The ClC-1 chloride channel inhibitor NMD670 improves skeletal muscle function in rat models and patients with myasthenia gravis.

Myasthenia gravis (MG) is a neuromuscular disease that results in compromised transmission of electrical signals at the neuromuscular junction (NMJ) from motor neurons to skeletal muscle fibers. As a result, patients with MG have reduced skeletal muscle function and present with symptoms of severe muscle weakness and fatigue. ClC-1 is a skeletal muscle specific chloride (Cl-) ion channel that plays important roles in regulating neuromuscular transmission and muscle fiber excitability during intense exercise. Here, we show that partial inhibition of ClC-1 with an orally bioavailable small molecule (NMD670) can restore muscle function in rat models of MG and in patients with MG. In severely affected MG rats, ClC-1 inhibition enhanced neuromuscular transmission, restored muscle function, and improved mobility after both single and prolonged administrations of NMD670. On this basis, NMD670 was progressed through nonclinical safety pharmacology and toxicology studies, leading to approval for testing in clinical studies. After successfully completing phase 1 single ascending dose in healthy volunteers, NMD670 was tested in patients with MG in a randomized, placebo-controlled, single-dose, three-way crossover clinical trial. The clinical trial evaluated safety, pharmacokinetics, and pharmacodynamics of NMD670 in 12 patients with mild MG. NMD670 had a favorable safety profile and led to clinically relevant improvements in the quantitative myasthenia gravis (QMG) total score. This translational study spanning from single muscle fiber recordings to patients provides proof of mechanism for ClC-1 inhibition as a potential therapeutic approach in MG and supports further development of NMD670.

A lentiviral vector-based genetic sensor system for comparative analysis of permeability and activity of vitamin D3 analogues in xenotransplanted human skin.

Vitamin D3 analogues are widely used topical and oral remedies for various ailments such as psoriasis, osteoporosis and secondary hyperparathyroidism. In topical treatment, high skin permeability and cellular uptake are key criteria for beneficial effects due to the natural barrier properties of skin. In this study, we wish to establish an in vivo model that allows the comparison of permeability and activity of vitamin D3 analogues in human skin. We generate a bipartite, genetic sensor technology that combines efficient lentivirus-directed gene delivery to xenotransplanted human skin with vitamin D3-induced expression of a luciferase reporter gene and live imaging of animals by bioluminescence imaging. Based on the induction of a transcriptional activator consisting of the vitamin D receptor fused to the Gal4 DNA-binding domain, the vitamin D3-responsive sensor facilitates non-invasive and rapid assessment of permeability and functional properties of vitamin D3 analogues. By topical application of a panel of vitamin D3 analogues onto 'sensorized' human skin, the sensor produces a drug-induced readout with a magnitude and persistence that allow a direct comparative analysis of different analogues. This novel genetic tool has great potential as a non-invasive in vivo screening system for further development and refinement of vitamin D3 analogues.

A Sleeping Beauty DNA transposon-based genetic sensor for functional screening of vitamin D3 analogues.

BACKGROUND: Analogues of vitamin D3 are extensively used in the treatment of various illnesses, such as osteoporosis, inflammatory skin diseases, and cancer. Functional testing of new vitamin D3 analogues and formulations for improved systemic and topical administration is supported by sensitive screening methods that allow a comparative evaluation of drug properties. As a new tool in functional screening of vitamin D3 analogues, we describe a genomically integratable sensor for sensitive drug detection. This system facilitates assessment of the pharmacokinetic and pharmadynamic properties of vitamin D3 analogues. The tri-cistronic genetic sensor encodes a drug-sensoring protein, a reporter protein expressed from an activated sensor-responsive promoter, and a resistance marker. RESULTS: The three expression cassettes, inserted in a head-to-tail orientation in a Sleeping Beauty DNA transposon vector, are efficiently inserted as a single genetic entity into the genome of cells of interest in a reaction catalyzed by the hyperactive SB100X transposase. The applicability of the sensor for screening purposes is demonstrated by the functional comparison of potent synthetic analogues of vitamin D3 designed for the treatment of psoriasis and cancer. In clones of human keratinocytes carrying from a single to numerous insertions of the vitamin D3 sensor, a sensitive sensor read-out is detected upon exposure to even low concentrations of vitamin D3 analogues. In comparative studies, the sensor unveils superior potency of new candidate drugs in comparison with analogues that are currently in clinical use. CONCLUSIONS: Our findings demonstrate the use of the genetic sensor as a tool in first-line evaluation of new vitamin D3 analogues and pave the way for new types of drug delivery studies in sensor-transgenic animals.

Extraction of high-quality epidermal RNA after ammonium thiocyanate-induced dermo-epidermal separation of 4 mm human skin biopsies.

To obtain a separation of the epidermal and dermal compartments to examine compartment specific biological mechanisms in the skin, we incubated 4 mm human skin punch biopsies in ammonium thiocyanate. We wanted to test (i) the histological quality of the dermo-epidermal separation obtained by different incubation times; (ii) the amount and quality of extractable epidermal RNA and (iii) its impact on sample RNA expression profiles assessed by large-scale gene expression microarray analysis in both normal and inflamed skin. At 30-min incubation, the split between dermis and epidermis was not always histologically well-defined (i.e. occurred partly intra-epidermally), but also varied between subjects. Consequently, curettage along the dermal surface of the biopsy was added to the procedure. This modified method resulted in an almost perfect separation of the epidermal and dermal compartments, and satisfactory amounts of high-quality RNA were obtained. Hybridization to Affymetrix HG_U133A 2.0 GeneChips showed that ammonium thiocyanate incubation had a minute effect on gene expression resulting in only one significantly downregulated gene (cystatin E/M). We conclude that epidermis can be reproducibly and almost completely separated from the dermis of 4 mm skin biopsies by 30 min incubation in 3.8% ammonium thiocyanate combined with curettage of the dermal surface, producing high-quality RNA suitable for transcriptional analysis. Our refined method of dermo-epidermal separation will undoubtedly prove valuable in the many different settings, where the epidermal and dermal compartments need to be evaluated separately.

Translational dermatology in drug discovery: perspectives for integrating humanized xenograft models and experimental clinical studies.

Application of humanized xenotransplantation disease models and experimental clinical studies in the context of translational research in drug discovery in dermatology is an opportunity to reduce failure due to lack of efficacy in clinical development stage.

Role of alpha4 integrin and VCAM-1 in CD18-independent neutrophil migration across mouse cardiac endothelium.

Myocardial damage due to reperfusion of ischemic tissue is caused primarily by infiltrating neutrophils. Although leukocyte beta2 integrins (CD18) play a critical role, significant neutrophil emigration persists when CD18 is neutralized or absent. This study examined the role of leukocyte beta1 integrin (alpha4) and its endothelial ligand VCAM-1 in CD18-independent neutrophil migration across cardiac endothelium. In a mouse model of myocardial ischemia and reperfusion, we show that compared with wild-type mice, neutrophil infiltration efficiency was reduced by 50% in CD18-null mice; in both types of mice, myocardial VCAM-1 staining increased after reperfusion. In wild-type mice, antibodies against CD18, ICAM-1 (an endothelial ligand for CD18), or VCAM-1 given 30 minutes before ischemia did not block neutrophil emigration at 3 hours reperfusion. Although anti-VCAM-1 attenuated neutrophil emigration by 90% in CD18-null mice, it did not diminish myocardial injury. To determine if CD18-independent neutrophil emigration was a tissue-specific response, we used isolated peripheral blood neutrophils from wild-type or CD18-null mice and showed neutrophil migration across lipopolysaccharide-activated cultured cardiac endothelium is CD18-independent, whereas migration across endothelium obtained from inferior vena cava is CD18-dependent. Consistent with our in vivo findings, migration of CD18-deficient neutrophils on cardiac endothelial monolayers is blocked by antibodies against alpha4 integrin or VCAM-1. We conclude tissue-specific differences in endothelial cells account, at least partially, for CD18-independent neutrophil infiltration in the heart.

In vivo pharmacological disease models for psoriasis and atopic dermatitis in drug discovery.

In order to perform relevant in vivo pharmacological investigations in drug discovery within dermatology it is fundamental to master or have access to relevant skin disease models that makes it possible to identify and validate targets and to screen and discover drugs in vivo. There is a strong need for highly predictive in vivo models in order to introduce the right drug candidates into the clinical phases of development. Recent advances in dermatological in vivo pharmacological disease models for psoriasis and atopic dermatitis are reviewed together with a discussion of the rationale for their application in drug discovery.

Development of transgenic cloned pig models of skin inflammation by DNA transposon-directed ectopic expression of human ß1 and α2 integrin.

Integrins constitute a superfamily of transmembrane signaling receptors that play pivotal roles in cutaneous homeostasis by modulating cell growth and differentiation as well as inflammatory responses in the skin. Subrabasal expression of integrins α2 and/or ß1 entails hyperproliferation and aberrant differentiation of keratinocytes and leads to dermal and epidermal influx of activated T-cells. The anatomical and physiological similarities between porcine and human skin make the pig a suitable model for human skin diseases. In efforts to generate a porcine model of cutaneous inflammation, we employed the Sleeping Beauty DNA transposon system for production of transgenic cloned Göttingen minipigs expressing human ß1 or α2 integrin under the control of a promoter specific for subrabasal keratinocytes. Using pools of transgenic donor fibroblasts, cloning by somatic cell nuclear transfer was utilized to produce reconstructed embryos that were subsequently transferred to surrogate sows. The resulting pigs were all transgenic and harbored from one to six transgene integrants. Molecular analyses on skin biopsies and cultured keratinocytes showed ectopic expression of the human integrins and localization within the keratinocyte plasma membrane. Markers of perturbed skin homeostasis, including activation of the MAPK pathway, increased expression of the pro-inflammatory cytokine IL-1α, and enhanced expression of the transcription factor c-Fos, were identified in keratinocytes from ß1 and α2 integrin-transgenic minipigs, suggesting the induction of a chronic inflammatory phenotype in the skin. Notably, cellular dysregulation obtained by overexpression of either ß1 or α2 integrin occurred through different cellular signaling pathways. Our findings mark the creation of the first cloned pig models with molecular markers of skin inflammation. Despite the absence of an overt psoriatic phenotype, these animals may possess increased susceptibility to severe skin damage-induced inflammation and should be of great potential in studies aiming at the development and refinement of topical therapies for cutaneous inflammation including psoriasis.

A growth curve model with fractional polynomials for analysing incomplete time-course data in microarray gene expression studies.

Identifying the various gene expression response patterns is a challenging issue in expression microarray time-course experiments. Due to heterogeneity in the regulatory reaction among thousands of genes tested, it is impossible to manually characterize a parametric form for each of the time-course pattern in a gene by gene manner. We introduce a growth curve model with fractional polynomials to automatically capture the various time-dependent expression patterns and meanwhile efficiently handle missing values due to incomplete observations. For each gene, our procedure compares the performances among fractional polynomial models with power terms from a set of fixed values that offer a wide range of curve shapes and suggests a best fitting model. After a limited simulation study, the model has been applied to our human in vivo irritated epidermis data with missing observations to investigate time-dependent transcriptional responses to a chemical irritant. Our method was able to identify the various nonlinear time-course expression trajectories. The integration of growth curves with fractional polynomials provides a flexible way to model different time-course patterns together with model selection and significant gene identification strategies that can be applied in microarray-based time-course gene expression experiments with missing observations.

Genome-wide expression analysis of human in vivo irritated epidermis: differential profiles induced by sodium lauryl sulfate and nonanoic acid.

The pathogenesis of irritant contact dermatitis (ICD) is poorly understood, and genes participating in the epidermal response to chemical irritants are only partly known. It is commonly accepted that different irritants have different mechanisms of action in the development of ICD. To define the differential molecular events induced in the epidermis by different irritants, we collected sequential biopsies ((1/2), 4, and 24 hours after a single exposure and at day 11 after repeated exposure) from human volunteers exposed to either sodium lauryl sulfate (SLS) or nonanoic acid (NON). Gene expression analysis using high-density oligonucleotide microarrays (representing 47,000 transcripts) revealed essentially different pathway responses (1/2)hours after exposure: NON transiently induced the IL-6 pathway as well as a number of mitogen-activated signaling cascades including extracellular signal-regulated kinase and growth factor receptor signaling, whereas SLS transiently downregulated cellular energy metabolism pathways. Differential expression of the cyclooxygenase-2 and matrix metalloproteinase 3 transcripts was confirmed immunohistochemically. After cumulative exposure, 883 genes were differentially expressed, whereas we identified 23 suggested common biomarkers for ICD. In conclusion, we bring new insights into two hitherto less well-elucidated phases of skin irritancy: the very initial as well as the late phase after single and cumulative mild exposures, respectively.

Establishment of a superficial skin infection model in mice by using Staphylococcus aureus and Streptococcus pyogenes.

A new animal model for the purpose of studying superficial infections is presented. In this model an infection is established by disruption of the skin barrier by partial removal of the epidermal layer by tape stripping and subsequent application of the pathogens Staphylococcus aureus and Streptococcus pyogenes. The infection and the infection route are purely topical, in contrast to those used in previously described animal models in mice, such as the skin suture-wound model, where the infection is introduced into the deeper layers of the skin. Thus, the present model is considered more biologically relevant for the study of superficial skin infections in mice and humans. Established topical antibiotic treatments are shown to be effective. The procedures involved in the model are simple, a feature that increases throughput and reproducibility. This new model should be applicable to the evaluation of novel antimicrobial treatments of superficial infections caused by S. aureus and S. pyogenes.

Preterm birth affects the intestinal response to parenteral and enteral nutrition in newborn pigs.

Maturation of gastrointestinal (GI) function in neonates is stimulated by enteral nutrition, whereas parenteral nutrition induces GI atrophy and malfunction. We investigated whether preterm birth alters the GI responses to parenteral and enteral nutrition. Pigs were delivered either preterm (107 d gestation) or at term (115 d gestation) and fed total parenteral nutrition (TPN) or enteral sow's milk (ENT) for 6 d after birth. Immaturity of the preterm pigs was documented by reduced blood pH, oxygen saturation and neutrophil granulocyte function, impaired intestinal immunoglobulin G uptake from colostrum, and altered relative weights of visceral organs (small intestine, liver, spleen, pancreas, and adrenals). For both ages at delivery, increases occurred in pancreatic weight (30-75%) and amylase activity (0.5- to 13-fold) after birth, but much more in ENT than in TPN pigs (P < 0.05). Six days of TPN feeding was associated with reduced intestinal weight for both delivery groups (60% of values in ENT, P < 0.001), but only in term TPN pigs was the weight lower than at birth (-20%, P < 0.05). Likewise, it was only in term TPN pigs that intestinal maltase activity increased, compared with ENT, and the absorption of glucose and proline decreased. Only in preterm pigs did TPN feeding increase lactase activity (+50% compared with ENT, P < 0.05). For both delivery ages, the mRNA of lactase-phloridzin hydrolase and sodium-coupled glucose transporter 1 were increased in TPN, compared with ENT. In conclusion, the trophic effect of enteral vs. parenteral nutrition on the GI tract is also present after preterm birth, but the postnatal maturation of many GI functions is modified, compared with term birth. The effects of nutritional regimen on the maturation of the gut epithelium in neonates depend on gestational age at birth.

Preterm birth affects the intestinal response to parenteral and enteral nutrition in newborn pigs.

Maturation of gastrointestinal (GI) function in neonates is stimulated by enteral nutrition, whereas parenteral nutrition induces GI atrophy and malfunction. We investigated whether preterm birth alters the GI responses to parenteral and enteral nutrition. Pigs were delivered either preterm (107 d gestation) or at term (115 d gestation) and fed total parenteral nutrition (TPN) or enteral sow's milk (ENT) for 6 d after birth. Immaturity of the preterm pigs was documented by reduced blood pH, oxygen saturation and neutrophil granulocyte function, impaired intestinal immunoglobulin G uptake from colostrum, and altered relative weights of visceral organs (small intestine, liver, spleen, pancreas, and adrenals). For both ages at delivery, increases occurred in pancreatic weight (30-75%) and amylase activity (0.5- to 13-fold) after birth, but much more in ENT than in TPN pigs (P < 0.05). Six days of TPN feeding was associated with reduced intestinal weight for both delivery groups (60% of values in ENT, P < 0.001), but only in term TPN pigs was the weight lower than at birth (-20%, P < 0.05). Likewise, it was only in term TPN pigs that intestinal maltase activity increased, compared with ENT, and the absorption of glucose and proline decreased. Only in preterm pigs did TPN feeding increase lactase activity (+50% compared with ENT, P < 0.05). For both delivery ages, the mRNA of lactase-phloridzin hydrolase and sodium-coupled glucose transporter 1 (SGLT-1) were increased in TPN, compared with ENT. In conclusion, the trophic effect of enteral vs. parenteral nutrition on the GI tract is also present after preterm birth, but the postnatal maturation of many GI functions is modified, compared with term birth. The effects of nutritional regimen on the maturation of the gut epithelium in neonates depend on gestational age at birth.