Lipoxin B4 Enhances Human Memory B Cell Antibody Production via Upregulating Cyclooxygenase-2 Expression.

Vaccination has been the most effective way to prevent or reduce infectious diseases; examples include the eradication of smallpox and attenuation of tetanus and measles. However, there is a large segment of the population that responds poorly to vaccines, in part because they are immunocompromised because of disease, age, or pharmacologic therapy and are unable to generate long-term protection. Specialized proresolving mediators are endogenously produced lipids that have potent proresolving and anti-inflammatory activities. Lipoxin B4 (LXB4) is a member of the lipoxin family, with its proresolving effects shown in allergic airway inflammation. However, its effects on the adaptive immune system, especially on human B cells, are not known. In this study, we investigated the effects of LXB4 on human B cells using cells from healthy donors and donors vaccinated against influenza virus in vitro. LXB4 promoted IgG Ab production in memory B cells and also increased the number of IgG-secreting B cells. LXB4 enhanced expression of two key transcription factors involved in plasma cell differentiation, BLIMP1 and XBP1. Interestingly, LXB4 increased expression of cyclooxygenase-2 (COX2), an enzyme that is required for efficient B cell Ab production. The effects of LXB4 are at least partially COX2-dependent as COX2 inhibitors attenuated LXB4-stimulated BLIMP1 and Xpb-1 expression as well as IgG production. Thus, our study reveals for the first time, to our knowledge, that LXB4 boosts memory B cell activation through COX2 and suggests that LXB4 can serve as a new vaccine adjuvant.

Activated Human Lung Fibroblasts Produce Extracellular Vesicles with Antifibrotic Prostaglandins.

The differentiation of interstitial lung fibroblasts into contractile myofibroblasts that proliferate and secrete excessive extracellular matrix is critical for the pathogenesis of pulmonary fibrosis. Certain lipid signaling molecules, such as prostaglandins (PGs), can inhibit myofibroblast differentiation. However, the sources and delivery mechanisms of endogenous PGs are undefined. Activated primary human lung fibroblasts (HLFs) produce PGs such as PGE2. We report that activation of primary HLFs with IL-1ß inhibited transforming growth factor ß-induced myofibroblast differentiation in both the IL-1ß-treated cells themselves (autocrine signal) and adjacent naive HLFs in cocultures (paracrine signal). Additionally, we demonstrate for the first time that at least some of the antifibrotic effect of activated fibroblasts on nearby naive fibroblasts is carried by exosomes and other extracellular vesicles that contain several PGs, including high levels of the antifibrotic PGE2. Thus, activated fibroblasts communicate with surrounding cells to limit myofibroblast differentiation and maintain homeostasis. This work opens the way for future research into extracellular vesicle-mediated intercellular signaling in the lung and may inform the development of novel therapies for fibrotic lung diseases.

Activated human T lymphocytes inhibit TGFß-induced fibroblast to myofibroblast differentiation via prostaglandins D2 and E2.

In pulmonary fibrosis (PF), fibroblasts and myofibroblasts proliferate and deposit excessive extracellular matrix in the interstitium, impairing normal lung function. Because most forms of PF have a poor prognosis and limited treatment options, PF represents an urgent unmet need for novel, effective therapeutics. Although the role of immune cells in lung fibrosis is unclear, recent studies suggest that T lymphocyte (T cell) activation may be impaired in PF patients. Furthermore, we have previously shown that activated T cells can produce prostaglandins with anti-scarring potential. Here, we test the hypothesis that activated T cells directly inhibit myofibroblast differentiation using a coculture system. Coculture with activated primary blood-derived T cells, from both healthy human donors and PF patients, inhibited transforming growth factor ß-induced myofibroblast differentiation in primary human lung fibroblasts isolated from either normal or PF lung tissue. Coculture supernatants contained anti-fibrotic prostaglandins D2 and E2, and the inhibitory effect of coculture on myofibroblast differentiation was largely reversed when prostaglandin production was abrogated either by resting the T cells before coculture or via specific pharmacological inhibitors. Moreover, coculture conditions induced COX-2 in HLFs but not in T cells, suggesting that T cells deliver an activating signal to HLFs, which in turn produce anti-fibrotic prostaglandins. We show for the first time that coculture with activated primary human T lymphocytes strongly inhibits myofibroblast differentiation, revealing a novel cell-to-cell communication network with therapeutic implications for fibrotic lung diseases.

Toll-Like Receptor Signaling Contributes to Proinflammatory Mediator Production in Localized Provoked Vulvodynia.

OBJECTIVES: Localized provoked vulvodynia (LPV) afflicts approximately 8% of women in the United States and represents a huge financial, physical, and psychological burden. Women with LPV experience intense pain localized to the vulvar vestibule (area immediately surrounding vaginal opening). We have identified mechanisms involved in the development of LPV whereby vulvar fibroblasts respond to proinflammatory stimuli to perpetuate an inflammatory response that causes pain. However, these mechanisms are not fully elucidated. Therefore, we explored the role of toll-like receptors (TLRs), a class of innate immune receptors that rapidly respond to microbial assaults. MATERIALS AND METHODS: To determine whether TLRs are expressed by vulvar fibroblasts and whether these contribute to proinflammatory mediator production and pain in LPV, we examined TLR expression and innate immune responses in fibroblasts derived from painful vestibular regions compared with nonpainful external vulvar regions. RESULTS: Human vulvar fibroblasts express functional TLRs that trigger production of inflammatory mediators associated with chronic pain. We focused on the TLR-7-imiquimod proinflammatory interaction, because imiquimod, a ligand of TLR-7, may exacerbate pain in women during treatment of human papillomavirus-associated disease. CONCLUSIONS: Human vulvar fibroblasts express a broad spectrum of TLRs (a new finding). A significantly higher TLR-mediated proinflammatory response was observed in LPV case vestibular fibroblasts, and with respect to the imiquimod-TLR 7 interaction, development of chronic vestibular pain and inflammation may be a possible sequelae of treatment of vulvar human papillomavirus-associated disease. Suppressing enhanced TLR-associated innate immune responses to a spectrum of pathogen-associated molecular patterns may represent a new/effective therapeutic approach for vulvodynia.

Endogenous ligands of the aryl hydrocarbon receptor regulate lung dendritic cell function.

The aryl hydrocarbon receptor (AhR) is a transcription factor that has been extensively studied as a regulator of toxicant metabolism. However, recent evidence indicates that the AhR also plays an important role in immunity. We hypothesized that the AhR is a novel, immune regulator of T helper type 2 (Th2) -mediated allergic airway disease. Here, we report that AhR-deficient mice develop increased allergic responses to the model allergen ovalbumin (OVA), which are driven in part by increased dendritic cell (DC) functional activation. AhR knockout (AhR(-/-) ) mice sensitized and challenged with OVA develop an increased inflammatory response in the lung compared with wild-type controls, with greater numbers of inflammatory eosinophils and neutrophils, greater T-cell proliferation, greater production of Th2 cytokines, and higher levels of OVA-specific IgE and IgG1. Lung DCs from AhR(-/-) mice stimulated antigen-specific proliferation and Th2 cytokine production by naive T cells in vitro. Additionally, AhR(-/-) DCs produced higher levels of tumour necrosis factor-α and interleukin-6, which promote Th2 differentiation, and expressed higher cell surface levels of stimulatory MHC Class II and CD86 molecules. Overall, loss of the AhR was associated with enhanced T-cell activation by pulmonary DCs and heightened pro-inflammatory allergic responses. This suggests that endogenous AhR ligands are involved in the normal regulation of Th2-mediated immunity in the lung via a DC-dependent mechanism. Therefore, the AhR may represent an important target for therapeutic intervention in allergic airways inflammation.

Thy1 (CD90) controls adipogenesis by regulating activity of the Src family kinase, Fyn.

Worldwide obesity rates are at epidemic levels, and new insight into the regulation of obesity and adipogenesis are required. Thy1 (CD90), a cell surface protein with an enigmatic function, is expressed on subsets of fibroblasts and stem cells. We used a diet-induced obesity model to show that Thy1-null mice gain weight at a faster rate and gain 30% more weight than control C57BL/6 mice. During adipogenesis, Thy1 expression is lost in mouse 3T3-L1 cells. Overexpression of Thy1 blocked adipocyte formation and reduced mRNA and protein expression of an adipocyte marker, fatty acid-binding protein 4, by 5-fold. Although preadipocyte fibroblasts expressed Thy1 mRNA and protein, adipocytes from mouse and human fat tissue had almost undetectable Thy1 levels. Thy1 decreases the activity of the adipogenic transcription factor PPARγ by more than 60% as shown by PPARγ-dependent reporter assays. Using both genetic and pharmacologic approaches, we show Thy1 expression dampens PPARγ by inhibiting the activity of the Src-family kinase, Fyn. Thus, these studies reveal Thy1 blocks adipogenesis and PPARγ by inhibiting Fyn and support the idea that Thy1 is a novel therapeutic target in obesity.

Identification of novel mechanisms involved in generating localized vulvodynia pain.

OBJECTIVE: Our goal was to gain a better understanding of the inflammatory pathways affected during localized vulvodynia, a poorly understood, common, and debilitating condition characterized by chronic pain of the vulvar vestibule. STUDY DESIGN: In a control matched study, primary human fibroblast strains were generated from biopsies collected from localized provoked vulvodynia (LPV) cases and from age- and race-matched controls. We then examined intracellular mechanisms by which these fibroblasts recognize pathogenic Candida albicans; >70% of vulvodynia patients report the occurrence of prior chronic Candida infections, which is accompanied by localized inflammation and elevated production of proinflammatory/pain-associated interleukin (IL)-6 and prostaglandin E2 (PGE2). We focused on examining the signaling pathways involved in recognition of yeast components that are present and abundant during chronic infection. RESULTS: Dectin-1, a surface receptor that binds C albicans cell wall glucan, was significantly elevated in vestibular vs external vulvar cells (from areas without pain) in both cases and controls, while its abundance was highest in LPV cases. Blocking Dectin-1 signaling significantly reduced pain-associated IL-6 and PGE2 production during the response to C albicans. Furthermore, LPV patient vestibular cells produced inflammatory mediators in response to low numbers of C albicans cells, while external vulvar fibroblasts were nonresponsive. Inhibition of nuclear factor kappa-light-chain-enhancer of activated B cells (proinflammatory transcription factor) nearly abrogated IL-6 and PGE2 production induced by C albicans, in keeping with observations that Dectin-1 signals through the nuclear factor kappa-light-chain-enhancer of activated B cells pathway. CONCLUSION: These findings implicate that a fibroblast-mediated proinflammatory response to C albicans contributes to the induction of pain in LPV cases. Targeting this response may be an ideal strategy for the development of new vulvodynia therapies.

Isoprostane and isofuran lipid mediators accumulate in stored red blood cells and influence platelet function in vitro.

BACKGROUND: Stored red blood cells (RBCs) release hemoglobin (Hb) that leads to oxidative damage, which may contribute to thrombosis in susceptible transfusion recipients. Oxidative stress stimulates the generation of a new class of lipid mediators called F2 -isoprostanes (F2 -IsoPs) and isofurans (IsoFs) that influence cellular behavior. This study investigated RBC-derived F2 -IsoPs and IsoFs during storage and their influence on human platelets (PLTs). STUDY DESIGN AND METHODS: F2 -IsoP and IsoF levels in RBC supernatants were measured by mass spectrometry during storage and after washing. The effects of stored supernatants, cell-free Hb, or a key F2 -IsoP, 8-iso-prostaglandin F2α (PGF2α ), on PLT function were examined in vitro. RESULTS: F2 -IsoPs, IsoFs, and Hb accumulated in stored RBC supernatants. Prestorage leukoreduction reduced supernatant F2 -IsoPs and IsoFs levels, which increased again over storage time. Stored RBC supernatants and 8-iso-PGF2α induced PLT activation marker CD62P (P-selectin) expression and prothrombotic thromboxane A2 release. Cell-free Hb did not alter PLT mediator release, but did inhibit PLT spreading. Poststorage RBC washing reduced F2 -IsoP and IsoF levels up to 24 hours. CONCLUSIONS: F2 -IsoPs and IsoFs are produced by stored RBCs and induce adverse effects on PLT function in vitro, supporting a potential novel role for bioactive lipids in adverse transfusion outcomes. F2 -IsoP and IsoF levels could be useful biomarkers for determining the suitability of blood components for transfusion. A novel finding is that cell-free Hb inhibits PLT spreading and could adversely influence wound healing. Poststorage RBC washing minimizes harmful lipid mediators, and its use could potentially reduce transfusion complications.

Neu-164 and Neu-107, two novel antioxidant and anti-myeloperoxidase compounds, inhibit acute cigarette smoke-induced lung inflammation.

Cigarette smoke is a profound proinflammatory stimulus that causes acute lung inflammation and chronic lung disease, including chronic obstructive pulmonary disease (COPD, emphysema, and chronic bronchitis), via a variety of mechanisms, including oxidative stress. Cigarette smoke contains high levels of free radicals, whereas inflammatory cells, including macrophages and neutrophils, express enzymes, including NADPH oxidase, nitric oxide synthase, and myeloperoxidase, that generate reactive oxygen species in situ and contribute to inflammation and tissue damage. Neu-164 and Neu-107 are small-molecule inhibitors of myeloperoxidase, as well as potent antioxidants. We hypothesized that Neu-164 and Neu-107 would inhibit acute cigarette smoke-induced inflammation. Adult C57BL/6J mice were exposed to mainstream cigarette smoke for 3 days to induce acute inflammation and were treated daily by inhalation with Neu-164, Neu-107, or dexamethasone as a control. Inflammatory cells and cytokines were assessed by bronchoalveolar lavage and histology. mRNA levels of endogenous antioxidant genes heme oxygenase-1 and glutamate-cysteine ligase modifier subunit were determined by qPCR. Cigarette smoke exposure induced acute lung inflammation with accumulation of neutrophils and upregulation of proinflammatory cytokines, including IL-6, macrophage inflammatory protein-2, and keratinocyte-derived cytokine. Both Neu-164 and Neu-107 significantly reduced the accumulation of inflammatory cells and the expression of inflammatory cytokines as effectively as dexamethasone. Upregulation of endogenous antioxidant genes was dampened. Neu-164 and Neu-107 inhibit acute cigarette smoke-induced inflammation by scavenging reactive oxygen species in cigarette smoke and by inhibiting further oxidative stress caused by inflammatory cells. These compounds may have promise in preventing or treating lung disease associated with chronic smoke exposure, including COPD.

Alterations of platelet function and clot formation kinetics after in vitro exposure to anti-A and -B.

BACKGROUND: ABO-mismatched platelets (PLTs) are commonly transfused despite reported complications. We hypothesized that because PLTs possess A and B antigens on their surface, ABO-mismatched transfused or recipient PLTs could become activated and/or dysfunctional after exposure to anti-A or -B in the transfused or recipient plasma. We present here in vitro modeling data on the functional effects of exposure of PLTs to ABO antibodies. STUDY DESIGN AND METHODS: PLT functions of normal PLTs of all ABO types were assessed before and after incubation with normal saline, ABO-identical plasma samples, or O plasma samples with varying titers of anti-A and anti-B (anti-A/B). Assays used for this assessment include PLT aggregation, clot kinetics, thrombin generation, PLT cytoskeletal function, and mediator release. RESULTS: Exposure of antigen-bearing PLTs to O plasma with moderate to high titers of anti-A/B significantly inhibits aggregation, prolongs PFA-100 epinephrine closure time, disrupts clot formation kinetics, accelerates thrombin generation, reduces total thrombin production, alters PLT cytoskeletal function, and influences proinflammatory and prothrombotic mediator release. CONCLUSIONS: Our findings demonstrate a wide range of effects that anti-A/B have on PLT function, clot formation, thrombin generation, PLT cytoskeletal function, and mediator release. These data provide potential explanations for clinical observations of increased red blood cell utilization in trauma and surgical patients receiving ABO-nonidentical blood products. Impaired hemostasis caused by anti-A/B interacting with A and B antigens on PLTs, soluble proteins, and perhaps even endothelial cells is a potential contributing factor to hemorrhage in patients receiving larger volumes of ABO-nonidentical transfusions.

Genetic ablation of the aryl hydrocarbon receptor causes cigarette smoke-induced mitochondrial dysfunction and apoptosis.

Cigarette smoke is the primary risk factor for chronic obstructive pulmonary disease (COPD). Alterations in the balance between apoptosis and proliferation are involved in the etiology of COPD. Fibroblasts and epithelial cells are sensitive to the oxidative properties of cigarette smoke, and whose loss may precipitate the development of COPD. Fibroblasts express the aryl hydrocarbon receptor (AhR), a transcription factor that attenuates pulmonary inflammation and may also regulate apoptosis. We hypothesized the AhR would prevent apoptosis caused by cigarette smoke. Using genetically deleted in vitro AhR expression models and an established method of cigarette smoke exposure, we report that AhR expression regulates fibroblasts proliferation and prevents morphological features of apoptosis, including membrane blebbing and chromatin condensation caused by cigarette smoke extract (CSE). Absence of AhR expression results in cleavage of PARP, lamin, and caspase-3. Mitochondrial dysfunction, including cytochrome c release, was associated with loss of AhR expression, indicating activation of the intrinsic apoptotic cascade. Heightened sensitivity of AhR-deficient fibroblasts was not the result of alterations in GSH, Nrf2, or HO-1 expression. Instead, AhR(-/-) cells had significantly less MnSOD and CuZn-SOD expression, enzymes that protects against oxidative stress. The ability of the AhR to suppress apoptosis was not restricted to fibroblasts, as siRNA-mediated knockdown of the AhR in lung epithelial cells also increased sensitivity to smoke-induced apoptosis. Collectively, these results suggest that cigarette smoke induced loss of lung structural support (i.e. fibroblasts, epithelial cells) caused by aberrations in AhR expression may explain why some smokers develop lung diseases such as COPD.

The aryl hydrocarbon receptor ligand ITE inhibits TGFß1-induced human myofibroblast differentiation.

Fibrosis can occur in any human tissue when the normal wound healing response is amplified. Such amplification results in fibroblast proliferation, myofibroblast differentiation, and excessive extracellular matrix deposition. Occurrence of these sequelae in organs such as the eye or lung can result in severe consequences to health. Unfortunately, medical treatment of fibrosis is limited by a lack of safe and effective therapies. These therapies may be developed by identifying agents that inhibit critical steps in fibrotic progression; one such step is myofibroblast differentiation triggered by transforming growth factor-ß1 (TGFß1). In this study, we demonstrate that TGFß1-induced myofibroblast differentiation is blocked in human fibroblasts by a candidate endogenous aryl hydrocarbon receptor (AhR) ligand 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE). Our data show that ITE disrupts TGFß1 signaling by inhibiting the nuclear translocation of Smad2/3/4. Although ITE functions as an AhR agonist, and biologically persistent AhR agonists, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin, cause severe toxic effects, ITE exhibits no toxicity. Interestingly, ITE effectively inhibits TGFß1-driven myofibroblast differentiation in AhR(-/-) fibroblasts: Its ability to inhibit TGFß1 signaling is AhR independent. As supported by the results of this study, the small molecule ITE inhibits myofibroblast differentiation and may be useful clinically as an antiscarring agent.

Platelets and megakaryocytes contain functional nuclear factor-kappaB.

OBJECTIVE: To investigate the presence and role of NF-kappaB proteins in megakaryocytes and platelets. The nuclear factor-kappaB (NF-kappaB) transcription factor family is well known for its role in eliciting inflammation and promoting cell survival. We discovered that human megakaryocytes and platelets express the majority of NF-kappaB family members, including the regulatory inhibitor-kappaB (I-kappaB) and I-kappa kinase (IKK) molecules. METHODS AND RESULTS: Anucleate platelets exposed to NF-kappaB inhibitors demonstrated impaired fundamental functions involved in repairing vascular injury and thrombus formation. Specifically, NF-kappaB inhibition diminished lamellapodia formation, decreased clot retraction times, and reduced thrombus stability. Moreover, inhibition of I-kappaB-alpha phosphorylation (BAY-11-7082) reverted fully spread platelets back to a spheroid morphology. Addition of recombinant IKK-beta or I-kappaB-alpha protein to BAY inhibitor-treated platelets partially restored platelet spreading in I-kappaB-alpha inhibited platelets, and addition of active IKK-beta increased endogenous I-kappaB-alpha phosphorylation levels. CONCLUSIONS: These novel findings support a crucial and nonclassical role for the NF-kappaB family in modulating platelet function and reveal that platelets are sensitive to NF-kappaB inhibitors. As NF-kappaB inhibitors are being developed as antiinflammatory and anticancer agents, they may have unintended effects on platelets. On the basis of these data, NF-kappaB is also identified as a new target to dampen unwanted platelet activation.

Novel anti-adipogenic activity produced by human fibroblasts.

Fatty tissue is generally found in distinct "depots" distributed throughout the human body. Adipocytes from each of the various depots differ in their metabolic capacities and their responses to environmental stimuli. Although a general understanding of the factors responsible for adipogenic transformation has been achieved, much is not understood about the mechanisms of adipose tissue deposition and the phenotypes of the adipocytes found within each depot. A clue to the factors regulating fat deposition may come from studies of adipogenesis using primary human orbital fibroblasts from patients with thyroid eye disease, a condition in which intense inflammation leads to expansion of orbital adipose tissue via differentiation of fibroblasts to adipocytes. We have previously demonstrated that adipogenesis of orbital fibroblasts is negatively correlated with cellular expression of the Thy-1 surface marker. In this study, we developed a novel imaging flow cytometric approach for the assessment of adipogenesis to test the hypothetical dependence of adipogenic potential on lack of Thy-1 expression. Using this technique, we learned that Thy-1-positive fibroblasts are, in fact, capable of differentiating into adipocytes but are less likely to do so because they secrete a paracrine anti-adipogenic factor. It is possible that such a factor plays an important role in the prevention of excess fat deposition in the normal orbit and may even be exploited as a therapy for the treatment of obesity, a major worldwide health concern.

Discovery of Novel Small Molecules that Block Myofibroblast Formation: Implications for Capsular Contracture Treatment.

Capsular contracture is a devastating complication that occurs in patients undergoing implant-based breast reconstruction. Ionizing radiation drives and exacerbates capsular contracture in part by activating cytokines, including transforming growth factor-beta (TGF-ß). TGF-ß promotes myofibroblast differentiation and proliferation, leading to excessive contractile scar formation. Therefore, targeting the TGF-ß pathway may attenuate capsular contracture. METHODS: A 20,000 small molecule library was screened for anti-TGF-ß activity. Structurally diverse anti-TGF-ß agents were identified and then tested on primary human capsular fibroblasts. Fibroblasts were irradiated or not, and then treated with both TGF-ß and candidate molecules. Resulting cells were then analyzed for myofibroblast activity using myofibroblast markers including alpha-smooth muscle actin, collagen I, Thy1, and periostin, using Western Blot, quantitative real-time polymerase chain reaction, and immunofluorescence. RESULTS: Human capsular fibroblasts treated with TGF-ß showed a significant increase in alpha-smooth muscle actin, collagen I, and periostin levels (protein and/or mRNA). Interestingly, fibroblasts treated with latent TGF-ß and 10 Gy radiation also showed significantly increased levels of myofibroblast markers. Cells that were treated with the novel small molecules showed a significant reduction in myofibroblast activation, even in the presence of radiation. CONCLUSIONS: Several novel small molecules with anti-TGF-ß activity can effectively prevent human capsular fibroblast to myofibroblast differentiation in vitro, even in the presence of radiation. These results highlight novel therapeutic options that may be utilized in the future to prevent radiation-induced capsular contracture.

Peroxisome proliferator-activated receptor gamma and retinoid X receptor transcription factors are released from activated human platelets and shed in microparticles.

Peroxisome proliferator-activated receptor gamma (PPARgamma) and its ligands are important regulators of lipid metabolism, inflammation, and diabetes. We previously demonstrated that anucleate human platelets express the transcription factor PPARgamma and that PPARgamma ligands blunt platelet activation. To further understand the nature of PPARgamma in platelets, we determined the platelet PPARgamma isoform(s) and investigated the fate of PPARgamma following platelet activation. Our studies demonstrated that human platelets contain only the PPARgamma1 isoform and after activation with thrombin, TRAP, ADP or collagen PPARgamma is released from internal stores. PPARgamma release was blocked by a cytoskeleton inhibitor, Latrunculin A. Platelet-released PPARgamma was complexed with the retinoid X receptor (RXR) and retained its ability to bind DNA. Interestingly, the released PPARgamma and RXR were microparticle associated and the released PPARgamma/RXR complex retained DNA-binding ability. Additionally, a monocytic cell line, THP-1, is capable of internalizing PMPs. Further investigation following treatment of these cells with the PPARgamma agonist, rosiglitazone and PMPs revealed a possible transcellular mechanism to attenuate THP-1 activation. These new findings are the first to demonstrate transcription factor release from platelets, revealing the complex spectrum of proteins expressed and expelled from platelets, and suggests that platelet PPARgamma has an undiscovered role in human biology.

In Vitro Characterization of Variable Porosity Wound Dressing With Anti-Scar Properties.

Introduction: New options are needed to improve wound healing while preventing excessive scar formation. Temporary primary dressings are important options in topical wound management that allow the natural healing process. Methods: We evaluated a novel primary dressing consisting of a biosynthetic, variable porosity, matrix-containing gelatin and Aloe Vera extract and a derivative dressing coated with the anti-scarring agent salinomycin for their ability to promote cell growth, reduce myofibroblast formation, and regulate cytokine production. In addition, salinomycin-coated primary dressings were tested for antimicrobial activity. Results: Both primary wound dressings permitted cell growth and attenuated TGFß-induced scar-forming myofibroblast formation. The primary wound dressings also reduced IL-6 production by 50%, IL-8 by 20%, MCP-1 by 75%, and GRO by 60% in human mesenchymal stem cells treated with TGFß. Salinomycin coating of the dressing showed antimicrobial activity by preventing Staphylococcus aureus growth. Conclusions: Both primary wound dressings support the growth of human fibroblasts and stem cells, as well as reduce inflammatory cytokine production, demonstrating their potential to serve as temporary wound dressings.

Evaluating a Variable Porosity Wound Dressing With Anti-Scar Properties in a Porcine Model of Wound Healing.

Introduction: New treatments that promote wound healing while preventing scar formation are needed. One option in topical wound healing is the use of temporary dressings that allow the natural healing process with minimal scar formation. Methods: We evaluated the temporary wound dressings PermeaDerm C, and a PermeaDerm C derivative coated with the anti-scarring agent, salinomycin (PermeaDerm D) in a pig model of wound healing to show the efficacy of these wound dressings in vivo. Results: Porcine fibroblasts grow well in the presence of PermeaDerm C or PermeaDerm A, and salinomycin reduces excessive myofibroblast formation in porcine fibroblasts in vitro. In vivo, wounds treated with PermeaDerm C and PermeaDerm A did not show abnormal or unwanted healing patterns up to 8 weeks post-wound formation. Wounds covered with either PermeaDerm C or PermeaDerm A showed a more mature wound-healing phenotype than the control wounds. Conclusions: PermeaDerm C and PermeaDerm A allowed wound healing, revealing the potential of both PermeaDerm C and PermeaDerm A to promote effective healing while preventing excessive scar formation.

Editor's Highlight: Thy1 (CD90) Expression is Reduced by the Environmental Chemical Tetrabromobisphenol-A to Promote Adipogenesis Through Induction of microRNA-103.

Environmental chemicals termed "obesogens" disrupt the endocrine system to promote adipogenesis and obesity. Tetrabromobisphenol-A (TBBPA) has been reported to increase adipogenesis; however, the mechanism(s) of action are unclear. Thy1 (CD90) is a glycophosphatidylinositol-anchored membrane protein that serves as a marker for stem cells and also plays an important role in regulating adipogenesis and obesity. We investigated whether or not TBBPA promotes adipogenesis in human and mouse cells by reducing Thy1 levels. We further sought to identify the molecular mechanism(s) whereby TBBPA targets Thy1 expression. Mouse and human cells were exposed to TBBPA, and Thy1 expression was analyzed using flow cytometry, Western blotting, and qPCR. We tested whether microRNAs predicted to target Thy1 (miR-103 and miR-107) were upregulated by TBBPA using quantitative PCR assays. We also determined if Thy1 mRNA was a bona fide miR-103/107 target. Our results show that Thy1 expression was reduced in both human and mouse cells after exposure to TBBPA. Both Thy1 mRNA and protein levels were decreased by low-dose TBBPA exposure. TBBPA reduced Thy1 levels and further increased adipogenesis when an adipogenic medium was used. Mechanistically, we show that miR-103 and miR-107 are induced by TBBPA and that miR-103 targets Thy1 to reduce its expression. Our results reveal for the first time that Thy1 is a target of TBBPA. Furthermore, our data support the concept that Thy1 is a key marker targeted by environmental chemicals that promote adipogenesis and obesity.

A Role for Bradykinin Signaling in Chronic Vulvar Pain.

Chronic vulvar pain is alarmingly common in women of reproductive age and is often accompanied by psychological distress, sexual dysfunction, and a significant reduction in quality of life. Localized provoked vulvodynia (LPV) is associated with intense vulvar pain concentrated in the vulvar vestibule (area surrounding vaginal opening). To date, the origins of vulvodynia are poorly understood, and treatment for LPV manages pain symptoms, but does not resolve the root causes of disease. Until recently, no definitive disease mechanisms had been identified; our work indicates LPV has inflammatory origins, although additional studies are needed to understand LPV pain. Bradykinin signaling is one of the most potent inducers of inflammatory pain and is a candidate contributor to LPV. We report that bradykinin receptors are expressed at elevated levels in LPV patient versus healthy control vestibular fibroblasts, and patient vestibular fibroblasts produce elevated levels of proinflammatory mediators with bradykinin stimulation. Inhibiting expression of one or both bradykinin receptors significantly reduces proinflammatory mediator production. Finally, we determined that bradykinin activates nuclear factor (NF)κB signaling (a major inflammatory pathway), whereas inhibition of NFκB successfully ablates this response. These data suggest that therapeutic agents targeting bradykinin sensing and/or NFκB may represent new, more specific options for LPV therapy. PERSPECTIVE: There is an unmet need for the development of more effective vulvodynia therapies. As we explore the mechanisms by which human vulvar fibroblasts respond to proinflammatory/propain stimuli, we move closer to understanding the origins of chronic vulvar pain and identifying new therapeutic targets, knowledge that could significantly improve patient care.