Association between a genetic index for digital dermatitis resistance and the presence of digital dermatitis, heel horn erosion, and interdigital hyperplasia in Holstein cows.

Digital dermatitis (DD) is a polybacterial disease endemic to most UK dairy farms. It poses a major financial and welfare threat and is characterized by high incidence and recurrence rates. We aimed to investigate the association between the UK EBV for resistance to digital dermatitis, the digital dermatitis index (DDI), and the frequency of DD, heel horn erosion (HHE), and interdigital hyperplasia (IH) in a population of Holstein dairy cows. We enrolled and genotyped 2,352 cows from 4 farms in a prospective cohort study. Foot lesion records were recorded by veterinary surgeons for each animal at 4 time points during a production cycle, starting at approximately 2 mo before calving and ending in late lactation. Importantly, these records were not used in the calculation of the DDI. Lesion records were matched to the animal's own DDI (n = 2,101) and their sire's DDI (n = 1,812). Digital dermatitis index values in our study population ranged from -1.41 to +1.2 and were transformed to represent distance from the mean expressed in SD. The relationship between the DDI and the presence of DD was investigated using a logistic regression model, with farm, parity, and a farm-parity interaction fitted as covariates. A multivariable logistic regression model was fitted to evaluate the relationship between HHE and DDI with farm fitted as a covariate. Finally, a univariable logistic regression model with DDI as explanatory variable was used to investigate the relationship between IH and DDI. The odds ratio of an animal being affected by DD was 0.69 for 1 SD increase in the animal's DDI (95% CI = 0.63-0.76). The odds of HHE and IH were 0.69 (95% CI = 0.62-0.76) and 0.58 (95% CI = 0.49-0.68) respectively for 1 SD increase in DDI. The adjusted probability of DD was 32% (95% CI = 27-36%) for cows with mean DDI value of 0, while it was 24% (95% CI = 20-29%) in cows with a DDI value of +1. Sire DDI breeding values were standardized in the same way and then binned into terciles creating an ordinal variable representing bulls of high, medium, and low genetic merit for DD resistance. The daughters of low genetic merit bulls were at 2.05 (95% CI = 1.60-2.64), 1.96 (95% CI = 1.53-2.50), and 2.85 (95% CI = 1.64-5.16) times greater odds of being affected by DD, HHE, and IH, respectively, compared with the daughters of high genetic merit bulls. The results of this study highlight the potential of digital dermatitis genetic indexes to aid herd management of DD, and suggest that breeding for resistance to DD, alongside environmental and management control practices, could reduce the prevalence of the disease.

Genome-wide association and functional genomic analyses for various hoof health traits in North American Holstein cattle.

Hoof diseases are a major welfare and economic issue in the global dairy cattle production industry, which can be minimized through improved management and breeding practices. Optimal genetic improvement of hoof health could benefit from a deep understanding of the genetic background and biological underpinning of indicators of hoof health. Therefore, the primary objectives of this study were to perform genome-wide association studies, using imputed high-density genetic markers data from North American Holstein cattle, for 8 hoof-related traits: digital dermatitis, sole ulcer, sole hemorrhage, white line lesion, heel horn erosion, interdigital dermatitis, interdigital hyperplasia, and toe ulcer, and a hoof health index. De-regressed estimated breeding values from 25,580 Holstein animals were used as pseudo-phenotypes for the association analyses. The genomic quality control, genotype phasing, and genotype imputation were performed using the PLINK (version 1.9), Eagle (version 2.4.1), and Minimac4 software, respectively. The functional genomic analyses were performed using the GALLO R package and the DAVID platform. We identified 22, 34, 14, 22, 28, 33, 24, 43, and 15 significant markers for digital dermatitis, heel horn erosion, interdigital dermatitis, interdigital hyperplasia, sole hemorrhage, sole ulcer, toe ulcer, white line lesion disease, and the hoof health index, respectively. The significant markers were located across all autosomes, except BTA10, BTA12, BTA20, BTA26, BTA27, and BTA28. Moreover, the genomic regions identified overlap with various previously reported quantitative trait loci for exterior, health, meat and carcass, milk, production, and reproduction traits. The enrichment analyses identified 44 significant gene ontology terms. These enriched genomic regions harbor various candidate genes previously associated with bone development, metabolism, and infectious and immunological diseases. These findings indicate that hoof health traits are highly polygenic and influenced by a wide range of biological processes.

Genome-wide association study between copy number variants and hoof health traits in Holstein dairy cattle.

Genome-wide association studies based on SNP have been completed for multiple traits in dairy cattle; however, copy number variants (CNV) could add genomic information that has yet to be harnessed. The objectives of this study were to identify CNV in genotyped Holstein animals and assess their association with hoof health traits using deregressed estimated breeding values as pseudophenotypes. A total of 23,256 CNV comprising 1,645 genomic regions were identified in 5,845 animals. Fourteen genomic regions harboring structural variations, including 9 deletions and 5 duplications, were associated with at least 1 of the studied hoof health traits. This group of traits included digital dermatitis, interdigital dermatitis, heel horn erosion, sole ulcer, white line lesion, sole hemorrhage, and interdigital hyperplasia; no regions were associated with toe ulcer. Twenty candidate genes overlapped with the regions associated with these traits including SCART1, NRXN2, KIF26A, GPHN, and OR7A17. In this study, an effect on infectious hoof lesions could be attributed to the PRAME (Preferentially Expressed Antigen in Melanoma) gene. Almost all genes detected in association with noninfectious hoof lesions could be linked to known metabolic disorders. The knowledge obtained considering information of associated CNV to the traits of interest in this study could improve the accuracy of estimated breeding values. This may further increase the genetic gain for these traits in the Canadian Holstein population, thus reducing the involuntary animal losses due to lameness.

Paraneoplastic Secretion of Multiple Phosphatonins From a Deep Fibrous Histiocytoma Causing Oncogenic Osteomalacia.

CONTEXT: Literature suggests that oncogenic osteomalacia is usually caused by a benign mesenchymal tumor secreting fibroblast growth factor subtype-23 (FGF-23), but the involvement of other phosphatonins has only been scarcely reported. We have previously published a seemingly typical case of oncogenic osteomalacia. Following curative neoplasm resection, we now report unique molecular characteristics and biology of this tumor. CASE DESCRIPTION: A 25-year-old man had been diagnosed with severe oncogenic osteomalacia that gradually crippled him over 6 years. 68Ga-DOTA-TATE positron emission tomography/computed tomography scan localized the culprit tumor to his left sole, which on resection revealed a deep fibrous histiocytoma displaying a proliferation of spindle cells with storiform pattern associated with multinucleated giant cells resembling osteoclasts. Circulating FGF-23, which was elevated more than 2-fold, declined to undetectable levels 24 h after surgery. Microarray analysis revealed increased tumor gene expression of the phosphatonins FGF-23, matrix extracellular phosphoglycoprotein (MEPE) and secreted frizzled-related protein subtype 4, with elevated levels of all 3 proteins confirmed through immunoblot analysis. Differential expression of genes involved in bone formation and bone mineralization were further identified. The patient made an astonishing recovery from being wheelchair bound to fully self-ambulant 2 months postoperatively. CONCLUSION: This report describes oncogenic osteomalacia due to a deep fibrous histiocytoma, which coincidentally has been found to induce profound muscle weakness via the overexpression of 3 phosphatonins, which resolved fully upon radical resection of the tumor. Additionally, genes involved in bone formation and bone remodeling contribute to the molecular signature of oncogenic osteomalacia.

Pediatric fibromyxoid soft tissue tumor with PLAG1 fusion: A novel entity?

The classification of undifferentiated soft tissue tumors continues to evolve with the expanded application of molecular analysis in clinical practice. We report three cases of a unique soft tissue tumor in young children (5 months to 2 years old) displaying a purely fibromyxoid histology, with positive staining for desmin and CD34. In two cases, RNA sequencing detected a YWHAZ-PLAG1 gene fusion, while in the third case, a previously unreported EEF1A1-PLAG1 fusion was identified. PLAG1 fusions have been reported in several pathologic entities including pleomorphic adenoma, myoepithelial tumors of skin and soft tissue, and lipoblastoma, the latter occurring preferentially in young children. In these tumors, expression of a full length PLAG1 protein comes under the control of the constitutively active promoter of the partner gene in the fusion, and the current cases conform to that model. Overexpression of PLAG1 was confirmed by diffusely positive immunostaining for PLAG1 in all three cases. Our findings raise the possibility of a novel fibromyxoid neoplasm in childhood associated with these rare PLAG1 fusion variants. The only other report of a PLAG1-YWHAZ fusion occurred in a pediatric tumor diagnosed as a "fibroblastic lipoblastoma." This finding raises the possibility of a relationship with our three cases, even though our cases lacked any fat component. Further studies with regard to a shared pathogenesis are required.

Congenital spindle cell rhabdomyosarcoma.

Spindle cell and sclerosing rhabdomyosarcoma (ssRMS) is a rare variant of rhabdomyosarcoma, which includes three distinct subtypes. In infants, these tumors are commonly associated with recurring fusions involving VGLL2 or NCOA2 and have a favorable prognosis. We present four cases of ssRMS and 16 additional cases from the literature, which show that these patients present with localized disease and have an excellent prognosis regardless of surgical margin or lack of radiation therapy. Molecularly defined spindle cell rhabdomyosarcoma in infants is likely a biologically distinct entity which may not require the aggressive multimodal treatment used for other subtypes of rhabdomyosarcoma.

Genomic analysis of claw lesions in Holstein cows: Opportunities for genomic selection, quantitative trait locus detection, and gene identification.

Claw lesions are the third most important health issue in dairy cattle, after mastitis and reproductive disorders, and genomic selection is a key component for long-term improvement of claw health. The objectives of this study were to assess the feasibility of a genomic evaluation for claw health in French Holstein cows, explore possibilities to increase evaluation accuracy, and gain a better understanding of the genetic determinism of claw health traits. The data set consisted of 48,685 trimmed Holstein cows, including 9,646 that were genotyped; 478 genotyped sires were also used. Seven claw lesion traits were evaluated using BLUP, genomic BLUP, BayesC, and single-step genomic BLUP, and the accuracies obtained using these approaches were measured through a validation study. The BayesC approach was used to detect quantitative trait locus (QTL) regions associated with the 7 individual traits (digital dermatitis, heel horn erosion, interdigital hyperplasia, sole hemorrhage circumscribed, sole hemorrhage diffused, sole ulcer, and white line fissure) based on their Bayes factor. Annotated genes on these regions were reported. Genomic evaluation approaches generally did not allow for greater accuracies than BLUP, except for single-step genomic BLUP. Accuracies were moderate, but best and worst validation animals were correctly discriminated and showed significant differences in lesion frequencies. A total of 192 QTL regions were identified, including 13 with major evidence or involved for 2 of the traits. A high number of genes were present on these regions, and several had functions associated with the immune system. In particular, the EPYC gene is located close to a major evidence QTL for resistance to digital dermatitis that is also a QTL for interdigital hyperplasia (on chromosome 5, around 20.9 MB) and has been associated with Ehlers-Danlos syndrome in cattle. Genomic selection can be used to improve resistance to individual claw lesions, and several possibilities exist to improve accuracies of genomic evaluations.

BAP1: case report and insight into a novel tumor suppressor.

BACKGROUND: BRCA1-Associated-Protein 1 (BAP1) is a dynamic tumor suppressor which, when mutated, has been associated with an increased risk of uveal melanoma, cutaneous melanoma, mesothelioma, and several other cancers. Germline BAP1 mutations have been extensively studied, where they have been found to cause hereditary cancer susceptibility. However, their sporadic counterparts, tumors that display a loss of BAP1 expression due to somatically arising mutations in the BAP1 gene, remain a poorly described entity. CASE PRESENTATION: Here we present the case of a 49-year-old female who presented with an asymptomatic dome-shaped pink papule on the dorsal foot which was found on biopsy to be deficient in the BAP1 tumor suppressor. While the patient's family history did not suggest the presence of a familial cancer syndrome, germline genetic testing was performed and was negative. The patient underwent surgical excision of this sporadically appearing "BAPoma" by Mohs surgery. CONCLUSIONS: Given the relatively banal clinical appearance of these dome-shaped neoplasms, sporadic BAPomas may often be overlooked by clinicians and dermatologists. In addition to providing a representative case, here we also provide a synopsis of the current understanding of these neoplasms, both in terms of the histopathological features, as well as the molecular mechanisms underlying BAP1 function and its ability to prevent tumorigenesis.

Acral melanoma foot lesions. Part 1: epidemiology, aetiology, and molecular pathology.

Acral melanoma (AM) is a rare subtype of cutaneous malignant melanoma (MM) found on acral skin, primarily on the soles of the feet. Although rare, it is the most common subtype of MM found in patients of African or East Asian ethnicity and has a poor prognosis, often because of the more advanced stage of presentation at diagnosis. The pathogenesis of AM is unclear, but genetic alterations, including mutations in BRAF, NRAS, and KIT have been implicated. Early diagnosis of AM is important for a better prognosis, but its identification is often challenging, leading to easy misdiagnosis. In the first of this two-part review, we review the history, epidemiology, aetiology and molecular pathology of AM; in part 2 we will review diagnosis and management.

Mutational profiling of acral melanomas in Korean populations.

The proportion of acral melanoma (AM) is much higher in Asian populations than in Caucasian populations. Although mutational profiles associated with AM have been discovered in Caucasian populations, knowledge of its genetic alterations in Asian populations is limited. To describe the molecular nature of AM in Korean patients, we performed mutational profiling of AM and matched normal tissues in patients. Fifty-one formalin-fixed paraffin-embedded AM samples and 32 matched pairs from patients' saliva DNA were analysed by next-generation sequencing. Only mutations confirmed via digital droplet PCR or in BRAF, KIT and NRAS, the most frequently altered cancer genes in cutaneous melanoma, were considered as positive. The relationship between mutational status and clinicopathological features were examined. Of the 47 AM patients screened, alteration of BRAF, NRAS and KIT genes was observed in 6.4%, 4.3% and 8.5%, respectively. We also tested matched normal tissues of patients to identify tumor-specific mutations. Examination of the mutational profile in a cohort of 28 primary melanomas and matched normal controls found BRAF mutations in two cases (7.1%), KIT mutations in three cases (10.7%) and CTNNB1 mutations in one case (3.6%). The BRAF, NRAS and KIT mutation status did not correlate with clinicopathological characteristics. Our results show that KIT, NRAS and BRAF hotspot mutations occur at a low frequency in Korean populations. We also observed a case with the CTNNB1 mutation, which raises the possibility that other pathways are associated with AM development.

Malignant melanoma of sun-protected sites: a review of clinical, histological, and molecular features.

In most cases of cutaneous melanoma, ultraviolet (UV) radiation is recognized as a prominent risk factor. Less is known regarding the mechanisms of mutagenesis for melanoma arising in sun-protected sites, such as acral and mucosal melanoma. Acral and mucosal melanoma share many common features, including a late age of onset, a broad radial growth phase with prominent lentiginous growth, the presence of field cancerization cells, and, in most cases, lack of a precursor nevus. In addition to early chromosomal instability, many of the same genes are also involved in these two distinct melanoma subtypes. To better understand non-UV-mediated pathogenesis in melanoma, we conducted a joint literature review of clinical, histological, and molecular features in acral and mucosal melanoma. We also reviewed the current literature regarding aberrations in KIT, PDGFRA, TERT, and other commonly involved genes. By comparing common features of these two subtypes, we suggest potential mechanisms underlying acral and/or mucosal melanoma and offer direction for future investigations.

Association analysis for feet and legs disorders with whole-genome sequence variants in 3 dairy cattle breeds.

Identification of genetic variants associated with feet and legs disorders (FLD) will aid in the genetic improvement of these traits by providing knowledge on genes that influence trait variations. In Denmark, FLD in cattle has been recorded since the 1990s. In this report, we used deregressed breeding values as response variables for a genome-wide association study. Bulls (5,334 Danish Holstein, 4,237 Nordic Red Dairy Cattle, and 1,180 Danish Jersey) with deregressed estimated breeding values were genotyped with the Illumina Bovine 54k single nucleotide polymorphism (SNP) genotyping array. Genotypes were imputed to whole-genome sequence variants, and then 22,751,039 SNP on 29 autosomes were used for an association analysis. A modified linear mixed-model approach (efficient mixed-model association eXpedited, EMMAX) and a linear mixed model were used for association analysis. We identified 5 (3,854 SNP), 3 (13,642 SNP), and 0 quantitative trait locus (QTL) regions associated with the FLD index in Danish Holstein, Nordic Red Dairy Cattle, and Danish Jersey populations, respectively. We did not identify any QTL that were common among the 3 breeds. In a meta-analysis of the 3 breeds, 4 QTL regions were significant, but no additional QTL region was identified compared with within-breed analyses. Comparison between top SNP locations within these QTL regions and known genes suggested that RASGRP1, LCORL, MOS, and MITF may be candidate genes for FLD in dairy cattle.

Ramps and hybrid effects on keel bone and foot pad disorders in modified aviaries for laying hens.

Non-cage systems provide laying hens with considerable space allowance, perches and access to litter, thereby offering opportunities for natural species-specific behaviors. Conversely, these typical characteristics of non-cage systems also increase the risk of keel bone and foot pad disorders. The aim of this study was twofold: 1) to investigate if providing ramps between perches (housing factor) reduces keel bone and foot pad disorders and 2) to test for genetic predisposition by comparing 2 different layer hybrids. In a 2 × 2 design, 16 pens were equipped either with or without ramps between perches and nest boxes (8 pens/treatment), and housed with either 25 ISA Brown or Dekalb White birds per pen (in total 200 birds/hybrid). Keel bone injuries and foot health were repeatedly measured via palpation and visual assessment between 17 and 52 wk of age and daily egg production was recorded. The relationships between the dependent response variables (keel bone and footpad disorders, egg production) and independent factors (age, ramps, hybrid) were analyzed using generalized linear mixed models and corrected for repeated measures. Ramps reduced keel bone fractures (F1,950 = 45.80, P < 0.001), foot pad hyperkeratosis (F1,889 = 10.40, P = 0.001), foot pad dermatitis (F1,792 = 20.48, P < 0.001) and bumble foot (F1,395 = 8.52, P < 0.001) compared to pens without ramps. ISA Brown birds sustained more keel bone fractures (F1,950 = 33.26, P < 0.001), had more foot pad hyperkeratosis (F1,889 = 44.69, P < 0.001) and laid more floor eggs (F1,1883 = 438.80, P < 0.001), but had fewer keel bone deviations (F1,1473 = 6.73, P < 0.001), fewer cases of foot pad dermatitis (F1,792 = 19.84, P < 0.001) and no bumble foot as compared to Dekalb White birds. Age, housing and hybrid showed several interaction effects. Providing ramps proved to be very effective in both reducing keel bone and foot pad problems in non-cage systems. Keel bone and foot pad disorders are related to genetic predisposition. These results indicate that adaptation of the housing systems and hybrid selection may be effective measures in improving laying hen welfare.

Genome-wide association study for claw disorders and trimming status in dairy cattle.

Performing a genome-wide association study (GWAS) might add to a better understanding of the development of claw disorders and the need for trimming. Therefore, the aim of the current study was to perform a GWAS on claw disorders and trimming status and to validate the results for claw disorders based on an independent data set. Data consisted of 20,474 cows with phenotypes for claw disorders and 50,238 cows with phenotypes for trimming status. Recorded claw disorders used in the current study were double sole (DS), interdigital hyperplasia (IH), sole hemorrhage (SH), sole ulcer (SU), white line separation (WLS), a combination of infectious claw disorders consisting of (inter-)digital dermatitis and heel erosion, and a combination of laminitis-related claw disorders (DS, SH, SU, and WLS). Of the cows with phenotypes for claw disorders, 1,771 cows were genotyped and these cow data were used for the GWAS on claw disorders. A SNP was considered significant when the false discovery rate≤0.05 and suggestive when the false discovery rate≤0.20. An independent data set of 185 genotyped bulls having at least 5 daughters with phenotypes (6,824 daughters in total) for claw disorders was used to validate significant and suggestive SNP detected based on the cow data. To analyze the trait "trimming status" (i.e., the need for claw trimming), a data set with 327 genotyped bulls having at least 5 daughters with phenotypes (18,525 daughters in total) was used. Based on the cow data, in total 10 significant and 45 suggestive SNP were detected for claw disorders. The 10 significant SNP were associated with SU, and mainly located on BTA8. The suggestive SNP were associated with DS, IH, SU, and laminitis-related claw disorders. Three of the suggestive SNP were validated in the data set of 185 bulls, and were located on BTA13, BTA14, and BTA17. For infectious claw disorders, SH, and WLS, no significant or suggestive SNP associations were detected. For trimming status, 1 significant and 1 suggestive SNP were detected, both located close to each other on BTA15. Some significant and suggestive SNP were located close to SNP detected in studies on feet and leg conformation traits. Genes with major effects could not be detected and SNP associations were spread across the genome, indicating that many SNP, each explaining a small proportion of the genetic variance, influence claw disorders. Therefore, to reduce the incidence of claw disorders by breeding, genomic selection is a promising approach.

Dual-color, break-apart fluorescence in situ hybridization probe for distinguishing clear cell sarcoma of soft tissue from malignant melanoma.

BACKGROUND: Clear cell sarcoma (CCS) of soft tissue is a rare soft tissue sarcoma with melanocytic differentiation and shares morphologic, immunohistochemical, ultrastructural, and molecular features with malignant melanoma (MM). Because the prognosis of CCS is much different from MM, it is important to distinguish each other by selective method. CCS is well-recognized as having the t(12;22)(q13;q12) translocation, on the other hand MM is not. Therefore, detecting Ewing sarcoma region 1 (ESWR1) gene rearrangement can serve as a crucial diagnostic determinant. METHODS: Biopsy was taken from a 52-year-old man who reported a 3-year history of a gradually enlarging nodule on the sole of his left foot. Routine and special stains for melanocytic markers and fluorescence in situ hybridization (FISH) evaluation using dual color break-apart rearrangement probes specific for ESWR1 was performed for formalin-fixed tissue. RESULTS: Neoplastic cells expressed diffuse but strong positivity for HMB45 and S100 but not for Melan-A. Dual color, break-apart interphase FISH revealed EWS(22q12) gene rearrangements in CCS tumor cells. CONCLUSION: Fluorescence in situ hybridization evaluation using ESWR1 gene probe for CCS sharing clinical and histopathological characteristics with MM is a valuable tool to distinguish each other.

Multiple primary acral melanomas in two young caucasian patients.

BACKGROUND: Acral melanoma (AM) is still one of the most poorly studied melanomas. It generally presents beyond the fifth decade of life and usually is a BRAF wild-type melanoma. OBJECTIVE: To report the first cases of multiple primary AM in Caucasians. METHODS: Clinical, dermoscopic, pathological and molecular profiles. RESULTS: A healthy 34- year-old male presented an in situ subungual melanoma on his finger, and 22 months later a fast-growing nodular melanoma appeared in an existing nevus on the sole. Both melanomas carried the V600E BRAF mutation. A 19-year-old female patient presented 2 in situ melanomas on different parts of her left foot within a 6-year period of time. The patients have neither familiar melanoma nor germline mutations in CDKN2A/CDK4 genes. CONCLUSION: Multiple AM in Caucasians is very rare. BRAF mutations are possible, especially in a high-risk set of patients with multiple nevi. Specific acral examination must be recommended since AM still suffers delayed detection.

Soft tissue perineurioma of the foot with 10q24 rearrangements: unique MRI features with histopathologic correlation.

Perineurioma is an uncommon benign peripheral nerve sheath tumor with advanced perineurial differentiation. Two distinct subtypes are recognized: intraneural and soft tissue. We herein present a unique case of soft tissue perineurioma in the right foot of a 43-year-old man. Radiographs showed a non-specific soft tissue mass. On computed tomography scan, the mass was iso- to slightly hypodense relative to muscle. On T1- and T2-weighted images, the mass exhibited iso- to slightly low signal intensity relative to muscle with foci of high signal intensity. Slight contrast enhancement was noted on enhanced T1-weighted images with fat suppression. A marginal excision of the tumor was performed and histopathologic examination confirmed the diagnosis of soft tissue perineurioma. The clinicopathologic, radiologic, and cytogenetic findings are described, and the relevant literature is reviewed.

Toll-like receptor and pro-inflammatory cytokine expression during prolonged hyperinsulinaemia in horses: implications for laminitis.

Equine laminitis, a disease of the lamellar structure of the horse's hoof, can be incited by numerous factors that include inflammatory and metabolic aetiologies. However, the role of inflammation in hyperinsulinaemic laminitis has not been adequately defined. Toll-like receptor (TLR) activation results in up-regulation of inflammatory pathways and the release of pro-inflammatory cytokines, including interleukin-6 (IL-6) and tumour necrosis factor-alpha (TNF-α), and may be a pathogenic factor in laminitis. The aim of this study was to determine whether TLR4 expression and subsequent pro-inflammatory cytokine production is increased in lamellae and skeletal muscle during equine hyperinsulinaemia. Standardbred horses were treated with either a prolonged, euglycaemic hyperinsulinaemic clamp (p-EHC) or a prolonged, glucose infusion (p-GI), which induced marked and moderate hyperinsulinaemia, respectively. Age-matched control horses were treated simultaneously with a balanced electrolyte solution. Treated horses developed clinical (p-EHC) or subclinical (p-GI) laminitis, whereas controls did not. Skeletal muscle and lamellar protein extracts were analysed by Western blotting for TLR4, IL-6, TNF-α and suppressor of cytokine signalling 3 (SOCS3) expression. Lamellar protein expression of TLR4 and TNF-α, but not IL-6, was increased by the p-EHC, compared to control horses. A significant positive correlation was found between lamellar TLR4 and SOCS3. Skeletal muscle protein expression of TLR4 signalling parameters did not differ between control and p-EHC-treated horses. Similarly, the p-GI did not result in up-regulation of lamellar protein expression of any parameter. The results suggest that insulin-sensitive tissues may not accurately reflect lamellar pathology during hyperinsulinaemia. While TLR4 is present in the lamellae, its activation appears unlikely to contribute significantly to the developmental pathogenesis of hyperinsulinaemic laminitis. However, inflammation may have a role to play in the later stages (e.g., repair or remodelling) of the disease.