Proline Isomerization and Molten Globular Property of TgPDCD5 Secreted from Toxoplasma gondii Confers Its Regulation of Heparin Sulfate Binding.

Toxoplasmosis, caused by Toxoplasma gondii, poses risks to vulnerable populations. TgPDCD5, a secreted protein of T. gondii, induces apoptosis through heparan sulfate-mediated endocytosis. The entry mechanism of TgPDCD5 has remained elusive. Here, we present the solution structure of TgPDCD5 as a helical bundle with an extended N-terminal helix, exhibiting molten globule characteristics. NMR perturbation studies reveal heparin/heparan sulfate binding involving the heparan sulfate/heparin proteoglycans-binding motif and the core region, influenced by proline isomerization of P107 residue. The heterogeneous proline recruits a cyclophilin TgCyp18, accelerating interconversion between conformers and regulating heparan/heparin binding. These atomic-level insights elucidate the binary switch's functionality, expose novel heparan sulfate-binding surfaces, and illuminate the unconventional cellular entry of pathogenic TgPDCD5.

Screening Implications for Distribution of Colorectal Cancer Subsite by Age and Role of Flexible Sigmoidoscopy.

Objectives: The effectiveness of colonoscopy to reduce colorectal cancer (CRC) mortality is extrapolated from cohort studies in the absence of randomized controlled trial (RCT) data, whereas flexible sigmoidoscopy is supported by RCT data and may be easier to implement in practice. We characterized the anatomic distribution of CRC to determine the proportion that is visible with sigmoidoscopy. Methods: Patients with a primary diagnosis of colorectal adenocarcinoma were identified in the Surveillance, Epidemiology, and End Results program (2000-2020). Tumors from the rectum to the descending colon were categorized as visible by sigmoidoscopy, whereas more proximal tumors required colonoscopy. Differential prognosis between tumor locations, stratified by age groups and stage, was assessed using the overall restricted mean survival time (RMST) at 2, 5, and 10 years. Results: Among 309,466 patients, 58% had tumors visible by sigmoidoscopy, including 73% of those under age 50 (OR 2.10, 95% CI 2.03-2.16 age < 45, OR 2.20, 95% CI 2.13-2.27 age 45-49 versus age ≥ 50). Male sex (OR 1.54, 95% CI 1.51-1.56) and Asian or Pacific Islander race (OR 1.60, 95% CI 1.56-1.64) were also positively associated with tumors visualizable by sigmoidoscopy. Across age groups, for local disease, RMST was comparable for tumors visible versus not visible on sigmoidoscopy. For regional and metastatic cancer, patients with tumors visible by sigmoidoscopy had improved RMST versus those with more proximal tumors. Conclusions: 58% of CRC arises in locations visible by flexible sigmoidoscopy. Flexible sigmoidoscopy should be considered as a viable option for CRC screening, particularly in younger patients unwilling or unable to undergo colonoscopy.

Targeting CD47-SIRPa axis shows potent preclinical anti-tumor activity as monotherapy and synergizes with PARP inhibition.

The objective was to correlate CD47 gene expression with resistance to immune checkpoint inhibitors (ICI) in tumor tissue of gynecological cancer (GC). Further, we sought to assess the efficacy of targeting CD47 pathway alone and in combination in pre-clinical ovarian cancer (OC) models. We performed transcriptomic analyses in GC treated with ICI. Signaling pathway enrichment analysis was performed using Ingenuity Pathway Analysis. Immune cell abundance was estimated. CD47 expression was correlated with other pathways, objective response, and progression-free survival (PFS). Anti-tumor efficacy of anti-CD47 therapy alone and in combination was investigated both in-vitro and in-vivo using cell-line derived xenograft (CDX) and patient-derived xenograft (PDX) models. High CD47 expression associated with lower response to ICI and trended toward lower PFS in GC patients. Higher CD47 associated negatively with PDL1 and CTLA4 expression, as well as cytotoxic T-cells and dendritic cells but positively with TGF-ß, BRD4 and CXCR4/CXCL12 expression. Anti-CD47 significantly enhanced macrophage-mediated phagocytosis of OC cells in-vitro and exhibited potent anti-tumor activity in-vivo in OC CDX and PDX models. In-vitro treatment with PARPi increased CD47 expression. Anti-CD47 led to significantly enhanced in-vitro phagocytosis, enhanced STING pathway and synergized in-vivo when combined with PARP inhibitors in BRCA-deficient OC models. This study provides insight on the potential role of CD47 in mediating immunotherapy resistance and its association with higher TGF-ß, BRD4 and CXCR4/CXCL12 expression. Anti-CD47 showed potent anti-tumor activity and synergized with PARPi in OC models. These data support clinical development of anti-CD47 therapy with PARPi in OC.

Screening Implications for Distribution of Colorectal Cancer Subsite by Age.

Objectives: The effectiveness of colonoscopy to reduce colorectal cancer (CRC) mortality is extrapolated from cohort studies in the absence of randomized controlled trial (RCT) data, whereas flexible sigmoidoscopy is supported by RCT data and may be easier to implement in practice. We characterized the anatomic distribution of CRC to determine the proportion that is visible with sigmoidoscopy. Methods: Patients with a primary diagnosis of colorectal adenocarcinoma were identified in the Surveillance, Epidemiology, and End Results program (2000-2020). Tumors from the rectum to descending colon were categorized as visible by sigmoidoscopy, whereas more proximal tumors as requiring colonoscopy. Differential prognosis between tumor locations, stratified by age groups and stage, were assessed using overall restricted mean survival time (RMST) at 2, 5, and 10 years. Results: Among 309,466 patients, 58% had tumors visible by sigmoidoscopy, including 73% of those under age 50 (OR 2.10, 95%CI 2.03-2.16 age <45, OR 2.20, 95%CI 2.13-2.27 age 45-49 versus age > 50). Male sex (OR 1.54, 95%CI 1.51-1.56) and Asian or Pacific Islander race (OR 1.60, 95%CI 1.56-1.64) were also positively associated with tumors visualizable by sigmoidoscopy. Across age groups, for local disease, RMST was comparable for tumors visible versus not visible on sigmoidoscopy. For regional and metastatic cancer, patients with tumors visible by sigmoidoscopy had improved RMST versus those with more proximal tumors. Conclusions: Most CRC arise in locations visible by flexible sigmoidoscopy. Flexible sigmoidoscopy should be considered as a viable option for CRC screening, particularly in younger patients unwilling or unable to undergo colonoscopy.

Structural and regulatory insights into the glideosome-associated connector from Toxoplasma gondii.

The phylum of Apicomplexa groups intracellular parasites that employ substrate-dependent gliding motility to invade host cells, egress from the infected cells, and cross biological barriers. The glideosome-associated connector (GAC) is a conserved protein essential to this process. GAC facilitates the association of actin filaments with surface transmembrane adhesins and the efficient transmission of the force generated by myosin translocation of actin to the cell surface substrate. Here, we present the crystal structure of Toxoplasma gondii GAC and reveal a unique, supercoiled armadillo repeat region that adopts a closed ring conformation. Characterisation of the solution properties together with membrane and F-actin binding interfaces suggests that GAC adopts several conformations from closed to open and extended. A multi-conformational model for assembly and regulation of GAC within the glideosome is proposed.

Viral vector gene delivery of the novel chaperone protein SRCP1 to modify insoluble protein in in vitro and in vivo models of ALS.

Protein misfolding and aggregation are shared features of neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), and protein quality control disruption contributes to neuronal toxicity. Therefore, reducing protein aggregation could hold therapeutic potential. We previously identified a novel chaperone protein, serine-rich chaperone protein 1 (SRCP1), that effectively prevents protein aggregation in cell culture and zebrafish models of Huntington's disease. Here we tested whether this benefit extends to aggregated proteins found in ALS. We used viral-mediated expression of SRCP1 in in vitro and in vivo models of ALS. We found that SRCP1 reduced insoluble SOD1 protein levels in HEK293T cells overexpressing either the A4V or G93R mutant SOD1. However, the reduction of insoluble protein was not observed in either mutant C9orf72 or SOD1 ALS iPSC-derived motor neurons infected with a lentivirus expressing SRCP1. SOD1-G93A ALS mice injected with AAV-SRCP1 showed a small but significant reduction in insoluble and soluble SOD1 in both the brain and spinal cord, but SRCP1 expression did not improve mouse survival. These data indicate that SRCP1 likely reduces insoluble protein burden in a protein and/or context-dependent manner indicating a need for additional insight into SRCP1 function and therapeutic potential.

Dose-dependent effects of enzyme replacement therapy on skeletal disease progression in mucopolysaccharidosis VII dogs.

Mucopolysaccharidosis (MPS) VII is an inherited lysosomal storage disorder characterized by deficient activity of the enzyme ß-glucuronidase. Skeletal abnormalities are common in patients and result in diminished quality of life. Enzyme replacement therapy (ERT) for MPS VII using recombinant human ß-glucuronidase (vestronidase alfa) was recently approved for use in patients; however, to date there have been no studies evaluating therapeutic efficacy in a large animal model of MPS VII. The objective of this study was to establish the effects of intravenous ERT, administered at either the standard clinical dose (4 mg/kg) or a high dose (20 mg/kg), on skeletal disease progression in MPS VII using the naturally occurring canine model. Untreated MPS VII animals exhibited progressive synovial joint and vertebral bone disease and were no longer ambulatory by age 6 months. Standard-dose ERT-treated animals exhibited modest attenuation of joint disease, but by age 6 months were no longer ambulatory. High-dose ERT-treated animals exhibited marked attenuation of joint disease, and all were still ambulatory by age 6 months. Vertebral bone disease was recalcitrant to ERT irrespective of dose. Overall, our findings indicate that ERT administered at higher doses results in significantly improved skeletal disease outcomes in MPS VII dogs.

Engineered multivalent DNA capsules for multiplexed detection of genotoxicants via versatile controlled release mechanisms.

Assessing the risks associated with genotoxic compounds is challenging because of their complex genotoxicity and the difficulty in the dynamic monitoring of coexisting hazards. In this paper, DNA-assembly-based multistimulus responsive capsules that can detect multiple genotoxic agents simultaneously are presented. By exploiting the sequence- and reactivity-editable properties of DNA, DNA sequences in a DNA shell are designed to exhibit multivalent susceptibility against ultraviolet B radiation, aflatoxin B1, and styrene oxide. Upon exposure to genotoxicants, the developed DNA capsules dissociate because of the production of DNA adducts or aptamer-ligand complex-activated dehybridization, which results in the release of encapsulated fluorophores for a measure of the genotoxicant level. The fluorophore release kinetics for each genotoxicant is investigated. Moreover, the destruction behaviors of the developed capsules are evaluated in binary and ternary toxin mixtures. Multiple linear regression indicates the existence of a strong relationship between the fluorescent response and the genotoxicant level; the result highlights the significance of particular genotoxicant and the antagonistic effect of interacting genotoxic substances on capsule destruction. This DNA architecture allows the monitoring of human exposure to genotoxic agents, which enables the timely adoption of remedial measures, and benefits development of an endogenous genotoxin-responsive drug delivery system.

Clinical Response to Anti-CD47 Immunotherapy Is Associated with Rapid Reduction of Exhausted Bystander CD4+ BTLA+ T Cells in Tumor Microenvironment of Mycosis Fungoides.

Cancer progression in mycosis fungoides, the most common form of cutaneous T-cell lymphoma, occurs in a predictable, sequential pattern that starts from patches and that evolves to plaques and later to tumors. Therefore, unlocking the relationship between the microarchitecture of mycosis fungoides and the clinical counterparts of that microstructure represents important steps for the design of targeted therapies. Using multispectral fluorescent imaging, we show that the progression of mycosis fungoides from plaque to tumor parallels the cutaneous expansion of the malignant CD4+ T cells that express TOX. The density of exhausted BTLA+ CD4+ T cells around malignant CD4+TOX+ cells was higher in tumors than it was in plaques, suggesting that undesired safeguards are in place within the tumor microenvironment that prevent immune activation and subsequent cancer eradication. Overriding the CD47 checkpoint with an intralesional SIRPαFc fusion decoy receptor induced the resolution of mycosis fungoides in patients that paralleled an amplified expansion of NK and CD8+ T cells in addition to a reduction of the exhausted BTLA+ CD4+ T cells that were engaged in promiscuous intercellular interactions. These therapeutic benefits of the CD47 blockade were further unleashed by adjuvant interferon-α, which stimulates cytotoxic cells, underscoring the importance of an inflamed microenvironment in facilitating the response to immunotherapy. Collectively, these findings support CD47 as a therapeutic target in treating mycosis fungoides and demonstrate a synergistic role of interferon-α in exploiting these clinical benefits.

Effects of tobacco and vaping on the skin.

Cigarette and electronic cigarette use are significant public health concerns across the United States. Tobacco use remains the single most preventable cause of morbidity and mortality in the world. Electronic cigarettes initially emerged as a better alternative to conventional cigarettes and for promoting smoking cessation; however, current evidence reveals similar deleterious health implications caused by both products on almost all organ systems, including the skin. Recognition of the cutaneous manifestations associated with cigarette and electronic cigarette use is essential for dermatologists in current clinical practice. Dermatologists play a vital role in educating and counseling patients on smoking cessation. We specifically highlight the cutaneous consequences of conventional cigarette smoking and electronic cigarettes on dermatologic disease.

Nutrition and nail disease.

The nail is a specialized keratinous skin appendage that is often overlooked, even though nail disorders comprise approximately 10% of all dermatologic conditions. We provide an overview on the basic anatomy of the nail and function of each structure. We examine the chemical profile, including the keratin and mineral composition, of the nail plate. Subsequently, nail manifestations are reviewed, as virtually every nutritional deficiency can affect nail growth in some manner. We focus on how each nutritional deficiency can affect the different anatomic structures of the nail unit. The terminology and the differential diagnoses of the many different nail plate and nail bed abnormalities are reviewed. Finally, we focus on the evidence behind nutrition-based treatments in the setting of several nail disorders.

The skin microbiome and the gut-skin axis.

The microbiome plays a significant role in human health, homeostasis, immune system, and disease pathogenesis. Disrupted communication between the microbiome and host has been extensively studied in gastrointestinal diseases. To a lesser extent, there is emerging research on the skin microbiome and its connection with the gut, referred to as the gut-skin axis and its effects on dermatologic conditions. A basic overview will be provided of the gut and skin microbiome with a focus on the impact of this connection on cutaneous diseases, such as psoriasis, atopic dermatitis, rosacea, acne vulgaris, photoaging, and cutaneous wounds. In addition, we shall discuss nutrition-based approaches mediated through the gut-skin axis and topical treatments that could serve as potential adjunctive management by manipulation of the microbiome. In particular, there is a growing body of research on oral probiotics, prebiotics, and dietary modifications that may help improve symptoms for a variety of dermatologic conditions in select demographic groups.

Geographic distribution and characteristics of the pediatric dermatology workforce in the United States.

BACKGROUND/OBJECTIVES: Up to 30% of pediatric primary care visits include a cutaneous complaint, yet the pediatric dermatology workforce has historically been too small to provide adequate specialized care. This study assesses the geographic distribution of pediatric dermatologists to determine physician-to-patient ratios, analyzes urban-rural disparities, and determines post-fellowship migration patterns. METHODS: Board-certified pediatric dermatologists were identified using the Society for Pediatric Dermatology's public database, and their demographics and credentials were subsequently verified by an online search. Analysis included physician density per 100 000 children for each state and region, along with geographic distribution for rural and urban areas, based on the United States Census Bureau's definitions. The distances between practice locations and the American Board of Dermatology-approved Pediatric Dermatology fellowship training sites were reviewed. RESULTS: An estimated 336 board-certified pediatric dermatologists currently work in the United States with 76.8% being women and 71.1% practicing within 50 miles of the nearest fellowship program. 96.4% are located in urban areas and 3.6% in rural areas with an average ratio of 0.54 and 0.09 per 100 000 children, respectively. The average ratio of pediatric dermatologists in the United States was 0.46 per 100 000 children. On average (standard deviation), there are 6.6 (8.8) pediatric dermatologists per state but with 7 states having zero. CONCLUSIONS: The demand for pediatric dermatologists continues to outpace the current physician availability with a disparity between urban and rural areas. Further awareness and emphasis on training and recruitment of additional pediatric dermatologists are essential to addressing this important issue.

The impact of gender in mentor-mentee success: Results from the Women's Dermatologic Society Mentorship Survey.

BACKGROUND: Mentorship can have a profound impact on the success and happiness of a mentee while also providing a sense of fulfillment and enrichment for the mentor. Both officially designated and spontaneously chosen mentors can be useful for protégés as they navigate through their training and professional work environment while striving to obtain the optimal work-life balance. Different genders can have variable experiences, in both their personal lives managing family obligations and their professional lives as dermatologists, which may affect the advice and guidance offered. OBJECTIVE: We studied the impact of gender on the mentor-mentee relationship for both official and spontaneous mentorships through a voluntary survey with a focus on reported outcomes from the perspective of the mentee. METHODS: Participants were selected through e-mail invitation via the Women's Dermatologic Society and program directors of the Association of Professors of Dermatology membership lists and given a link to the anonymous survey tool. The survey included 13 questions looking at official and spontaneous mentorships, the role of gender, and success in the dermatology field. RESULTS: Of the 288 respondents, 202 (69.9%) were women, 86 (29.8%) were men, and one identified as other. Of the survey participants, 81% had official mentors and 91% had spontaneous mentors, with the overlap indicating that there may have been a history of multiple mentors per individual. Mentoring had an overall significant positive impact, and 98.5% of those in the spontaneous-mentor group rated the mentor as helpful compared with 87.6% in the official-mentor cohort. For official mentorships, 60.1% involved gender-similar mentors, and of those who had officially designated mentors of any type, 55% indicated a preference for mentors of the same gender. When specifically looking at respondents who participated in same-gender official mentorships, 65.5% preferred this type; of those who had a gender-dissimilar equivalent, only 36.7% indicated a preference for gender similarity in a mentor. Comparably, 59% of protégés with spontaneous mentors had a gender-similar one, and of those who had spontaneous mentors of any type, 59.2% preferred gender similarity. When considering only those in gender-similar spontaneous mentorships, 74.5% favored a same-gender pairing compared with 32.9% of those in the gender-dissimilar group. For female-female official mentorships, 75% preferred a female mentor, similar to 80.5% of the spontaneous-mentor cohort. CONCLUSION: Spontaneous mentors may provide a greater benefit than officially designated ones. For the majority of the categories, there was no statistical difference between female same-gender mentorships and gender-dissimilar relationships, which is in contrast with previously published literature. Overall, based on the feedback provided, the respondents believed that the quality of the relationship was the most important defining factor, but some noted that same-gender mentorships can provide additional benefit geared toward similar interests and experiences in life.

Intralesional TTI-621, a novel biologic targeting the innate immune checkpoint CD47, in patients with relapsed or refractory mycosis fungoides or Sézary syndrome: a multicentre, phase 1 study.

BACKGROUND: Intravenous TTI-621 (SIRPα-IgG1 Fc) was previously shown to have activity in relapsed or refractory haematological malignancies. This phase 1 study evaluated the safety and activity of TTI-621 in patients with percutaneously accessible relapsed or refractory mycosis fungoides, Sézary syndrome, or solid tumours. Here we report the clinical and translational results among patients with mycosis fungoides or Sézary syndrome. METHODS: This multicentre, open-label, phase 1 study was conducted at five academic health-care and research centres in the USA. Eligible patients were aged 18 years or older; had injectable, histologically or cytologically confirmed relapsed or refractory cutaneous T-cell lymphoma (CTCL) or solid tumours; Eastern Cooperative Oncology Group performance status of 2 or less; and adequate haematological, renal, hepatic, and cardiac function. TTI-621 was injected intralesionally in a sequential dose escalation (cohorts 1-5; single 1 mg, 3 mg, or 10 mg injection or three 10 mg injections weekly for 1 or 2 weeks) and in expansion cohorts (cohorts 6-9; 2 week induction at the maximum tolerated dose; weekly continuation was allowed). In cohort 6, patients were injected with TTI-621 in a single lesion and in cohort 7, they were injected in multiple lesions. In cohort 8, TTI-621 was combined with pembrolizumab 200 mg injections per product labels. In cohort 9, TTI-621 was combined with the standard labelled dose of subcutaneous pegylated interferon alpha-2a 90 µg. The primary endpoint was the incidence and severity of adverse events. The study is registered with ClinicalTrials.gov, NCT02890368, and was closed by the sponsor to focus on intravenous studies with TTI-621. FINDINGS: Between Jan 30, 2017, and March 31, 2020, 66 patients with mycosis fungoides, Sézary syndrome, other CTCL, or solid tumours were screened, 35 of whom with mycosis fungoides or Sézary syndrome were enrolled and received intralesional TTI-621 (escalation, n=13; expansion, n=22). No dose-limiting toxicities occurred; the maximum tolerated dose was not established. In the dose expansion cohorts, the maximally assessed regimen (10 mg thrice weekly for 2 weeks) was used. 25 (71%) patients had treatment-related adverse events; the most common (occurring in ≥10% of patients) were chills (in ten [29%] patients), injection site pain (nine [26%]), and fatigue (eight [23%]). No treatment-related adverse events were grade 3 or more or serious. There were no treatment-related deaths. Rapid responses (median 45 days, IQR 17-66) occurred independently of disease stage or injection frequency. 26 (90%) of 29 evaluable patients had decreased Composite Assessment of Index Lesion Severity (CAILS) scores; ten (34%) had a decrease in CAILS score of 50% or more (CAILS response). CAILS score reductions occurred in adjacent non-injected lesions in eight (80%) of ten patients with paired assessments and in distal non-injected lesions in one additional patient. INTERPRETATION: Intralesional TTI-621 was well tolerated and had activity in adjacent or distal non-injected lesions in patients with relapsed or refractory mycosis fungoides or Sézary syndrome, suggesting it has systemic and locoregional abscopal effects and potential as an immunotherapy for these conditions. FUNDING: Trillium Therapeutics.

Evidence concerning the accusation that melanoma is overdiagnosed.

BACKGROUND: Melanoma is one of the most commonly diagnosed malignancies in the United States and is responsible for the majority of deaths from skin cancer. OBJECTIVE: Since the 1970s, the incidence of melanoma has risen appreciably while melanoma-specific mortality has remained stable. This has raised a debate about potential overdiagnosis of melanoma. Herein, we review temporal trends in melanoma incidence and mortality and explore factors that may contribute to observed trends, including an aging population in the United States, ultraviolet exposure, increased numbers of biopsies by dermatologists and physician extenders, skin cancer screenings, histopathology criteria, and historic underdiagnosis. Additionally, we discuss melanoma overdiagnosis and the extent to which it may contribute to current trends. METHODS: The literature was reviewed. RESULTS: Several factors may contribute to an increased incidence of melanoma, including an aging population, ultraviolet exposure, increased skin biopsies, skin cancer screenings, histopathologic criteria, historic underdiagnosis, and current overdiagnosis. LIMITATIONS: Further studies are required to determine exactly which tumors are being overdiagnosed, and how to improve patient outcomes with adjustment to physician's practice. CONCLUSION: The rise in the incidence of melanoma observed since the 1970s is likely multifactorial.

Clinical pearl: topical timolol for refractory hypergranulation.

Hypergranulation is a frequent complication of dermatologic surgery. Management of postoperative hypergranulation with routine wound care and/or topical silver nitrate often is effective, but refractory cases can be challenging given the paucity of treatment options. We present an alternative treatment of refractory hypergranulation using timolol ophthalmic gel forming solution.

Choline acetyltransferase-expressing T cells are required to control chronic viral infection.

Although widely studied as a neurotransmitter, T cell-derived acetylcholine (ACh) has recently been reported to play an important role in regulating immunity. However, the role of lymphocyte-derived ACh in viral infection is unknown. Here, we show that the enzyme choline acetyltransferase (ChAT), which catalyzes the rate-limiting step of ACh production, is robustly induced in both CD4+ and CD8+ T cells during lymphocytic choriomeningitis virus (LCMV) infection in an IL-21-dependent manner. Deletion of Chat within the T cell compartment in mice ablated vasodilation in response to infection, impaired the migration of antiviral T cells into infected tissues, and ultimately compromised the control of chronic LCMV clone 13 infection. Our results reveal a genetic proof of function for ChAT in T cells during viral infection and identify a pathway of T cell migration that sustains antiviral immunity.

Mucopolysaccharidosis Type VI in a Great Dane Caused by a Nonsense Mutation in the ARSB Gene.

Mucopolysaccharidoses are inherited metabolic disorders that result from a deficiency of lysosomal enzymes required for the catabolism of glycosaminoglycans. Lysosomal glycosaminoglycan accumulation results in cell and organ dysfunction. This study characterized the phenotype and genotype of mucopolysaccharidosis VI in a Great Dane puppy with clinical signs of stunted growth, facial dysmorphia, skeletal deformities, corneal opacities, and increased respiratory sounds. Clinical and pathologic evaluations, urine glycosaminoglycan analyses, lysosomal enzyme assays, and ARSB sequencing were performed. The urine mucopolysaccharide spot test was strongly positive predominantly due to the accumulation of dermatan sulfate. Enzyme assays in leukocytes and tissues indicated a deficiency of arylsulfatase B (ARSB) activity. Histologic examination revealed cytoplasmic vacuoles in many tissues. Analysis of the exonic ARSB DNA sequences from the affected puppy compared to the published canine genome sequence revealed a homozygous nonsense mutation (c.295C>T) in exon 1, replacing glutamine with a premature stop codon (p.Gln99*), predicting no enzyme synthesis. A polymerase chain reaction-based restriction fragment length polymorphism test was established to assist with the clinical diagnosis and breeding of Great Danes. This genotyping test revealed that the clinically healthy parents and some other relatives of the puppy were heterozygous for the mutant allele, but all 200 clinically healthy dogs screened including 15 Great Danes were homozygous for the normal allele. This ARSB mutation is the fourth identified genetic variant causing canine mucopolysaccharidosis VI. Mucopolysaccharidosis VI is the first lysosomal storage disorder described in Great Danes but does not appear to be widespread in this breed.