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1.
Immunity ; 37(4): 747-58, 2012 Oct 19.
Article in English | MEDLINE | ID: mdl-23063331

ABSTRACT

Scratching triggers skin flares in atopic dermatitis. We demonstrate that scratching of human skin and tape stripping of mouse skin cause neutrophil influx. In mice, this influx was largely dependent on the generation of leukotriene B4 (LTB4) by neutrophils and their expression of the LTB4 receptor BLT1. Allergic skin inflammation in response to epicutaneous (EC) application of ovalbumin to tape-stripped skin was severely impaired in Ltb4r1(-/-) mice and required expression of BLT1 on both T cells and non-T cells. Cotransfer of wild-type (WT) neutrophils, but not neutrophils deficient in BLT1 or the LTB4-synthesizing enzyme LTA4H, restored the ability of WT CD4(+) effector T cells to transfer allergic skin inflammation to Ltb4r1(-/-) recipients. Pharmacologic blockade of LTB4 synthesis inhibited allergic skin inflammation elicited by cutaneous antigen challenge in previously EC-sensitized mice. Our results demonstrate that a neutrophil-T cell axis reliant on LTB4-BLT1 interaction is required for allergic skin inflammation.


Subject(s)
Dermatitis/immunology , Leukotriene B4/immunology , Neutrophil Infiltration , Neutrophils/immunology , Animals , Biopsy , Dermatitis/pathology , Disease Models, Animal , Humans , Leukotriene B4/biosynthesis , Mice , Mice, Inbred C57BL , Receptors, Leukotriene B4/deficiency , Receptors, Leukotriene B4/immunology
2.
Gen Dent ; 63(4): e18-22, 2015.
Article in English | MEDLINE | ID: mdl-26147176

ABSTRACT

The purpose of this in vitro study was to compare the penetration of 2 resin-based and 2 glass ionomer sealants into fissures after either conventional or mechanical preparation. All the materials placed in the conventionally prepared and mechanically prepared fissures penetrated the fissures beyond the standard (0.5-mm) reference line used in this study. For 1 glass ionomer sealant, the number of specimens showing complete penetration of sealant was significantly greater (P < 0.05) in mechanically prepared fissures than in conventionally prepared ones. No significant differences were found between mechanically and conventionally prepared fissures for the other sealants.


Subject(s)
Pit and Fissure Sealants/therapeutic use , Tooth Preparation/methods , Acrylic Resins/therapeutic use , Dental Caries/prevention & control , Humans , In Vitro Techniques , Resin Cements/therapeutic use , Silicon Dioxide/therapeutic use , Treatment Outcome
3.
PLoS Pathog ; 8(11): e1003047, 2012.
Article in English | MEDLINE | ID: mdl-23209417

ABSTRACT

Neutrophil abscess formation is critical in innate immunity against many pathogens. Here, the mechanism of neutrophil abscess formation was investigated using a mouse model of Staphylococcus aureus cutaneous infection. Gene expression analysis and in vivo multispectral noninvasive imaging during the S. aureus infection revealed a strong functional and temporal association between neutrophil recruitment and IL-1ß/IL-1R activation. Unexpectedly, neutrophils but not monocytes/macrophages or other MHCII-expressing antigen presenting cells were the predominant source of IL-1ß at the site of infection. Furthermore, neutrophil-derived IL-1ß was essential for host defense since adoptive transfer of IL-1ß-expressing neutrophils was sufficient to restore the impaired neutrophil abscess formation in S. aureus-infected IL-1ß-deficient mice. S. aureus-induced IL-1ß production by neutrophils required TLR2, NOD2, FPR1 and the ASC/NLRP3 inflammasome in an α-toxin-dependent mechanism. Taken together, IL-1ß and neutrophil abscess formation during an infection are functionally, temporally and spatially linked as a consequence of direct IL-1ß production by neutrophils.


Subject(s)
Abscess/immunology , Interleukin-1beta/immunology , Neutrophils/immunology , Staphylococcal Skin Infections/immunology , Staphylococcus aureus/immunology , Abscess/genetics , Abscess/metabolism , Abscess/microbiology , Abscess/pathology , Adoptive Transfer , Animals , Carrier Proteins/genetics , Carrier Proteins/immunology , Carrier Proteins/metabolism , Inflammasomes/genetics , Inflammasomes/immunology , Inflammasomes/metabolism , Interleukin-1beta/biosynthesis , Interleukin-1beta/genetics , Mice , Mice, Mutant Strains , NLR Family, Pyrin Domain-Containing 3 Protein , Neutrophils/metabolism , Neutrophils/pathology , Nod2 Signaling Adaptor Protein/genetics , Nod2 Signaling Adaptor Protein/immunology , Nod2 Signaling Adaptor Protein/metabolism , Receptors, Formyl Peptide/genetics , Receptors, Formyl Peptide/immunology , Receptors, Formyl Peptide/metabolism , Staphylococcal Skin Infections/genetics , Staphylococcal Skin Infections/metabolism , Staphylococcal Skin Infections/microbiology , Staphylococcal Skin Infections/pathology , Toll-Like Receptor 2/genetics , Toll-Like Receptor 2/immunology , Toll-Like Receptor 2/metabolism
4.
J Immunol ; 188(7): 2977-80, 2012 Apr 01.
Article in English | MEDLINE | ID: mdl-22387558

ABSTRACT

Nitrogen-containing bisphosphonates (NBPs) are taken by millions for bone disorders but may cause serious inflammatory reactions. In this study, we used a murine peritonitis model to characterize the inflammatory mechanisms of these agents. At dosages comparable to those used in humans, injection of NBPs into the peritoneum caused recruitment of neutrophils, followed by an influx of monocytes. These cellular changes corresponded to an initial increase in IL-1α, which preceded a rise in multiple other proinflammatory cytokines. IL-1R, IL-1α, and IL-1ß were required for neutrophil recruitment, whereas other MyD88-dependent signaling pathways were needed for the monocyte influx. Mice deficient in mast cells, but not mice lacking lymphocytes, were resistant to NBP-induced inflammation, and reconstitution of these mice with mast cells restored sensitivity to NBPs. These results document the critical role of mast cells and IL-1 in NBP-mediated inflammatory reactions.


Subject(s)
Alendronate/toxicity , Diphosphonates/toxicity , Imidazoles/toxicity , Interleukin-1alpha/physiology , Interleukin-1beta/physiology , Mast Cells/physiology , Peritonitis/chemically induced , Animals , Chemotaxis/physiology , Clodronic Acid/toxicity , Interleukin-1alpha/deficiency , Interleukin-1alpha/genetics , Interleukin-1beta/deficiency , Interleukin-1beta/genetics , Leukocytes/immunology , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Mutant Strains , Myeloid Differentiation Factor 88/deficiency , Myeloid Differentiation Factor 88/genetics , Myeloid Differentiation Factor 88/physiology , Neutrophils/immunology , Pamidronate , Peritonitis/immunology , Peritonitis/pathology , Receptors, Interleukin-1/deficiency , Receptors, Interleukin-1/genetics , Receptors, Interleukin-1/physiology , Zoledronic Acid
5.
Antimicrob Agents Chemother ; 57(2): 855-63, 2013 Feb.
Article in English | MEDLINE | ID: mdl-23208713

ABSTRACT

Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) frequently causes skin and soft tissue infections, including impetigo, cellulitis, folliculitis, and infected wounds and ulcers. Uncomplicated CA-MRSA skin infections are typically managed in an outpatient setting with oral and topical antibiotics and/or incision and drainage, whereas complicated skin infections often require hospitalization, intravenous antibiotics, and sometimes surgery. The aim of this study was to develop a mouse model of CA-MRSA wound infection to compare the efficacy of commonly used systemic and topical antibiotics. A bioluminescent USA300 CA-MRSA strain was inoculated into full-thickness scalpel wounds on the backs of mice and digital photography/image analysis and in vivo bioluminescence imaging were used to measure wound healing and the bacterial burden. Subcutaneous vancomycin, daptomycin, and linezolid similarly reduced the lesion sizes and bacterial burden. Oral linezolid, clindamycin, and doxycycline all decreased the lesion sizes and bacterial burden. Oral trimethoprim-sulfamethoxazole decreased the bacterial burden but did not decrease the lesion size. Topical mupirocin and retapamulin ointments both reduced the bacterial burden. However, the petrolatum vehicle ointment for retapamulin, but not the polyethylene glycol vehicle ointment for mupirocin, promoted wound healing and initially increased the bacterial burden. Finally, in type 2 diabetic mice, subcutaneous linezolid and daptomycin had the most rapid therapeutic effect compared with vancomycin. Taken together, this mouse model of CA-MRSA wound infection, which utilizes in vivo bioluminescence imaging to monitor the bacterial burden, represents an alternative method to evaluate the preclinical in vivo efficacy of systemic and topical antimicrobial agents.


Subject(s)
Anti-Bacterial Agents/administration & dosage , Anti-Bacterial Agents/therapeutic use , Luminescent Measurements , Methicillin-Resistant Staphylococcus aureus/drug effects , Staphylococcal Infections/drug therapy , Acetamides/administration & dosage , Acetamides/therapeutic use , Administration, Oral , Administration, Topical , Animals , Bacterial Load , Bridged Bicyclo Compounds, Heterocyclic/administration & dosage , Bridged Bicyclo Compounds, Heterocyclic/therapeutic use , Clindamycin/administration & dosage , Clindamycin/therapeutic use , Community-Acquired Infections/drug therapy , Community-Acquired Infections/microbiology , Daptomycin/administration & dosage , Daptomycin/therapeutic use , Diabetes Mellitus, Type 2 , Disease Models, Animal , Diterpenes , Doxycycline/administration & dosage , Doxycycline/therapeutic use , Linezolid , Male , Mice , Mice, Inbred C57BL , Mupirocin/administration & dosage , Mupirocin/therapeutic use , Oxazolidinones/administration & dosage , Oxazolidinones/therapeutic use , Skin/injuries , Skin/microbiology , Soft Tissue Infections/drug therapy , Soft Tissue Infections/microbiology , Staphylococcal Infections/microbiology , Staphylococcal Skin Infections/drug therapy , Staphylococcal Skin Infections/microbiology , Trimethoprim, Sulfamethoxazole Drug Combination/administration & dosage , Trimethoprim, Sulfamethoxazole Drug Combination/therapeutic use , Vancomycin/administration & dosage , Vancomycin/therapeutic use , Wound Healing/drug effects
6.
Angew Chem Int Ed Engl ; 52(2): 674-8, 2013 Jan 07.
Article in English | MEDLINE | ID: mdl-23161823

ABSTRACT

Fighting the resistance: biodegradable and injectable/moldable hydrogels with hierarchical nanostructures were made with broad-spectrum antimicrobial activities and biofilm-disruption capability. They demonstrate no cytotoxicity in vitro, and show excellent skin biocompatibility in animals. These hydrogels have great potential for clinical use in prevention and treatment of various multidrug-resistant infections.


Subject(s)
Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Biofilms/drug effects , Hydrogels/chemistry , Hydrogels/pharmacology , Microscopy, Electron
7.
Cancer Immunol Immunother ; 58(7): 1057-69, 2009 Jul.
Article in English | MEDLINE | ID: mdl-19018533

ABSTRACT

The systemic administration of an agonist antibody against glucocorticoid-induced tumor necrosis factor receptor related (GITR) protein has been shown to be effective in overcoming immune tolerance and promoting tumor rejection in a variety of murine tumor models. However, little is known regarding the functional consequence of ligation of GITR with its natural ligand (GITR-L) in the context of regulatory T cell (Treg) suppression in vivo. To determine the mechanism of GITR-L action in vivo, we generated a panel of tumor cell clones that express varying levels of GITR-L. The ectopic expression of GITR-L on the tumor cell surface was sufficient to enhance anti-tumor immunity and delay tumor growth in syngeneic BALB/c mice. Within the range examined, the extent of anti-tumor activity in vivo did not correlate with the level of GITR-L expression, as all clones tested exhibited a similar delay in tumor growth. The localized expression of GITR-L on tumor cells led to a significant increase in CD8+ T cell infiltration compared to the levels seen in control tumors. The increased proportion of CD8+ T cells was only observed locally at the tumor site and was not seen in the tumor draining lymph node. Depletion studies showed that CD8+ T cells, but not CD4+ T cells, were required for GITR-L mediated protection against tumor growth. These studies demonstrate that signaling between GITR-L and GITR in the tumor microenvironment promotes the infiltration of CD8+ T cells, which are essential for controlling tumor growth.


Subject(s)
CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Colonic Neoplasms/immunology , Receptors, Nerve Growth Factor/metabolism , Receptors, Tumor Necrosis Factor/metabolism , T-Lymphocytes, Regulatory/immunology , Tumor Necrosis Factors/metabolism , Animals , CD4-Positive T-Lymphocytes/metabolism , CD8-Positive T-Lymphocytes/metabolism , Cell Line, Tumor , Female , Genetic Vectors , Glucocorticoid-Induced TNFR-Related Protein , Immune Tolerance , Mice , Mice, Inbred BALB C , Receptors, Nerve Growth Factor/immunology , Receptors, Tumor Necrosis Factor/immunology , T-Lymphocytes, Regulatory/metabolism , Transfection , Tumor Necrosis Factors/immunology
8.
PLoS One ; 11(9): e0162758, 2016.
Article in English | MEDLINE | ID: mdl-27618690

ABSTRACT

Systemic sclerosis (SSc) is a chronic autoimmune disorder that can result in extensive tissue damage in the skin and, in advanced cases, internal organs. Vasculopathy, aberrant immune activation, and tissue fibrosis are three hallmarks of the disease that have been identified, with vasculopathy and aberrant immunity being amongst the earliest events. However, a mechanistic link between these processes has not been established. Here, we have identified a novel role of platelet derived growth factor-BB (PDGF-BB)/PDGFRß activation in combination with dermal injury induced by bleomycin as a driver of early, aberrant expression of interferon stimulatory genes (ISGs) and inflammatory monocyte infiltration. Activation of PDGFRß in combination with bleomycin-induced dermal injury resulted in increased dermal thickness, vascular density, monocyte/macrophage infiltration, and exacerbation of tissue injury. Many of these features were dependent on IFNAR-signaling, and an increase in the number of interferon-beta (IFN-ß) producing monocytes cells was found in the skin lesions. Taken together, these results identify a novel link between PDGFRß activation, and Type I IFN-driven vascular maintenance and monocyte/macrophage cell recruitment, and provide a potential explanation linking key features of SSc that were previously thought to be unrelated.


Subject(s)
Blood Vessels/pathology , Disease Models, Animal , Interferon Type I/physiology , Monocytes/pathology , Proto-Oncogene Proteins c-sis/physiology , Scleroderma, Systemic/pathology , Skin Diseases/pathology , Animals , Becaplermin , Bleomycin/pharmacology , Fibrosis , Mice , Mice, Inbred C57BL , Receptor, Platelet-Derived Growth Factor beta/metabolism
9.
J Orthop Res ; 30(3): 335-40, 2012 Mar.
Article in English | MEDLINE | ID: mdl-21837686

ABSTRACT

Post-arthroplasty infections are a devastating problem in orthopaedic surgery. While acute infections can be treated with a single stage washout and liner exchange, chronic infections lead to multiple reoperations, prolonged antibiotic courses, extended disability, and worse clinical outcomes. Unlike previous mouse models that studied an acute infection, this work aimed to develop a model of a chronic post-arthroplasty infection. To achieve this, a stainless steel implant in the knee joints of mice was inoculated with a bioluminescent Staphylococcus aureus strain (1 × 10(2) -1 × 10(4) colony forming units, CFUs) and in vivo imaging was used to monitor the bacterial burden for 42 days. Four different S. aureus strains were compared in which the bioluminescent construct was integrated in an antibiotic selection plasmid (ALC2906), the bacterial chromosome (Xen29 and Xen40), or a stable plasmid (Xen36). ALC2906 had increased bioluminescent signals through day 10, after which the signals became undetectable. In contrast, Xen29, Xen40, and Xen36 had increased bioluminescent signals through 42 days with the highest signals observed with Xen36. ALC2906, Xen29, and Xen40 induced significantly more inflammation than Xen36 as measured by in vivo enhanced green fluorescence protein (EGFP)-neutrophil flourescence of LysEGFP mice. All four strains induced comparable biofilm formation as determined by variable-pressure scanning electron microscopy. Using a titanium implant, Xen36 had higher in vivo bioluminescence signals than Xen40 but had similar biofilm formation and adherent bacteria. In conclusion, Xen29, Xen40, and especially Xen36, which had stable bioluminescent constructs, are feasible for long-term in vivo monitoring of bacterial burden and biofilm formation to study chronic post-arthroplasty infections and potential antimicrobial interventions.


Subject(s)
Arthritis, Infectious/microbiology , Joint Prosthesis/microbiology , Luminescent Measurements , Postoperative Complications/microbiology , Staphylococcus aureus , Animals , Arthroplasty, Replacement, Knee/adverse effects , Bacterial Load , Biofilms , Chronic Disease , Disease Models, Animal , Gene Transfer Techniques , Green Fluorescent Proteins , Male , Mice , Mice, Inbred C57BL , Neutrophils/cytology , Titanium
10.
Nat Rev Immunol ; 11(8): 505-18, 2011 Jul 01.
Article in English | MEDLINE | ID: mdl-21720387

ABSTRACT

Complications arising from cutaneous and soft tissue infections with Staphylococcus aureus are a major clinical problem owing to the high incidence of these infections and the widespread emergence of antibiotic-resistant bacterial strains. If prophylactic vaccines or immunotherapy for certain patient populations are to be developed as an alternative to antibiotics, it will be essential to better understand the immune mechanisms that provide protection against S. aureus skin infections. Recent discoveries have identified a key role for interleukin-1 (IL-1)- and IL-17-mediated immune responses in promoting neutrophil recruitment to the site of infection in the skin, a process that is required for host defence and bacterial clearance. This Review describes these new insights and discusses their potential impact on immune-based therapies and vaccination strategies.


Subject(s)
Staphylococcal Skin Infections/immunology , Staphylococcus aureus/immunology , Animals , Humans , Immunotherapy/methods , Interleukins/metabolism , Neutrophils/cytology , Neutrophils/immunology , Staphylococcal Skin Infections/complications , Staphylococcal Skin Infections/metabolism , Staphylococcal Skin Infections/therapy
11.
J Orthop Res ; 29(10): 1621-6, 2011 Oct.
Article in English | MEDLINE | ID: mdl-21445990

ABSTRACT

MyD88 is an adapter molecule that is used by both IL-1R and TLR family members to initiate downstream signaling and promote immune responses. Given that IL-1ß is induced after Staphylococcus aureus infections and TLR2 is activated by S. aureus lipopeptides, we hypothesized that IL-1ß and TLR2 contribute to MyD88-dependent protective immune responses against post-arthroplasty S. aureus infections. To test this hypothesis, we used a mouse model of a post-arthroplasty S. aureus infection to compare the bacterial burden, biofilm formation and neutrophil recruitment in IL-1ß-deficient, TLR2-deficient and wild-type (wt) mice. By using in vivo bioluminescence imaging, we found that the bacterial burden in IL-1ß-deficient mice was 26-fold higher at 1 day after infection and remained 3- to 10-fold greater than wt mice through day 42. In contrast, the bacterial burden in TLR2-deficient mice did not differ from wt mice. In addition, implants harvested from IL-1ß-deficient mice had more biofilm formation and 14-fold higher adherent bacteria compared with those from wt mice. Finally, IL-1ß-deficient mice had ∼50% decreased neutrophil recruitment to the infected postoperative joints than wt mice. Taken together, these findings suggest a mechanism by which IL-1ß induces neutrophil recruitment to help control the bacterial burden and the ensuing biofilm formation in a post-surgical joint.


Subject(s)
Interleukin-1beta/metabolism , Prosthesis-Related Infections/immunology , Staphylococcal Infections/immunology , Toll-Like Receptor 2/metabolism , Animals , Arthroplasty , Biofilms/growth & development , Bone Wires/microbiology , Male , Mice , Mice, Congenic , Mice, Inbred C57BL , Myeloid Differentiation Factor 88/metabolism , Neutrophil Infiltration , Prosthesis-Related Infections/metabolism , Staphylococcal Infections/metabolism , Staphylococcus aureus
12.
J Invest Dermatol ; 131(4): 907-15, 2011 Apr.
Article in English | MEDLINE | ID: mdl-21191403

ABSTRACT

Staphylococcus aureus skin infections represent a significant public health threat because of the emergence of antibiotic-resistant strains such as methicillin-resistant S. aureus (MRSA). As greater understanding of protective immune responses and more effective antimicrobial therapies are needed, a S. aureus skin wound infection model was developed in which full-thickness scalpel cuts on the backs of mice were infected with a bioluminescent S. aureus (methicillin sensitive) or USA300 community-acquired MRSA strain and in vivo imaging was used to noninvasively monitor the bacterial burden. In addition, the infection-induced inflammatory response was quantified using in vivo fluorescence imaging of LysEGFP mice. Using this model, we found that both IL-1α and IL-1ß contributed to host defense during a wound infection, whereas IL-1ß was more critical during an intradermal S. aureus infection. Furthermore, treatment of a USA300 MRSA skin infection with retapamulin ointment resulted in up to 85-fold reduction in bacterial burden and a 53% decrease in infection-induced inflammation. In contrast, mupirocin ointment had minimal clinical activity against this USA300 strain, resulting in only a 2-fold reduction in bacterial burden. Taken together, this S. aureus wound infection model provides a valuable preclinical screening method to investigate cutaneous immune responses and the efficacy of topical antimicrobial therapies.


Subject(s)
Anti-Bacterial Agents/pharmacology , Dermoscopy/methods , Methicillin-Resistant Staphylococcus aureus/immunology , Microscopy, Fluorescence/methods , Mupirocin/pharmacology , Staphylococcal Skin Infections , Administration, Topical , Animals , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Community-Acquired Infections/drug therapy , Community-Acquired Infections/immunology , Community-Acquired Infections/pathology , Dermatitis/drug therapy , Dermatitis/immunology , Dermatitis/pathology , Disease Models, Animal , Diterpenes , Drug Monitoring/methods , Green Fluorescent Proteins/genetics , Interleukin-1alpha/immunology , Interleukin-1beta/immunology , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Staphylococcal Skin Infections/drug therapy , Staphylococcal Skin Infections/immunology , Staphylococcal Skin Infections/pathology
13.
J Invest Dermatol ; 130(12): 2703-6, 2010 Dec.
Article in English | MEDLINE | ID: mdl-21068735

ABSTRACT

Staphylococcus aureus colonization is a major risk factor for infection. In this issue, Simanski et al. demonstrate that the antimicrobial peptide RNase 7 is essential for preventing S. aureus colonization in human skin. These findings suggest that therapeutic interventions aimed at targeting RNase 7 production in the skin may be a novel strategy to protect against S. aureus infections.


Subject(s)
Ribonucleases/immunology , Staphylococcal Skin Infections/immunology , Staphylococcal Skin Infections/prevention & control , Staphylococcus aureus/immunology , Epidermis/immunology , Epidermis/microbiology , Humans , Risk Factors , Staphylococcal Skin Infections/epidemiology
14.
PLoS One ; 5(9): e12580, 2010 Sep 07.
Article in English | MEDLINE | ID: mdl-20830204

ABSTRACT

BACKGROUND: Post-arthroplasty infections represent a devastating complication of total joint replacement surgery, resulting in multiple reoperations, prolonged antibiotic use, extended disability and worse clinical outcomes. As the number of arthroplasties in the U.S. will exceed 3.8 million surgeries per year by 2030, the number of post-arthroplasty infections is projected to increase to over 266,000 infections annually. The treatment of these infections will exhaust healthcare resources and dramatically increase medical costs. METHODOLOGY/PRINCIPAL FINDINGS: To evaluate novel preventative therapeutic strategies against post-arthroplasty infections, a mouse model was developed in which a bioluminescent Staphylococcus aureus strain was inoculated into a knee joint containing an orthopaedic implant and advanced in vivo imaging was used to measure the bacterial burden in real-time. Mice inoculated with 5x10(3) and 5x10(4) CFUs developed increased bacterial counts with marked swelling of the affected leg, consistent with an acute joint infection. In contrast, mice inoculated with 5x10(2) CFUs developed a low-grade infection, resembling a more chronic infection. Ex vivo bacterial counts highly correlated with in vivo bioluminescence signals and EGFP-neutrophil fluorescence of LysEGFP mice was used to measure the infection-induced inflammation. Furthermore, biofilm formation on the implants was visualized at 7 and 14 postoperative days by variable-pressure scanning electron microscopy (VP-SEM). Using this model, a minocycline/rifampin-impregnated bioresorbable polymer implant coating was effective in reducing the infection, decreasing inflammation and preventing biofilm formation. CONCLUSIONS/SIGNIFICANCE: Taken together, this mouse model may represent an alternative pre-clinical screening tool to evaluate novel in vivo therapeutic strategies before studies in larger animals and in human subjects. Furthermore, the antibiotic-polymer implant coating evaluated in this study was clinically effective, suggesting the potential for this strategy as a therapeutic intervention to combat post-arthroplasty infections.


Subject(s)
Anti-Bacterial Agents/therapeutic use , Arthroplasty/adverse effects , Joint Diseases/microbiology , Postoperative Complications/prevention & control , Staphylococcal Infections/prevention & control , Staphylococcus aureus/physiology , Animals , Disease Models, Animal , Humans , Joint Diseases/drug therapy , Joint Diseases/surgery , Joints/microbiology , Joints/surgery , Male , Mice , Mice, Inbred C57BL , Minocycline/therapeutic use , Postoperative Complications/drug therapy , Postoperative Complications/microbiology , Prostheses and Implants , Rifampin/therapeutic use , Staphylococcal Infections/drug therapy , Staphylococcal Infections/microbiology , Staphylococcal Infections/surgery , Staphylococcus aureus/drug effects , Staphylococcus aureus/genetics
15.
J Clin Invest ; 120(5): 1762-73, 2010 May.
Article in English | MEDLINE | ID: mdl-20364087

ABSTRACT

Staphylococcus aureus is the most common cause of skin and soft tissue infections, and rapidly emerging antibiotic-resistant strains are creating a serious public health concern. If immune-based therapies are to be an alternative to antibiotics, greater understanding is needed of the protective immune response against S. aureus infection in the skin. Although neutrophil recruitment is required for immunity against S. aureus, a role for T cells has been suggested. Here, we used a mouse model of S. aureus cutaneous infection to investigate the contribution of T cells to host defense. We found that mice deficient in gammadelta but not alphabeta T cells had substantially larger skin lesions with higher bacterial counts and impaired neutrophil recruitment compared with WT mice. This neutrophil recruitment was dependent upon epidermal Vgamma5+ gammadelta T cell production of IL-17, but not IL-21 and IL-22. Furthermore, IL-17 induction required IL-1, TLR2, and IL-23 and was critical for host defense, since IL-17R-deficient mice had a phenotype similar to that of gammadelta T cell-deficient mice. Importantly, gammadelta T cell-deficient mice inoculated with S. aureus and treated with a single dose of recombinant IL-17 had lesion sizes and bacterial counts resembling those of WT mice, demonstrating that IL-17 could restore the impaired immunity in these mice. Our study defines what we believe to be a novel role for IL-17-producing epidermal gammadelta T cells in innate immunity against S. aureus cutaneous infection.


Subject(s)
Interleukin-17/physiology , Skin/microbiology , Staphylococcal Skin Infections/immunology , Staphylococcus aureus/metabolism , Animals , Interferon-gamma/metabolism , Interleukin-17/metabolism , Interleukins/metabolism , Male , Mice , Mice, Inbred C57BL , Models, Biological , Neutrophils/metabolism , Stem Cells , T-Lymphocytes/immunology , Interleukin-22
16.
PLoS One ; 4(9): e7029, 2009 Sep 15.
Article in English | MEDLINE | ID: mdl-19753301

ABSTRACT

BACKGROUND: The events required to initiate host defenses against invading pathogens involve complex signaling cascades comprised of numerous adaptor molecules, kinases, and transcriptional elements, ultimately leading to the production of proinflammatory cytokines, such as tumor necrosis factor alpha (TNF-alpha). How these signaling cascades are regulated, and the proteins and regulatory elements participating are still poorly understood. RESULTS: We report here the development a completely random short-hairpin RNA (shRNA) library coupled with a novel forward genetic screening strategy to identify inhibitors of Toll-like receptor (TLR) dependent proinflammatory responses. We developed a murine macrophage reporter cell line stably transfected with a construct expressing diphtheria toxin-A (DT-A) under the control of the TNF-alpha-promoter. Stimulation of the reporter cell line with the TLR ligand lipopolysaccharide (LPS) resulted in DT-A induced cell death, which could be prevented by the addition of an shRNA targeting the TLR adaptor molecule MyD88. Utilizing this cell line, we screened a completely random lentiviral short hairpin RNA (shRNA) library for sequences that inhibited TLR-mediated TNF-alpha production. Recovery of shRNA sequences from surviving cells led to the identification of unique shRNA sequences that significantly inhibited TLR4-dependent TNF-alpha gene expression. Furthermore, these shRNA sequences specifically blocked TLR2 but not TLR3-dependent TNF-alpha production. CONCLUSIONS: Thus, we describe the generation of novel tools to facilitate large-scale forward genetic screens in mammalian cells and the identification of potent shRNA inhibitors of TLR2 and TLR4- dependent proinflammatory responses.


Subject(s)
Gene Expression Regulation , Myeloid Differentiation Factor 88/metabolism , RNA Interference , Animals , Cytokines/metabolism , Gene Library , Humans , Inflammation , Mice , Promoter Regions, Genetic , Signal Transduction , Toll-Like Receptor 2/metabolism , Toll-Like Receptor 3 , Toll-Like Receptors/metabolism , Tumor Necrosis Factor-alpha/metabolism
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