Combination prophylactic therapy with rifampin increases efficacy against an experimental Staphylococcus epidermidis subcutaneous implant-related infection.

The incidence of infections related to cardiac devices (such as permanent pacemakers) has been increasing out of proportion to implantation rates. As management of device infections typically requires explantation of the device, optimal prophylactic strategies are needed. Cefazolin and vancomycin are widely used as single agents for surgical prophylaxis against cardiac device-related infections. However, combination antibiotic prophylaxis may further reduce infectious complications. To model a localized subcutaneous implant-related infection, a bioluminescent strain of Staphylococcus epidermidis was inoculated onto a medical-procedure-grade titanium disc, which was placed into a subcutaneous pocket in the backs of mice. In vivo bioluminescence imaging, quantification of ex vivo CFU from the capsules and implants, variable-pressure scanning electron microscopy (VP-SEM), and neutrophil enhanced green fluorescent protein (EGFP) fluorescence in LysEGFP mice were employed to monitor the infection. This model was used to evaluate the efficacies of low- and high-dose cefazolin (50 and 200 mg/kg of body weight) and vancomycin (10 and 110 mg/kg) intravenous prophylaxis with or without rifampin (25 mg/kg). High-dose cefazolin and high-dose vancomycin treatment resulted in almost complete bacterial clearance, whereas both low-dose cefazolin and low-dose vancomycin reduced the in vivo and ex vivo bacterial burden only moderately. The addition of rifampin to low-dose cefazolin and vancomycin was highly effective in further reducing the CFU harvested from the implants. However, vancomycin-rifampin was more effective than cefazolin-rifampin in further reducing the CFU harvested from the surrounding tissue capsules. Future studies in humans will be required to determine whether the addition of rifampin has improved efficacy in preventing device-related infections in clinical practice.

Neutrophil-derived IL-1ß is sufficient for abscess formation in immunity against Staphylococcus aureus in mice.

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.

In vivo bioluminescence imaging to evaluate systemic and topical antibiotics against community-acquired methicillin-resistant Staphylococcus aureus-infected skin wounds in mice.

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.

Vancomycin-rifampin combination therapy has enhanced efficacy against an experimental Staphylococcus aureus prosthetic joint infection.

Treatment of prosthetic joint infections often involves a two-stage exchange, with implant removal and antibiotic spacer placement followed by systemic antibiotic therapy and delayed reimplantation. However, if antibiotic therapy can be improved, one-stage exchange or implant retention may be more feasible, thereby decreasing morbidity and preserving function. In this study, a mouse model of prosthetic joint infection was used in which Staphylococcus aureus was inoculated into a knee joint containing a surgically placed metallic implant extending from the femur. This model was used to evaluate whether combination therapy of vancomycin plus rifampin has increased efficacy compared with vancomycin alone against these infections. On postoperative day 7, vancomycin with or without rifampin was administered for 6 weeks with implant retention. In vivo bioluminescence imaging, ex vivo CFU enumeration, X-ray imaging, and histologic analysis were carried out. We found that there was a marked therapeutic benefit when vancomycin was combined with rifampin compared with vancomycin alone. Taken together, our results suggest that the mouse model used could serve as a valuable in vivo preclinical model system to evaluate and compare efficacies of antibiotics and combinatory therapy for prosthetic joint infections before more extensive studies are carried out in human subjects.

Daptomycin and tigecycline have broader effective dose ranges than vancomycin as prophylaxis against a Staphylococcus aureus surgical implant infection in mice.

Vancomycin is widely used for intravenous prophylaxis against surgical implant infections. However, it is unclear whether alternative antibiotics used to treat methicillin-resistant Staphylococcus aureus (MRSA) infections are effective as prophylactic agents. The aim of this study was to compare the efficacies of vancomycin, daptomycin, and tigecycline as prophylactic therapy against a methicillin-sensitive S. aureus (MSSA) or MRSA surgical implant infection in mice. MSSA or MRSA was inoculated into the knee joints of mice in the presence of a surgically placed medical-grade metallic implant. The efficacies of low- versus high-dose vancomycin (10 versus 110 mg/kg), daptomycin (1 versus 10 mg/kg), and tigecycline (1 versus 10 mg/kg) intravenous prophylaxis were compared using in vivo bioluminescence imaging, ex vivo bacterial counts, and biofilm formation. High-dose vancomycin, daptomycin, and tigecycline resulted in similar reductions in bacterial burden and biofilm formation. In contrast, low-dose daptomycin and tigecycline were more effective than low-dose vancomycin against the implant infection. In this mouse model of surgical implant MSSA or MRSA infection, daptomycin and tigecycline prophylaxis were effective over a broader dosage range than vancomycin. Future studies in humans will be required to determine whether these broader effective dose ranges for daptomycin and tigecycline in mice translate to improved efficacy in preventing surgical implant infections in clinical practice.

Urine Cell-Free MicroRNAs in Localized Prostate Cancer Patients.

Prostate cancer (PCa) is the most common cancer in men. Prostate-specific antigen screening is recommended for the detection of PCa. However, its specificity is limited. Thus, there is a need to find more reliable biomarkers that allow non-invasive screening for early-stage PCa. This study aims to explore urine microRNAs (miRs) as diagnostic biomarkers for PCa. We assessed cell-free miR (cfmiR) profiles of urine and plasma samples from pre- and post-operative PCa patients (n = 11) and normal healthy donors (16 urine and 24 plasma) using HTG EdgeSeq miRNA Whole Transcriptome Assay based on next-generation sequencing. Furthermore, tumor-related miRs were detected in formalin-fixed paraffin-embedded tumor tissues obtained from patients with localized PCa. Specific cfmiRs signatures were found in urine samples of localized PCa patients using differential expression analysis. Forty-two cfmiRs that were detected were common to urine, plasma, and tumor samples. These urine cfmiRs may have potential utility in diagnosing early-stage PCa and complementing or improving currently available PCa screening assays. Future studies may validate the findings.

Interleukin enhancer-binding factor 2 promotes cell proliferation and DNA damage response in metastatic melanoma.

BACKGROUND: 1q21.3 amplification, which is frequently observed in metastatic melanoma, is associated with cancer progression. Interleukin enhancer-binding factor 2 (ILF2) is located in the 1q21.3 amplified region, but its functional role or contribution to tumour aggressiveness in cutaneous melanoma is unknown. METHODS: In silico analyses were performed using the TCGA SKCM dataset with clinical annotations and three melanoma microarray cohorts from the GEO datasets. RNA in situ hybridisation and immunohistochemistry were utilised to validate the gene expression in melanoma tissues. Four stable melanoma cell lines were established for in vitro ILF2 functional characterisation. RESULTS: Our results showed that the ILF2 copy number variation (CNV) is positively correlated with ILF2 mRNA expression (r = 0.68, p < .0001). Additionally, ILF2 expression is significantly increased with melanoma progression (p < .0001), and significantly associated with poor overall survival for metastatic melanoma patients (p = .026). The overexpression of ILF2 (ILF2-OV) promotes proliferation in metastatic melanoma cells, whereas ILF2 knockdown decreases proliferation by blocking the cell cycle. Mechanistically, we demonstrated the interaction between ILF2 and the splicing factor U2AF2, whose knockdown reverses the proliferation effects mediated by ILF2-OV. Stage IIIB-C melanoma patients with high ILF2-U2AF2 expression showed significantly shorter overall survival (p = .024). Enhanced ILF2/U2AF2 expression promotes a more efficient DNA-damage repair by increasing RAD50 and ATM mRNA expression. Paradoxically, metastatic melanoma cells with ILF2-OV were more sensitive to ATM inhibitors. CONCLUSION: Our study uncovered that ILF2 amplification of the 1q21.3 chromosome is associated with melanoma progression and triggers a functional downstream pathway in metastatic melanoma promoting drug resistance.

Acetylated DNMT1 Downregulation and Related Regulatory Factors Influence Metastatic Melanoma Patients Survival.

The role of post-translational modifications (PTM) of the key epigenetic factor DNMT1 protein has not been well explored in cutaneous metastatic melanoma progression. The acetylated DNMT1 (ac-DNMT1) protein level was assessed using an anti-acetylated lysine antibody in a clinically annotated melanoma patient tumor specimen cohort. In this study, we showed that surgically resected tumors have significantly higher DNMT1 protein expression in metastatic melanoma (stage III metastasis n = 17, p = 0.0009; stage IV metastasis n = 164, p = 0.003) compared to normal organ tissues (n = 19). Additionally, reduced ac-DNMT1 protein levels were associated with melanoma progression. There was a significant inverse correlation between ac-DNMT1 and DNMT1 protein levels in stage IV metastatic melanoma (r = -0.18, p = 0.02, n = 164). Additionally, ac-DNMT1 protein levels were also significantly positively correlated with TIP60 (r = 0.6, p < 0.0001) and USP7 (r = 0.74, p < 0.0001) protein levels in stage IV metastatic melanoma (n = 164). Protein analysis in metastatic melanoma tumor tissues showed that with high ac-DNMT1 (p = 0.006, n = 59), or concurrent high ac-DNMT1 with low DNMT1 (p = 0.05, n = 27), or high TIP60 (p = 0.007, n = 41), or high USP7 (p = 0.01, n = 48) consistently showed better 4-year melanoma-specific survival (MSS). Multivariate Cox proportional hazard analysis showed that ac-DNMT1 level is a significant independent factor associated with MSS (HR, 0.994; 95% confidential interval (CI), 0.990-0.998; p = 0.002). These results demonstrated that low ac-DNMT1 levels may represent an important regulatory factor in controlling metastatic melanoma progression and a promising factor for stratifying aggressive stage IV metastasis.

The melanoma brain metastatic microenvironment: aldolase C partakes in shaping the malignant phenotype of melanoma cells - a case of inter-tumor heterogeneity.

Previous studies indicated that microglia cells upregulate the expression of aldolase C (ALDOC) in melanoma cells. The present study using brain-metastasizing variants from three human melanomas explores the functional role of ALDOC in the formation and maintenance of melanoma brain metastasis (MBM). ALDOC overexpression impacted differentially the malignant phenotype of these three variants. In the first variant, ALDOC overexpression promoted cell viability, adhesion to and transmigration through a layer of brain endothelial cells, and amplified brain micrometastasis formation. The cross-talk between this MBM variant and microglia cells promoted the proliferation and migration of the latter cells. In sharp contrast, ALDOC overexpression in the second brain-metastasizing melanoma variant reduced or did not affect the same malignancy features. In the third melanoma variant, ALDOC overexpression augmented certain characteristics of malignancy and reduced others. The analysis of biological functions and disease pathways in the ALDOC overexpressing variants clearly indicated that ALDOC induced the expression of tumor progression promoting genes in the first variant and antitumor progression properties in the second variant. Overall, these results accentuate the complex microenvironment interactions between microglia cells and MBM, and the functional impact of intertumor heterogeneity. Since intertumor heterogeneity imposes a challenge in the planning of cancer treatment, we propose to employ the functional response of tumors with an identical histology, to a particular drug or the molecular signature of this response, as a predictive indicator of response/nonresponse to this drug.

Genetic Variants in Immune Related Genes as Predictors of Responsiveness to BCG Immunotherapy in Metastatic Melanoma Patients.

Adjuvant immunotherapy in melanoma patients improves clinical outcomes. However, success is unpredictable due to inherited heterogeneity of immune responses. Inherent immune genes associated with single nucleotide polymorphisms (SNPs) may influence anti-tumor immune responses. We assessed the predictive ability of 26 immune-gene SNPs genomic panels for a clinical response to adjuvant BCG (Bacillus Calmette-Guérin) immunotherapy, using melanoma patient cohorts derived from three phase III multicenter clinical trials: AJCC (American Joint Committee on Cancer) stage IV patients given adjuvant BCG (pilot cohort; n = 92), AJCC stage III patients given adjuvant BCG (verification cohort; n = 269), and AJCC stage III patients that are sentinel lymph node (SLN) positive receiving no immunotherapy (control cohort; n = 80). The SNP panel analysis demonstrated that the responder patient group had an improved disease-free survival (DFS) (hazard ratio [HR] 1.84, 95% CI 1.09-3.13, p = 0.021) in the pilot cohort. In the verification cohort, an improved overall survival (OS) (HR 1.67, 95% CI 1.07-2.67, p = 0.025) was observed. No significant differences of SNPs were observed in DFS or OS in the control patient cohort. This study demonstrates that SNP immune genes can be utilized as a predictive tool for identifying melanoma patients that are inherently responsive to BCG and potentially other immunotherapies in the future.

Downregulation of the Ubiquitin-E3 Ligase RNF123 Promotes Upregulation of the NF-κB1 Target SerpinE1 in Aggressive Glioblastoma Tumors.

This study examined the role of the ubiquitin E3-ligase RNF123 in modulating downstream NF-κB1 targets in glioblastoma (GB) tumor progression. Our findings revealed an oncogenic pathway (miR-155-5p-RNF123-NF-κB1-p50-SerpinE1) that may represent a new therapeutic target pathway for GB patients with isocitrate dehydrogenase 1 and 2 (IDH) WT (wild type). Mechanistically, we demonstrated that RNF123 is downregulated in IDH WT GB patients and leads to the reduction of p50 levels. RNA-sequencing, reverse-phase protein arrays, and in vitro functional assays on IDH WT GB cell lines with RNF123 overexpression showed that SerpinE1 was a downstream target that is negatively regulated by RNF123. SERPINE1 knockdown reduced the proliferation and invasion of IDH WT GB cell lines. Both SerpinE1 and miR-155-5p overexpression negatively modulated RNF123 expression. In clinical translational analysis, RNF123, SerpinE1, and miR-155-5p were all associated with poor outcomes in GB patients. Multivariable analysis in IDH WT GB patients showed that concurrent low RNF123 and high SerpinE1 was an independent prognostic factor in predicting poor overall survival (p < 0.001, hazard ratio (HR) = 2.93, 95% confidence interval (CI) 1.7-5.05), and an increased risk of recurrence (p < 0.001, relative risk (RR) = 3.56, 95% CI 1.61-7.83).

Upregulation of cell surface GD3 ganglioside phenotype is associated with human melanoma brain metastasis.

Melanoma metastasis to the brain is one of the most frequent extracranial brain tumors. Cell surface gangliosides are elevated in melanoma metastasis; however, the metabolic regulatory mechanisms that govern these specific changes are poorly understood in melanoma particularly brain metastases (MBM) development. We found ganglioside GD3 levels significantly upregulated in MBM compared to lymph node metastasis (LNM) but not for other melanoma gangliosides. Moreover, we demonstrated an upregulation of ST8SIA1 (GD3 synthase) as melanoma progresses from melanocytes to MBM cells. Using RNA-ISH on FFPE specimens, we evaluated ST8SIA1 expression in primary melanomas (PRM) (n = 23), LNM and visceral metastasis (n = 45), and MBM (n = 39). ST8SIA1 was significantly enhanced in MBM compared to all other specimens. ST8SIA1 expression was assessed in clinically well-annotated melanoma patients from multicenters with AJCC stage III B-D LNM (n = 58) with 14-year follow-up. High ST8SIA1 expression was significantly associated with poor overall survival (HR = 3.24; 95% CI, 1.19-8.86, P = 0.02). In a nude mouse human xenograft melanoma brain metastasis model, MBM variants had higher ST8SIA1 expression than their respective cutaneous melanoma variants. Elevated ST8SIA1 expression enhances levels of cell surface GD3, a phenotype that favors MBM development, hence associated with very poor prognosis. Functional assays demonstrated that ST8SIA1 overexpression enhanced cell proliferation and colony formation, whereby ST8SIA1 knockdown had opposite effects. Icaritin a plant-derived phytoestrogen treatment significantly inhibited cell growth in high GD3-positive MBM cells through targeting the canonical NFκB pathway. The study demonstrates GD3 phenotype associates with melanoma progression and poor outcome.

Hypomethylation of CNTFRα is associated with proliferation and poor prognosis in lower grade gliomas.

Ciliary neurotrophic factor receptor α subunit (CNTFRα) and CNTF play important roles in neuron survival, glial differentiation and brain tumor growth. However, the molecular mechanisms of CNTFRα regulation and its clinical significance in glioma remain largely unknown. Here, we found CNTFRα was overexpressed in lower grade gliomas (LGG) compared with glioblastoma (GBM) and normal brain specimens in TCGA datasets and in an independent cohort. Bioinformatics analysis revealed a CpG shore of the CNTFRα gene regulated its mRNA expression in TCGA datasets. This observation was further validated with clinical specimens and functionally verified using demethylating agents. Additionally, we observed that independent of IDH mutation status, methylation of CNTFRα was significantly correlated with down-regulated CNTFRα gene expression and longer LGG patient survival. Interestingly, combination of CNTFRα methylation and IDH mutation significantly (p < 0.05) improved the prognostic prediction in LGG patients. Furthermore, the role of CNTFRα in glioma proliferation and apoptosis through the PI3K/AKT pathways was demonstrated by supplementation with exogenous CNTF  in vitro and siRNA knockdown in vivo. Our study demonstrated that hypomethylation leading to CNTFRα up-regulation, together with autocrine expression of CNTF, was involved in glioma growth regulation. Importantly, DNA methylation of CNTFRα might serve as a potential epigenetic theranostic target for LGG patients.

Insights into Local Tumor Microenvironment Immune Factors Associated with Regression of Cutaneous Melanoma Metastases by Mycobacterium bovis Bacille Calmette-Guérin.

Mycobacterium bovis bacille Calmette-Guérin (BCG) is listed as an intralesional (IL) therapeutic option for inoperable stage III in-transit melanoma in the National Comprehensive Cancer Network Guidelines. Although the mechanism is unknown, others have reported up to 50% regression of injected lesions, and 17% regression of uninjected lesions in immunocompetent patients after direct injection of BCG into metastatic melanoma lesions in the skin. BCG and other mycobacteria express ligands capable of stimulating the γ9δ2 T cells. Therefore, we hypothesized that γ9δ2 T cells play a role in promoting BCG-mediated antitumor immunity in patients treated with IL-BCG for in-transit cutaneous melanoma metastases. Indeed, we found γ9δ2 T cell infiltration in melanoma skin lesions during the course of IL-BCG treatment. Gene expression analysis revealed that BCG injection elicits the expression of a vast array of chemokines in tumor lesions, including strong expression of CXCL9, 10, and 11, a set of chemokines that attract T cells expressing the CXCR3 chemokine receptor. In corroboration with our hypothesis, approximately 85% of γδ T cells express high levels of CXCR3 on their surface. Importantly, the injected tumor lesions also express genes whose protein products are the antigenic ligands for γδ T cells (BTN3A1 and MICB), and the cytokines that are the typical products of activated γδ T cells. Interestingly, we also found that γδ T cells infiltrate the regressed lesions that did not receive BCG injections. Our study suggests that γ9δ2 T cells may contribute to melanoma regression induced by IL-BCG treatment.

Clinical Benefit from Ipilimumab Therapy in Melanoma Patients may be Associated with Serum CTLA4 Levels.

Stage IV metastatic melanoma patients historically have a poor prognosis with 5-10% 5-year survival. Ipilimumab, a monoclonal antibody against cytotoxic T-lymphocyte antigen 4 (CTLA4), is one of the first treatments to provide beneficial durable responses in advanced melanoma. However, less than 25% of those treated benefit, treatment is expensive, and side effects can be fatal. Since soluble (s) CTLA4 may mediate inhibitory effects previously ascribed to the membrane-bound isoform (mCTLA4), we hypothesized patients benefiting from ipilimumab have higher serum levels of sCTLA4. We found that higher sCTLA4 levels correlated both with response and improved survival in patients treated with ipilimumab in a small patient cohort [patients with (n = 9) and without (n = 5) clinical benefit]. sCTLA4 levels were statistically higher in ipilimumab-treated patients with response to ipilimumab. In contrast, sCTLA4 levels did not correlate with survival in patients who did not receive ipilimumab (n = 11). These preliminary observations provide a previously unrecognized link between serum sCTLA4 levels and response to ipilimumab as well as to improved survival in ipilimumab-treated melanoma patients and a potential mechanism by which ipilimumab functions.

Mouse model of chronic post-arthroplasty infection: noninvasive in vivo bioluminescence imaging to monitor bacterial burden for long-term study.

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.

Monitoring bacterial burden, inflammation and bone damage longitudinally using optical and µCT imaging in an orthopaedic implant infection in mice.

BACKGROUND: Recent advances in non-invasive optical, radiographic and µCT imaging provide an opportunity to monitor biological processes longitudinally in an anatomical context. One particularly relevant application for combining these modalities is to study orthopaedic implant infections. These infections are characterized by the formation of persistent bacterial biofilms on the implanted materials, causing inflammation, periprosthetic osteolysis, osteomyelitis, and bone damage, resulting in implant loosening and failure. METHODOLOGY/PRINCIPAL FINDINGS: An orthopaedic implant infection model was used in which a titanium Kirshner-wire was surgically placed in femurs of LysEGFP mice, which possess EGFP-fluorescent neutrophils, and a bioluminescent S. aureus strain (Xen29; 1×10(3) CFUs) was inoculated in the knee joint before closure. In vivo bioluminescent, fluorescent, X-ray and µCT imaging were performed on various postoperative days. The bacterial bioluminescent signals of the S. aureus-infected mice peaked on day 19, before decreasing to a basal level of light, which remained measurable for the entire 48 day experiment. Neutrophil EGFP-fluorescent signals of the S. aureus-infected mice were statistically greater than uninfected mice on days 2 and 5, but afterwards the signals for both groups approached background levels of detection. To visualize the three-dimensional location of the bacterial infection and neutrophil infiltration, a diffuse optical tomography reconstruction algorithm was used to co-register the bioluminescent and fluorescent signals with µCT images. To quantify the anatomical bone changes on the µCT images, the outer bone volume of the distal femurs were measured using a semi-automated contour based segmentation process. The outer bone volume increased through day 48, indicating that bone damage continued during the implant infection. CONCLUSIONS/SIGNIFICANCE: Bioluminescent and fluorescent optical imaging was combined with X-ray and µCT imaging to provide noninvasive and longitudinal measurements of the dynamic changes in bacterial burden, neutrophil recruitment and bone damage in a mouse orthopaedic implant infection model.

Protective role of IL-1ß against post-arthroplasty Staphylococcus aureus infection.

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.

Noninvasive in vivo imaging to evaluate immune responses and antimicrobial therapy against Staphylococcus aureus and USA300 MRSA skin infections.

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.

IL-17 is essential for host defense against cutaneous Staphylococcus aureus infection in mice.

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.