Identifying a Minor Histocompatibility Antigen in Mauritian Cynomolgus Macaques Encoded by APOBEC3C.

Allogeneic hematopoietic stem cell transplants can lead to dramatic reductions in human immunodeficiency virus (HIV) reservoirs. This effect is partially mediated by donor T cells recognizing lymphocyte-expressed minor histocompatibility antigens (mHAgs). The potential to mark malignant and latently infected cells for destruction makes mHAgs attractive targets for cellular immunotherapies. However, testing such HIV reservoir reduction strategies will likely require preclinical studies in non-human primates (NHPs). In this study, we used a combination of alloimmunization, whole exome sequencing, and bioinformatics to identify an mHAg in Mauritian cynomolgus macaques (MCMs). We mapped the minimal optimal epitope to a 10-mer peptide (SW10) in apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3C (APOBEC3C) and determined the major histocompatibility complex class I restriction element as Mafa-A1∗063, which is expressed in almost 90% of MCMs. APOBEC3C SW10-specific CD8+ T cells recognized immortalized B cells but not fibroblasts from an mHAg-positive MCM. These results provide a framework for identifying mHAgs in a non-transplant setting and suggest that APOBEC3C SW10 could be used as a model antigen to test mHAg-targeted therapies in NHPs.

The effect of extra-osseous talotarsal stabilization (EOTTS) to reduce medial knee compartment forces - An in vivo study.

BACKGROUND: Excessive hindfoot pronation, talotarsal joint (TTJ) instability, has been attributed to an increase in medial knee compartment pathology. Advocacy for hindfoot realignment has been the subject of research. An internal solution for TTJ instability, extra-osseous talotarsal stabilization (EOTTS), exists but its effect on knee forces is unknown. This is the first study to measure the in vivo forces acting within the medial knee compartment before and after EOTTS. We hypothesized that following EOTTS there should be a reduction of force acting on the medial knee compartment. METHODS: 10 fresh frozen cadaver lower extremities exhibiting clinical and radiographic evidence of TTJ instability were evaluated. The proximal femur segment was mounted to a mechanical testing unit. Pressure sensors were placed within the medial knee compartment. A force of 1000 newtons was then applied, and the femur was internally rotated 10 degrees. Measurements were recorded before and after the insertion of a type II EOTTS stent. RESULTS: Pre-EOTTS resulted in an average of 842 ± 247N acting within the medial knee joint compartment. These forces then decreased to an average of 565 ± 260N (<0.05) following EOTTS, representing an average reduction of force by 32.8%. CONCLUSION: EOTTS has been shown to decrease the in vivo forces action within the medial knee compartment. This helps to further illustrate the importance of realigning and stabilizing the hindfoot for the prevention and treatment of chronic knee pain.

Characterization of Mauritian Cynomolgus Macaque FcγR Alleles Using Long-Read Sequencing.

The FcγRs are immune cell surface proteins that bind IgG and facilitate cytokine production, phagocytosis, and Ab-dependent, cell-mediated cytotoxicity. FcγRs play a critical role in immunity; variation in these genes is implicated in autoimmunity and other diseases. Cynomolgus macaques are an excellent animal model for many human diseases, and Mauritian cynomolgus macaques (MCMs) are particularly useful because of their restricted genetic diversity. Previous studies of MCM immune gene diversity have focused on the MHC and killer cell Ig-like receptor. In this study, we characterize FcγR diversity in 48 MCMs using PacBio long-read sequencing to identify novel alleles of each of the four expressed MCM FcγR genes. We also developed a high-throughput FcγR genotyping assay, which we used to determine allele frequencies and identify FcγR haplotypes in more than 500 additional MCMs. We found three alleles for FcγR1A, seven each for FcγR2A and FcγR2B, and four for FcγR3A; these segregate into eight haplotypes. We also assessed whether different FcγR alleles confer different Ab-binding affinities by surface plasmon resonance and found minimal difference in binding affinities across alleles for a panel of wild type and Fc-engineered human IgG. This work suggests that although MCMs may not fully represent the diversity of FcγR responses in humans, they may offer highly reproducible results for mAb therapy and toxicity studies.

Novel full-length major histocompatibility complex class I allele discovery and haplotype definition in pig-tailed macaques.

Pig-tailed macaques (Macaca nemestrina, Mane) are important models for human immunodeficiency virus (HIV) studies. Their infectability with minimally modified HIV makes them a uniquely valuable animal model to mimic human infection with HIV and progression to acquired immunodeficiency syndrome (AIDS). However, variation in the pig-tailed macaque major histocompatibility complex (MHC) and the impact of individual transcripts on the pathogenesis of HIV and other infectious diseases is understudied compared to that of rhesus and cynomolgus macaques. In this study, we used Pacific Biosciences single-molecule real-time circular consensus sequencing to describe full-length MHC class I (MHC-I) transcripts for 194 pig-tailed macaques from three breeding centers. We then used the full-length sequences to infer Mane-A and Mane-B haplotypes containing groups of MHC-I transcripts that co-segregate due to physical linkage. In total, we characterized full-length open reading frames (ORFs) for 313 Mane-A, Mane-B, and Mane-I sequences that defined 86 Mane-A and 106 Mane-B MHC-I haplotypes. Pacific Biosciences technology allows us to resolve these Mane-A and Mane-B haplotypes to the level of synonymous allelic variants. The newly defined haplotypes and transcript sequences containing full-length ORFs provide an important resource for infectious disease researchers as certain MHC haplotypes have been shown to provide exceptional control of simian immunodeficiency virus (SIV) replication and prevention of AIDS-like disease in nonhuman primates. The increased allelic resolution provided by Pacific Biosciences sequencing also benefits transplant research by allowing researchers to more specifically match haplotypes between donors and recipients to the level of nonsynonymous allelic variation, thus reducing the risk of graft-versus-host disease.

Oropharyngeal mucosal transmission of Zika virus in rhesus macaques.

Zika virus is present in urine, saliva, tears, and breast milk, but the transmission risk associated with these body fluids is currently unknown. Here we evaluate the risk of Zika virus transmission through mucosal contact in rhesus macaques. Application of high-dose Zika virus directly to the tonsils of three rhesus macaques results in detectable plasma viremia in all animals by 2 days post-exposure; virus replication kinetics are similar to those observed in animals infected subcutaneously. Three additional macaques inoculated subcutaneously with Zika virus served as saliva donors to assess the transmission risk from contact with oral secretions from an infected individual. Seven naive animals repeatedly exposed to donor saliva via the conjunctivae, tonsils, or nostrils did not become infected. Our results suggest that there is a risk of Zika virus transmission via the mucosal route, but that the risk posed by oral secretions from individuals with a typical course of Zika virus infection is low.Zika virus (ZIKV) is present in body fluids, including saliva, but transmission risk through mucosal contact is not well known. Here, the authors show that oropharyngeal mucosal infection of macaques with a high ZIKV dose results in viremia, but that transmission risk from saliva of infected animals is low.

Highly efficient maternal-fetal Zika virus transmission in pregnant rhesus macaques.

Infection with Zika virus (ZIKV) is associated with human congenital fetal anomalies. To model fetal outcomes in nonhuman primates, we administered Asian-lineage ZIKV subcutaneously to four pregnant rhesus macaques. While non-pregnant animals in a previous study contemporary with the current report clear viremia within 10-12 days, maternal viremia was prolonged in 3 of 4 pregnancies. Fetal head growth velocity in the last month of gestation determined by ultrasound assessment of head circumference was decreased in comparison with biparietal diameter and femur length within each fetus, both within normal range. ZIKV RNA was detected in tissues from all four fetuses at term cesarean section. In all pregnancies, neutrophilic infiltration was present at the maternal-fetal interface (decidua, placenta, fetal membranes), in various fetal tissues, and in fetal retina, choroid, and optic nerve (first trimester infection only). Consistent vertical transmission in this primate model may provide a platform to assess risk factors and test therapeutic interventions for interruption of fetal infection. The results may also suggest that maternal-fetal ZIKV transmission in human pregnancy may be more frequent than currently appreciated.

Improved full-length killer cell immunoglobulin-like receptor transcript discovery in Mauritian cynomolgus macaques.

Killer cell immunoglobulin-like receptors (KIRs) modulate disease progression of pathogens including HIV, malaria, and hepatitis C. Cynomolgus and rhesus macaques are widely used as nonhuman primate models to study human pathogens, and so, considerable effort has been put into characterizing their KIR genetics. However, previous studies have relied on cDNA cloning and Sanger sequencing that lack the throughput of current sequencing platforms. In this study, we present a high throughput, full-length allele discovery method utilizing Pacific Biosciences circular consensus sequencing (CCS). We also describe a new approach to Macaque Exome Sequencing (MES) and the development of the Rhexome1.0, an adapted target capture reagent that includes macaque-specific capture probe sets. By using sequence reads generated by whole genome sequencing (WGS) and MES to inform primer design, we were able to increase the sensitivity of KIR allele discovery. We demonstrate this increased sensitivity by defining nine novel alleles within a cohort of Mauritian cynomolgus macaques (MCM), a geographically isolated population with restricted KIR genetics that was thought to be completely characterized. Finally, we describe an approach to genotyping KIRs directly from sequence reads generated using WGS/MES reads. The findings presented here expand our understanding of KIR genetics in MCM by associating new genes with all eight KIR haplotypes and demonstrating the existence of at least one KIR3DS gene associated with every haplotype.

Major histocompatibility complex haplotyping and long-amplicon allele discovery in cynomolgus macaques from Chinese breeding facilities.

Very little is currently known about the major histocompatibility complex (MHC) region of cynomolgus macaques (Macaca fascicularis; Mafa) from Chinese breeding centers. We performed comprehensive MHC class I haplotype analysis of 100 cynomolgus macaques from two different centers, with animals from different reported original geographic origins (Vietnamese, Cambodian, and Cambodian/Indonesian mixed-origin). Many of the samples were of known relation to each other (sire, dam, and progeny sets), making it possible to characterize lineage-level haplotypes in these animals. We identified 52 Mafa-A and 74 Mafa-B haplotypes in this cohort, many of which were restricted to specific sample origins. We also characterized full-length MHC class I transcripts using Pacific Biosciences (PacBio) RS II single-molecule real-time (SMRT) sequencing. This technology allows for complete read-through of unfragmented MHC class I transcripts (~1100 bp in length), so no assembly is required to unambiguously resolve novel full-length sequences. Overall, we identified 311 total full-length transcripts in a subset of 72 cynomolgus macaques from these Chinese breeding facilities; 130 of these sequences were novel and an additional 115 extended existing short database sequences to span the complete open reading frame. This significantly expands the number of Mafa-A, Mafa-B, and Mafa-I full-length alleles in the official cynomolgus macaque MHC class I database. The PacBio technique described here represents a general method for full-length allele discovery and genotyping that can be extended to other complex immune loci such as MHC class II, killer immunoglobulin-like receptors, and Fc gamma receptors.

Inherent Strength of the osteo-WEDGE(™) Bone Plate Locking System for Arthrodesis of the First Metatarsocuneiform Joint: A Biomechanical Study.

First metatarsocuneiform joint arthrodesis with a locking bone plate and screw system has been effectively used to correct metatarsus primus varus and instability of the first ray. The goal of the present cadaveric biomechanical study was to quantify and compare the inherent strength of the first metatarsocuneiform joint and surrounding bones fixated with the osteo-WEDGE(™) bone plate locking system (OW) with that of intact specimens. Fourteen fresh-frozen adult human cadaveric foot specimens consisting of the first metatarsal and medial cuneiform bones with intact joint capsules and ligaments were used. The OW was implanted in 7 of these specimens at the first metatarsal cuneiform joint (MCJ), and the remaining 7 specimens were left intact. Each of the specimens was then subjected to axial force to simulate dorsiflexion of the first metatarsal using a cantilever bending test setup. Load was applied on the plantar aspect of the first metatarsal head until failure of the construct. The mean load and bending moment on the first MCJ at failure for the implanted specimens were 119.98 ± 56.76 N and 5.57 ± 2.71 Nm, respectively. For the intact specimens, the mean load and bending moment on the first MCJ at failure were 107.93 ± 60.90 N and 6.07 ± 3.18 Nm, respectively. None of the specimens showed catastrophic failure within the physiologic loading limits. These results imply that the mechanical strength of the OW is comparable to that of intact specimens. Thus, the first MCJ and surrounding bones fixated with an OW should be able to effectively withstand the vertical ground reaction forces the same as intact specimens.

Congenital talotarsal joint displacement and pes planovalgus: evaluation, conservative management, and surgical management.

The diagnosis of and preferred treatment regimens for pediatric flatfoot, a complex and ambiguous deformity, continues to be debated. Incongruence of the talotarsal joint, whether flexible or rigid, is always present in pes planovalgus. However, it is important to note that talotarsal dislocation can occur without a flatfoot. The displacement of the talus on the hindfoot bones serves as the apex of the deformity. External measures, such as conservative care, are limited in providing correction to this internal deformity. Extraosseous talotarsal stabilization provides a minimally invasive internal option that should be considered before more radical surgical intervention is decided upon.

The epiphytic microbiota of the globally widespread macroalga Cladophora glomerata (Chlorophyta, Cladophorales).

PREMISE OF THE STUDY: The filamentous chlorophyte Cladophora produces abundant nearshore populations in marine and freshwaters worldwide, often dominating periphyton communities and producing nuisance growths under eutrophic conditions. High surface area and environmental persistence foster such high functional and taxonomic diversity of epiphytic microfauna and microalgae that Cladophora has been labeled an ecological engineer. We tested the hypotheses that (1) Cladophora supports a structurally and functionally diverse epiphytic prokaryotic microbiota that influences materials cycling and (2) mutualistic host-microbe interactions occur. Because previous molecular sequencing-based analyses of the microbiota of C. glomerata found as western Lake Michigan beach drift had identified pathogenic associates such as Escherichia coli, we also asked if actively growing lentic C. glomerata harbors known pathogens. METHODS: We used 16S rRNA gene amplicon pyrosequencing to examine the microbiota of C. glomerata of Lake Mendota, Dane, Wisconsin, United States, during the growing season of 2011, at the genus- or species-level to infer functional phenotypes. We used correlative scanning electron and fluorescence microscopy to describe major prokaryotic morphotypes. KEY RESULTS: We found microscopic evidence for diverse bacterial morphotypes, and molecular evidence for ca. 100 distinct sequence types classifiable to genus at the 80% confidence level or species at the 96-97% level within nine bacterial phyla, but not E. coli or related human pathogens. CONCLUSIONS: We inferred that bacterial epiphytes of lentic C. glomerata have diverse functions in materials cycling, with traits that indicate the occurrence of mutualistic interactions with the algal host.

Extraosseous talotarsal stabilization devices: a new classification system.

Displacement of the articular facets of talus on the tarsal mechanism, or partial talotarsal dislocation, is a condition seen in children, adult, and geriatric populations. A characteristic of this pathologic condition is a prolonged period of and excessive amount of pronation (hyperpronation) on weightbearing. The ill effects of this condition may lead to a multitude of other foot pathologies and to pathologies associated with the proximal lower extremity musculoskeletal structures. A variety of conservative and operative treatment options have been used to eliminate or minimize hyperpronation. Extraosseous talotarsal stabilization (EOTTS) devices have been used to realign and stabilize the articular facets of the talus on the tarsal mechanism, thereby attempting to restore the normal range of hindfoot motion while eliminating hyperpronation. A multitude of such devices, which are intended for the same purpose, are available for the surgeon to choose from. However, there is no literature discussing the differences among these devices, or the benefits of one device over the other. Based on current understanding and available knowledge base, the goal of this article was to classify EOTTS devices based on their design features and biomechanical functioning. A theoretical description of how these different types of devices function is laid out in an attempt to understand the reason for their success or failure. This new classification system is intended to help researchers and surgeons appreciate the subtle yet important differences among these devices, and to thus help them design future research studies when using these devices.

Surgical treatment of hyperpronation using an extraosseous talotarsal stabilization device: radiographic outcomes in 70 adult patients.

The purpose of this study was to determine radiographic correction achieved in adult patients treated with an extraosseous talotarsal stabilization (EOTTS) procedure. Patients diagnosed with flexible/reducible talotarsal joint dislocation (partial) underwent surgical correction with the HyProCure(®) EOTTS device. Preoperative and postoperative weightbearing radiographs taken in the anteroposterior (AP) and lateral views for a total 95 feet (in 70 patients) were analyzed to determine standardized radiographic angles, and to quantify the correction obtained after the EOTTS procedure. Postoperative radiographs were taken at an average follow-up of 17 days from the surgery date. The mean preoperative and postoperative talar second metatarsal angles (measured from the AP radiographs) were 24.8° ± 1.0° and 5.8° ± 0.9°, respectively, that is, mean decrease by 19°. The mean preoperative and postoperative talar declination angles (measured from the lateral radiographs) were 25.1° ± 0.7° and 19.4° ± 0.5°, respectively, that is, mean decrease by 5.7°. The mean preoperative and postoperative calcaneal inclination angles (measured from the lateral radiographs) were 21° ± 0.7° and 21.8° ± 0.7°, respectively, that is, mean increase by 0.8°. Postoperatively, the talar second metatarsal and talar declination angles were reduced to average values reported in the literature for normal feet. This study shows the efficacy of a minimally invasive EOTTS procedure in restoring the normal angular relationships between hindfoot and forefoot osseous structures on weightbearing, in both the transverse and sagittal planes. This indicates stabilization of the talotarsal joint complex and elimination of hyperpronation, which may lead to reduced pain, improved foot functional abilities, and patient satisfaction.

Extraosseous talotarsal stabilization using HyProCure® in adults: a 5-year retrospective follow-up.

The purpose of this retrospective study was to determine long-term functional outcomes and device tolerance achieved in adult patients who chose to undergo an extraosseous talotarsal stabilization procedure HyProCure(®) for the treatment of flexible talotarsal joint deformity. Eighty-three adult patients participated in this study. Postoperative subjective assessment of device performance was evaluated using Maryland Foot Scores, which were collected at a mean follow-up period of 51 months. The mean postoperative Maryland Foot Score was 88 out of 100; postoperatively, 52% of cases reported complete alleviation of foot pain, 69% of cases had no limitations on their foot functional abilities, and 80% of cases reported complete satisfaction with the appearance of their feet. The implant was removed in 7 out of 117 cases (removal rate: 6%) due to prolonged pain of the anterior talofibular ligament (4 cases), psychogenic reaction (2 cases), and postoperative infection (1 case). The long-term positive subjective outcomes and excellent patient satisfaction obtained in this study may imply that extraosseous talotarsal stabilization was effective in stabilizing the talotarsal joint complex and eliminating excessive abnormal pronation, thus reducing pain and improving quality of life of the patients; it represents a possible treatment option for partial talotarsal dislocation in cases with flexible and reducible deformity.

Validation of the talar-second metatarsal angle as a standard measurement for radiographic evaluation.

BACKGROUND: Radiographs provide valuable information for assessing osseous foot deformities and aid in accurate diagnosis. The radiographic angular measurements can be used to establish a relationship between the forefoot and the hindfoot that will present valuable information about normal versus pathologic alignment of the foot. The talar-first metatarsal (T1M) angle is frequently used as one of these angles in this capacity; however, there are limitations to the anteroposterior T1M angle. We present a more consistent, reproducible, and accurate measurement for determining foot abnormalities in the transverse plane using the T2M angle instead of the T1M angle. METHODS: Seventy feet in 35 participants (12 men and 23 women) were considered for this study. Individuals were selected on the basis of the established inclusion and exclusion criteria. Anteroposterior radiographs were taken in the angle and base of gait, the neutral calcaneal stance position (NCSP), and the resting calcaneal stance position (RCSP). Three observers measured these angles using three different methods. RESULTS: The mean ± SD T2M angle was 2.95° ± 7.16° in NCSP and 18.61° ± 7.21° in RCSP. No significant differences were found among the measurements made by the three observers using slightly varying procedures in NCSP and RCSP (P > .05). The intraclass correlation coefficients among the measurements were 0.905 in NCSP and 0.937 in RCSP. Bland-Altman plots showed very good agreement between the measurements made by the three observers. CONCLUSIONS: The anteroposterior T2M angle gives a consistent and reproducible measurement that provides accurate information about foot alignment.

Evaluating plantar fascia strain in hyperpronating cadaveric feet following an extra-osseous talotarsal stabilization procedure.

Abnormal talotarsal joint mechanics leading to hyperpronation is implicated as one of the most common causes of plantar fasciopathy. In patients with hyperpronating feet, the plantar fascia experiences excessive tensile forces during static and dynamic weight-bearing activities because of excessive medial longitudinal arch depression. For the purposes of this study, we hypothesized that plantar fascia strain in hyperpronating cadaveric feet would decrease after intervention with an extra-osseous talotarsal stabilization (EOTTS) device. A miniature differential variable reluctance transducer was used to quantify the plantar fascia strain in 6 fresh-frozen cadaver foot specimens exhibiting flexible instability of the talotarsal joint complex (i.e., hyperpronation). The strain was measured as the foot was moved from its neutral to maximally pronated position, before and after intervention using the HyProCure(®) EOTTS device. The mean plantar fascia elongation was 0.83 ± 0.27 mm (strain 3.62% ± 1.17%) and 0.56 ± 0.2 mm (strain 2.42% ± 0.88%) before and after intervention, respectively (N = 18, variation reported is ± 1 SD). The average plantar fascia strain decreased by 33%, and the difference was statistically significant with p < .001. From this cadaveric experiment, the reduction in plantar fascia strain suggests that an EOTTS device might be effective in stabilizing the pathologic talotarsal joint complex and the medial longitudinal arch and in eliminating hyperpronation. An EOTTS procedure might offer a possible treatment option for plantar fasciopathy in cases in which the underlying etiology is abnormal talotarsal biomechanics.

Stabilization of joint forces of the subtalar complex via HyProCure sinus tarsi stent.

BACKGROUND: The foot is the foundation of the body. The stability of the osseous hindfoot structure is crucial in bipedal locomotion. The subtalar joint is responsible for conversion of the rotatory forces of the lower extremities and dictates the movements of the midtarsal joints and the forefoot. In a hyperpronated foot, excessive abnormal pronation results in partial to full obliteration of the sinus tarsi. The hypothesis is that the HyProCure device will prevent obliteration of the sinus tarsi, thereby stabilizing the subtalar joint complex and eliminating the excessive forces to the proximal and distal musculoskeletal system that occur with every step. METHODS: Five fresh-frozen human foot cadaver specimens that represented pathologic hindfoot and midfoot instability and partial to full obliteration of the sinus tarsi were axially compressed to 2,000 N and internally rotated by 15° to produce maximum pronation of the subtalar joint using a biaxial materials testing machine. The forces were measured across the posterior and anterior talocalcaneal joint facets before and after placement of the HyProCure stent. RESULTS: The subtalar joint stabilized after placement of the HyProCure sinus tarsi stent. The mean ± 1 SD forces in the posterior talocalcaneal joint increased from 795.88 ± 106 N to 1,004.86 ± 72.41 N (P < 0.05, two-tailed paired t test). The mean ± SD forces at the anterior talocalcaneal joint decreased from 520.15 ± 127.18 N to 394.56 ± 73.83 N (P < 0.05), shifting the contact area posteriorly. CONCLUSIONS: Placement of the HyProCure subtalar stabilization stent in a hyperpronated foot prevents excessive talar subluxation and assists in proper distribution of the axial loads on the subtalar facet joints.

Effect of extra-osseous talotarsal stabilization on posterior tibial nerve strain in hyperpronating feet: a cadaveric evaluation.

Excessive abnormal strain or tension on the posterior tibial nerve in feet exhibiting talotarsal instability has been considered one of the possible etiologic factors of tarsal tunnel syndrome. The suggested treatment options in such cases include stabilization of the talotarsal joint complex in a corrected position, which might help minimize the abnormal forces placed on the posterior tibial nerve due to over stretching. The primary goal of this study was to quantify strain on the posterior tibial nerve in feet exhibiting hyperpronation caused by talotarsal instability, before and after an extra-osseous talotarsal stabilization (EOTTS) procedure. We hypothesized that the excessive strain placed on the posterior tibial nerve in hyperpronating cadaveric feet would be reduced significantly after intervention using the HyProCure(®) EOTTS device. Posterior tibial nerve strain was quantified in 9 fresh-frozen cadaver specimens. A miniature differential variable reluctance transducer was used to measure nerve elongation as the foot was moved from its neutral to a maximally pronated position, before and after intervention. The mean elongation of the posterior tibial nerve (with respect to a fixed reference point) decreased by 43% after the EOTTS procedure (i.e., from 5.91 ± 0.91 mm to 3.38 ± 1.20 mm; N = 27). The reduction was statistically significant at p < .001. HyProCure(®) was effective in stabilizing the talotarsal joint complex, thus reducing the excessive amount of strain placed on the posterior tibial nerve. Clinical implications of this study suggest the use of EOTTS devices in the treatment of tarsal tunnel syndrome.