Mouse Footpad Research Articles

Alphaherpesvirus infection of mice primes PNS neurons to an inflammatory state regulated by TLR2 and type I IFN signaling.

Pseudorabies virus (PRV), an alphaherpesvirus closely related to Varicella-Zoster virus (VZV) and Herpes simplex type 1 (HSV1) infects mucosa epithelia and the peripheral nervous system (PNS) of its host. We previously demonstrated that PRV infection induces a specific and lethal inflammatory response, contributing to severe neuropathy in mice. So far, the mechanisms that initiate this neuroinflammation remain unknown. Using a mouse footpad inoculation model, we found that PRV infection rapidly and simultaneously induces high G-CSF and IL-6 levels in several mouse tissues, including the footpad, PNS and central nervous system (CNS) tissues. Interestingly, this global increase occurred before PRV had replicated in dorsal root ganglia (DRGs) neurons and also was independent of systemic inflammation. These high G-CSF and IL-6 levels were not caused by neutrophil infiltration in PRV infected tissues, as we did not detect any neutrophils. Efficient PRV replication and spread in the footpad was sufficient to activate DRGs to produce cytokines. Finally, by using knockout mice, we demonstrated that TLR2 and IFN type I play crucial roles in modulating the early neuroinflammatory response and clinical outcome of PRV infection in mice. Overall, these results give new insights into the initiation of virus-induced neuroinflammation during herpesvirus infections.

Anthrax Edema Toxin Co-Opts IGF1R and EGFR Signaling to Promote Cell-Cell Barrier Dysfunction

Cell-cell barrier dysfunction induced by the anthrax edema toxin (ET) is central to bacterial dissemination and lethal vascular collapse during disease progression. Available treatments including antimicrobial and early-stage antitoxin measures do not prevent lethal anthrax toxemia during fulminant stages of infection. This study identifies cell-based therapeutic measures that target the late-stage intracellular activities of ET and their potential to mitigate consequences of ET toxemia. We uncover a critical involvement of IGF1R and EGFR in promoting ET-dependent barrier disruption through effects on cellular F-actin network that are separate from, but have an impact upon, previously characterized cAMP-mediated inhibition of vesicular trafficking. ET rapidly trans-activates IGF1R and EGFR, resulting in the activation of downstream effectors PI3K and MEK, and subsequently a Rac1- and cofilin-dependent actin remodeling. Finally, we provide in vivo pre-clinical validation of the therapeutic potential of PI3K, MEK and Rac1 inhibitors in preventing ET-dependent edema in a mouse footpad model.

344 Thimerosal induces skin pseudo-allergic reaction via Mas-related G-protein coupled receptor B2

Thimerosal has been used as a preservative in a variety of products which may cause contact dermatitis. It is the second most common allergen in positive patch test reactions, though being a clinical irrelevant allergen. It is believed that thimerosal may cause contact dermatitis through delayed-type hypersensitivity response. However, recent studies have demonstrated the association between Mas-related G protein coupled receptor X2 (MRGPRX2) on mast cells and pseudo-allergic reaction. It is possible that thimerosal may cause contact dermatitis via MRGPRX2 mediated mechanism. To investigate the role of MRGPRX2/MrgprB2 in contact dermatitis induced by thimerosal, we developed a novel skin pseudo-allergic reaction mouse model, footpad swelling and extravasation assays in vivo and mast cell degranulation assay in vitro were studied. The results showed that thimerosal induced contact dermatitis in dorsal skin and footpad swelling in wild-type mice, but had no significant effect in MrgprB2-knockout mice. Thimerosal-induced dermatitis is characterized by infiltration of inflammatory cells and elevation of serum histamine and inflammatory cytokines, rather than elevation of serum IgE level. Moreover, thimerosal increased the intracellular Ca2+ concentration in HEK293 cells overexpressing MrgprB2/MRGPRX2. Downregulation of MRGPRX2 resulted in the reduced degranulation of human mast cells from LAD2 cell line. Collectively, we demonstrate that MrgprB2 mediated thimerosal-induced mast cell degranulation and pseudo-allergic reaction in mice, thimerosal also activated human mast cell via MRGPRX2, indicating that MRGPRX2 may be a key contributor to human contact dermatitis.

Anti-Inflammatory Substances in Wheat Malt Inducing Antisecretory Factor

Extensively malted cereals counteract enterotoxic diarrhea and inflammatory bowel diseases. This effect depends on a protein called antisecretory factor (AF), which is secreted into the blood as a larger complex known as the compleasome. In this study, we identified anti-inflammatory substances in malt and assayed their capacity to induce AF. Guaiacol and quercetin inhibited inflammation in a mouse footpad model, while catechin, sinapic acid, ferulic acid, and quercetin inhibited nitric oxide formation in RAW 264.7 cells. The proteasome activity in these cells was inhibited by vanillic acid and quercetin but not by the other tested phenols. As the transient receptor potential vanilloid 1 (TRPV1) might be involved in AF induction, the TRPV1 antagonist capsazepine was tested and shown to inhibit inflammation in mouse paw and nitric oxide formation. Catechin, ferulic acid, and sinapic acid induced AF in rat blood, and these substances were all increased in malt compared to control wheat. These phenols might therefore be of particular importance for the beneficial effect of malted cereals on inflammatory diseases. Our results further suggest that TRPV1 might play a role in the anti-inflammatory activity of phenols via the induction of AF.

DNA gyrase could be a crucial regulatory factor for growth and survival of Mycobacterium leprae

Leprosy, an important infectious disease in humans caused by Mycobacterium leprae (Mle), remains endemic in many countries. Notably, the pathogen cannot be cultured in vitro, except in mouse footpads in vivo. The molecular basis of these characteristics and the mechanisms remain unknown. Consequently, analysis of Mle growth and survival is urgently needed to develop novel therapies against leprosy, including rapid, simple, and specific methods to detect infection. Here, we demonstrated the functional role and contribution of Mle-DNA gyrase, which regulates DNA topology, DNA replication, and chromosome segregation to promote bacterial growth and survival, in Mle growth and survival in vitro and in vivo. The optimum temperature for Mle-DNA gyrase activity was 30 °C. When the DNA gyrB-gyrA genes in Mycobacterium smegmatis were replaced with the Mle gyrase genes by allelic exchange, the recombinants could not grow at 37 °C. Moreover, using radiorespirometry analysis for viability of Mle bacilli, we found that Mle growth was more vigorous at 25–30 °C than at 37 °C, but was inhibited above 40 °C. These results propose that DNA gyrase is a crucial factor for Mle growth and survival and its sensitivity to temperature may be exploited in heat-based treatment of leprosy.

Thimerosal induces skin pseudo-allergic reaction via Mas-related G-protein coupled receptor B2

BackgroundThimerosal has been used as a preservative in many products which may cause contact dermatitis. It is the second most common allergen in positive patch test reactions, though being a clinical irrelevant allergen. Thimerosal-induced contact dermatitis is generally considered to be a delayed-type hypersensitivity reaction, but it is difficult to explain the fact that most patients develop an allergic reaction upon first encounter with thimerosal. Recent studies have demonstrated the association between Mas-related G protein coupled receptor X2 (MRGPRX2) and pseudo-allergic reactions which occur at the first contact with stimulation. This suggests the possibility that thimerosal may cause contact dermatitis via MRGPRX2 mediated mechanism. ObjectivesTo investigate the role of Mas-related G-protein coupled receptor B2 (MrgprB2)/MRGPRX2 in contact dermatitis induced by thimerosal. MethodsThimerosal induced pseudo-allergic reactions via MrgprB2/ MRGPRX2 were investigated using a novel skin pseudo-allergic reaction mouse model, footpad swelling and extravasation assays in vivo and mast cell degranulation assay in vitro. ResultsThimerosal induced contact dermatitis in dorsal skin and footpad swelling in wild-type mice, but had no significant effect in MrgprB2-knockout mice. Thimerosal-induced dermatitis is characterized by infiltration of inflammatory cells and elevation of serum histamine and inflammatory cytokines, rather than elevation of serum IgE level. Thimerosal increased the intracellular Ca2+ concentration in HEK293 cells overexpressing MrgprB2/MRGPRX2. Downregulation of MRGPRX2 resulted in the reduced degranulation of LAD2 human mast cells. ConclusionsMrgprB2 mediates thimerosal-induced mast cell degranulation and pseudo-allergic reaction in mice. MRGPRX2 may be a key contributor to human contact dermatitis.

The Transcription Factor Deaf1 Modulates Engrailed-1 Expression to Regulate Skin Appendage Fate

The Transcription Factor Deaf1 Modulates Engrailed-1 Expression to Regulate Skin Appendage Fate

Mycolactone as Analgesic: Subcutaneous Bioavailability Parameters.

Mycobacterium ulcerans is the bacillus responsible for Buruli ulcer, an infectious disease and the third most important mycobacterial disease worldwide, after tuberculosis and leprosy. M. ulcerans infection is a type of panniculitis beginning mostly with a nodule or an oedema, which can progress to large ulcerative lesions. The lesions are caused by mycolactone, the polyketide toxin of M. ulcerans. Mycolactone plays a central role for host colonization as it has immunomodulatory and analgesic effects. On one hand, mycolactone induces analgesia by targeting type-2 angiotensin II receptors (AT2R), causing cellular hyperpolarization and neuron desensitization. Indeed, a single subcutaneous injection of mycolactone into the mouse footpad induces a long-lasting hypoesthesia up to 48 h. It was suggested that the long-lasting hypoesthesia may result from the persistence of a significant amount of mycolactone locally following its injection, which could be probably due to its slow elimination from tissues. To verify this hypothesis, we investigated the correlation between hypoesthesia and mycolactone bioavailability directly at the tissue level. Various quantities of mycolactone were then injected in mouse tissue and hypoesthesia was recorded with nociception assays over a period of 48 h. The hypoesthesia was maximal 6 h after the injection of 4 μg mycolactone. The basal state was reached 48 h after injection, which demonstrated the absence of nerve damage. Surprisingly, mycolactone levels decreased strongly during the first hours with a reduction of 70 and 90% after 4 and 10 h, respectively. Also, mycolactone did not diffuse in neighboring skin tissue and only poorly into the bloodstream upon direct injection. Nevertheless, the remaining amount was sufficient to induce hypoesthesia during 24 h. Our results thus demonstrate that intact mycolactone is rapidly eliminated and that very small amounts of mycolactone are sufficient to induce hypoesthesia. Taken together, our study points out that mycolactone ought to be considered as a promising analgesic.

Norfloxacin, a Fluoroquinolone Antibiotic, Inhibits Langerhans Cell-Mediated Th1 and Th2 Cell Development.

It is widely acknowledged that Langerhans cells (LCs) play a primary role in the polarization of T helper type 1 (Th1) or T helper type 2 (Th2) immune responses. Our aim was to find fluoroquinolone ("new quinolone") antibiotics that would inhibit LC-mediated Th2 cell development. Expression of LC surface molecules was investigated using the reverse transcriptase polymerase chain reaction. The effects of fluoroquinolone antibiotics on T-cell immunoglobulin and mucin domain-containing protein (TIM)-4 expression in LCs were examined to predict whether they would inhibit Th2 cell development. Mice were primed via the hind footpad with ovalbumin (OVA) peptide-pulsed LCs that had been treated with a selected fluoroquinolone antibiotic, then 5 days later the cytokine response in popliteal lymph nodes was examined by enzyme-linked immunosorbent assay. Norfloxacin was selected as a candidate inhibitor of Th2 cell development. As expected, OVA peptide-pulsed LCs that had been treated with norfloxacin and injected into the hind footpads of mice inhibited Th2 cell development, as represented by down-regulation of interleukin (IL)-4 production, as well as Th1 cell development, as represented by down-regulation of interferon (IFN)- g production. This additional inhibition of Th1 cell development was accompanied by suppression of CD40 expression in LCs. In addition, Staphylococcus aureus strains isolated from skin lesions of patients with atopic dermatitis (AD) were more susceptible to norfloxacin than to gentamicin. Topical treatment with norfloxacin significantly suppressed the increase in the skin severity score in NC/Nga mice with AD-like skin lesions. This suppressive effect was associated with a decrease in the production of IFN-g and IL-4 in auricular lymph node cells. The present results show that topical application of norfloxacin inhibits the development of AD-like skin lesions in NC/Nga mice. This suggests that topical application of norfloxacin to AD lesions colonized with S. aureus would act on both superficial S. aureus and epidermal LCs, thus possibly inhibiting the development of Th1 and Th2 cells in vivo, and controlling the severity of AD.

Design and synthesis of multivalent α-1,2-trimannose-linked bioerodible microparticles for applications in immune response studies of Leishmania major infection.

Leishmaniasis, a neglected tropical disease, currently infects approximately 12 million people worldwide with 1 to 2 million new cases each year in predominately underdeveloped countries. The treatment of the disease is severely underdeveloped due to the ability of the Leishmania pathogen to evade and abate immune responses. In an effort to develop anti-leishmaniasis vaccines and adjuvants, novel carbohydrate-based probes were made to study the mechanisms of immune modulation. In this study, a new bioerodible polyanhydride microparticle was designed and conjugated with a glycodendrimer molecular probe. This molecular probe incorporates a pathogen-like multivalent display of α-1,2-trimannose, for which a more efficient synthesis was designed, with a tethered fluorophore. Further attachment of the glycodendrimer to a biocompatible, surface eroding microparticle allows for targeted uptake and internalization of the pathogen-associated oligosaccharide by phagocytic immune cells. The α-1,2-trimannose-linked bioerodible microparticles were found to be safe after administration into the footpad of mice and demonstrated a similar response to α-1,2-trimannose-coated latex beads during L. major footpad infection. Furthermore, the bioerodible microparticles allowed for investigation of the role of pathogen-associated oligosaccharides for recognition by pathogen-recognition receptors during L. major-induced leishmaniasis.

A new Mycobacterium leprae dihydropteroate synthase variant (V39I) from Papua, Indonesia

Indonesia had the third highest number of new leprosy cases worldwide in 2017. This disease is still prevalent in Papua province, where the number of new cases in 2014 (3.0 cases per 10,000 people) is considered highly endemic and is well above the World Health Organization's (WHO) cutoff of <1 new case per 10,000 people. Since 1995, the WHO has supplied Papua province with a multi-drug therapy (MDT) in which multibacillary (MB) patients are treated with rifampicin, clofazimine, and dapsone and paucibacillary (PB) patients are treated with rifampicin and dapsone. Recent published data on global drug resistance reported cases of dapsone resistance in relapsed and newly diagnosed cases in Indonesia during this period. The detection of specific point mutations in folP1 that encode dihydropteroate synthases (DHPS) is used exclusively to identify dapsone resistant strains of Mycobacterium leprae. The purpose of this study was to test for the presence of folP1 mutations in M. leprae strains isolated from patients residing in Papua Island, Indonesia who responded less effectively to dapsone. This study identified a folP1 point mutation that changed a valine (V) residue at amino acid position 39 (from the N-terminus) to isoleucine (I) (V39I) of DHPS. The V39I variant is located within an α-helix motif that may not much affect its structure. Molecular docking analysis indicated that the binding affinity of the V39I variant was slightly reduced as compared to the wildtype of DHPS. The decreasing of affinity may have a consequence of increasing inhibition constants (Ki) of dapsone on the variant V39I of DHPS. The data suggest that the DHPS V39I variant might cause less sensitive to dapsone. However, in vivo studies (e.g., mouse footpad model) are needed to confirm the effect of this DHPS variant on dapsone therapy.

A genetically engineered Fc-binding amphiphilic polypeptide for congregating antibodies in vivo

We report herein an affinity-based hydrogel used in creating subcutaneous depots of antibodies in vivo. The biomaterials design centered on pG_EAK, a polypeptide we designed and expressed in E. coli. The sequence consists of a truncated protein G (pG) genetically fused with repeats of the amphiphilic sequence AEAEAKAK (“EAK”). Capture of IgG was demonstrated in vitro in gels prepared from admixing pG_EAK and EAK (“pG_EAK/EAK gel”). The binding affinities and kinetics of pG for IgG were recapitulated in the pG_EAK polypeptide. Injecting IgG antibodies formulated with pG_EAK/EAK gel into subcutaneous space resulted in retention of the antibodies at the site for at least six days, whereas only signal at background levels was detected in grafts injected with IgG formulated in saline or diffusion-driven gel. The local retention of IgG in pG_EAK/EAK gel was correlated with limited distribution of the antibody in liver, spleen and lymph nodes, in contrast to those injected with antibodies formulated in saline or non-Fc binding EAK gel. In addition, antibodies formulated with pG_EAK/EAK gel and injected in mouse footpads were found to retain at the site for 19 days. As a demonstration of potential bioengineering applications, thymic epithelial cells (TECs), the primary population of thymic stromal cells that are critical for the development of T-lymphocytes, were mixed with pG_EAK/EAK gel formulated with TEC-specific anti-EpCAM antibodies and injected subcutaneously into athymic nude mice. The injected TECs congregated into functional thymic units in vivo, supporting the development of both CD4+ and CD8+ T cells as well as Foxp3+ regulatory T cells in the mice. In conclusion, pG_EAK/EAK gel can be used to retain IgG locally in vivo, and can be tailored as scaffolds for controlling deposition of molecular and/or cellular therapeutics. Statement of significanceThe unique concept of the work centers on the genetic fusion of an Fc-binding domain and a self-assembling domain into a single polypeptide. To our knowledge, such bi-functional peptide has not been reported in the literature. The impact of the work lies in the ability to display IgG antibodies and Fc-fusion proteins of any specificity. The data shown demonstrate the platform can be used to localize IgG in vivo, and can be tailored for controlling deposition of primary thymic epithelial cells (TECs). The results support a biomaterials-based strategy by which TECs can be delivered as functional units to support T-lymphocyte development in vivo. The platform described in the study may serve as an important tool for immune engineering.

The compound TB47 is highly bactericidal against Mycobacterium ulcerans in a Buruli ulcer mouse model

Buruli ulcer (BU) is an emerging infectious disease that causes disfiguring skin ulcers. The causative agent, Mycobacterium ulcerans, secretes toxin called mycolactone that triggers inflammation and immunopathology. Existing treatments are lengthy and consist of drugs developed for tuberculosis. Here, we report that a pyrazolo[1,5-a]pyridine-3-carboxamide, TB47, is highly bactericidal against M. ulcerans both in vitro and in vivo. In the validated mouse model of BU, TB47 alone reduces M. ulcerans burden in mouse footpads by more than 2.5 log10 CFU compared to the standard BU treatment regimen recommended by the WHO. We show that mutations of ubiquinol-cytochrome C reductase cytochrome subunit B confer resistance to TB47 and the dissimilarity of CydABs from different mycobacteria may account for their differences in susceptibility to TB47. TB47 is highly potent against M. ulcerans and possesses desirable pharmacological attributes and low toxicity that warrant further assessment of this agent for treatment of BU.

External temperature control of lymphatic drainage of thermo-sensitive nanomaterials.

Nano-carrier-facilitated delivery of bioactive molecules into lymph nodes (LNs) has found application in the treatment and diagnosis of numerous immune-related diseases. Much work has focused on optimization of physicochemical properties of the nano-carrier to enhance lymphatic drainage passively, whereas active modulation of the quantity and timing of lymphatic delivery remains a significant challenge. Here, inspired by the success of thermo-modulation of tumor targeting, we have developed a simple external temperature control strategy to regulate the distribution of thermo-sensitive nanomaterials between the injection site and draining LNs. To demonstrate feasibility of this strategy, we injected Rhodamine-B-labeled poly(N-isopropylacrylamide) (RhB-PNIPAm) (2.5 kDa) into the footpad of mice at different initial temperatures - either below or above the lower critical solution temperature (LCST), followed by physical cooling of the injection site. We show that RhB-PNIPAm drained efficiently into the popliteal and inguinal nodes (pLNs, iLNs, respectively) with low levels of accumulation in major internal organs. Within the first two hours post-injection the rate of drainage was primarily dependent on the initial temperature of RhB-PNIPAm. However, over the course of 24 h, temperature gradient due to local cooling affected significantly the draining of the injection site, resulting in differential accumulation of RhB-PNIPAm in the proximal (pLNs) versus the distal (iLNs) nodes. This study provides a new methodology and insights for modulating in vivo lymphatic distribution of thermo-sensitive nanomaterials with implications in immune regulation and immunotherapy.

Toward reducing biomaterial antigenic potential: a miniaturized Fc-binding domain for local deposition of antibodies.

A peptide derived from staphylococcal protein A (SpA) was developed as an affinity module for antibody delivery applications. The miniaturized protein consists of the first helix of the engineered SpA Z domain fused with the self-assembling peptide (SAP) AEAEAKAKAEAEAKAK, or EAK. The resulting peptide, named Z15_EAK, was shown to possess fibrillization properties and an Fc-binding function. The peptide induced a red shift in the Congo red absorbance characteristic of peptide fibrils, also evidenced in transmission electron microscopy images. The one-site binding affinity (Kd) of a gel-like coacervate generated by admixing Z15_EAK with EAK for IgG was determined to be 1.27 ± 0.14 μM based on a microplate-based titration assay. The coacervate was found to localize IgG subcutaneously in mouse footpads for 8 to 28 days. A set of in vivo data was fit to a one-compartment model for simulating the relative fractions of IgG dissociated from the materials in the depot. The model predicted that close to 27% of the antibodies injected were available unbound for the duration of the experiment. Z15_EAK did not appear to induce innate immune responses; injecting Z15_EAK into mouse footpads elicited neither interleukin-6 (IL-6) nor tumor necrosis factor-alpha (TNF-α) from splenocytes isolated from the animals one day, seven days, or eleven days afterward. The antigenic potential of Z15 was analyzed using a bioinformatic approach in predicting sequences in SpA and Z15 dually presented by class I and class II human MHC alleles covering the majority of the population. A peptide in SpA identified as a potential T cell epitope cross reacting with a known epitope in a microbial antigen was eliminated by miniaturization. These results demonstrate that Z15_EAK is a potential platform for generating antibody depots by which the impacts of Fc-based biotherapeutics can be enhanced through spatiotemporal control.

Inhibition of multiple voltage-gated calcium channels may contribute to spinally mediated analgesia by epipregnanolone in a rat model of surgical paw incision

ABSTRACTVoltage-activated calcium channels play an important role in excitability of sensory nociceptive neurons in acute and chronic pain models. We have previously shown that low-voltage-activated calcium channels, or T-type channels (T-channels), increase excitability of sensory neurons after surgical incision in rats. We have also found that endogenous 5β-reduced neuroactive steroid epipregnanolone [(3β,5β)-3-hydroxypregnan-20-one] blocked isolated T-currents in dorsal root ganglion (DRG) cells in vitro, and reduced nociceptive behavior in vivo, after local intraplantar application into the foot pads of heathy rats and mice. Here, we investigated if epipregnanolone exerts an antinociceptive effect after intrathecal (i.t.) application in healthy rats, as well as an antihyperalgesic effect in a postsurgical pain model. We also studied if this endogenous neurosteroid blocks currents originating from high voltage-activated (HVA) calcium channels in rat sensory neurons. In in vivo studies, we found that epipregnanolone alleviated thermal and mechanical nociception in healthy rats after i.t. administration without affecting their sensory-motor abilities. Furthermore, epipregnanolone effectively reduced mechanical hyperalgesia after i.t application in rats after surgery. In subsequent in vitro studies, we found that epipregnanolone blocked isolated HVA currents in nociceptive sensory neurons with an IC50 of 3.3 μM in a G-protein-dependent fashion. We conclude that neurosteroids that have combined inhibitory effects on T-type and HVA calcium currents may be suitable for development of novel pain therapies during the perioperative period.

Chemical composition and evaluation of the anti-inflammatory and antinociceptive activities of Duguetia furfuracea essential oil: Effect on edema, leukocyte recruitment, tumor necrosis factor alpha production, iNOS expression, and adenosinergic and opioidergic systems

Ethnopharmacological relevanceDuguetia furfuracea (A. St. -Hil.) Saff. (Annonaceae) is commonly known in Brazil as “araticum-seco,” and its root is used in folk medicine to treat inflammatory and painful disorders. However, no studies have been performed to evaluate these therapeutic activities. Aim of the studyInvestigate the chemical composition, anti-inflammatory and antinociceptive effects, and elucidate the possible antinociceptive mechanisms of action from the essential oil of D. furfuracea (EODf) underground stem bark. Materials and methodsChemical composition was determined by gas chromatography and mass spectrometry (GC/MS). The paw edema induced by LPS, formalin-induced nociception, LPS-induced thermal hyperalgesia and rota-rod tests in vivo were used to evaluate the anti-inflammatory and antinociceptive effects in addition to the alteration on motor coordination. Histological analyses and an immunohistochemistry assay for iNOS were performed on mouse footpads of naive, control, 10 mg/kg EODf, and 10 mg/kg indomethacin (Ind) groups. The samples were removed at 1, 3, and 6 h after subplantar injection of LPS. In addition, the involvement of the adenosinergic, opioidergic, serotonergic, and cholinergic systems were investigated, in order to elucidate possible antinociceptive mechanisms. ResultsTwenty-four volatile constituents were detected and identified. (E)-asarone (21.9%), bicyclogermacrene (16.7%), 2,4,5-trimethoxystyrene (16.1%), α-gurjunene (15%), cyperene (7.8%), and (E)-caryophyllene (4.6%) were major compounds found in EODf. Oral treatment (p.o.) with EODf (1, 3, and 10 mg/kg) significantly inhibited the paw edema induced by LPS. At 10 mg/kg EODf promoted inhibition of tumor necrosis factor alpha (TNF-α) production, recruitment of polymorphonuclear (PMN) leukocytes and inducible nitric oxide synthase (iNOS) expression in paw tissue. EODf (10 and 30 mg/kg, p.o.) also reduced licking time in both phases of the formalin test and it had a significant effect on the LPS-induced thermal hyperalgesia model. The administration of caffeine (Caf) and naloxone (Nal) reversed the antinociceptive activity of EODf, in the first phase of the formalin test and in the LPS-induced thermal hyperalgesia model. Moreover, Nal was also able to abolish the antinociception caused by EODf, in the second phase of formalin test. In the rota-rod test, EODf-treated animals did not show any alteration of motor coordination. ConclusionsOur findings indicate that EODf underground stem bark produces anti-inflammatory and both central and peripheral antinociceptive effects. Furthermore, the antinociceptive activity of EODf underground stem bark is possibly mediated by adenosinergic and opioidergic pathways, and its properties do not induce effects on motor coordination. These results support the use of the folk medicine, D. furfuracea root, to treat inflammation and painful conditions.

DsRNA Sensing Induces Loss of Cell Identity

How cell and tissue identity persist despite constant cell turnover is an important biologic question with cell therapy implications. Although many mechanisms exist, we investigated the controls for site-specific gene expression in skin, given its diverse structures and functions. For example, the transcriptome of invivo palmoplantar (i.e., volar) epidermis is globally unique, including Keratin 9 (KRT9). Although volar fibroblasts have the capacity to induce KRT9 in nonvolar keratinocytes, we show here that volar keratinocytes continue to express KRT9 in invitro solo cultures. Despite this, KRT9 expression is lost with volar keratinocyte passaging, despite stable hypomethylation of its promoter. Coincident with KRT9 loss is a gain of the primitive keratin 7 and a signature of dsRNA sensing, including the double-stranded RNA (dsRNA) receptor DExD/H-Box Helicase 58 (DDX58/RIG-I). Exogenous dsRNA inhibits KRT9 expression in early passage volar keratinocytes or invivo footpads of wild-type mice. Loss of DDX58 in passaged volar keratinocytes rescues KRT9 and inhibits KRT7 expression. Additionally, DDX58-null mice are resistant to the ability of dsRNA to inhibit KRT9 expression. These results show that the sensing of dsRNA is critical for loss of cell-specific gene expression; our results have important implications for how dsRNA sensing is important outside of immune pathways.

Shorter-course treatment for Mycobacterium ulcerans disease with high-dose rifamycins and clofazimine in a mouse model of Buruli ulcer.

Starting in 2004, the standard regimen for treatment of Buruli ulcer (BU) recommended by the World Health Organization has been daily treatment for eight weeks with rifampin (RIF) and streptomycin. Based on recent clinical trials, treatment with an all-oral regimen of RIF and clarithromycin (CLR) may be an effective alternative. With the achievement of an all-oral regimen, a new goal is to find a regimen that can shorten the duration of treatment without compromising efficacy. We recently observed that increasing the dose of RIF from the standard 10 mg/kg dose to 20 or 40 mg/kg, or replacing RIF with the more potent long-acting rifamycin, rifapentine (RPT) at 10 mg/kg or 20 mg/kg increased the bactericidal activity of the RIF+CLR regimen in a mouse model of BU. We also recently showed that replacing CLR with clofazimine(CFZ) at 25 mg/kg may have greater sterilizing activity than the RIF+CLR regimen. Here, we demonstrate that combining high-dose rifamycins with CFZ at a lower dose of 12.5 mg/kg results in similar reductions in swelling, bacterial burden and mycolactone concentrations in mouse footpads compared to the standard regimens and more rapid sterilization of footpads as determined by the proportions of footpads harboring viable bacteria three months after completion of treatment. The potential of these high-dose rifamycin and CFZ combinations to shorten BU treatment to four weeks warrants evaluation in a clinical trial.Trial registrationClinicalTrials.gov NCT03474198, NCT01659437

Immunization by Replication-Competent Controlled Herpesvirus Vectors.

Replication-competent controlled virus vectors were derived from the virulent herpes simplex virus 1 (HSV-1) wild-type strain 17syn+ by placing one or two replication-essential genes under the stringent control of a gene switch that is coactivated by heat and an antiprogestin. Upon activation of the gene switch, the vectors replicate in infected cells with an efficacy that approaches that of the wild-type virus from which they were derived. Essentially no replication occurs in the absence of activation. When administered to mice, localized application of a transient heat treatment in the presence of systemic antiprogestin results in efficient but limited virus replication at the site of administration. The immunogenicity of these viral vectors was tested in a mouse footpad lethal challenge model. Unactivated viral vectors-which may be regarded as equivalents of inactivated vaccines-induced detectable protection against lethality caused by wild-type virus challenge. Single activation of the viral vectors at the site of administration (rear footpads) greatly enhanced protective immune responses, and a second immunization resulted in complete protection. Once activated, vectors also induced far better neutralizing antibody and HSV-1-specific cellular immune responses than unactivated vectors. To find out whether the immunogenicity of a heterologous antigen was also enhanced in the context of efficient transient vector replication, a virus vector constitutively expressing an equine influenza virus hemagglutinin was constructed. Immunization of mice with this recombinant induced detectable antibody-mediated neutralization of equine influenza virus, as well as a hemagglutinin-specific cellular immune response. Single activation of viral replication resulted in a severalfold enhancement of these immune responses.IMPORTANCE We hypothesized that vigorous replication of a pathogen may be critical for eliciting the most potent and balanced immune response against it. Hence, attenuation/inactivation (as in conventional vaccines) should be avoided. Instead, the necessary safety should be provided by placing replication of the pathogen under stringent control and by activating time-limited replication of the pathogen strictly in an administration region in which pathology cannot develop. Immunization will then occur in the context of highly efficient pathogen replication and uncompromised safety. We found that localized activation in mice of efficient but limited replication of a replication-competent controlled herpesvirus vector resulted in a greatly enhanced immune response to the virus or an expressed heterologous antigen. This finding supports the above-mentioned hypothesis and suggests that the vectors may be promising novel agents worth exploring for the prevention/mitigation of infectious diseases for which efficient vaccination is lacking, in particular in immunocompromised patients.