Imaging in osteogenesis imperfecta: Where we are and where we are going.

Osteogenesis imperfecta (OI) is a rare phenotypically and genetically heterogeneous group of inherited skeletal dysplasias. The hallmark features of OI include bone fragility and susceptibility to fractures, bone deformity, and diminished growth, along with a plethora of associated secondary features (both skeletal and extraskeletal). The diagnosis of OI is currently made on clinical grounds and may be confirmed by genetic testing. However, imaging remains pivotal in the evaluation of this disease. The aim of this article is to review the current role played by the various radiologic techniques in the diagnosis and monitoring of OI in the postnatal setting as well as to discuss recent advances and future perspectives in OI imaging. Conventional Radiography and Dual-energy X-ray Absorptiometry (DXA) are currently the two most used imaging modalities in OI. The cardinal radiographic features of OI include generalized osteopenia/osteoporosis, bone deformities, and fractures. DXA is currently the most available technique to assess Bone Mineral Density (BMD), specifically areal BMD (aBMD). However, DXA has important limitations and cannot fully characterize bone fragility in OI based on aBMD. Novel DXA-derived parameters, such as Trabecular Bone Score (TBS), may provide further insight into skeletal changes induced by OI, but evidence is still limited. Techniques like Computed Tomography (CT) and Magnetic Resonance Imaging (MRI) can be useful as problem-solvers or in specific settings, including the evaluation of cranio-cervical abnormalities. Recent evidence supports the use of High-Resolution peripheral Quantitative Computed Tomography (HR-pQCT) as a promising tool to improve the characterization of bone fragility in OI. However, HR-pQCT remains a primarily research technique at present. Quantitative Computed Tomography (QCT) is an alternative to DXA for the determination of BMD at central sites, with distinct advantages but considerably higher radiation exposure. Quantitative Ultrasound (QUS) is a portable, inexpensive, and radiation-free modality that may complement DXA evaluation, providing information on bone quality. However, evidence of usefulness of QUS in OI is poor. Radiofrequency Echographic Multi Spectrometry (REMS) is an emerging non-ionizing imaging method that holds promise for the diagnosis of low BMD and for the prediction of fracture risk, but so far only one published study has investigated its role in OI. To conclude, several different radiologic techniques have proven to be effective in the diagnosis and monitoring of OI, each with their own specificities and peculiarities. Clinicians should be aware of the strategic role of the various modalities in the different phases of the patient care process. In this scenario, the development of international guidelines including recommendations on the role of imaging in the diagnosis and monitoring of OI, accompanied by continuous active research in the field, could significantly improve the standardization of patient care.

Transcriptomic analysis of paired healthy human skeletal muscles to identify modulators of disease severity in DMD.

Muscle damage and fibro-fatty replacement of skeletal muscles is a main pathologic feature of Duchenne muscular dystrophy (DMD) with more proximal muscles affected earlier and more distal affected later in the disease course, suggesting that different skeletal muscle groups possess distinctive characteristics that influence their susceptibility to disease. To explore transcriptomic factors driving differential gene expression and modulating DMD skeletal muscle severity, we characterized the transcriptome of vastus lateralis (VL), a more proximal and susceptible muscle, relative to tibialis anterior (TA), a more distal and protected muscle, in 15 healthy individuals using bulk RNA sequencing to identify gene expression differences that may mediate their relative susceptibility to damage with loss of dystrophin. Matching single nuclei RNA sequencing data was generated for 3 of the healthy individuals, to infer cell composition in the bulk RNA sequencing dataset and to improve mapping of differentially expressed genes to their cell source of expression. A total of 3,410 differentially expressed genes were identified and mapped to cell type using single nuclei RNA sequencing of muscle, including long non-coding RNAs and protein coding genes. There was an enrichment of genes involved in calcium release from the sarcoplasmic reticulum, particularly in the myofibers and these myofiber genes were higher in the VL. There was an enrichment of genes in "Collagen-Containing Extracellular Matrix" expressed by fibroblasts, endothelial, smooth muscle and pericytes, with most genes higher in the TA, as well as genes in "Regulation Of Apoptotic Process" expressed across all cell types. Previously reported genetic modifiers were also enriched within the differentially expressed genes. We also identify 6 genes with differential isoform usage between the VL and TA. Lastly, we integrate our findings with DMD RNA sequencing data from the TA, and identify "Collagen-Containing Extracellular Matrix" and "Negative Regulation Of Apoptotic Process" as differentially expressed between DMD compared to healthy. Collectively, these findings propose novel candidate mechanisms that may mediate differential muscle susceptibility in muscular dystrophies and provide new insight into potential therapeutic targets.

Single nuclei transcriptomics of muscle reveals intra-muscular cell dynamics linked to dystrophin loss and rescue.

In Duchenne muscular dystrophy, dystrophin loss leads to chronic muscle damage, dysregulation of repair, fibro-fatty replacement, and weakness. We develop methodology to efficiently isolate individual nuclei from minute quantities of frozen skeletal muscle, allowing single nuclei sequencing of irreplaceable archival samples and from very small samples. We apply this method to identify cell and gene expression dynamics within human DMD and mdx mouse muscle, characterizing effects of dystrophin rescue by exon skipping therapy at single nuclei resolution. DMD exon 23 skipping events are directly observed and increased in myonuclei from treated mice. We describe partial rescue of type IIa and IIx myofibers, expansion of an MDSC-like myeloid population, recovery of repair/remodeling M2-macrophage, and repression of inflammatory POSTN1 + fibroblasts in response to exon skipping and partial dystrophin restoration. Use of this method enables exploration of cellular and transcriptomic mechanisms of dystrophin loss and repair within an intact muscle environment. Our initial findings will scaffold our future work to more directly examine muscular dystrophies and putative recovery pathways.

Multicenter Registry of Patients Receiving Systemic Mold-Active Triazoles for the Management of Invasive Fungal Infections.

INTRODUCTION: 'Real-world' data for mold-active triazoles (MATs) in the treatment of invasive fungal infections (IFIs) are lacking. This study evaluated usage of MATs in a disease registry for the management of IFIs. METHODS: Data were collected for this multicenter, observational, prospective study from 55 US centers, between March 2017 and April 2020. Eligible patients received isavuconazole, posaconazole, or voriconazole as MAT monotherapy (one MAT) or multiple/sequenced MAT therapy (more than one MAT) for prophylaxis or treatment. Patients were enrolled within 60 days of MAT initiation. The primary objective was to characterize patients receiving a MAT and their patterns of therapy. The full analysis set (FAS) included eligible patients for the relevant enrollment protocol, and the safety analysis set (SAF) included patients who received ≥ 1 MAT dose. RESULTS: Overall, 2009 patients were enrolled in the SAF. The FAS comprised 1993 patients (510 isavuconazole; 540 posaconazole; 491 voriconazole; 452 multiple/sequenced MAT therapies); 816 and 1177 received treatment and prophylaxis at study index/enrollment, respectively. Around half (57.8%) of patients were male, and median age was 59 years. Among patients with IFIs during the study, the most common pathogens were Aspergillus fumigatus in the isavuconazole (18.2% [10/55]) and voriconazole (25.5% [12/47]) groups and Candida glabrata in the posaconazole group (20.9% [9/43]); the lungs were the most common infection site (58.2% [166/285]). Most patients were maintained on MAT monotherapy (77.3% [1541/1993]), and 79.4% (1520/1915) completed their MAT therapies. A complete/partial clinical response was reported in 59.1% (591/1001) of patients with a clinical response assessment. Breakthrough IFIs were reported in 7.1% (73/1030) of prophylaxis patients. Adverse drug reactions (ADRs) were reported in 14.7% (296/2009) of patients (3.9% [20/514] isavuconazole; 11.3% [62/547] posaconazole; 14.2% [70/494] voriconazole). CONCLUSIONS: In this 'real-world' study, most patients remained on their initial therapy and completed their MAT therapy. Over half of patients receiving MATs for IFIs had a successful response, and most receiving prophylaxis did not develop breakthrough IFIs. ADRs were uncommon.

Modeling Patient-Specific Muscular Dystrophy Phenotypes and Therapeutic Responses in Reprogrammed Myotubes Engineered on Micromolded Gelatin Hydrogels.

In vitro models of patient-derived muscle allow for more efficient development of genetic medicines for the muscular dystrophies, which often present mutation-specific pathologies. One popular strategy to generate patient-specific myotubes involves reprogramming dermal fibroblasts to a muscle lineage through MyoD induction. However, creating physiologically relevant, reproducible tissues exhibiting multinucleated, aligned myotubes with organized striations is dependent on the introduction of physicochemical cues that mimic the native muscle microenvironment. Here, we engineered patient-specific control and dystrophic muscle tissues in vitro by culturing and differentiating MyoD-directly reprogrammed fibroblasts isolated from one healthy control subject, three patients with Duchenne muscular dystrophy (DMD), and two Limb Girdle 2A/R1 (LGMD2A/R1) patients on micromolded gelatin hydrogels. Engineered DMD and LGMD2A/R1 tissues demonstrated varying levels of defects in α-actinin expression and organization relative to control, depending on the mutation. In genetically relevant DMD tissues amenable to mRNA reframing by targeting exon 44 or 45 exclusion, exposure to exon skipping antisense oligonucleotides modestly increased myotube coverage and alignment and rescued dystrophin protein expression. These findings highlight the value of engineered culture substrates in guiding the organization of reprogrammed patient fibroblasts into aligned muscle tissues, thereby extending their value as tools for exploration and dissection of the cellular and molecular basis of genetic muscle defects, rescue, and repair.

Impact of self-ligating orthodontic brackets on dental biofilm and periodontal pathogens in adolescents.

The aim of the present prospective study was to evaluate periodontal health and subgingival microbiological alterations in adolescents treated with fixed self ligating orthodontic brackets in comparison to subject without any orthodontic appliance. A total of 40 adolescents (23 females and 17 males; mean age: 13.2 ± 3.2 years) were included: 30 subjects with self ligating brackets (test group) and 10 patients without orthodontic appliances (control group). Follow-ups were as follows: T1 (1 month), T2 (3 months), T3 (6 months) from the beginning of the orthodontic therapy. Clinical parameters (plaque index, gingival index and clinical attachment level) were measured for every patient and a microbiological analysis was performed. Mann Whitney test was performed to evaluate clinical parameters between test and control group and Friedman test and Fisher test were adopted to evaluate intra group differences at different follow-ups. Student T-test was performed to compare clinical attachment level between the two groups. Significance level was set at p<0.05. No periodontal pathogens and no clinical attachment loss were found in the whole sample. A slightly higher plaque index and gingival inflammation were recorded in the test group in comparison to the controls.

Oral manifestations in paediatric patients with hepatobiliary diseases: a review.

It is well known that greenish pigmentation of the teeth is seen in children following remission of severe jaundice and clinical and serum bilirubin, a degradation product of haemoglobin, may be permanently trapped in forming dental hard tissues causing discolouration and enamel and dentine hypoplasia. Neonatal jaundice is the most common cause of hyperbilirubinemia and pigmentation of the deciduous teeth is the consequence of this condition. Various hepatobiliary pathologies may have a clinical finding in the oral cavity; furthermore, oral manifestations of hepatic pathologies are not just limited to the pigmentation of the deciduous teeth but also the permanent dentition and the mucous membranes can be affected.

Quantitative immuno-mass spectrometry imaging of skeletal muscle dystrophin.

Emerging and promising therapeutic interventions for Duchenne muscular dystrophy (DMD) are confounded by the challenges of quantifying dystrophin. Current approaches have poor precision, require large amounts of tissue, and are difficult to standardize. This paper presents an immuno-mass spectrometry imaging method using gadolinium (Gd)-labeled anti-dystrophin antibodies and laser ablation-inductively coupled plasma-mass spectrometry to simultaneously quantify and localize dystrophin in muscle sections. Gd is quantified as a proxy for the relative expression of dystrophin and was validated in murine and human skeletal muscle sections following k-means clustering segmentation, before application to DMD patients with different gene mutations where dystrophin expression was measured up to 100 µg kg-1 Gd. These results demonstrate that immuno-mass spectrometry imaging is a viable approach for pre-clinical to clinical research in DMD. It rapidly quantified relative dystrophin in single tissue sections, efficiently used valuable patient resources, and may provide information on drug efficacy for clinical translation.

A well-tolerated core needle muscle biopsy process suitable for children and adults.

Serial muscle biopsies within clinical trials for Duchenne muscular dystrophy (DMD) are critical to document therapeutic responses. Less invasive means of sampling muscle are needed. We analyzed a retrospective consecutive case-series cohort of vacuum-assisted core needle muscle biopsy procedures performed on healthy and dystrophic individuals at a single institution assessing for safety and reliability of obtaining sufficient high-quality biopsy tissue for histologic assessment in adult and pediatric subjects. Of 471 muscle cores from 128 biopsy procedures, 377-550 mg of total muscle tissue was obtained per procedure with mean core weight of 129 mg (SD, 25.1 mg). All biopsies were adequate for histological assessment. There were no significant adverse events. This core needle biopsy approach, when combined with improved sample processing, provides a safe means to consistently obtain muscle samples for diagnostic and clinical trial applications.

Impact of Sjögren's syndrome on oral health and quality of life: an observational cross-sectional study.

Sjögren's Syndrome is a complex disease, due to an autoimmune physiopathology, that strongly impacts both patients' primary needs (nutrition and speaking), and patients' relationship life related factors (psychological health and quality of life). In Literature, few studies had investigated oral health status in Sjögren's syndrome and its impact on patients' quality of life, so the aim of this study was to analyse that issue. 30 patients were enrolled, within the Department of Rheumatology (University of Pisa), both first diagnosis patients' and both patients who had been diagnosed with Sjögren's syndrome in the past. For each patient, a medical record was filled out together with the compilation of the Oral Health Impact Profile questionnaire. Then, during a specialistic rheumatologic visit, Sjögren's Syndrome Disease Damage Index Score (SSDDI) was determined. Results showed a direct proportion between years from diagnosis and severity of oral health issues. It was found that these issues were related to soft tissue damage and an overall worse, reported quality of life and psychological health.

Voriconazole plus terbinafine combination antifungal therapy for invasive Lomentospora prolificans infections: analysis of 41 patients from the FungiScope® registry 2008-2019.

OBJECTIVES: Lomentospora prolificans is an emerging cause of serious invasive fungal infections. Optimal treatment of these infections is unknown, although voriconazole-containing treatment regimens are considered the treatment of choice. The objective of this study was to evaluate the role of combination antifungal therapy for L. prolificans infections. METHODS: We performed a retrospective review of medical records of patients with invasive L. prolificans infection diagnosed between 1 January 2008 and 9 September 2019 that were documented in the FungiScope® registry of rare invasive fungal infections. We compared clinical outcomes between antifungal treatment strategies. RESULTS: Over the study period, 41 individuals with invasive L. prolificans infection from eight different countries were documented in the FungiScope® registry. Overall, 17/40 (43%) had treatment response/stable disease and 21/40 (53%) had a fatal outcome attributed to invasive fungal infection. Combination antifungal therapy was associated with increased 28-day survival (15/24 survived versus 4/16 receiving monotherapy; p 0.027) and the combination voriconazole plus terbinafine trended to be associated with higher rates of treatment success (10/16, 63%, 95% CI 35%-85%) compared with other antifungal treatment regimens (7/24, 29%, 95% CI 13%-51%, p 0.053). In Kaplan-Meier survival analysis there was a higher survival probability in individuals receiving the voriconazole/terbinafine combination compared with other antifungal regimens (median survival 150 days versus 17 days). CONCLUSIONS: While overall mortality was high, combination antifungal treatment, and in particular combination therapy with voriconazole plus terbinafine may be associated with improved treatment outcomes compared with other antifungal regimens for the treatment of invasive L. prolificans infections.

Large in-frame 5' deletions in DMD associated with mild Duchenne muscular dystrophy: Two case reports and a review of the literature.

Duchenne muscular dystrophy is caused by mutations in the dystrophin-encoding DMD gene. While Duchenne is most commonly caused by large intragenic deletions that cause frameshift and complete loss of dystrophin expression, in-frame deletions in DMD can result in the expression of internally truncated dystrophin proteins and may be associated with a milder phenotype. In this study, we describe two individuals with large in-frame 5' deletions (exon 3-23 and exon 3-28) that remove the majority of the N-terminal region, including part of the actin binding and central rod domains. Both patients had progressive muscle weakness during childhood but are observed to have a relatively mild disease course compared to typical Duchenne. We show that in muscle biopsies from both patients, truncated dystrophin is expressed at the sarcolemma. We have additionally developed a patient-specific fibroblast-derived cell model, which can be inducibly reprogrammed to form myotubes that largely recapitulate biopsy findings for the patient with the exon 3-23 deletion, providing a culture model for future investigation of this unusual case. We discuss these mutations in the context of previously reported 5' in-frame DMD deletions and relevant animal models, and review the spectrum of phenotypes associated with these deletions.

Targeting RyR Activity Boosts Antisense Exon 44 and 45 Skipping in Human DMD Skeletal or Cardiac Muscle Culture Models.

Systemic delivery of antisense oligonucleotides (AO) for DMD exon skipping has proven effective for reframing DMD mRNA, rescuing dystrophin expression, and slowing disease progression in animal models. In humans with Duchenne muscular dystrophy treated with AOs, low levels of dystrophin have been induced, and modest slowing of disease progression has been observed, highlighting the need for improved efficiency of human skipping drugs. Here, we demonstrate that dantrolene and Rycals S107 and ARM210 potentiate AO-mediated exon skipping of exon 44 or exon 45 in patient-derived myotube cultures with appropriate mutations. Further, dantrolene is shown to boost AO-mediated exon skipping in patient-derived, induced cardiomyocyte cultures. Our findings further validate the ryanodine receptors (RyR) as the likely target responsible for exon skip boosting and demonstrate potential applicability beyond exon 51 skipping. These data provide preclinical support of dantrolene trial as an adjuvant to AO-mediated exon-skipping therapy in humans and identify a novel Rycal, ARM210, for development as a potential exon-skipping booster. Further, they highlight the value of mutation-specific DMD culture models for basic discovery, preclinical drug screening and translation of personalized genetic medicines.

Longitudinal study on the effect of oral hygiene measures on the salivary count of microbial species with cariogenic potential.

The effect of oral hygiene education measures and professional tooth cleaning on the salivary levels of microbial species with high cariogenic potential (i.e. Streptococcus mutans, Lactobacillus spp. and Candida albicans) was evaluated at different time points. At time 0, high salivary carriage rates were recorded in the study group (n=30). Fifty percent of the subjects harbored all three species in their saliva, 27% harbored 2 species, and 23% only one species. At 3 months after oral hygiene measures, a statistically significant reduction was observed in salivary count of S. mutans and Lactobacillus spp. The percentage of subjects harboring all three species was also highly reduced, along with an overall improvement of clinical and risk factors parameters. At 8 months after oral hygiene measures, S. mutans and Lactobacillus spp. load was still statistically lower than that recorded at time 0, although an increment in bacterial load and a partial worsening of clinical and risk factors parameters were observed. S. mutans count in saliva inversely correlated with salivary pH, while it positively correlated with C. albicans salivary levels. The results obtained suggest that strengthening of the motivation and administration of oral hygiene instructions and professional tooth cleaning every 6-8 months, might be necessary to control salivary levels of cariogenic species.

Validation and Detection of Exon Skipping Boosters in DMD Patient Cell Models and mdx Mouse.

Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene. Most deletions, duplications, or indels lead to shift of mRNA reading frame, which prevent the production of dystrophin protein. DMD is the leading fatal genetic disorder in childhood. One therapeutic strategy aims to skip one or more exons to restore reading frame to enable the production of internally truncated proteins with partial functionality. However, to date the efficiency of this strategy is suboptimal. Here we present methods for assessing exon skipping using AON alone or in combination with skip booster in the context of human DMD patient fibroblast derived myotubes and in the mdx mouse model of DMD.

DMD genotype correlations from the Duchenne Registry: Endogenous exon skipping is a factor in prolonged ambulation for individuals with a defined mutation subtype.

Antisense oligonucleotide (AON)-mediated exon skipping is an emerging therapeutic for individuals with Duchenne muscular dystrophy (DMD). Skipping of exons adjacent to common exon deletions in DMD using AONs can produce in-frame transcripts and functional protein. Targeted skipping of DMD exons 8, 44, 45, 50, 51, 52, 53, and 55 is predicted to benefit 47% of affected individuals. We observed a correlation between mutation subgroups and age at loss of ambulation in the Duchenne Registry, a large database of phenotypic and genetic data for DMD (N = 765). Males amenable to exon 44 (N = 74) and exon 8 skipping (N = 18) showed prolonged ambulation compared to other exon skip groups and nonsense mutations (P = 0.035 and P < 0.01, respectively). In particular, exon 45 deletions were associated with prolonged age at loss of ambulation relative to the rest of the exon 44 skip amenable cohort and other DMD mutations. Exon 3-7 deletions also showed prolonged ambulation relative to all other exon 8 skippable mutations. Cultured myotubes from DMD patients with deletions of exons 3-7 or exon 45 showed higher endogenous skipping than other mutations, providing a potential biological rationale for our observations. These results highlight the utility of aggregating phenotypic and genotypic data for rare pediatric diseases to reveal progression differences, identify potentially confounding factors, and probe molecular mechanisms that may affect disease severity.

Repurposing Dantrolene for Long-Term Combination Therapy to Potentiate Antisense-Mediated DMD Exon Skipping in the mdx Mouse.

Duchenne muscular dystrophy (DMD) is caused by mutations in DMD, resulting in loss of dystrophin, which is essential to muscle health. DMD "exon skipping" uses anti-sense oligo-nucleotides (AONs) to force specific exon exclusion during mRNA processing to restore reading frame and rescue of partially functional dystrophin protein. Although exon-skipping drugs in humans show promise, levels of rescued dystrophin protein remain suboptimal. We previously identified dantrolene as a skip booster when combined with AON in human DMD cultures and short-term mdx dystrophic mouse studies. Here, we assess the effect of dantrolene/AON combination on DMD exon-23 skipping over long-term mdx treatment under conditions that better approximate potential human dosing. To evaluate the dantrolene/AON combination treatment effect on dystrophin induction, we assayed three AON doses, with and without oral dantrolene, to assess multiple outcomes across different muscles. Meta-analyses of the results of statistical tests from both the quadriceps and diaphragm assessing contributions of dantrolene beyond AON, across all AON treatment groups, provide strong evidence that dantrolene modestly boosts exon skipping and dystrophin rescue while reducing muscle pathology in mdx mice (p < 0.0087). These findings support a trial of combination dantrolene/AON to increase exon-skipping efficacy and highlight the value of combinatorial approaches and Food and Drug Administration (FDA) drug re-purposing for discovery of unsuspected therapeutic application and rapid translation.

Voluntary alcohol intake after noise exposure in adolescent rats: Hippocampal-related behavioral alterations.

Different physical or chemical agents, such as noise or alcohol, can induce diverse behavioral and biochemical alterations. Considering the high probability of young people to undergo consecutive or simultaneous exposures, the aim of the present work was to investigate in an animal model if noise exposure at early adolescence could induce hippocampal-related behavioral changes that might be modified after alcohol intake. Male Wistar rats (28-days-old) were exposed to noise (95-97 dB, 2 h). Afterwards, animals were allowed to voluntarily drink alcohol (10% ethanol in tap water) for three consecutive days, using the two-bottle free choice paradigm. After that, hippocampal-related memory and anxiety-like behavior tests were performed. Results show that whereas noise-exposed rats presented deficits in habituation memory, those who drank alcohol exhibited impairments in associative memory and anxiety-like behaviors. In contrast, exposure to noise followed by alcohol intake showed increases in exploratory and locomotor activities as well as in anxiety-like behaviors, unlike what was observed using each agent separately. Finally, lower levels of alcohol intake were measured in these animals when compared with those that drank alcohol and were not exposed to noise. Present findings demonstrate that exposure to physical and chemical challenges during early adolescence might induce behavioral alterations that could differ depending on the schedule used, suggesting a high vulnerability of rat developing brain to these socially relevant agents.

A phase 3 randomized placebo-controlled trial of tadalafil for Duchenne muscular dystrophy.

OBJECTIVE: To conduct a randomized trial to test the primary hypothesis that once-daily tadalafil, administered orally for 48 weeks, lessens the decline in ambulatory ability in boys with Duchenne muscular dystrophy (DMD). METHODS: Three hundred thirty-one participants with DMD 7 to 14 years of age taking glucocorticoids were randomized to tadalafil 0.3 mg·kg-1·d-1, tadalafil 0.6 mg·kg-1·d-1, or placebo. The primary efficacy measure was 6-minute walk distance (6MWD) after 48 weeks. Secondary efficacy measures included North Star Ambulatory Assessment and timed function tests. Performance of Upper Limb (PUL) was a prespecified exploratory outcome. RESULTS: Tadalafil had no effect on the primary outcome: 48-week declines in 6MWD were 51.0 ± 9.3 m with placebo, 64.7 ± 9.8 m with low-dose tadalafil (p = 0.307 vs placebo), and 59.1 ± 9.4 m with high-dose tadalafil (p = 0.538 vs placebo). Tadalafil also had no effect on secondary outcomes. In boys >10 years of age, total PUL score and shoulder subscore declined less with low-dose tadalafil than placebo. Adverse events were consistent with the known safety profile of tadalafil and the DMD disease state. CONCLUSIONS: Tadalafil did not lessen the decline in ambulatory ability in boys with DMD. Further studies should be considered to confirm the hypothesis-generating upper limb data and to determine whether ambulatory decline can be slowed by initiation of tadalafil before 7 years of age. CLINICALTRIALSGOV IDENTIFIER: NCT01865084. CLASSIFICATION OF EVIDENCE: This study provides Class I evidence that tadalafil does not slow ambulatory decline in 7- to 14-year-old boys with Duchenne muscular dystrophy.

Lectin-binding characterizes the healthy human skeletal muscle glycophenotype and identifies disease-specific changes in dystrophic muscle.

Our understanding of muscle glycosylation to date has derived from studies in mouse models and a limited number of human lectin histochemistry studies. As various therapeutic approaches aimed at treating patients with muscular dystrophies are being translated from rodent models to human, it is critical to better understand human muscle glycosylation and relevant disease-specific differences between healthy and dystrophic muscle. Here, we report the first quantitative characterization of human muscle glycosylation, and identify differentiation- and disease-specific differences in human muscle glycosylation. Utilizing a panel of 13 lectins with varying glycan specificities, we surveyed lectin binding to primary and immortalized myoblasts and myotubes from healthy and dystrophic sources. Following differentiation of primary and immortalized healthy human muscle cells, we observed increased binding of Narcissus pseudonarcissus agglutinin (NPA), PNA, MAA-II and WFA to myotubes compared to myoblasts. Following differentiation of immortalized healthy and dystrophic human muscle cells, we observed disease-specific differences in binding of NPA, Jac and Tricosanthes japonica agglutinin-I (TJA-I) to differentiated myotubes. We also observed differentiation- and disease-specific differences in binding of NPA, Jac, PNA, TJA-I and WFA to glycoprotein receptors in muscle cells. Additionally, Jac, PNA and WFA precipitated functionally glycosylated α-DG, that bound laminin, while NPA and TJA-I did not. Lectin histochemistry of healthy and dystrophic human muscle sections identified disease-specific differences in binding of O-glycan and sialic acid-specific lectins between healthy and dystrophic muscle. These results indicate that specific and discrete changes in glycosylation occur following differentiation, and identify specific lectins as potential biomarkers sensitive to changes in healthy human muscle glycosylation.