Filaggrin loss-of-function variants are associated with atopic dermatitis phenotypes in a diverse, early-life prospective cohort.

Loss-of-function (LoF) variants in the filaggrin (FLG) gene are the strongest known genetic risk factor for atopic dermatitis (AD), but the impact of these variants on AD outcomes is poorly understood. We comprehensively identified genetic variants through targeted region sequencing of FLG in children participating in the Mechanisms of Progression of Atopic Dermatitis to Asthma in Children cohort. Twenty FLG LoF variants were identified, including 1 novel variant and 9 variants not previously associated with AD. FLG LoF variants were found in the cohort. Among these children, the presence of 1 or more FLG LoF variants was associated with moderate/severe AD compared with those with mild AD. Children with FLG LoF variants had a higher SCORing for Atopic Dermatitis (SCORAD) and higher likelihood of food allergy within the first 2.5 years of life. LoF variants were associated with higher transepidermal water loss (TEWL) in both lesional and nonlesional skin. Collectively, our study identifies established and potentially novel AD-associated FLG LoF variants and associates FLG LoF variants with higher TEWL in lesional and nonlesional skin.

Temporal relationships between Staphylococcus aureus colonization, filaggrin expression, and pediatric atopic dermatitis.

BACKGROUND: Atopic dermatitis (AD) is characterized by Staphylococcus aureus (S. aureus) colonization. Longitudinal early life data delineating relationships of S. aureus colonization, barrier function, and AD outcomes are lacking. We define longitudinal S. aureus endotypes and AD pathogenesis in early life. METHODS: We defined longitudinal S. aureus skin colonization phenotypes across two annual visits (non-colonized: V1- V2- , early transient: V1+ V2- , late-onset: V1- V2+ , persistent: V1+ V2+ ) in the Mechanisms of Progression of Atopic Dermatitis to Asthma in Children cohort. We analyzed AD severity, sensitization, and skin barrier function across phenotypes, and performed mediation analyses between colonization and FLG expression. RESULTS: Persistent S. aureus colonization was associated with increased SCORAD at V1 (33.5 vs. 19.0, p = .004) and V2 (40.1 vs.16.9, p < .001), and lower non-lesional (NL) FLG at V2 (1.77 vs. 4.09, p = .029) compared to the non-colonized phenotype, with early transient and late-onset colonization as intermediate phenotypes. Children colonized at V2 demonstrated a decrease in NL-FLG expression from V1 to V2 compared to those non-colonized at V2 (p = .0012), who maintained expression. This effect remained significant even after adjusting for V1 colonization and SCORAD (p = .011). CONCLUSIONS: Our findings are the first to present longitudinal quantitative FLG expression and S. aureus skin colonization in early life and suggest that a decrease in NL-FLG drives later colonization. Hence, therapies to maintain NL-FLG expression may prevent S. aureus colonization. Further, a longitudinal AD endotype of persistent colonization is characterized by increased AD severity, sensitization, and decreasing NL-FLG.

Heat-killed probiotic Lactobacillus plantarum affects the function of neutrophils but does not improve survival in murine burn injury.

Probiotics have become of interest as therapeutics in trauma or sepsis-induced inflammation due to their ability to affects the immune response. However, their use is still under debate due to the potential risk of septicemia. Therefore, heat-killed probiotics offer a potential alternative, with recent research suggesting a comparable immunomodulating potential and increased safety. In a previous study, we demonstrated decreased mortality by administration of live Lactobacillus plantarum in a mouse burn-sepsis model. Neutrophils are an essential innate defense against pathogens. Therefore, our present study aims to understand the impact of heat-killed probiotic L. plantarum (HKLP) on neutrophil function. Utilizing an in vitro stimulation with HKLP and a burn-infection in vivo model, we determined that administration of HKLP induced significant release of granulocyte-colony stimulating factor (G-CSF) and stimulated the release of pro-and anti-inflammatory cytokines. HKLP had no impact on neutrophil function, such as phagocytosis, oxidative burst, and NETosis, but increased apoptosis and activated neutrophils. HKLP did not improve survival. Together, contrary to our hypothesis, heat-killed probiotics did not improve neutrophil function and survival outcome in a murine severe burn injury model.

Pirfenidone as a potential antifibrotic injectable for Dupuytren's disease.

Dupuytren's disease is a progressive fibrotic condition of the hand that causes contracture of fingers in later stages. Our previous in vitro studies suggest that the transformation of fibroblasts to myofibroblasts induced by transforming growth factor-beta can be inhibited by the addition of the antifibrotic drug, pirfenidone (PFD). We hypothesize that the local delivery of PFD directly to nodules can potentially prevent the progression to cords and, furthermore, that injection of PFD after the resection of cords can limit the recurrence of the disease. The purpose of this research was to develop a PFD injectable solution and to assess its safety in mice. Based on preformulation observations, a sterile solution containing up to 8 mg/0.4 mL of PFD was prepared in a phosphate buffer with and without 15%v/v N-methyl-2-pyrrolidone. Accelerated stability studies suggested that the product should be kept at refrigerated temperature (2-8 °C) for long-term storage. Safety studies involving subcutaneous administration to mice showed that 2-4 mg of PFD in 0.4 mL aqueous buffer did not elicit a significant inflammatory reaction. However, 4 mg PFD in 0.4 mL (FB) of buffer: NMP cosolvent system led to a significant increase in the influx of inflammatory cells and 8 mg PFD (FA) in the cosolvent system was lethal to the animals.

AB569, a Novel, Topical Bactericidal Gel Formulation, Kills Pseudomonas aeruginosa and Promotes Wound Healing in a Murine Model of Burn Wound Infection.

Cutaneous thermal injuries from burns/explosives are a major cause of morbidity and mortality and represent a monumental burden on our current health care system. Injury severity is predominantly due to potentially lethal sepsis caused by multidrug-resistant (MDR) bacteria such as Pseudomonas aeruginosa (MDR-PA). Thus, there is a critical need to develop novel and effective antimicrobials for the (i) prevention, (ii) treatment, and (iii) healing of such wounds that are complicated by MDR-P. aeruginosa and other bacterial infections. AB569 is a novel bactericidal tandem consisting of acidified NaNO2 (A-NO2-) and Na2-EDTA. Here, we first show that AB569 acts synergistically to kill all human burn wound strains of P. aeruginosa in vitro. This was found to be due, in part, to the generation of A-NO2--mediated nitric oxide (NO) formation coupled with the metal chelating properties of Na2-EDTA. Using a murine scald burn wound model of P. aeruginosa infection, an AB569-Solosite gel formulation eradicated all bacteria. Futher, we also demonstrate enhanced AB569-mediated wound healing by not only accelerating wound contraction, but also by reducing levels of the proinflammatory cytokines interleukin-6 (IL-6) and IL-1ß while increasing the levels of anti-inflammatory cytokine, IL-10, and granulocyte-colony-stimulating factor (G-CSF). We also observed better epidermal restoration in AB569-treated wounds. Taken together, we conclude that this study provides solid foundational evidence that AB569 can be used topically to treat highly problematic dermal insults, including wound, burn, blast, and likely, diabetic infections in civilian and military populations, and help relieve the economical burden that MDR organisms have on the global health care system.

The Bactericidal Tandem Drug, AB569: How to Eradicate Antibiotic-Resistant Biofilm Pseudomonas aeruginosa in Multiple Disease Settings Including Cystic Fibrosis, Burns/Wounds and Urinary Tract Infections.

The life-threatening pandemic concerning multi-drug resistant (MDR) bacteria is an evolving problem involving increased hospitalizations, billions of dollars in medical costs and a remarkably high number of deaths. Bacterial pathogens have demonstrated the capacity for spontaneous or acquired antibiotic resistance and there is virtually no pool of organisms that have not evolved such potentially clinically catastrophic properties. Although many diseases are linked to such organisms, three include cystic fibrosis (CF), burn/blast wounds and urinary tract infections (UTIs), respectively. Thus, there is a critical need to develop novel, effective antimicrobials for the prevention and treatment of such problematic infections. One of the most formidable, naturally MDR bacterial pathogens is Pseudomonas aeruginosa (PA) that is particularly susceptible to nitric oxide (NO), a component of our innate immune response. This susceptibility sets the translational stage for the use of NO-based therapeutics during the aforementioned human infections. First, we discuss how such NO therapeutics may be able to target problematic infections in each of the aforementioned infectious scenarios. Second, we describe a recent discovery based on years of foundational information, a novel drug known as AB569. AB569 is capable of forming a "time release" of NO from S-nitrosothiols (RSNO). AB569, a bactericidal tandem consisting of acidified NaNO2 (A-NO2 -) and Na2-EDTA, is capable of killing all pathogens that are associated with the aforementioned disorders. Third, we described each disease state in brief, the known or predicted effects of AB569 on the viability of PA, its potential toxicity and highly remote possibility for resistance to develop. Finally, we conclude that AB569 can be a viable alternative or addition to conventional antibiotic regimens to treat such highly problematic MDR bacterial infections for civilian and military populations, as well as the economical burden that such organisms pose.

AB569, a non-toxic combination of acidified nitrite and EDTA, is effective at killing the notorious Iraq/Afghanistan combat wound pathogens, multi-drug resistant Acinetobacter baumannii and Acinetobacter spp.

Multi-drug resistant (MDR) Acinetobacter baumannii (Ab) and Acinetobacter spp. present monumental global health challenges. These organisms represent model Gram-negative pathogens with known antibiotic resistance and biofilm-forming properties. Herein, a novel, nontoxic biocide, AB569, consisting of acidified nitrite (A-NO2-) and ethylenediaminetetraacetic acid (EDTA), demonstrated bactericidal activity against all Ab and Acinetobacter spp. strains, respectively. Average fractional inhibitory concentrations (FICs) of 0.25 mM EDTA plus 4 mM A-NO2- were observed across several clinical reference and multiple combat wound isolates from the Iraq/Afghanistan wars. Importantly, toxicity testing on human dermal fibroblasts (HDFa) revealed an upper toxicity limit of 3 mM EDTA plus 64 mM A-NO2-, and thus are in the therapeutic range for effective Ab and Acinetobacter spp. treatment. Following treatment of Ab strain ATCC 19606 with AB569, quantitative PCR analysis of selected genes products to be responsive to AB569 revealed up-regulation of iron regulated genes involved in siderophore production, siderophore biosynthesis non-ribosomal peptide synthetase module (SBNRPSM), and siderophore biosynthesis protein monooxygenase (SBPM) when compared to untreated organisms. Taken together, treating Ab infections with AB569 at inhibitory concentrations reveals the potential clinical application of preventing Ab from gaining an early growth advantage during infection followed by extensive bactericidal activity upon subsequent exposures.

Biological Targets for Dupuytren Disease.

ABSTRACT: Dupuytren disease is a connective tissue disorder occurring on the palm, causing flexion contractures of fingers. There is a pressing need for therapeutic interventions that can slow, stop, or even incrementally reverse the progression of the disease. Numerous in vitro studies have shed light on cellular and molecular agents that contribute to contractures. This article comprehensively reviews various growth factors that can be targeted to prevent and limit the progression and recurrence of Dupuytren contracture (DC). Fibroblasts are the major cell population that has been reported for the contractures in DC, and they are also known to exacerbate the cytokine production. Limiting the fibroblast function by targeting the growth factor production will be of great benefit in treating DC. This review will focus on the studies that have shown to limit the exaggerated function of fibroblasts by reducing the expression of profibrotic growth factors by using antagonizing agents.

Protease levels are significantly altered in pediatric burn wounds.

Burn wounds contain high levels of protease activity due to the need to remodel the damaged extracellular matrix proteins. While necessary, excessive protease activity can lead to improper wound healing and is associated with increased contraction and fibrosis. No studies to date have investigated the expression changes of all the collagenases and elastases in burn wounds. The present study compares gene expression changes and changes in collagenase and elastase activity between burn wound eschar and normal skin in a pediatric population. Deidentified pediatric tissues were used for these experiments. Burn wound tissue was excised as part of normal standard care within a week from injury; normal skin was removed during elective plastic surgery procedures. RNA-sequencing was performed and significant results were confirmed with qRT-PCR. Activity assays showed a significant increase in both collagenase and elastase activity in the burn wound tissue compared to the normal skin. Western blotting and substrate zymography of tissue homogenates evaluated the results at the protein levels. Four elastases and three collagenases were determined to be significantly upregulated in the wound tissues by both RNA-sequencing and qRT-PCR. Cathepsin V was the only protease that was significantly downregulated. All but one metalloproteinase studied was significantly upregulated. None of the serine proteases were significantly altered in the wound tissues. In conclusion, matrix metalloproteinases appear to be the most highly elevated proteases after a pediatric burn wound injury, at least within the first 3-7 days. The data warrant further investigation into the effects of MMPs on burn wound healing.

Pirfenidone inhibits epithelial-mesenchymal transition in keloid keratinocytes.

BACKGROUND: Keloids are benign fibroproliferative skin lesions that are difficult to treat and become a lifetime predicament for patients. Several treatment modalities have been put forth, but as yet no satisfactory approach to the prevention or treatment of keloids has been identified. The process of epithelial-to-mesenchymal transition (EMT) has been implicated in keloid scarring, as keloid keratinocytes display an EMT-like phenotype. This study investigated the potential of pirfenidone, an antifibrotic agent, to counteract EMT-like alterations in keloid keratinocytes, including gene expression, cell migratory and proliferative functions. METHODS: Normal and keloid keratinocytes were isolated from discarded normal skin tissues and from resected keloid tissues, respectively. Cells were quiesced for 24 h without epidermal growth factor DS-Qi1MCDigital and were exposed to transforming growth factor-beta1 (TGF-ß1; 10 ng/mL), with or without pirfenidone (400 µg/mL), for an additional 24 h. The effects of pirfenidone on cytotoxicity, cell migration, cell proliferation, and on expression of genes and proteins involved in EMT were assayed. Statistical significance was determined by two-way ANOVA using Sigma Plot. RESULTS: We found that pirfenidone did not elicit any cytotoxic effect at concentrations up to 1000 µg/mL. A statistically significant dose-dependent decrease in basal cell proliferation rate was noted in both normal and keloid keratinocytes when exposed to pirfenidone at concentrations ranging from 200 to 1000 µg/mL. Pirfenidone significantly decreased basal cell migration in both normal and keloid keratinocytes, but a significant decrease in TGF-ß1-induced cell migration was seen only in keloid keratinocytes. Significant inhibition of the expression of TGF-ß1-induced core EMT genes, namely hyaluronan synthase 2, vimentin, cadherin-11, and wingless-type MMTV integration site family, member 5A along with fibronectin-1, was observed in both normal and keloid keratinocytes treated with pirfenidone. In addition, the protein levels of vimentin and fibronectin were significantly reduced by pirfenidone (400 µg/mL) in both normal and keloid keratinocytes. CONCLUSIONS: For the first time, this study shows the efficacy of pirfenidone in inhibiting the EMT-like phenotype in keratinocytes derived from keloids, suggesting that pirfenidone may counteract a critical contributor of keloid progression and recurrence.

Characterization of Burn Eschar Pericytes.

Pericytes are cells that reside adjacent to microvasculature and regulate vascular function. Pericytes gained great interest in the field of wound healing and regenerative medicine due to their multipotential fate and ability to enhance angiogenesis. In burn wounds, scarring and scar contractures are the major pathologic feature and cause loss of mobility. The present study investigated the influence of burn wound environment on pericytes during wound healing. Pericytes isolated from normal skin and tangentially excised burn eschar tissues were analyzed for differences in gene and protein expression using RNA-seq., immunocytochemistry, and ELISA analyses. RNA-seq identified 443 differentially expressed genes between normal- and burn eschar-derived pericytes. Whereas, comparing normal skin pericytes to normal skin fibroblasts identified 1021 distinct genes and comparing burn eschar pericytes to normal skin fibroblasts identified 2449 differential genes. Altogether, forkhead box E1 (FOXE1), a transcription factor, was identified as a unique marker for skin pericytes. Interestingly, FOXE1 levels were significantly elevated in burn eschar pericytes compared to normal. Additionally, burn wound pericytes showed increased expression of profibrotic genes periostin, fibronectin, and endosialin and a gain in contractile function, suggesting a contribution to scarring and fibrosis. Our findings suggest that the burn wound environment promotes pericytes to differentiate into a myofibroblast-like phenotype promoting scar formation and fibrosis.

Investigating the effects of Pirfenidone on TGF-ß1 stimulated non-SMAD signaling pathways in Dupuytren's disease -derived fibroblasts.

BACKGROUND: Dupuytren's disease (DD) is a progressive, debilitating condition of the hand that can eventually cause contractures of the affected fingers. Transforming growth factor- ß1 (TGF-ß1) has been reported to play a key role in DD pathology. Increased expression of TGF-ß1 has shown to be the main stimulator of myofibroblast activity and in DD contractures. Pirfenidone (PFD), a small active molecule possess the ability to inhibit TGF-ß1-mediated action in various fibrotic disorders. Our recent published findings show that PFD reduced TGF-ß1-mediated cellular functions implicated in DD through SMAD signaling pathways. In the present study, the effect of PFD on TGF-ß1-mediated non-SMAD signaling pathways were investigated in both carpal tunnel (CT) - and DD-derived fibroblasts. METHODS: Fibroblasts harvested from Dupuytren's disease (DD) and carpal tunnel (CT) tissues were cultured in the presence or absence of TGF-ß1 (10 ng/ml) and/or PFD (800 µg/ml). Cell lysates were analyzed using Western blots. Equal amounts of proteins were loaded to determine the phosphorylation levels of phosphatidylinositol-3 kinase (PI3K/AKT), extracellular regulated kinases (ERK1/2), p38 mitogen-activated protein kinase and Rho family related myosin light chain (MLC). RESULTS: We show that the TGF-ß1-induced phosphorylation of AKT was significantly decreased by the addition of PFD (800 µg/mL) in both CT- and DD-derived fibroblasts. Interestingly, there was no significant difference in the phosphorylation levels of both ERK and p38 on TGF-ß1- induced cells in both CT-and DD-derived fibroblasts. But, PFD significantly decreased the TGF- ß1-induced phosphorylation levels of ERK1/2 in both CT- and DD- cells. In contrast, PFD significantly decreased the basal and TGF- ß1-induced phosphorylation levels of p38 in DD-derived fibroblasts. TGF- ß1-induced phosphorylation levels of MLC was decreased by PFD in DD-derived fibroblasts. CONCLUSIONS: These in-vitro results indicate for the first time that PFD has the potential to inhibit TGF-ß1-induced non-SMAD signaling pathways in both CT- and DD-derived fibroblasts but pronounced statistically significant inhibition on all molecules was observed only in DD-derived fibroblasts. Our previous studies show that PFD can inhibit TGF-ß1- induced SMAD signaling pathway proteins, namely p- SMAD2/SMAD3. These broad and complementary actions suggest PFD as a promising candidate to inhibit the TGF-ß1- mediated molecular mechanisms leading to DD fibrosis.

Pericytes reduce inflammation and collagen deposition in acute wounds.

BACKGROUND: Pericytes have been shown to have mesenchymal stromal cell-like properties and play a role in tissue regeneration. The goal of this study was to determine whether the addition of a pericyte sheet to a full-thickness dermal wound would enhance the healing of an acute wound. METHODS: Human muscle-derived pericytes and human dermal fibroblasts were formed into cell sheets, then applied to full-thickness excisional wounds on the dorsum of nu/nu mice. Histology was performed to evaluate epidermal and dermal reformation, inflammation and fibrosis. In addition, real-time reverse transcriptase-polymerase chain reaction (RT-PCR) was used to determine cytokine response. RESULTS: Pericytes were detected in the wounds until day 16 but not fibroblasts. Decrease in wound size was noted in pericyte sheet-treated wounds. Enhanced neo-vascularization and healthy granulation tissue formation were noted in the pericyte-treated wounds. Expression of type I collagen messenger RNA (mRNA) was significantly higher in the fibroblast-treated group, whereas Type III collagen mRNA showed significant increase in the pericyte group at days 3, 6 and 9 compared with the fibroblast and no-cell groups. Trichrome staining revealed thick unorganized collagen fibrils in the fibroblast-treated wounds, whereas pericyte-treated wounds contained thinner and more alligned collagen fibrils. Tumor necrosis factor (TNF)-α mRNA levels were increased in the fibroblast-treated wounds compared with pericyte-treated wounds. DISCUSSION: The addition of pericytes may confer beneficial effects to wound healing resulting in reduced recruitment of inflammatory cells and collagen I deposition, potential to enhance wound closure and better collagen alignment promoting stronger tissue.

Adipose stem cells enhance excisional wound healing in a porcine model.

BACKGROUND: Adipose-derived stem cells (ASCs) are capable of secreting regenerative growth factors and replacing multiple tissue types. Although current literature suggests that ASCs accelerate wound healing and reduce scarring, the dose-response relationship has not been adequately investigated in large animals. We sought to establish a porcine model to optimize dose and delivery. METHODS: Four-centimeter circular, full thickness excisional wounds were created on the backs of Yorkshire pigs. Fluorescently labeled allogeneic porcine ASCs were injected into the superficial wound bed and around the wound perimeter at high (3.0 × 106 cells/cm2; n = 8), medium (1.0 × 106 cells/cm2; n = 8), and low (0.3 × 106 cells/cm2; n = 8) doses. Control wounds received saline injections (n = 8) or no treatment (n = 8). Dressings were changed twice per week, and wound closure was tracked by surface area tracing. Animals were sacrificed at 1 and 2 wk. Wounds were harvested for real-time quantitative reverse transcriptase polymerase chain reaction, immunohistochemistry, and ASC tracking. RESULTS: Labeled ASCs integrated into treated wounds by 1 wk in a dose-dependent fashion. Epithelial coverage was achieved by 14 d in all wounds. Wounds receiving high-dose ASCs exhibited thicker granulating neodermis at 7 d and greater wound contraction at 14 d. real-time quantitative reverse transcriptase polymerase chain reaction revealed improved collagen 1:collagen 3 (Col1:Col3) ratio in the medium-dose group and enhanced α-smooth muscle actin in the high-dose group at 14 d. Western blot demonstrated increased cluster of differentiation 31 protein at 2 wk in wounds receiving >106 cells/cm2. CONCLUSIONS: Doses up to 3.0 × 106 cells/cm2 were well-tolerated. High-dose ASCs accelerate wound contraction, enhance neovascularization, and may improve scar quality in excisional wounds healing by secondary intention. Doses greater than those previously used may be necessary to achieve desired effects.

Local Probiotic Therapy with Lactobacillus plantarum Mitigates Scar Formation in Rabbits after Burn Injury and Infection.

BACKGROUND: Infection is the most common complication in burn-injured patients and is believed to contribute to the hypertrophic scarring frequently observed in such injury. Pseudomonas aeruginosa is a common pathogen in burn wound infection. We examined the effect of local probiotic therapy with Lactobacillus plantarum on the severity of the scarring following burn wounding and infection with P. aeruginosa in a rabbit model. METHODS: Full-thickness burn wounds were inoculated with control vehicle or L. plantarum; wounds were then challenged with bioluminescent P. aeruginosa. The time course of the ensuing infection was monitored by quantification of the emitted light. After allowing wounds to contract to near completion, they were harvested and analyzed for markers of scar formation. RESULTS: Application of L. plantarum curtailed both the severity and the length of the pseudomonal infection. Probiotic therapy significantly reduced both Type I collagen mRNA concentrations and total collagen protein accumulation in infected wounds, consistent with reduced scarring. Surprisingly, the probiotic showed a nearly equivalent effect in uninfected wounds. Masson's trichrome staining confirmed these findings histologically. CONCLUSIONS: Lactobacillus plantarum shows exciting potential as a therapeutic agent to both counteract burn wound infection and to alleviate scarring even in the absence of infection.

Anti-fibrotic action of pirfenidone in Dupuytren's disease-derived fibroblasts.

BACKGROUND: Dupuytren's disease (DD) is a complex fibro-proliferative disorder of the hand that is often progressive and eventually can cause contractures of the affected fingers. Transforming growth factor beta (TGF-ß1) has been implicated as a key stimulator of myofibroblast activity and fascial contraction in DD. Pirfenidone (PFD) is an active small molecule shown to inhibit TGF-ß1-mediated action in other fibrotic disorders. This study investigates the efficacy of PFD in vitro in inhibiting TGF-ß1-mediated cellular functions leading to Dupuytren's fibrosis. METHODS: Fibroblasts harvested from (DD) and carpal tunnel (CT)- tissues were treated with or without TGF-ß1 and/or PFD and were subjected to cell migration, cell proliferation and cell contraction assays. ELISA; western blots and real time RT-PCR assays were performed to determine the levels of fibronectin; p-Smad2/Smad3; alpha-smooth muscle actin (α-SMA), α2 chain of type I collagen and α1 chain of type III collagen respectively. RESULTS: Our results show that PFD effectively inhibits TGF-ß1-induced cell migration, proliferation and cell contractile properties of both CT- and DD-derived fibroblasts. TGF-ß1-induced α-SMA mRNA and protein levels were inhibited at the higher concentration of PFD (800 µg/ml). Interestingly, TGF-ß1 induction of type I and type III collagens and fibronectin was inhibited by PFD in both CT- and DD- derived fibroblasts, but the effect was more prominent in DD cells. PFD down-regulated TGF-ß1-induced phosphorylation of Smad2/Smad3, a key factor in the TGF-ß1 signaling pathway. CONCLUSION: Taken together these results suggest the PFD can potentially prevent TGF-ß1-induced fibroblast to myofibroblast transformation and inhibit ECM production mainly Type I- and Type III- collagen and fibronectin in DD-derived fibroblasts. Further in-vivo studies with PFD may lead to a novel therapeutic application in preventing the progression or recurrence of Dupuytren's disease.

Local Application of Probiotic Bacteria Prophylaxes against Sepsis and Death Resulting from Burn Wound Infection.

OBJECTIVE: To determine if local prophylactic application of probiotic bacteria to burn wounds will prevent death in a mouse model of burn wound sepsis. BACKGROUND: Infection remains the most common complication after burn injury and can result in sepsis and death, despite the use of topical and systemic antibiotics. Pseudomonas aeruginosa is a frequently implicated pathogen. Local application of probiotics directly to burn wounds is an attractive novel intervention that avoids the pitfalls of standard antibiotic therapies. METHODS: A burn-sepsis model was established using a sub-eschar injection of bioluminescent P. aeruginosa; infection was tracked using a charge-coupled camera. Full-thickness burn injuries were placed on the dorsums of adult mice; the injured sites were then treated with vehicle (burn wound control), probiotics (Lactobacillus plantarum only), pathogenic bacteria (Pseudomonas aeruginosa only), or probiotics plus pathogen (Lactobacillus plus Pseudomonas). Animals were monitored until death/moribundity or for one week, then sacrificed. Harvested tissues were subjected to imaging and molecular assays. RESULTS: Control and probiotic-only animals showed no mortality (100% survival) at one week. Pseudomonas-only animals showed > 90% mortality within 40 hours of infection. In contrast, animals treated with probiotics plus Pseudomonas showed less than 10% mortality. Use of bioluminescent Pseudomonas bacteria demonstrated that probiotic therapy inhibited septicemic accumulation of the pathogen in remote organs. In addition, probiotic therapy successfully suppressed the infection-dependent induction of TNF-α and interleukins 6 and 10 in the liver. CONCLUSIONS: Local probiotic therapy shows great potential as a valuable adjunct in the management of complicated burn injury.

Impact of individualized yoga therapy on perceived quality of life performance on cognitive tasks and depression among Type II diabetic patients.

CONTEXT: An individualized approach of providing yoga support can address many of the disease-related concerns indicated in the management of diabetes, specifically the impact on other life activities and long-term functional wellbeing. AIM: To analyze the role of regular yoga practice as a self-management approach to achieve glycemic control and psychological wellbeing in Type II diabetic patients. METHODS: Ninety-one subjects of both sexes responded to the announcement and consented to participate in the study. This was a single group, before and after yoga evaluation without control comparison. The fasting and postprandial blood sugar, glycosylated hemoglobin (HBA1c), cognitive tasks, depression, cognitive failure, and diabetic-related quality of life (QOL) were measured as pretest. The subjects underwent one-to-one individualized yoga therapy sessions, which included 12 supervised sessions spread over a 3-month period. The posttest data were analyzed using paired t-test and Wilcoxon paired rank test. RESULTS: Showed significant reduction in fasting blood sugar. QOL of the diabetic patients had improved significantly. There was a significant reduction in the frequency (mean difference of 7.58, P > 0.01) of depressive symptoms and intensity of depression (mean difference 1.66, P > 0.05). Concentration and attention span improved significantly and mean discrepancy score reduced (mean difference 3.42, P > 0.01). There were no marked changes in the postprandial blood sugar and HBA1c. CONCLUSION: Yoga practice enhances the subjective wellbeing, QOL, improves mood and concentration, and facilitates achievement of adequate glycemic control among Type II diabetic patients.

Administration of adipose-derived stem cells enhances vascularity, induces collagen deposition, and dermal adipogenesis in burn wounds.

Current treatment options for severe burn wounds are often insufficient in reconstructing skin and soft tissue defects. Adipose-derived stem cells (ASCs), a readily available source of multipotent stem cells, represent a promising therapy for the treatment of full-thickness burn wounds. Full-thickness burn wounds were created on the paraspinal region of athymic mice. A one-time, sub-eschar injection of 6.8×10(6) ASCs in PBS or PBS alone was administered at 24-h postoperatively. Time to healing was quantified using Image J analysis. At days 4, 7, 14, and 21, mice were sacrificed and tissues were excised for molecular and histological analysis. ASCs were able to survive in burn wounds as determined by the presence of PKH labeling and human PPARγ expression within the wounds. CD-31 staining demonstrated increased vascularity in ASC-treated wounds at POD 4 (p<0.05). Molecular studies showed enhanced adipogenesis, as well as type III and type I collagen deposition in the ASC treated group (p<0.05). An increase in the mRNA expression ratio of type III to type I collagen was also observed following ASC treatment (p<0.05). By enhancing vascularity, collagen deposition, and adipogenesis, ASCs show promise as an adjunctive therapy for the current treatment of full thickness burn wounds.