Thomas K. Petersen

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 sows 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.

Development of Transgenic Cloned Pig Models of Skin Inflammation by DNA Transposon-Directed Ectopic Expression of Human β1 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 β1 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 β1 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 β1 and α2 integrin-transgenic minipigs, suggesting the induction of a chronic inflammatory phenotype in the skin. Notably, cellular dysregulation obtained by overexpression of either β1 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.

Anti-irritants I: Dose-response in acute irritation

The term ‘anti‐irritant’ (AI) was coined in 1965 by Goldemberg to describe a diverse group of topical product ingredients, which were able to reduce the irritation potential of other more irritating ingredients in the same product. ‘AIs’ are being added to cosmetic formulations in order, allegedly, to benefit tolerability of the products and allow claims such as ‘soothing’ and ‘healing’ ingredients. Limited documentation in favour of the efficacy of AIs is published. We studied the dose‐related effect of 4 alleged AIs (nifedipine, (–)‐α‐bisabolol, canola oil and glycerol) on experimentally induced acute irritation in healthy volunteers. Each AI was used in 3 concentrations. Acute irritation was induced by occlusive tests with 1% sodium lauryl sulfate and 20% nonanoic acid in N‐propanol. The irritant reactions were treated twice daily with AI‐containing formulations from the time of removal of the patches. Evaluation of skin irritation and efficacy of treatments were performed daily for 4 days using clinical scoring, evaporimetry (transepidermal water loss), hydration measurement and colourimetry. Only glycerol showed dose–response and effects potentially better than no treatment. There was no significant effect and no difference between the three other AIs.

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.

Anti-irritants II: efficacy against cumulative irritation

So‐called anti‐irritants (AI) are widely used in cosmetic formulations, with the aim of reducing irritation from substances in the formulation. It may also be claimed that they are ‘soothing’ and ‘healing’ ingredients. However, the proof for these claims is circumstantial. The dose–response effect of 4 alleged AI (nifedipine, (‐)‐α‐bisabolol, canola oil and glycerol) was studied on experimentally induced acute irritation in healthy volunteers, and only glycerol showed dose‐related response and effects potentially better than no treatment. The acute irritation model only allowed a small window of opportunity in which to demonstrate efficacy. Therefore, the effect of AI was studied in a cumulative irritation model by inducing irritant dermatitis with 10 min daily exposures for 5 + 4 days (no irritation on weekend) to 1% sodium lauryl sulfate on the right and 20% nonanoic acid on the left volar forearm. AI ointments were applied twice daily. Clinical scoring was performed daily, evaporimetry (Trans Epidermal Water Loss), hydration and colourimetry were measured at baseline (D0), in the middle and at the end of treatment. The glycerol ointment was the only treatment statistically better than both ‘no treatment’ and vehicle.

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

Abstract:  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.

Induced keratinocyte hyper-proliferation in α2β1 integrin transgenic mice results in systemic immune cell activation

alpha2beta1 integrins are normally confined to the proliferating basal layers of the epidermis. However, during wound healing and in psoriasis, these integrins are expressed on keratinocytes in suprabasal layers correlating with a less differentiated phenotype. Transgenic mice expressing alpha2beta1 integrins under the involucrine promoter have previously been demonstrated, to various degrees, spontaneously develop a skin disorder resembling psoriasis. Herein, we show that a mild epidermal wounding induces a uniform acanthosis together with an influx of immune cells. The disease initiates as a normal wound healing process and is completely restored in wildtype mice by day 14. However, in the integrin transgenic mice a chronic inflammation develops, a process that can be compared to the Koebner phenomenon in psoriatic patients. In this study, we have followed the integrin transgenic mice for five weeks, where substantial keratinocyte hyper-proliferation, inflammatory infiltration and high cytokine levels within the skin can still be observed. In addition, draining lymph nodes were dramatically increased in size and contained highly activated T cells, as well as APCs secreting large amounts of pro-inflammatory cytokines. Furthermore, the systemic immune response was affected with increased spleen size, elevated cytokine levels in the serum and altered lymphocyte trafficking patterns, very much resembling what is seen in psoriasis patients. Finally, CD4(+) T cell depletion was not able to affect the onset or progression of skin inflammation. This suggests that altered keratinocyte differentiation and proliferation can drive a skin inflammation and cause chronic immune cell activation both at a local and systemic level.

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

BackgroundAnalogues 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.ResultsThe 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.ConclusionsOur 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.

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.

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.