Tony Huynh

VBP15, a novel anti-inflammatory and membrane-stabilizer, improves muscular dystrophy without side effects.

Absence of dystrophin makes skeletal muscle more susceptible to injury, resulting in breaches of the plasma membrane and chronic inflammation in Duchenne muscular dystrophy (DMD). Current management by glucocorticoids has unclear molecular benefits and harsh side effects. It is uncertain whether therapies that avoid hormonal stunting of growth and development, and/or immunosuppression, would be more or less beneficial. Here, we discover an oral drug with mechanisms that provide efficacy through anti‐inflammatory signaling and membrane‐stabilizing pathways, independent of hormonal or immunosuppressive effects. We find VBP15 protects and promotes efficient repair of skeletal muscle cells upon laser injury, in opposition to prednisolone. Potent inhibition of NF‐κB is mediated through protein interactions of the glucocorticoid receptor, however VBP15 shows significantly reduced hormonal receptor transcriptional activity. The translation of these drug mechanisms into DMD model mice improves muscle strength, live‐imaging and pathology through both preventive and post‐onset intervention regimens. These data demonstrate successful improvement of dystrophy independent of hormonal, growth, or immunosuppressive effects, indicating VBP15 merits clinical investigation for DMD and would benefit other chronic inflammatory diseases.

Disorders of sex development: Insights from targeted gene sequencing of a large international patient cohort

BackgroundDisorders of sex development (DSD) are congenital conditions in which chromosomal, gonadal, or phenotypic sex is atypical. Clinical management of DSD is often difficult and currently only 13% of patients receive an accurate clinical genetic diagnosis. To address this we have developed a massively parallel sequencing targeted DSD gene panel which allows us to sequence all 64 known diagnostic DSD genes and candidate genes simultaneously.ResultsWe analyzed DNA from the largest reported international cohort of patients with DSD (278 patients with 46,XY DSD and 48 with 46,XX DSD). Our targeted gene panel compares favorably with other sequencing platforms. We found a total of 28 diagnostic genes that are implicated in DSD, highlighting the genetic spectrum of this disorder. Sequencing revealed 93 previously unreported DSD gene variants. Overall, we identified a likely genetic diagnosis in 43% of patients with 46,XY DSD. In patients with 46,XY disorders of androgen synthesis and action the genetic diagnosis rate reached 60%. Surprisingly, little difference in diagnostic rate was observed between singletons and trios. In many cases our findings are informative as to the likely cause of the DSD, which will facilitate clinical management.ConclusionsOur massively parallel sequencing targeted DSD gene panel represents an economical means of improving the genetic diagnostic capability for patients affected by DSD. Implementation of this panel in a large cohort of patients has expanded our understanding of the underlying genetic etiology of DSD. The inclusion of research candidate genes also provides an invaluable resource for future identification of novel genes.

Selective modulation through the glucocorticoid receptor ameliorates muscle pathology in mdx mice

The over‐expression of NF‐κB signalling in both muscle and immune cells contribute to the pathology in dystrophic muscle. The anti‐inflammatory properties of glucocorticoids, mediated predominantly through monomeric glucocorticoid receptor inhibition of transcription factors such as NF‐κB (transrepression), are postulated to be an important mechanism for their beneficial effects in Duchenne muscular dystrophy. Chronic glucocorticoid therapy is associated with adverse effects on metabolism, growth, bone mineral density and the maintenance of muscle mass. These detrimental effects result from direct glucocorticoid receptor homodimer interactions with glucocorticoid response elements of the relevant genes. Compound A, a non‐steroidal selective glucocorticoid receptor modulator, is capable of transrepression without transactivation. We confirm the in vitro NF‐κB inhibitory activity of compound A in H‐2Kb‐tsA58 mdx myoblasts and myotubes, and demonstrate improvements in disease phenotype of dystrophin deficient mdx mice. Compound A treatment in mdx mice from 18 days of post‐natal age to 8 weeks of age increased the absolute and normalized forelimb and hindlimb grip strength, attenuated cathepsin‐B enzyme activity (a surrogate marker for inflammation) in forelimb and hindlimb muscles, decreased serum creatine kinase levels and reduced IL‐6, CCL2, IFNγ, TNF and IL‐12p70 cytokine levels in gastrocnemius (GA) muscles. Compared with compound A, treatment with prednisolone, a classical glucocorticoid, in both wild‐type and mdx mice was associated with reduced body weight, reduced GA, tibialis anterior and extensor digitorum longus muscle mass and shorter tibial lengths. Prednisolone increased osteopontin (Spp1) gene expression and osteopontin protein levels in the GA muscles of mdx mice and had less favourable effects on the expression of Foxo1, Foxo3, Fbxo32, Trim63, Mstn and Igf1 in GA muscles, as well as hepatic Igf1 in wild‐type mice. In conclusion, selective glucocorticoid receptor modulation by compound A represents a potential therapeutic strategy to improve dystrophic pathology. Copyright

The association between ketoacidosis and 25(OH)-vitamin D-3 levels at presentation in children with type 1 diabetes mellitus

Background:  There is considerable evidence supporting the role of vitamin D deficiency in the pathogenesis of type 1 diabetes mellitus (T1DM). Vitamin D deficiency is also associated with impairment of insulin synthesis and secretion. There have been no formal studies looking at the relationship between 25(OH)‐vitamin D3 and the severity of diabetic ketoacidosis (DKA) in children at presentation with T1DM.

Omigapil Treatment Decreases Fibrosis and Improves Respiratory Rate in dy2J Mouse Model of Congenital Muscular Dystrophy

Introduction Congenital muscular dystrophy is a distinct group of diseases presenting with weakness in infancy or childhood and no current therapy. One form, MDC1A, is the result of laminin alpha-2 deficiency and results in significant weakness, respiratory insufficiency and early death. Modification of apoptosis is one potential pathway for therapy in these patients. Methods dy2J mice were treated with vehicle, 0.1 mg/kg or 1 mg/kg of omigapil daily via oral gavage over 17.5 weeks. Untreated age matched BL6 mice were used as controls. Functional, behavioral and histological measurements were collected. Results dy2J mice treated with omigapil showed improved respiratory rates compared to vehicle treated dy2J mice (396 to 402 vs. 371 breaths per minute, p<0.03) and similar to control mice. There were no statistical differences in normalized forelimb grip strength between dy2J and controls at baseline or after 17.5 weeks and no significant differences seen among the dy2J treatment groups. At 30–33 weeks of age, dy2J mice treated with 0.1 mg/kg omigapil showed significantly more movement time and less rest time compared to vehicle treated. dy2J mice showed normal cardiac systolic function throughout the trial. dy2J mice had significantly lower hindlimb maximal (p<0.001) and specific force (p<0.002) compared to the control group at the end of the trial. There were no statistically significant differences in maximal or specific force among treatments. dy2J mice treated with 0.1 mg/kg/day omigapil showed decreased percent fibrosis in both gastrocnemius (p<0.03) and diaphragm (p<0.001) compared to vehicle, and in diaphragm (p<0.013) when compared to 1 mg/kg/day omigapil treated mice. Omigapil treated dy2J mice demonstrated decreased apoptosis. Conclusion Omigapil therapy (0.1 mg/kg) improved respiratory rate and decreased skeletal and respiratory muscle fibrosis in dy2J mice. These results support a putative role for the use of omigapil in laminin deficient congenital muscular dystrophy patients.

The effects of MyD88 deficiency on disease phenotype in dysferlin-deficient A/J mice: Role of endogenous TLR ligands

An absence of dysferlin leads to activation of innate immune receptors such as Toll‐like receptors (TLRs) and skeletal muscle inflammation. Myeloid differentiation primary response gene 88 (MyD88) is a key mediator of TLR‐dependent innate immune signalling. We hypothesized that endogenous TLR ligands released from the leaking dysferlin‐deficient muscle fibres engage TLRs on muscle and immune cells and contribute to disease progression. To test this hypothesis, we generated and characterized dysferlin and MyD88 double‐deficient mice. Double‐deficient mice exhibited improved body weight, grip strength, and maximum muscle contractile force at 6–8 months of age when compared to MyD88‐sufficient, dysferlin‐deficient A/J mice. Double‐deficient mice also showed a decrease in total fibre number, which contributed to the observed increase in the number of central nuclei/fibres. These results indicate that there was less regeneration in the double‐deficient mice. We next tested the hypothesis that endogenous ligands, such as single‐stranded ribonucleic acids (ssRNAs), released from damaged muscle cells bind to TLR‐7/8 and perpetuate the disease progression. We found that injection of ssRNA into the skeletal muscle of pre‐symptomatic mice (2 months old) resulted in a significant increase in degenerative fibres, inflammation, and regenerating fibres in A/J mice. In contrast, characteristic histological features were significantly decreased in double‐deficient mice. These data point to a clear role for the TLR pathway in the pathogenesis of dysferlin deficiency and suggest that TLR‐7/8 antagonists may have therapeutic value in this disease. Copyright

VBP15, a glucocorticoid analogue, is effective at reducing allergic lung inflammation in mice.

Asthma is a chronic inflammatory condition of the lower respiratory tract associated with airway hyperreactivity and mucus obstruction in which a majority of cases are due to an allergic response to environmental allergens. Glucocorticoids such as prednisone have been standard treatment for many inflammatory diseases for the past 60 years. However, despite their effectiveness, long-term treatment is often limited by adverse side effects believed to be caused by glucocorticoid receptor-mediated gene transcription. This has led to the pursuit of compounds that retain the anti-inflammatory properties yet lack the adverse side effects associated with traditional glucocorticoids. We have developed a novel series of steroidal analogues (VBP compounds) that have been previously shown to maintain anti-inflammatory properties such as NFκB-inhibition without inducing glucocorticoid receptor-mediated gene transcription. This study was undertaken to determine the effectiveness of the lead compound, VBP15, in a mouse model of allergic lung inflammation. We show that VBP15 is as effective as the traditional glucocorticoid, prednisolone, at reducing three major hallmarks of lung inflammation—NFκB activity, leukocyte degranulation, and pro-inflammatory cytokine release from human bronchial epithelial cells obtained from patients with asthma. Moreover, we found that VBP15 is capable of reducing inflammation of the lung in vivo to an extent similar to that of prednisone. We found that prednisolone–but not VBP15 shortens the tibia in mice upon a 5 week treatment regimen suggesting effective dissociation of side effects from efficacy. These findings suggest that VBP15 may represent a potent and safer alternative to traditional glucocorticoids in the treatment of asthma and other inflammatory diseases.

Benefit of Early Commencement of Growth Hormone Therapy in Children with Prader-Willi Syndrome

Prader-Willi syndrome (PWS) is a chromosomal disorder and growth failure is a common presentation. Growth hormone (GH) treatment is beneficial in PWS although the optimal age for starting GH is unknown. We investigated whether GH response in PWS was associated with the age of GH commencement by comparing 16 children who commenced GH before 3 years of age (early group) with 40 children who commenced GH after 3 years of age (late group) from the Ozgrow database. Height SDS, body mass index (BMI) SDS, bone age (BA)-chronological age (CA) ratio, change in height (delta Ht) SDS and change in BMI during 4 years of GH treatment were compared between the groups. The early group had better height SDS and delta Ht SDS. BA delay was more pronounced in the early group but BA did not mature beyond CA with GH therapy in either group. Although the initial GH dose for the early group was lower than that of the late group, the former had better height outcome. The starting GH dose seen in the database is lower than the dose used by international centres.

Effects of dantrolene therapy on disease phenotype in dystrophin deficient mdx mice

Dystrophin deficiency causes contraction-induced injury and damage to the muscle fiber, resulting in sustained increase in intracellular calcium levels, activation of calcium-dependent proteases and cell death. It is known that the Ryanodine receptor (RyR1) on the sarcoplasmic reticular (SR) membrane controls calcium release. Dantrolene, an FDA approved skeletal muscle relaxant, inhibits the release of calcium from the SR during excitation-contraction and suppresses uncontrolled calcium release by directly acting on the RyR complex to limit its activation. This study examines whether Dantrolene can reduce the disease phenotype in the mdx mouse model of muscular dystrophy. We treated mdx mice (4 weeks old) with daily intraperitoneal injections of 40mg/kg of Dantrolene for 6 weeks and measured functional (grip strength, in vitro force contractions), behavioral (open field digiscan), imagining (optical imaging for inflammation), histological (H&E), and molecular (protein and RNA) endpoints in a blinded fashion. We found that treatment with Dantrolene resulted in decreased grip strength and open field behavioral activity in mdx mice. There was no significant difference in inflammation either by optical imaging analysis of cathepsin activity or histological (H&E) analysis. In vitro force contraction measures showed no changes in EDL muscle-specific force, lengthening-contraction force deficit, or fatigue resistance. We found Dantrolene treatment significantly reduces serum CK levels. Further, Dantrolene-treated mice showed decreased SERCA1 but not RyR1 expression in skeletal muscle. These results suggest that Dantrolene treatment alone has no significant beneficial effects at the tested doses in young mdx mice.

Urine metabonomic profiling of a female adolescent with PIT-1 mutation before and during growth hormone therapy: insights into the metabolic effects of growth hormone.

OBJECTIVE Growth hormone (GH) is a protein hormone with important roles in growth and metabolism. The objective of this study was to investigate the metabolism of a human subject with severe GH deficiency (GHD) due to a PIT-1 gene mutation and the metabolic effects of GH therapy using Nuclear Magnetic Resonance (NMR)-based metabonomics. NMR-based metabonomics is a platform that allows the metabolic profile of biological fluids such as urine to be recorded, and any alterations in the profile modulated by GH can potentially be detected. DESIGN Urine samples were collected from a female subject with severe GHD before, during and after GH therapy, and from healthy age- and sex-matched controls and analysed with NMR-based metabonomics. SETTING The samples were collected at a hospital and the study was performed at a research facility. PARTICIPANTS We studied a 17 year old female adolescent with severe GHD secondary to PIT-1 gene mutation who had reached final adult height and who had ceased GH therapy for over 3 years. The subject was subsequently followed for 5 years with and without GH therapy. Twelve healthy age-matched female subjects acted as control subjects. INTERVENTION The GH-deficient subject re-commenced GH therapy at a dose of 1 mg/day to normalise serum IGF-1 levels. MAIN OUTCOME MEASURES Urine metabolic profiles were recorded using NMR spectroscopy and analysed with multivariate statistics to distinguish the profiles at different time points and identify significant metabolites affected by GH therapy. RESULTS NMR-based metabonomics revealed that the metabolic profile of the GH-deficient subject altered with GH therapy and that her profile was different from healthy controls before, and during withdrawal of GH therapy. CONCLUSION This study illustrates the potential use of NMR-based metabonomics for monitoring the effects of GH therapy on metabolism by profiling the urine of GH-deficient subjects. Further controlled studies in larger numbers of GH-deficient subjects are required to determine the clinical benefits of NMR-based metabonomics in subjects receiving GH therapy.