suggest that PCTR1 is a novel regulator of host defense and tissue repair, which could inform new approaches for therapeutic management of delayed tissue repair and infection.

Inflammation is a critical phase of the tissue repair program that aids in containment of pathogens and clearance of dead tissue and debris.^1,2 Neutrophils are among the first innate immune cells recruited to injured tissue, and they possess an armament of chemical mediators that facilitate pathogen eradication. Monocytes are subsequently recruited and differentiate into macrophages that assist in pathogen detection and killing, as well as efferocytosis (ie, clearance of apoptotic cells). Distinct monocyte/macrophage subsets are temporally reprogrammed to directly promote tissue matrix remodeling, vascularization, and re-establishment of protective epithelial barriers through the release of growth factors, cytokines, and lipid mediators.3, 4, 5 If the tightly coordinated temporal dynamics of inflammation and its subsequent resolution are altered, delayed tissue repair can manifest and lead to necrosis and susceptibility to ongoing infection with pathogenic microbes.^2,6 Indeed, altered tissue repair is associated with several chronic diseases and thus new approaches to stimulate tissue repair are urgently needed.6, 7, 8, 9Lipid mediators are enzymatically generated from polyunsaturated fatty acids in injured tissues and govern both the initial phases of inflammation (eg, leukocyte recruitment, activation), as well as the resolution phase.^10,11 A superfamily of structurally diverse specialized pro-resolving mediators (SPM), which include lipoxins, resolvins, protectins, and maresins, have emerged as key mediators of active resolution that engage immune cells via specific receptors to blunt excessive neutrophil recruitment and to expedite macrophage efferocytosis.¹¹ Several SPM, including lipoxins, E-series resolvins, and D-series resolvins, also actively participate in tissue repair and regeneration in distinct contexts, including the skin, intestine, skeletal muscle, eye, gut, and periodontium.12, 13, 14, 15, 16, 17 Importantly, SPM facilitate host defense by stimulating macrophages and neutrophils to phagocytose and kill bacterial pathogens.¹⁸A novel series of cysteinyl-SPM (cys-SPM) comprising peptide conjugates within the resolvin, protectin, and maresin families were discovered and were coined conjugates in tissue regeneration (CTRs) based on their roles in promoting tissue regeneration in planaria.18, 19, 20 The complete structures of these novel mediators were systematically elucidated, and stereochemical assignments were performed, which was facilitated by total organic synthesis.18, 19, 20 They are biosynthesized via conjugation of glutathione to epoxide intermediates involved in protectin (16S,17S-epoxy protectin), maresin (13S,14S-epoxy maresin), and resolvin (7S,8S-epoxy resolvin) biosynthesis, yielding protectin CTRs (PCTRs), maresin CTRs (MCTRs), and resolvin CTRs (RCTRs), respectively¹⁸ (Figure 1A). The glutathione-conjugated mediators are designated PCTR1, MCTR1, and RCTR1, whereas cleavage of the γ-glutamyl group yields PCTR2, MCTR2, and RCTR2. These products are subsequently converted to cysteine-containing PCTR3, MCTR3, and RCTR3. These cys-SPM have been identified in human and mouse tissues, including spleen, lymph nodes, and self-resolving inflammatory exudates, and actively promote resolution of acute inflammation in vivo.¹⁸ They have direct actions on human leukocytes and promote macrophage efferocytosis, as well as bacterial phagocytosis, to facilitate host defense.²⁰ These pro-resolving roles act in concert with their roles in promoting tissue regeneration.18, 19, 20 Recent studies confirm and extend the potent inflammation-resolving actions of cys-SPM in multiple injury contexts.21, 22, 23, 24, 25Open in a separate windowFigure 1Structure-activity analysis of cys-SPM in promoting human keratinocyte migration. A: Cys-SPM biosynthetic pathways and structures, depicting key epoxide intermediates involved in the biosynthesis of protectins (PD1 and PCTRs), D-series resolvins (RvD1, RvD2, and RCTRs), and maresins (MaR1, MaR2, and MCTRs). Right panel, assessment of closure in scratch-wounded monolayers of human primary keratinocytes stimulated with EGF (100 ng/mL) or structurally distinct synthetic cys-SPM (1 and 10 nmol/L; 24 hours). B: Representative images of control and PCTR1-treated keratinocytes at baseline and 24 hours after wounding, with the black dotted line indicating the cell border. C: Assessment of proliferation by 5-ethynyl-2′-deoxyuridine (EdU) incorporation in keratinocytes in the presence of full serum medium or indicated concentrations of PCTR1 for 24 hours. D: Measurement of cAMP accumulation in keratinocytes stimulated with PCTR1 (10 nmol/L). E: Assessment of closure in wounded keratinocytes stimulated with PCTR1 in the presence or absence of PKA inhibitor, H89 (5 μmol/L). Data are expressed as means ± SEM. n = 5 independent experiments (A); n = 10 to 13 replicates from 2 independent experiments (C); n = 4 replicates from 2 independent assays (D); n = 15 to 17 replicates from 3 independent experiments (E). ∗P < 0.05 by one-way analysis of variance followed by Dunnett''s post hoc tests (A, C, and E), or unpaired t-test (D).The actions of cys-SPM in mammalian tissue repair programs, including re-establishment of epithelial barriers, have yet to be addressed. Here, evidence is presented that PCTR1 directly stimulates migration of human keratinocytes in vitro which translates to accelerated closure of full-thickness skin wounds in vivo. Importantly, PCTR1 was produced in wounds infected with the common skin pathogen, Staphylococcus aureus, and PCTR1 accelerated bacterial clearance, suggesting novel roles of this SPM in both facilitating host defense, as well as engaging in tissue repair programs.     

Human leukocytes selectively convert 4S,5S-epoxy-resolvin to resolvin D3, resolvin D4, and a cys-resolvin isomer

Human phagocytes have key functions in the resolution of inflammation. Here, we assessed the role of the proposed 4S,5S-epoxy-resolvin intermediate in the biosynthesis of both resolvin D3 and resolvin D4. We found that human neutrophils converted this synthetic intermediate to resolvin D3 and resolvin D4. M2 macrophages transformed this labile epoxide intermediate to resolvin D4 and a previously unknown cysteinyl-resolvin isomer without appreciable amounts of resolvin D3. M2 macrophages play critical roles in the resolution of inflammation and in wound healing. Human M2 macrophages also converted leukotriene A₄ to lipoxins. The cysteinyl-resolvin isomer significantly accelerated tissue regeneration of surgically injured planaria. In a model of human granuloma formation, the cysteinyl-resolvin isomer significantly inhibited granuloma development by human peripheral blood leukocytes. Together, these results provide evidence for a human cell type–specific role of 4S,5S-epoxy-resolvin in the biosynthesis of resolvin D3 by neutrophils, resolvin D4 by both M2 macrophages and neutrophils, and a unique cysteinyl-resolvin isomer produced by M2 macrophages that carries potent biological activities in granuloma formation and tissue regeneration.

The acute inflammatory response is an essential host-protective process in response to infection and injury. Its timely resolution is required for wound healing and tissue repair and unresolved inflammation is linked to the pathology of many human diseases. The resolution of inflammation is an active process driven, in part, by lipid mediator class switching from classic stimulatory eicosanoids (e.g., prostaglandins, leukotrienes) to the superfamily of specialized proresolving mediators (SPMs) that activate resolution programs (1–3). The SPM superfamily consists of D-series resolvins, maresins, and protectins, which are biosynthesized from docosahexaenoic acid (DHA), E-series resolvins, from eicosapentaenoic acid (EPA), and lipoxins, from arachidonic acid; all are stereochemically defined and each confirmed to stimulate resolution in vivo. (For a recent review see ref. 4).The functional criteria for a proresolving mediator, as initially defined by resolvins, are expurgatio reliquiorum (clearance of debris), expurgatio contagionem agentis (clearance of infective agents), doloris absentia (analgesia), muneris lucrum (gain of function), and increasing mucosal surface clearance of neutrophils (5–7). The cysteinyl-SPMs (cys-SPMs) expand these criteria further to also include increasing and accelerating tissue regeneration and wound repair (8, 9). Failed resolution may lead to chronic unresolved inflammation that underlies pathology in many human diseases (7) and is known to involve dysregulated lipidome signatures, such as in COVID-19 infections (10–12). The addition of specific SPMs diminishes inflammatory responses and activates endogenous resolution programs in diverse animal models (5, 9, 13–21).The complete stereochemistry for each member of the bioactive D-series resolvins is established (reviewed in refs. 2, 4). From the D-series resolvins, resolvin D3 (4S,11R,17S-trihydroxy-5Z,7E,9E,13Z,15E,19Z-docosahexaenoic acid [RvD3]) and resolvin D4 (4S,5R,17S-trihydroxy-6E,8E,10Z,13Z,15E,19Z-docosahexaenoic acid [RvD4]) evoke reparative and proresolving bioactions. RvD3 and its aspirin-triggered 17R-epimer are potent regulators of leukocyte functions (e.g., stopping neutrophil transmigration and enhancing macrophage phagocytosis and efferocytosis) and pain, as well as abating dermal inflammation and murine peritonitis (13, 22, 23). RvD4 modulates the severity of pathological thrombosis, stimulates biosynthesis of other resolvins (21), and protects from secondary organ injury (14). Both RvD3 and RvD4 are biosynthesized during the later phase of resolution compared to other SPMs, suggesting their production by macrophage subtypes that enter exudates in the later phase of resolution with high levels of 15-lipoxygenase (LOX) (14, 22). Methanol-trapping of zymosan-A activated human polymorphonuclear neutrophils (PMNs) provided evidence that resolvin D1 biosynthesis occurs via the formation of an epoxide intermediate (24). Evidence for the biosynthesis of RvD3 and RvD4 implicated their formation is via a transient 4S,5S-epoxy-resolvin intermediate (25). The potent functions of RvD3 (22) and RvD4 (26) were confirmed using materials prepared by total organic synthesis.Recently, cys-SPMs biosynthesized from DHA-derived SPM epoxide intermediates were identified and shown to possess potent tissue-regenerative properties with surgically injured planaria (2, 9). The stereochemistry of each was confirmed via matching to materials synthesized by total organic synthesis (9, 27). Upon exposure to Escherichia coli (E. coli), human M2 macrophages produced cys-SPMs from DHA (28). The organ protective functions of each cys-SPMs are independently confirmed by others in acute respiratory distress syndrome (19, 29) and in cardiac dysfunction (16, 17). Herein we report the cell type–specific biosynthesis of RvD3 and RvD4 via the 4S,5S-epoxy-resolvin intermediate. During these studies, a previously unidentified cys-resolvin (Rv) isomer derived from this 4S,5S-epoxy-resolvin intermediate was uncovered from human macrophages that carries potent bioactivities in tissue regeneration and granuloma formation, processes where M2 macrophages are well known to play pivotal roles (1–3). Also, we report that human M2 macrophages convert leukotriene A₄ (LTA₄) to lipoxins, a potential transcellular biosynthetic route to these proresolving mediators where these reparative cells are involved.     

Solution behavior of a graft copolymer in selective solvents for its backbone or grafts

Association of a graft copolymer polyisoprene-graft-polystyrene in solvent mixtures selective either for polyisoprene backbone (THF/heptane) or for polystyrene grafts (THF/ethanol; 1,4-dioxane/ethanol) was studied by static and dynamic light scattering and sedimentation velocity. Values of molar mass and dimensions of graft copolymer micelles were determined, and qualitative information on the dynamics of the unimer ? micelles equilibrium was obtained.     

Identification of high frequency of Y chromosome deletions in patients with sex chromosome mosaicism and correlation with the clinical phenotype and Y-chromosome instability

A mosaic karyotype consisting of a 45,X cell line and a second cell line containing a normal or an abnormal Y chromosome is relatively common and is associated with a wide spectrum of clinical phenotypes. The aim of this study was to investigate patients with such a mosaic karyotype for Y chromosome material loss and then study the possible association of the absence of these regions with the phenotype, diagnosis, and Y-chromosome instability. We studied 17 clinically well-characterized mosaic patients whose karyotype consisted of a 45,X cell line and a second cell line containing a normal or an abnormal Y chromosome. The presence of the Y chromosome centromere was verified by fluorescence in situ hybridization (FISH) and was then characterized by 44 Y-chromosome specific-sequence tagged site (STS) markers. This study identifies a high frequency of Yq chromosome deletions (47%). The deletions extend from interval 5 to 7 sharing a common deleted interval (6F), which overlaps with the azoospermia factor region (AZF) region. This study finds no association between Y-chromosome loci hosting genes other than SRY, and the phenotypic sex, the diagnosis, and the phenotype of the patients. Furthermore, this study shows a possible association of these deletions with Y-chromosome instability.     

Duplex evaluation of venous insufficiency

Duplex ultrasound is the most useful examination for the evaluation of venous valvular incompetence. Multi-frequency 4 to 7-MHz linear array transducers are typically used for this assessment of superficial and deep reflux. The examination is done with the patient standing and manual compression maneuvers are used to initiate reflux. Automatic rapid inflation and deflation cuffs may be used when a standard stimulus is needed. Cutoff values for reflux have been defined. Perforating veins must be identified and flow direction during compression recorded. When ulcers are present, duplex ultrasound is used to investigate veins of the ulcerated legs. Venous outflow obstruction is also studied by duplex ultrasound and chronic changes in deep and superficial veins following deep venous thrombosis noted. The main drawback in evaluation of chronic obstruction is inability to quantify hemodynamic significance. Anatomic variations in superficial and deep veins are common and their identification is necessary. Reporting results of duplex ultrasound studies must take into consideration the proper classification of venous disease as well as the new anatomic terms that have been accepted.     

Toxicity and outcome analysis of patients with recurrent head and neck cancer treated with hyperfractionated split-course reirradiation and concurrent cisplatin and paclitaxel chemotherapy from two prospective phase I and II studies

BACKGROUND: Patients with local recurrences or new head and neck primary tumors in previously irradiated tissues have few options for salvage treatment. One option for select patients is to undergo reirradiation with concurrent chemotherapy. The purpose of this study is to report the initial clinical results of the Fox Chase phase I and II prospective reirradiation and chemotherapy studies. METHODS: Between July 1996 and January 2002, 38 patients with locally recurrent unresectable squamous cell carcinoma of the head and neck were treated with concurrent chemotherapy and reirradiation on two prospective trials. All patients had received prior radiation therapy to the head and neck region (median dose, 64.2 Gy). Patients received cisplatin and paclitaxel along with hyperfractionated external beam radiation therapy to the site of recurrence. RESULTS: The median follow-up was 10 months. The median survival was 12.4 months, with actuarial rates of overall survival of 50% and 35% at 1 and 2 years, respectively. During follow-up, 63% of patients experienced local progression of disease, all in the irradiated field. Actuarial progression-free survival at 1 year was 33%, with a median time to progression of 7.3 months. Acute grade 3 to 4 toxicity included neutropenia, nausea, emesis, and mucositis. CONCLUSIONS: Hyperfractionated split-course reirradiation and concurrent cisplatin and paclitaxel chemotherapy demonstrates durable locoregional control in select patients, although late toxicity may occasionally be significant. Only sites of disease recurrence need to be covered in the reirradiation fields.     

Faster determination of membrane permeabilities without using the lag time method

A new method of data analysis is presented that allows the determination of membrane permeabilities. The method is applicable to data obtained from a common experimental setup, in which drug dissolved in an inert donor gel diffuses through a membrane, initially void of drug, into a receiver for which sink conditions are maintained. The equations developed can also be used to predict the release of drug from these systems. Fick's Laws are solved, and the early time behavior of the mathematical solution is used to develop the analysis methods. Limitations of the model and their relations to experimental design are determined, and the method of application to experimental data is presented. The method is tested numerically using simulated data generated by a 1-d finite difference program that was used to numerically solve Fick's Laws, and also applied to in vitro human cadaver skin transdermal data for the drugs doxepin, imipramine and amitriptyline. It is concluded that this method can be applied to determine membrane permeabilities and diffusion coefficients with accuracy comparable to other experimental setups, such as lag time experiments and steady state experiments, but requiring experiments that can be significantly shorter.