The Mark Coventry Award: PhotothermAA Gel Combined With Debridement, Antibiotics, and Implant Retention Significantly Decreases Implant Biofilm Burden and Soft-Tissue Infection in a Rabbit Model of Knee Periprosthetic Joint Infection.

BACKGROUND: Chronic periprosthetic joint infection (PJI) is a major complication of total joint arthroplasty. The underlying pathogenesis often involves the formation of bacterial biofilm that protects the pathogen from both host immune responses and antibiotics. The gold standard treatment requires implant removal, a procedure that carries associated morbidity and mortality risks. Strategies to preserve the implant while treating PJI are desperately needed. Our group has developed an anti-biofilm treatment, PhotothermAA gel, which has shown complete eradication of 2-week-old mature biofilm in vitro. In this study, we tested the anti-biofilm efficacy and safety of PhotothermAA in vivo when combined with debridement, antibiotics and implant retention (DAIR) in a rabbit model of knee PJI. METHODS: New Zealand white rabbits (n = 21) underwent knee joint arthrotomy, titanium tibial implant insertion, and inoculation with Xen36 (bioluminescent Staphylococcus aureus) after capsule closure. At 2 weeks, rabbits underwent sham surgery (n = 6), DAIR (n = 6), or PhotothermAA with DAIR (n = 9) and were sacrificed 2 weeks later to measure implant biofilm burden, soft-tissue infection, and tissue necrosis. RESULTS: The combination of anti-biofilm PhotothermAA with DAIR significantly decreased implant biofilm coverage via scanning electron microscopy compared to DAIR alone (1.8 versus 81.0%; P < .0001). Periprosthetic soft-tissue cultures were significantly decreased in the PhotothermAA with DAIR treatment group (log reduction: Sham 1.6, DAIR 2.0, combination 5.6; P < .0001). Treatment-associated necrosis was absent via gross histology of tissue adjacent to the treatment area (P = .715). CONCLUSIONS: The addition of an anti-biofilm solution like PhotothermAA as a supplement to current treatments that allow implant retention may prove useful in PJI treatment.

Mechanistic insights into bone remodelling dysregulation by human viral pathogens.

Bone-related diseases (osteopathologies) associated with human virus infections have increased around the globe. Recent findings have highlighted the intricate interplay between viral infection, the host immune system and the bone remodelling process. Viral infections can disrupt bone homeostasis, contributing to conditions such as arthritis and soft tissue calcifications. Osteopathologies can occur after arbovirus infections such as chikungunya virus, dengue virus and Zika virus, as well as respiratory viruses, such as severe acute respiratory syndrome coronavirus 2 and enteroviruses such as Coxsackievirus B. Here we explore how human viruses dysregulate bone homeostasis, detailing viral factors, molecular mechanisms, host immune response changes and bone remodelling that ultimately result in osteopathologies. We highlight model systems and technologies to advance mechanistic understanding of viral-mediated bone alterations. Finally, we propose potential prophylactic and therapeutic strategies, introduce 'osteovirology' as a research field highlighting the underestimated roles of viruses in bone-related diseases, and discuss research avenues for further investigation.

Periprosthetic joint infection and immunity: Current understanding of host-microbe interplay.

Periprosthetic joint infection (PJI) is a major complication of total joint arthroplasty. Even with current treatments, failure rates are unacceptably high with a 5-year mortality rate of 26%. Majority of the literature in the field has focused on development of better biomarkers for diagnostics and treatment strategies including innovate antibiotic delivery systems, antibiofilm agents, and bacteriophages. Nevertheless, the role of the immune system, our first line of defense during PJI, is not well understood. Evidence of infection in PJI patients is found within circulation, synovial fluid, and tissue and include numerous cytokines, metabolites, antimicrobial peptides, and soluble receptors that are part of the PJI diagnosis workup. Macrophages, neutrophils, and myeloid-derived suppressor cells (MDSCs) are initially recruited into the joint by chemokines and cytokines produced by immune cells and bacteria and are activated by pathogen-associated molecular patterns. While these cells are efficient killers of planktonic bacteria by phagocytosis, opsonization, degranulation, and recruitment of adaptive immune cells, biofilm-associated bacteria are troublesome. Biofilm is not only a physical barrier for the immune system but also elicits effector functions. Additionally, bacteria have developed mechanisms to evade the immune system by inactivating effector molecules, promoting killing or anti-inflammatory effector cell phenotypes, and intracellular persistence and dissemination. Understanding these shortcomings and the mechanisms by which bacteria can subvert the immune system may open new approaches to better prepare our own immune system to combat PJI. Furthermore, preoperative immune system assessment and screening for dysregulation may aid in developing preventative interventions to decrease PJI incidence.

The Use of Oral Antibiotics After Total Joint Arthroplasty: A Critical Analysis Review.

¼ There is conflicting and insufficient evidence that extended oral antibiotic (EOA) therapy prevents infection in high-risk patients undergoing primary total joint arthroplasty (TJA), limiting recommendation for or against the practice.¼ In the case of aseptic revision TJA, the evidence is also conflicting and limited by underlying confounders, preventing recommendation for use of EOA.¼ There is fair evidence that use of EOA after debridement antibiotic therapy and implant retention of the prosthesis prolongs infection-free survival, but randomized controlled trials are needed. On the other hand, there is strong evidence that patients undergoing 2-stage revision should receive a period of suppressive oral antibiotics after the second stage.¼ The optimal duration of EOA in primary TJA, aseptic revision, and debridement antibiotic therapy and implant retention of the prosthesis is unknown. However, there is strong evidence that 3 months of EOA suppression may be appropriate after reimplantation as part of 2-stage exchange arthroplasty.¼ Complications secondary to EOA are reported to be between 0% and 13.7%, yet are inconsistently reported and poorly defined. The risks associated with antibiotic use, including development of antimicrobial resistance, must be weighed against a possible decrease in infection rate.

Exploring Differences in Screening and Enrollment Metrics in Orthopaedic Clinical Trials.

The success of any clinical trial relies heavily on patient recruitment and retention. The purpose of this study was to review screening and enrollment metrics for orthopaedic clinical trials, comparing different patient populations to determine common challenges to recruitment and differences in rates of enrollment. Screening logs and study trackers were manually reviewed for four clinical trials at a single academic institution and included randomized controlled trials (RCTs) and an observational study. Data extracted from these documents included the number of patients screened, number excluded and reasons for exclusion, number enrolled, number of withdrawn and reason. Of the four trials reviewed, the point-of-care diagnostic test had the highest number of patients excluded and the lowest patient refusal rate. Refusal rates were highest in the venous thromboembolism prophylaxis study and enrollment rates were the lowest in the RCT of drug treatments and the highest rate in the observational study. The success of the trial relies on the ability to recruit patients and factors need to be considered when recruiting participants including sample size requirements and inclusion and exclusion criteria. These data provide some insights into the patient recruitment experience at our institution with different patient populations and study types, highlighting key points to be aware of when planning for an orthopaedic clinical trial.

Combination d-Amino Acid and Photothermal Hydrogel for the Treatment of Prosthetic Joint Infections.

Prosthetic joint infection (PJI) is a devastating complication requiring surgical intervention and prolonged antimicrobial treatment. The prevalence of PJI is on the rise, with an average incidence of 60,000 cases per year and a projected annual cost of $1.85 billion in the US. The underlying pathogenesis of PJI involves the formation of bacterial biofilms that protect the pathogen from the host immune response and antibiotics, making it difficult to eradicate such infections. Biofilms on implants are also resistant to mechanical brushing/scrubbing methods of removal. Since the removal of biofilms is currently only achievable by the replacement of the prosthesis, therapies aimed at eradicating biofilms while enabling retention of implants will revolutionize the management of PJIs. To address severe complications associated with biofilm-related infections on implants, we have developed a combination treatment that is based on a hydrogel nanocomposite system, containing d-amino acids (d-AAs) and gold nanorods, which can be delivered and transforms from a solution to a gel state at physiological temperature for sustained release of d-AAs and light-activated thermal treatment of infected sites. Using this two-step approach to utilize a near-infrared light-activated hydrogel nanocomposite system for thermal treatment, following initial disruption with d-AAs, we were able to successfully demonstrate in vitro the total eradication of mature Staphylococcus aureus biofilms grown on three-dimensional printed Ti-6Al-4V alloy implants. Using a combination of cell assays, computer-aided scanning electron microscopy analyses, and confocal microscopy imaging of the biofilm matrix, we could show 100% eradication of the biofilms using our combination treatment. In contrast, we were only able to see 25% eradication of the biofilms using the debridement, antibiotics, and implant retention method. Moreover, our hydrogel nanocomposite-based treatment approach is adaptable in the clinical setting and capable of combating chronic infections brought about by biofilms on medical implants.

Standardized quantification of biofilm in a novel rabbit model of periprosthetic joint infection.

Periprosthetic joint infection (PJI) is one of the most devastating complications of total joint arthroplasty. The underlying pathogenesis involves the formation of bacterial biofilm that protects the pathogen from the host immune response and antibiotics, making eradication difficult. The aim of this study was to develop a rabbit model of knee PJI that would allow reliable biofilm quantification and permit the study of treatments for PJI. In this work, New Zealand white rabbits ( n = 19 ) underwent knee joint arthrotomy, titanium tibial implant insertion, and inoculation with Xen36 (bioluminescent Staphylococcus aureus) or a saline control after capsule closure. Biofilm was quantified via scanning electron microscopy (SEM) of the tibial explant 14 d after inoculation ( n = 3 noninfected, n = 2 infected). Rabbits underwent debridement, antibiotics, and implant retention (DAIR) ( n = 6 ) or sham surgery ( n = 2 noninfected, n = 6 infected) 14 d after inoculation, and they were sacrificed 14 d post-treatment. Tibial explant and periprosthetic tissues were examined for infection. Laboratory assays supported bacterial infection in infected animals. No differences in weight or C-reactive protein (CRP) were detected after DAIR compared to sham treatment. Biofilm coverage was significantly decreased with DAIR treatment when compared with sham treatment (61.4 % vs. 90.1 %, p < 0 .0011) and was absent in noninfected control explants. In summary, we have developed an experimental rabbit hemiarthroplasty knee PJI model with bacterial infection that reliably produces quantifiable biofilm and provides an opportunity to introduce treatments at 14 d. This model may be used to better understand the pathogenesis of this condition and to measure treatment strategies for PJI.

Current treatments for biofilm-associated periprosthetic joint infection and new potential strategies.

Periprosthetic joint infection (PJI) remains a devastating complication after total joint arthroplasty. Bacteria involved in these infections are notorious for adhering to foreign implanted surfaces and generating a biofilm matrix. These biofilms protect the bacteria from antibiotic treatment and the immune system making eradication difficult. Current treatment strategies including debridement, antibiotics, and implant retention, and one- and two-stage revisions still present a relatively high overall failure rate. One of the main shortcomings that has been associated with this high failure rate is the lack of a robust approach to treating bacterial biofilm. Therefore, in this review, we will highlight new strategies that have the potential to combat PJI by targeting biofilm integrity, therefore giving antibiotics and the immune system access to the internal network of the biofilm structure. This combination antibiofilm/antibiotic therapy may be a new strategy for PJI treatment while promoting implant retention.

A Web-Based Interactive Patient-Provider Software Platform Does Not Increase Patient Satisfaction or Decrease Hospital Resource Utilization in Total Knee and Hip Arthroplasty Patients in a Single Large Hospital System.

BACKGROUND: Web-based platforms used to enhance patient-provider communication are being explored to improve patient satisfaction and care delivery, and decrease cost. This study tested a web-based interactive patient-provider software platform (IPSP), JointCOACH, which enabled patient communication with their care team and preparatory/recovery guidance. The aims of this study are to compare (1) patient satisfaction and (2) healthcare resource utilization by patients who underwent total knee and hip replacements and added IPSP to standard of care (SOC). METHODS: This study is a prospective, randomized clinical trial at a single large academic healthcare system. Between May 2018 and March 2020, 399 patients undergoing elective total hip or knee arthroplasty were randomized to SOC arm (n = 204) or SOC + IPSP arm (n = 195). Patient demographics, surgical details, and comorbidities were collected. Patient satisfaction was assessed using Visual Analog Scale and the Picker Patient Experience-15. Healthcare utilization was measured using length of stay, emergency department and office visits, office calls, readmissions, and reoperations at 30 and 90 days after surgery. RESULTS: No difference was found in length of stay between SOC and SOC + IPSP. No differences were found in 30-day or 90-day satisfaction or in healthcare resource utilization (P > .05) including number of office and emergency department visits, phone calls, and readmissions. CONCLUSION: Statistical differences were not found in satisfaction and healthcare utilization with the addition of IPSP to SOC. IPSP can be used to reinforce patient education and communication between the patient and provider, and should be evaluated as an element of virtual care rather than supplementing traditional in-office follow-up. CLINICALTRIALS.GOV: More information on this study can be found at clinicaltrials.gov NCT03499028.

Are Regulatory T Cells Defective in Type 1 Diabetes and Can We Fix Them?

Regulatory T cells (Tregs) are critical regulators of peripheral immune tolerance. Treg insufficiency can lead to autoimmune disorders, including type 1 diabetes (T1D). Increasing evidence in mouse models of T1D, as well as other autoimmune disorders, suggests that there are defects in Treg-mediated suppression. Indeed, whereas Treg frequency in the peripheral blood of T1D patients is unaltered, their suppressive abilities are diminished compared with Tregs in healthy controls. Although expression of the transcription factor Foxp3 is a prerequisite for Treg development and function, there are many additional factors that can alter their stability, survival, and function. Much has been learned in other model systems, such as tumors, about the mechanism and pathways that control Treg stability and function. This review poses the question of whether we can use these findings to develop new therapeutic approaches that might boost Treg stability, survival, and/or function in T1D and possibly other autoimmune disorders.

γδ T cells support gut Ag-reactive colitogenic effector T-cell generation by enhancing Ag presentation by CD11b(+) DCs in the mesenteric LN.

T cells expressing the γδ TCR are dominant T-cell subsets in the intestinal immune system. We previously demonstrated that γδ T cells play important roles in augmenting Th17-type colitogenic immune responses in a T-cell-induced colitic inflammation model. However, its underlying mechanism remains poorly understood. In this study, an in vitro coculture system using effector T cells enriched in gut Ag-reactive cells was employed as a readout tool to search for gut Ag presenting APCs. We found that the presence of γδ T cells dramatically enhances gut Ag presentation within the mLN in mice. Gut Ag presentation by CD11b(+) DC subsets was particularly controlled by γδ T cells. Interestingly, γδ T-cell entry to the lymph nodes was essential to improve the Ag presentation. Therefore, our results highlight that γδ T cells play a previously unrecognized role to support colitogenic immunity by regulating gut Ag presentation in the draining LN.

Cellular factors targeting APCs to modulate adaptive T cell immunity.

The fate of adaptive T cell immunity is determined by multiple cellular and molecular factors, among which the cytokine milieu plays the most important role in this process. Depending on the cytokines present during the initial T cell activation, T cells become effector cells that produce different effector molecules and execute adaptive immune functions. Studies thus far have primarily focused on defining how these factors control T cell differentiation by targeting T cells themselves. However, other non-T cells, particularly APCs, also express receptors for the factors and are capable of responding to them. In this review, we will discuss how APCs, by responding to those cytokines, influence T cell differentiation and adaptive immunity.

γδ T cells restrain extrathymic development of Foxp3+-inducible regulatory T cells via IFN-γ.

Inducible Treg (iTreg) cells generated from Ag-stimulated naïve CD4(+) T cells in the periphery play an important role in regulating immune responses. TGF-ß is a key cytokine that promotes this conversion process; however, how this process is regulated in vivo remains unclear. Here, we report that γδ T cells play a crucial role in controlling iTreg generation and suppressor function. Ag-induced iTreg generation was significantly enhanced in C57BL/6 mice in the absence of γδ T cells. Inhibition of iTreg conversion was mediated by IFN-γ produced by activated γδ T cells but not by activated CD4(+) T cells. BM chimera experiments further confirmed γδ-derived IFN-γ-dependent mechanism in regulating iTreg generation in vivo. Lastly, human peripheral blood γδ T cells also interfere with iTreg conversion via IFN-γ. Our results suggest a novel function of γδ T cells in limiting the generation of iTreg cells, potentially balancing immunity and tolerance.

Colitogenic effector T cells: roles of gut-homing integrin, gut antigen specificity and γδ T cells.

Disturbance of T-cell homeostasis could lead to intestinal inflammation. Naive CD4 T cells undergoing spontaneous proliferation, a robust proliferative response that occurs under severe lymphopenic conditions, differentiate into effector cells producing Th1- and/or Th17-type cytokines and induce a chronic inflammation in the intestine that resembles human inflammatory bowel disease. In this study, we investigated the key properties of CD4 T cells necessary to induce experimental colitis. α4ß7 upregulation was primarily induced by mesenteric lymph node (mLN) resident CD11b(+) dendritic cell subsets via transforming growth factor beta (TGFß)/retinoic acid-dependent mechanism. Interestingly, α4ß7 expression was essential but not sufficient to induce inflammation. In addition to gut-homing specificity, expression of gut Ag specificity was also crucial. T-cell acquisition of the specificity was dramatically enhanced by the presence of γδ T cells, a population previously shown to exacerbate T-cell-mediated colitis. Importantly, interleukin (IL)-23-mediated γδ T cell stimulation was necessary to enhance colitogenicity but not gut antigen reactivity of proliferating CD4 T cells. These findings demonstrate that T-cell colitogenicity is achieved through multiple processes, offering a therapeutic rationale by intervening these pathways.

Memory CD4 T cells induce selective expression of IL-27 in CD8+ dendritic cells and regulate homeostatic naive T cell proliferation.

Naive T cells undergo robust proliferation in lymphopenic conditions, whereas they remain quiescent in steady-state conditions. However, a mechanism by which naive T cells are kept from proliferating under steady-state conditions remains unclear. In this study, we report that memory CD4 T cells are able to limit naive T cell proliferation within lymphopenic hosts by modulating stimulatory functions of dendritic cells (DC). The inhibition was mediated by IL-27, which was primarily expressed in CD8(+) DC subsets as the result of memory CD4 T cell-DC interaction. IL-27 appeared to be the major mediator of inhibition, as naive T cells deficient in IL-27R were resistant to memory CD4 T cell-mediated inhibition. Finally, IL-27-mediated regulation of T cell proliferation was also observed in steady-state conditions as well as during Ag-mediated immune responses. We propose a new model for maintaining peripheral T cell homeostasis via memory CD4 T cells and CD8(+) DC-derived IL-27 in vivo.

CD4 T cells play important roles in maintaining IL-17-producing γδ T-cell subsets in naive animals.

A proportional balance between αß and γδ T-cell subsets in the periphery is exceedingly well maintained by a homeostatic mechanism. However, a cellular mechanism underlying the regulation remains undefined. We recently reported that a subset of developing γδ T cells spontaneously acquires interleukin (IL)-17-producing capacity even within naive animals through a transforming growth factor (TGF)ß1-dependent mechanism, thus considered 'innate' IL-17-producing cells. Here, we report that γδ T cells generated within αß T cell (or CD4 T cell)-deficient environments displayed altered cytokine profiles; particularly, 'innate' IL-17 expression was significantly impaired compared with those in wild-type mice. Impaired IL-17 production in γδ T cells was directly related to CD4 T-cell deficiency, because depletion of CD4 T cells in wild-type mice diminished and adoptive CD4 T-cell transfer into T-cell receptor ß-/- mice restored IL-17 expression in γδ T cells. CD4 T cell-mediated IL-17 expression required TGFß1. Moreover, Th17 but not Th1 or Th2 effector CD4 T cells were highly efficient in enhancing γδ T-cell IL-17 expression. Taken together, our results highlight a novel CD4 T cell-dependent mechanism that shapes the generation of IL-17+ γδ T cells in naive settings.

Cutting edge: Generation of colitogenic Th17 CD4 T cells is enhanced by IL-17+ γδ T cells.

Th 17 cells have been implicated in the pathogenesis of colitis; however, a cellular mechanism by which colitogenic Th17 immunity arises in vivo remains unclear. In this study, we report that a subset of IL-17(+) γδ T cells plays a crucial role in enhancing in vivo Th17 differentiation and T cell-mediated colitis. TCRß(-/-) mice were highly susceptible to T cell-mediated colitis, whereas TCRßδ(-/-) mice were resistant to the disease. Importantly, cotransfer of IL-17(+) but not of IL-17(-) γδ T cells with CD4 T cells was sufficient to enhance Th17 differentiation and induce full-blown colitis in TCRßδ(-/-) recipients. Collectively, our results provide a novel function of IL-17(+) γδ T cell subsets in supporting in vivo Th17 differentiation and possibly in fostering the development of intestinal inflammation.

Pyriform spidroin 1, a novel member of the silk gene family that anchors dragline silk fibers in attachment discs of the black widow spider, Latrodectus hesperus.

Spiders spin high performance threads that have diverse mechanical properties for specific biological applications. To better understand the molecular mechanism by which spiders anchor their threads to a solid support, we solubilized the attachment discs from black widow spiders and performed in-solution tryptic digests followed by MS/MS analysis to identify novel peptides derived from glue silks. Combining matrix-assisted laser desorption ionization tandem time-of-flight mass spectrometry and cDNA library screening, we isolated a novel member of the silk gene family called pysp1 and demonstrate that its protein product is assembled into the attachment disc silks. Alignment of the PySp1 amino acid sequence to other fibroins revealed conservation in the non-repetitive C-terminal region of the silk family. MS/MS analysis also confirmed the presence of MaSp1 and MaSp2, two important components of dragline silks, anchored within the attachment disc materials. Characterization of the ultrastructure of attachment discs using scanning electron microscopy studies support the localization of PySp1 to small diameter fibers embedded in a glue-like cement, which network with large diameter dragline silk threads, producing a strong, adhesive material. Consistent with elevated PySp1 mRNA levels detected in the pyriform gland, MS analysis of the luminal contents extracted from the pyriform gland after tryptic digestion support the assertion that PySp1 represents one of the major constituents manufactured in the pyriform gland. Taken together, our data demonstrate that PySp1 is spun into attachment disc silks to help affix dragline fibers to substrates, a critical function during spider web construction for prey capture and locomotion.