Transtibial osseointegration following unilateral traumatic amputation: An observational study of patients with at least two years follow-up.

IMPORTANCE: Most patients use a traditional socket prosthesis (TSP) to ambulate independently following transtibial amputation. However, these patients generally require prosthesis repairs more than twice annually and an entirely new prosthesis every two years. Furthermore, transtibial amputation patients have four times the skin ulceration rate of transfemoral patients, prompting more frequent prosthesis refitting and diminished use. Trans-Tibial osseointegration (TTOI) is a promising technique to address the limitations of TSP, but remains understudied with only four cohorts totaling 41 total procedures reported previously. Continued concerns regarding the risk of infection and questions as to functional capacity postoperatively have slowed adoption of TTOI worldwide. OBJECTIVE: This study reports the changes in mobility, quality of life (QOL), and the safety profile of the largest described cohort of patients with unilateral TTOI following traumatic amputation. DESIGN: Retrospective observational cohort study. The cohort consisted of patients with data outcomes collected before and after osseointegration intervention. SETTING: A large, tertiary referral, major metropolitan center. PARTICIPANTS: Twenty-one skeletally mature adults who had failed socket prosthesis rehabilitation, with at least two years of post-osseointegration follow-up. MAIN OUTCOMES AND MEASURES: Mobility was evaluated by K-level, Timed Up and Go (TUG), and Six Minute Walk Test (6MWT). QOL was assessed by survey: daily prosthesis wear hours, prosthesis problem experience, general contentment with prosthesis, and Short Form 36 (SF36). Adverse events included any relevant unplanned surgery such as for infection, fracture, implant loosening, or implant failure. RESULTS: All patients demonstrated statistically significant improvement post osseointegration surgery with respect to K-level, TUG, 6MWT, prosthesis wear hours, prosthesis problem experience, general prosthesis contentment score, and SF36 Physical Component Score (p < 0.01 for all). Three patients had four unplanned surgeries: two soft tissue refashionings, and one soft tissue debridement followed eventually by implant removal. No deaths, postoperative systemic complications, more proximal amputations, or periprosthetic fractures occurred. CONCLUSIONS AND RELEVANCE: TTOI is likely to confer mobility and QOL improvements to patients dissatisfied with TSP rehabilitation following unilateral traumatic transtibial amputation. Adverse events are relatively infrequent and not further disabling. Judicious use of TTOI seems reasonable for properly selected patients. LEVEL OF EVIDENCE: 2 (Therapeutic investigation, Observational study with dramatic effect).

Techniques to Remove Press-Fit Osseointegration Implants.

Background: Transcutaneous osseointegration for amputees (TOFA) has proven to consistently, significantly improve the quality of life and mobility for the vast majority of amputees, as compared with the use of a socket prosthesis1,2. As with any implant, situations such as infection, aseptic loosening, or implant fracture can occur, which may necessitate hardware removal. Although it may eventually occur, to date no osseointegration implant has ever required removal in the setting of periprosthetic fracture. Since TOFA implants are designed to facilitate robust bone integration, removal can be challenging. Even in cases in which portions of the implant are loose, other areas of the implant may remain strongly integrated and resist removal. Further, there can be cases in which an implant fractures, leaving the residual portion of the implant in place without the interface for an extraction tool. Although the outcomes of revision osseointegration has not been the primary focus of any publication, the fact that revision can be necessary and generally succeeds in restoring similar mobility has been documented3-5. As with any hardware removal, preserving healthy tissue and avoiding iatrogenic injury are critically important. This article demonstrates several techniques to remove press-fit osseointegration implants that we have found safe and effective. Description: The procedure is performed with the patient in the supine position and with the affected extremity prepared and draped in a typical sterile fashion. The use of a tourniquet can help reduce blood loss, but it may be safer to not use a tourniquet during the portions of the procedure that create increased or prolonged bone thermal exposure, such as during reaming or drilling. If patients are clinically stable, withholding antibiotics until cultures are obtained may improve diagnostic yield. The implant removal technique should proceed from conservative to aggressive, as necessary: slap hammer, thin wire-assisted slap hammer, and extended osteotomy. Trephine reaming is discouraged because of the need for and difficulty of removing the dual cone interface portion of the implant, along with the extensive damage often caused to the surrounding bone during reaming, which can be avoided with the osteotomy technique. Alternatives: It is important to emphasize that most infections related to transcutaneous osseointegration do not require implant removal; the use of antibiotics alone or soft-tissue and/or limited bone debridement is sufficient to resolve infection in the majority of cases. If a patient has a non-infectious indication for removal (such as a loose implant) but declines surgery, activity modification with close observation may be reasonable. If a patient has an infectious indication for removal but declines surgery, very close observation must be maintained to avoid potential osteomyelitis. The use of suppressive antibiotics (oral and/or intravenous) may help reduce the severity of the infection. An alternative surgery to manage implant-associated infection can be amputation above the implant (within the same bone, through-joint amputation, or through the femur for a transtibial osseointegration). This should be considered only if there is a tremendously compelling reason to do so, such as an emergency need to amputate due to a life-threatening infection. Rationale: Indications for implant removal include persistent pain, deep infection recalcitrant to soft-tissue or bone debridement and implant retention, or mechanical complications involving the implant, such as loosening or breakage. The following are specific examples of indications for removal: infection that cannot be resolved with oral and/or intravenous antibiotics or with debridement of soft tissue and/or bone, implants that have not achieved or not maintained stable integration (aseptic or loosening) and are causing pain, and implants that have fractured or have deformed and are a concern for fracture. Expected Outcomes: Most patients who require removal of press-fit osseointegration implants are suitable for reimplantation after a decontamination period involving a local antibiotic depot and intravenous antibiotics. This duration is often 6 to 12 weeks. Following revision osseointegration, patients generally achieve similar levels of performance as they had during the stable period prior to removal3-5. Infection does not appear to be associated with an increased risk of mortality6. Patients who decline revision osseointegration are able to return to the use of a socket prosthesis. Important Tips: Infection is often treatable with use of oral and/or intravenous antibiotics, with or without soft-tissue and/or bone debridement. Consider these options before reflexively removing an implant.Preserving bone stock and quality is important for an optimal revision to another osseointegration prosthesis or conversion to a socket prosthesis. Living bone, even if infected, can be decontaminated with antibiotics. Minimize thermal injury by releasing the tourniquet and using saline solution irrigation during reaming or similar portions of the procedure. If the implant can only be removed by fully removing a portion of bone (rather than through a single clamshell-type osteotomy), attempt screw osteosynthesis to preserve a canal for future osseointegration.Be gentle and patient during the removal techniques. Elevating bone quickly or violently may cause propagating fractures, increased morbidity, or splinter bone fragments. Carefully separating bone from implant will reduce bone loss and preserve the bone condition for a potential revision.Although patients may not appear to have an infection in some cases, it is advised to treat every removal as if it involves an infection. The first surgical stage should be for removal, culture acquisition, and decontamination with antibiotics. A separate second surgery can be performed after the antibiotics have eradicated infection.Many implants have been removed with slap hammer or fine wire techniques; osteotomy should be reserved for situations in which diligent attempts using these techniques have proven unsuccessful, in order to optimize bone integrity.

Transfemoral amputation versus knee arthrodesis for failed total knee replacement: A systematic review of outcomes.

BACKGROUND: The options available to salvage a failed total knee replacement (TKR) include transfemoral amputation (TFA) and knee arthrodesis (KA). This systematic review aims to evaluate outcomes following either TFA or KA, comparing ambulatory status, additional subsequent surgery, postoperative infection, pain, health-related quality of life (HRQoL), and mortality rate. METHODS: A literature search was conducted in EMBASE, Ovid Medline, and PubMed. Only primary research studies were included and data were independently extracted using a standardized form. The methodological quality of the studies was evaluated using Newcastle-Ottawa Scale. RESULTS: Forty-four papers were included, comprising 470 TFA and 1034 KA patients. The methodological quality of the studies was moderate. No TFA versus KA randomized controlled trials could be identified. Pooled data totals via subgroup analyses were performed, owing to inconsistent reporting methods in the included studies. Prosthesis use rate by TFA patients was 157/316 = 49.7%. Significant differences included that TFA patients had lower rates of ambulatory capacity than KA patients (139/294 = 45.6% versus 248/287 = 86.4%, p < 0.001), TFA ambulators were less likely to use an ambulatory aid (55/135 = 40.7% versus 167/232 = 72.0%, p < 0.001), and TFA was associated with a greater postoperative infection rate than KA (29/118 = 24.6% versus 129/650 = 17.2%, p = 0.054). There was a similar rate of revision surgery between TFA and KA (37/183 = 20.2% versus 145/780 = 18.6%, p = 0.612). Data on HRQoL for both TFA and KA were limited, contradictory, and heterogeneous. CONCLUSION: No randomized controlled trials comparing TFA versus KA exist;therefore, current data likely reflects substantial selection bias. The currently available evidence suggests that KA patients are significantly more likely to achieve independent bipedal ambulation than TFA patients. In both treatment cohorts, subsequent infection and revision surgery remain a relatively common occurrence.

Avoiding Compartment Syndrome, Vascular Injury, and Neurologic Deficit in Tibial Osteotomy: An Observational Study of 108 Limbs.

INTRODUCTION: Tibial deformities are common, but substantial concern may be associated with corrective osteotomy regarding major complications reported in classic literature. Such studies chiefly focused on high tibial osteotomy, with relatively little investigation of other areas and types of deformity. The primary aim of this study was to identify the rate of compartment syndrome, vascular injury, nerve injury, and other major complications after elective tibial osteotomy. METHODS: One hundred eight tibia osteotomies performed during 2019 to 2021 were evaluated, representing all tibia osteotomies except situations of existing infection. A retrospective chart review was performed to identify patient demographics, surgical indications, anatomic location of osteotomy, fixation used, and complications prompting additional surgery. RESULTS: The most common osteotomy locations were high tibial osteotomy (35/108 = 32%, 32/35 = 91% medial opening, and 3/35 = 9% medial closing), proximal metaphysis (30/108 = 28%), and diaphysis (32/108 = 30%). The most common fixation was plate and screw (38/108 = 35%) or dynamic frame (36/108 = 33%). Tranexamic acid was administered to 107/108 = 99% of patients and aspirin chemoprophylaxis was used for 83/108 = 86%. A total of 33/34= 97% of anterior compartment prophylactic fasciotomies were performed for diaphyseal or proximal metaphysis osteotomies. No events of compartment syndrome, vascular injury, nerve injury, or pulmonary embolism occurred. One patient required débridement to address infection. Additional surgery for delayed/nonunion occurred for nine segments (8%). Additional surgery for other reasons were performed for 10 segments (9%), none resulting in reduced limb function. CONCLUSION: Tibial osteotomy can be safely performed for a variety of indications in a diverse range of patients, without a notable risk of the most feared complications of compartment syndrome, vascular injury, and neurologic deficit. Prophylactic fasciotomy and reducing postoperative bleeding using tranexamic acid, along with location-specific safe surgical techniques, may help prevent major complications and thereby facilitate optimized deformity care.

Bone density changes after five or more years of unilateral lower extremity osseointegration: Observational cohort study.

Context: Rehabilitation following lower extremity amputation presents multiple challenges, many related to the traditional prosthesis (TP) socket. Without skeletal loading, bone density also rapidly decreases. Transcutaneous osseointegration for amputees (TOFA) surgically implants a metal prosthesis attachment directly into the residual bone, facilitating direct skeletal loading. Quality of life and mobility are consistently reported to be significantly superior with TOFA than TP. Objective: To investigate how femoral neck bone mineral density (BMD, g/cm2) changes for unilateral transfemoral and transtibial amputees at least five years following single-stage press-fit osseointegration. Methods: Registry review was performed of five transfemoral and four transtibial unilateral amputees who had dual x-ray absorptiometry (DXA) performed preoperatively and after at least five years. The average BMD was compared using Student's t-test (significance p < .05). First, all nine Amputated versus Intact limbs. Second, the five patients with local disuse osteoporosis (ipsilateral femoral neck T-score < -2.5) versus the four whose T-score was greater than -2.5. Results: The average Amputated Limb BMD was significantly less than the Intact Limb, both Before Osseointegration (0.658 ± 0.150 vs 0.929 ± 0.089, p < .001) and After Osseointegration (0.720 ± 0.096 vs 0.853 ± 0.116, p = .018). The Intact Limb BMD decreased significantly during the study period (0.929 ± 0.089 to 0.853 ± 0.116, p = .020), while the Amputated Limb BMD increased a not statistically significant amount (0.658 ± 0.150 to 0.720 ± 0.096, p = .347). By coincidence, all transfemoral amputees had local disuse osteoporosis (BMD 0.545 ± 0.066), and all transtibial patients did not (BMD 0.800 ± 0.081, p = .003). The local disuse osteoporosis cohort eventually had a greater average BMD (not statistically significant) than the cohort without local disuse osteoporosis (0.739 ± 0.100 vs 0.697 ± 0.101, p = .556). Conclusions: Single-stage press-fit TOFA may facilitate significant BMD improvement to unilateral lower extremity amputees with local disuse osteoporosis.

Association Between Osseointegration of Lower Extremity Amputation and Mortality Among Adults.

Importance: Transcutaneous osseointegration post amputation (TOPA) creates a direct linkage between residual bone and an external prosthetic limb, providing superior mobility and quality of life compared with a socket prosthesis. The causes and potential risks of mortality after TOPA have not been investigated. Objective: To investigate the association between TOPA and mortality and assess the potential risk factors. Design, Setting, and Participants: This observational cohort study included all patients with amputation of a lower extremity who underwent TOPA between November 1, 2010, and October 31, 2021, at a specialty orthopedic practice and tertiary referral hospital in a major urban center. Patients lived on several continents and were followed up as long as 10 years. Exposures: Transcutaneous osseointegration post amputation, consisting of a permanent intramedullary implant passed transcutaneously through a stoma and connected to an external prosthetic limb. Main Outcomes and Measures: Death due to any cause. The hypotheses tested-that patient variables (sex, age, level of amputation, postosseointegration infection, and amputation etiology) may be associated with subsequent mortality-were formulated after initial data collection identifying which patients had died. Results: A total of 485 patients were included in the analysis (345 men [71.1%] and 140 women [28.9%]), with a mean (SD) age at osseointegration of 49.1 (14.6) years among living patients or 61.2 (12.4) years among patients who had died. Nineteen patients (3.9%) died a mean (SD) of 2.2 (1.7) years (range, 58 days to 5 years) after osseointegration, including 17 (3.5%) who died of causes unrelated to osseointegration (most commonly cardiac issues) and 2 (0.4%) who died of direct osseointegration-related complications (infectious complications), of which 1 (0.2%) was coclassified as a preexisting health problem exacerbated by osseointegration (myocardial infarction after subsequent surgery to manage infection). No deaths occurred intraoperatively or during inpatient recuperation or acute recovery after index osseointegration (eg, cardiopulmonary events). Kaplan-Meier survival analysis with log-rank comparison and Cox proportional hazards regression modeling identified increased age (hazard ratio, 1.06 [95% CI, 1.02-1.09]) and vascular (odds ratio [OR], 4.73 [95% CI, 1.35-16.56]) or infectious (OR, 3.87 [95% CI, 1.31-11.40]) amputation etiology as risk factors. Notable factors not associated with mortality risk included postosseointegration infection and male sex. Conclusions and Relevance: These findings suggest that patients who have undergone TOPA rarely die of problems associated with the procedure but instead usually die of unrelated causes.

Periprosthetic Femur Fractures in Osseointegration Amputees: A Report of 2 Cases Using a Modified Traction Technique.

CASES: Two patients with periprosthetic femur fracture proximal to a femoral osseointegration had surgical stabilization. These cases produced operative challenges because commonly used intraoperative traction methods cannot be used for patients with transfemoral osseointegration. CONCLUSION: We describe a novel technique to apply traction and rotation to the osseointegrated extremity: using the implant's antirotation propeller handle. This allows for an otherwise familiar technique to be used for fracture fixation: open reduction with internal fixation using a proximal femur hook plate and eccentric peri-implant screws.

Osseointegration Following Transfemoral Amputation After Infected Total Knee Replacement: A Case Series of 10 Patients With a Mean Follow-up of 5 Years.

Background: Management of total knee replacement (TKR) infection may sometimes prompt knee fusion (KF) or transfemoral amputation (TFA), both associated with low mobility and quality of life (QOL). Transcutaneous osseointegration for amputees provides superior mobility and QOL vs traditional socket prostheses but has not been studied for patients with a history of infected TKR. This study investigates the following hypothesis: Patients who have had TFA or KF following infected TKR achieve better mobility and QOL following transfemoral osseointegration. Material and methods: A retrospective evaluation of the prospectively maintained registry identified 10 patients who had prior infected TKR. The mobility assessments (patient daily prosthesis wear time, K-level, Timed Up and Go, 6-Minute Walk Test) and QOL surveys (Questionnaire for Persons with a Transfemoral Amputation Global, Mobility, and Problem scores) were compared preoperatively and after at least 2 years. Complications requiring an additional surgery were also evaluated. Results: Daily wear hours, K-level, and 6-Minute Walk Test and Questionnaire for Persons with a Transfemoral Amputation Global and Problem scores significantly improved (P < .05). Through 1 year, 4 patients (40%) had additional surgeries. After several years, 7 patients (70%) had at least 1 additional surgery, and 5 (50%) had multiple, for an average of 1 debridement and 1.3 soft-tissue refashionings per patient. One patient died of newly diagnosed cancer 1 year after transcutaneous osseointegration for amputees. Conclusion: Transfemoral osseointegration confers significantly better mobility and QOL vs KF or a TFA with traditional socket prostheses following infected TKR. Technique improvements to prevent subsequent surgeries may provide an increasingly streamlined experience.

The Clinical History and Basic Science Origins of Transcutaneous Osseointegration for Amputees.

Transcutaneous osseointegration for amputees (TOFA) refers to an intramedullary metal endoprosthesis which passes transcutaneously to connect with a limb exoprosthesis. The first recognizably modern experiments and attempts occurred in the 1940s. Multiple researchers using a plethora of materials and techniques over the following 50 years identified principles and obstacles which informed the first long-term successful surgery in 1990. Unfortunately, the current mainstream TOFA literature presents almost exclusively subsequent developments, generally omitting prior research, leading to some historical mistakes being repeated. Given the increasing interest and surgical volume of TOFA, this literature review was performed to delineate TOFA's basic science and surgical origins and to integrate these early efforts within the contemporary understanding. Studying this research could protect and benefit future patients, surgeons, and implant developers as TOFA is entering a phase of increased attention and innovation. The aim of this article is to provide a focused reference of foundational research, much of which is difficult to identify and retrieve, for clinicians and researchers.

Transtibial Osseointegration for Patients with Peripheral Vascular Disease: A Case Series of 6 Patients with Minimum 3-Year Follow-up.

The management of peripheral vascular disease (PVD) can require amputation. Osseointegration surgery is an emerging rehabilitation strategy for amputees. In this study, we report on 6 patients who had PVD requiring transtibial amputation (PVD-TTA) and either simultaneous or subsequent osseointegration (PVD-TTOI). METHODS: Six patients (aged 36 to 84 years) with transtibial amputation and preexisting PVD underwent osseointegration between 2014 and 2016 and were followed for 3 to 5 years. Pre- and postoperative clinical and functional outcomes (pain, prosthesis wear time, mobility, walking ability, and quality of life) and adverse events (infection, fracture, implant failure, revision surgery, additional amputation, and death) were prospectively recorded. RESULTS: All patients' mobility improved following osseointegration. Three patients initially had required the use of a wheelchair, precluding baseline walking tests; the other 3 were classified as K level 1 or 2, with mean baseline Timed Up and Go (TUG) test = 14.0 ± 2.2 s and 6-Minute Walk Test (6MWT) = 262 ± 75 m. At the time of the latest follow-up, all patients were K level 2 or 3; mean TUG = 12.7 ± 7.2 s and 6MWT = 353 ± 148 m. Four patients wore their prosthesis ≥16 hours daily. Three patients had superficial soft-tissue infections. One other patient experienced recurrent infections 2.8 years after osseointegration requiring debridements and transfemoral amputation; the patient died 2 days following surgery from myocardial infarction caused by coronary atherosclerosis. CONCLUSIONS: All 6 patients who underwent PVD-TTOI in this case series survived through 2 years. Patients who initially had used a wheelchair achieved and maintained independent, unaided ambulation until PVD-related impairments in the contralateral leg occurred in 1 patient. Patients previously using a traditional socket prosthesis reported improvement in mobility and quality of life. One patient's death underscores the importance of careful patient selection. However, marked improvement in the other 5 patients suggests cautious optimism that PVD-TTA is not an absolute osseointegration contraindication. Conscientious further investigation seems appropriate. LEVEL OF EVIDENCE: Therapeutic Level IV. See Instructions for Authors for a complete description of levels of evidence.

Lengthening the Lower Extremities of Children with Ollier's and Maffucci's Enchondromatosis Using Implantable Lengthening Nails.

There are multiple forms of enchondromatosis with Ollier's and Maffucci's being the most prevalent types. Limb length discrepancy is a common problem in patients with Ollier's and Maffucci's enchondromatosis. There are multiple reports about lengthening bones in patients with enchondromatosis using external fixators. However, there are no case series regarding the use of implantable lengthening technology. The purpose of this paper is to describe our experience with implantable nail lengthening in patients with enchondromatosis. A retrospective chart and radiographic review of patients with enchondromatosis who underwent implantable nail limb lengthening was performed. Seven patients with 14 bony segments were reviewed. A total of 11/14 lengthenings were completed without difficulty. There were no issues in terms of fixation location in patients with Ollier's disease. One patient with Maffucci's syndrome experienced migration of the nail during two lengthenings due to a combination of intralesional fixation and preconsolidation. One patient with Ollier's disease developed a knee extension contracture requiring manipulation under anesthesia. No other complications were recorded. The use of implantable nail lengthening to resolve limb length discrepancies in patients with Ollier's disease appears to be safe and effective.

Difficult Management of a Femur Fracture in a Patient with Dyskeratosis Congenita: A Literature Review and Case Report.

CASE: A 14-year-old boy with dyskeratosis congenita (DKC), status-post unrelated bone marrow transplant (BMT), sustained a femoral shaft fracture. Despite immediate fixation with the widest possible titanium elastic nails, fixation construct rigidity was insufficient and malunion occurred with refracture 5 years later. Revision fixation with rigid intramedullary nailing has maintained position for 1 year, although callus formation remains meager. CONCLUSION: This is the first article to detail fracture care for a DKC patient. Although BMT increases lifespan, patients seemingly remain skeletally frail. Rigid intramedullary fixation is optimally durable and appears hematopoietically safe. Long-term follow-up is recommended.

Amputation With Osseointegration for Patients With Intractable Complex Regional Pain Syndrome: A Report of 3 Cases.

CASES: Three patients with knee-level complex regional pain syndrome type 1 (CRPS1), recalcitrant to conservative interventions, elected for transfemoral amputation and osseointegration. Two patients gained independent ambulation; the third remains on crutches after a disrupted sciatic nerve targeted reinnervation. One uses no pain medication, one is weaning off, and one requires a reduced regimen after revision nerve innervation. CONCLUSION: Osseointegration seems suitable to optimize rehabilitation after amputation for CRPS1.

Osseointegration for Amputees: Current Implants, Techniques, and Future Directions.

Osseointegrated prostheses provide a rehabilitation option for amputees offering greater mobility, better satisfaction, and higher use than traditional socket prostheses. There are several different osseointegrated implant designs, surgical techniques, and rehabilitation protocols with their own strengths and limitations. The 2 most prominent risks, infection and periprosthetic fracture, do not seem unacceptably frequent or insurmountable. Proximal amputations or situations leading to reduced mobility are exceptionally infrequent. Osseointegrated implants can be attached to advanced sensory and motor prostheses.

Tumor-to-Tumor Metastasis: Prostate Carcinoma Metastatic to Soft Tissue Sarcoma, a Previously Unreported Event.

Tumor-to-tumor metastasis is an unusual phenomenon wherein one distinct malignancy is present within the substance of another independent tumor. This event is rare, difficult to detect with imaging, and, due to conflicting terminology in the literature, can be challenging to classify. This article reports the first documented case of tumor-to-tumor metastasis involving prostatic adenocarcinoma and myxoid liposarcoma, reviews the available literature for carcinoma metastatic to sarcoma, and discusses the current situation within the context of the established criteria for the classification of combination tumors.

Pediatric Open Both-Bone Forearm Fracture Stabilized with Titanium and Stainless Steel Stacked Extension Internal Plating: A Case Report.

CASE: A 12-year-old male patient sustained a multisegmental both-bone forearm fracture. Definitive stabilization was ulna intramedullary pinning, with stainless steel plating of the radius followed by a titanium plate stacked atop and extending internal fixation. Currently, 36 months after surgery, he experiences no limitations or complications. All hardware remains in situ. CONCLUSION: We believe this is the first description of different material plates in direct contact, of stacked plates, and of 1 plate extending another's fixation. No adverse effects developed.

Combined TLR7/8 and TLR9 ligands potentiate the activity of a Schistosoma japonicum DNA vaccine.

BACKGROUND: Toll-like receptor (TLR) ligands have been explored as vaccine adjuvants for tumor and virus immunotherapy, but few TLR ligands affecting schistosoma vaccines have been characterized. Previously, we developed a partially protective DNA vaccine encoding the 26-kDa glutathione S-transferase of Schistosoma japonicum (pVAX1-Sj26GST). METHODOLOGY/PRINCIPAL FINDINGS: In this study, we evaluated a TLR7/8 ligand (R848) and a TLR9 ligand (CpG oligodeoxynucleotides, or CpG) as adjuvants for pVAX1-Sj26GST and assessed their effects on the immune system and protection against S. japonicum. We show that combining CpG and R848 with pVAX1-Sj26GST immunization significantly increases splenocyte proliferation and IgG and IgG2a levels, decreases CD4(+)CD25(+)Foxp3(+) regulatory T cells (Treg) frequency in vivo, and enhances protection against S. japonicum. CpG and R848 inhibited Treg-mediated immunosuppression, upregulated the production of interferon (IFN)-γ, tumor necrosis factor (TNF)-α, interleukin (IL)-4, IL-10, IL-2, and IL-6, and decreased Foxp3 expression in vitro, which may contribute to prevent Treg suppression and conversion during vaccination and allow expansion of antigen-specific T cells against pathogens. CONCLUSIONS: Our data shows that selective TLR ligands can increase the protective efficacy of DNA vaccines against schistosomiasis, potentially through combined antagonism of Treg-mediated immunosuppression and conversion.