Search

Psoriasis is a complex human-specific disease characterized by perturbed keratinocyte proliferation and a pro-inflammatory environment in the skin. Porcine skin architecture and immunity are very similar to that in humans, rendering the pig a suitable animal model for studying the biology and treatment of psoriasis. Expression of integrins, which is normally confined to the basal layer of the epidermis, is maintained in suprabasal keratinocytes in psoriatic skin, modulating proliferation and differentiation as well as leukocyte infiltration. Here, we generated minipigs co-expressing integrins α2 and β1 in suprabasal epidermal layers. Integrin-transgenic minipigs born into the project displayed skin phenotypes that correlated with the number of inserted transgenes. Molecular analyses were in good concordance with histological observations of psoriatic hallmarks, including hypogranulosis and T-lymphocyte infiltration. These findings mark the first creation of minipigs with a psoriasiform phenotype resembling human psoriasis and demonstrate that integrin signaling plays a key role in psoriasis pathology., Summary: A cloned porcine disease model to advance topical treatment in the debilitating skin disorder psoriasis.

Leptin is an adipocyte-derived cytokine secreted mostly by adipose tissue. Serum leptin levels are elevated in obese individuals and correlate positively with body mass index (BMI). Interestingly, serum leptin levels are also elevated in psoriasis patients and correlate positively with disease severity. Psoriasis is associated with obesity; psoriasis patients have a higher incidence of obesity and obese individuals have a higher risk of developing psoriasis. Additionally, obese psoriasis patients experience a more severe degree of psoriasis. In this study, we hypothesised that leptin may link psoriasis and obesity and plays an aggravating role in psoriasis. To investigate leptin's role in psoriasis, we applied the widely accepted imiquimod (IMQ) induced psoriasis-like skin inflammation mouse model on leptin-deficient (ob/ob) mice and evaluated psoriasis severity. Moreover, we stimulated human keratinocytes with leptin and investigated the effect on proliferation and expression of pro-inflammatory proteins. In ob/ob mice, clinical signs of erythema, infiltration and scales in dorsal skin and inflammation in ear skin, as measured by ear thickness, were attenuated compared with wt mice. Moreover, IL-17A and IL-22 mRNA expression levels, as well as increased epidermal thickness were significantly less induced. In vitro, the effect of leptin stimulation on human keratinocytes demonstrated increased proliferation and induced secretion of several pro-inflammatory proteins; two hallmarks of psoriasis. In conclusion, leptin-deficiency attenuated IMQ-induced psoriasis-like skin inflammation in a mouse model and leptin stimulation induced a pro-inflammatory phenotype in human keratinocytes, thus, supporting an aggravating role of leptin in psoriasis. This article is protected by copyright. All rights reserved.

Summary Background Valrubicin is a cytostatic anthracycline analogue, lacking toxicity by skin and tissue contact, and represents a new drug with potential for topical treatment of psoriasis and nonmelanoma skin cancer (NMSC); the beneficial effects have been partly explained by its antiproliferative and proapoptotic characteristics. Objectives To assess the effect of valrubicin on skin inflammation as inflammation also plays a key role in psoriasis and NMSC. Methods The effect of topical valrubicin treatment on skin inflammation in vivo was addressed in skin inflammation mouse models, where 12-O-tetradecanoylphorbol 13-acetate was used to induce irritant contact dermatitis. An acute and a chronic model were included, to investigate the effect of valrubicin in short-term inflammation and in more persistent inflammation. Inflammation-associated ear oedema was evaluated by measuring ear thickness, infiltration of neutrophil cells, and expression of inflammatory cytokines, interleukin (IL)-1β and IL-6. Results Topical valrubicin treatment effectively reduced the inflammatory response in the acute and the chronic models. Conclusions The present data document an anti-inflammatory effect of valrubicin, and may suggest an interesting new role for valrubicin in other debilitating skin diseases in which inflammation is a significant factor.

Summary Background The pathogenesis of hidradenitis suppurativa (HS) is not clearly understood. The nomenclature suggests an important role for the apocrine glands but recent evidence implicates the pilosebaceous unit as a more likely candidate to play a central role in the pathogenesis. Objectives Our aim was to estimate the volume of the follicular epithelium, the follicular lumen and the sebaceous glands of patients with HS and healthy controls by means of stereology. Methods Four-millimetre punch biopsies were taken from 21 patients with HS and nine healthy controls, fixed in formalin, embedded in paraffin and stained with haematoxylin and eosin prior to volume estimation using the Cavalieri principle. Results Sebaceous gland tissue could be visualized in only 10 of 15 suitable hair follicle biopsies from patients with HS but was present in all biopsies from healthy controls (P = 0·05) and the mean sebaceous gland volume per follicle was one-seventh of that of healthy controls (P = 0·03). There was no significant difference between patients with HS and healthy controls with regard to follicular epithelium and follicle lumen volume. Conclusions Our results suggest that the absence or reduced volume of the sebaceous gland may play a role in the pathogenesis of HS. The presence of fibrosis suggests that sebaceous glands are obliterated early in the pathogenesis of HS.

Psoriasis xenograft transplantation models where human skin is transplanted onto immune-deficient mice are generally accepted in psoriasis research. Over the last decade, they have been widely employed to screen for new therapeutics with a potential anti-psoriatic effect. However, experimental designs differ in several parameters. Especially, the number of donors and grafts per experimental design varies greatly; numbers that are directly related to the probability of detecting statistically significant drug effects. In this study, we performed a statistical evaluation of the effect of cyclosporine A, a recognized anti-psoriatic drug, to generate a statistical model employable to simulate different scenarios of experimental designs and to calculate the associated statistical study power, defined as the probability of detecting a statistically significant anti-psoriatic drug treatment effect. Results showed that to achieve a study power of 0.8, at least 20 grafts per treatment group and a minimum of five donors should be included in the chosen experimental setting. To our knowledge, this is the first time that study power calculations have been performed to evaluate treatment effects in a psoriasis xenograft transplantation model. This study was based on a defined experimental protocol, thus other parameters such as drug potency, treatment protocol, mouse strain and graft size should, also, be taken into account when designing an experiment. We propose that the results obtained in this study may lend a more quantitative support to the validity of results obtained when exploring new potential anti-psoriatic drug effects.

Quantitative morphological studies of the healthy epidermis are essential in providing a range of parameter estimates that can be considered within the range of normality. Stereology is a set of statistical tools that provides potentially unbiased and precise estimates of 3-dimensional tissue characteristics from 2-dimensional sections. We set out to establish reference values for the volume of the viable epidermis contained within a four-millimetre punch biopsy (Vepi), the volume of the stratum corneum (VSC) and the surface area of the dermo-epidermal junction(ADEJ) in 4 predetermined body regions by use of stereology. Four-millimetre punch biopsies were taken from 20 freshly diseased corpses, fixed in formalin and embedded in paraffin. Vepi, VSC and ADEJ were established stereologically for all 4 body locations followed by pairwise comparison of means after Bonferroni correction. Vepi was significantly larger in the sole compared to all other body locations (p < 0.01). Furthermore, linear regression analysis showed a strong linear relationship between Vepi and VSC in the sole (r = 0.70). Our results suggest that the viable layers of the epidermis might also serve a mechanical function, either directly or by providing the stratum corneum with keratinocytes to support the hyperkeratosis in the weight-bearing parts of the skin.

Udgivelsesdato: 2009-Jun-30 Tumor necrosis factor-alpha (TNF-alpha) is upregulated in psoriatic skin and represents a prominent target in psoriasis treatment. The level of TNF-alpha-encoding mRNA, however, is not increased in psoriatic skin, and it remains unclear whether intervention strategies based on RNA interference (RNAi) are therapeutically relevant. To test this hypothesis the present study describes first the in vitro functional screening of a panel of short hairpin RNAs (shRNAs) targeting human TNF-alpha mRNA and, next, the transfer of the most potent TNF-alpha shRNA variant, as assessed in vitro, to human skin in the psoriasis xenograft transplantation model by the use of lentiviral vectors. TNF-alpha shRNA treatment leads to amelioration of the psoriasis phentotype in the model, as documented by reduced epidermal thickness, normalization of the skin morphology, and reduced levels of TNF-alpha mRNA as detected in skin biopsies 3 weeks after a single vector injection of lentiviral vectors encoding TNF-alpha shRNA. Our data show efficient lentiviral gene delivery to psoriatic skin and therapeutic applicability of anti-TNF-alpha shRNAs in human skin. These findings validate TNF-alpha mRNA as a target molecule for a potential persistent RNA-based treatment of psoriasis and establish the use of small RNA effectors as a novel platform for target validation in psoriasis and other skin disorders.Molecular Therapy (2009); doi:10.1038/mt.2009.141.

Acne is a common skin disease involving colonization with Propionibacterium acnes (P. acnes), hyperproliferation of the follicular epithelium and inflammatory events. Valrubicin is a second-generation anthracycline, non-toxic upon contact, and available in a topical formulation. Valrubicin’s predecessor doxorubicin possesses antibacterial effects and previously we demonstrated that valrubicin inhibits keratinocyte proliferation and skin inflammation suggesting beneficial topical treatment of acne with valrubicin. This study aims to investigate valrubicin’s possible use in acne treatment by testing valrubicin’s antibacterial effects against P. acnes and P. acnes-induced skin inflammation in vitro and in vivo. Valrubicin was demonstrated not to possess antibacterial effects against P. acnes. Additionally, valrubicin was demonstrated not to reduce mRNA and protein expression levels of the inflammatory markers interleukin (IL)-1β, IL-8, and tumor necrosis factor (TNF)-α in vitro in human keratinocytes co-cultured with P. acnes. Moreover, in vivo, valrubicin, applied both topically and intra-dermally, was not able to reduce signs of inflammation in mouse ears intra-dermally injected with P. acnes. Taken together, this study does not support beneficial antibacterial and anti inflammatory effects of topical valrubicin treatment of acne.

Targeting more than one molecule in multifactorial diseases involving several disease mediators may provide improved therapeutic efficacy. Psoriasis is a multifactorial disease in which interleukin (IL)-6 and IL-23 are important disease mediators because they facilitate development of Th17 cells; widely accepted to be associated with psoriasis. To meet the need for new therapeutics, we aimed to create a clinically relevant bispecific drug, by combining the inhibitory properties of anti-IL-6 and anti-IL-23 antibodies, exhibiting high affinity, high stability and the ability to be produced in high yield. The bispecific molecule AZ17 was created by combining high affinity binding domains originating from monoclonal antibodies targeting human IL-6 and IL-23. To allow for high and efficient production, AZ17 was assembled by site-specific bioconjugation from two individual single chain fragment variables that were synthesized separately in Escherichia coli. To improve stability and extend pharmacokinetics, a flexible poly-ethylene glycol molecule was used as linker. In preclinical psoriasis models, AZ17 reduced IL-23-induced ear inflammation and improved psoriasis in a xenograft transplantation model where psoriasis skin is transplanted onto immune-deficient mice. The data presented here suggest AZ17 to be a promising drug candidate in psoriasis and other inflammatory diseases associated with Th17 cell development.

BACKGROUND: The polyphenol resveratrol has anti-inflammatory effects in various cells, tissues, animals and human settings of low-grade inflammation. Psoriasis is a disease of both localized and systemic low-grade inflammation. The Sirtuin1 enzyme thought to mediate the effects of resveratrol is present in skin and resveratrol is known to down regulate NF-κB; an important contributor in the development of psoriasis. Consequently we investigated whether resveratrol has an effect on an Imiquimod induced psoriasis-like skin inflammation in mice and sought to identify candidate genes, pathways and interleukins mediating the effects.METHODS: The study consisted of three treatment groups: A control group, an Imiquimod group and an Imiquimod+resveratrol group. Psoriasis severity was assessed using elements of the Psoriasis Area Severity Index, skin thickness measurements, and histological examination. We performed an RNA microarray from lesional skin and afterwards Ingenuity pathway analysis to identify affected signalling pathways. Our microarray was compared to a previously deposited microarray to determine if gene changes were psoriasis-like, and to a human microarray to determine if findings could be relevant in a human setting.RESULTS: Imiquimod treatment induced a psoriasis-like skin inflammation. Resveratrol significantly diminished the severity of the psoriasis-like skin inflammation. The RNA microarray revealed a psoriasis-like gene expression-profile in the Imiquimod treated group, and highlighted several resveratrol dependent changes in relevant genes, such as increased expression of genes associated with retinoic acid stimulation and reduced expression of genes involved in IL-17 dependent pathways. Quantitative PCR confirmed a resveratrol dependent decrease in mRNA levels of IL-17A and IL-19; both central in developing psoriasis.CONCLUSIONS: Resveratrol ameliorates psoriasis, and changes expression of retinoic acid stimulated genes, IL-17 signalling pathways, IL-17A and IL-19 mRNA levels in a beneficial manner, which suggests resveratrol, might have a role in the treatment of psoriasis and should be explored further in a human setting.

Osteoblasts and adipocytes share a common precursor cell in the bone marrow stroma, termed marrow stromal cell (MSC). As the volume of bone adipose tissue increases in vivo with age, we hypothesized that decreased bone formation observed during aging and in patients with osteoporosis (OP) is the result of enhanced adipogenesis and decreased osteoblastogenesis from the MSCs. Thus, cultures of MSCs were established from young donors (age 18-42, n = 34), elderly healthy donors (age 66-78, n = 20), and patients with OP (age 58-76, n = 15). Cells were cultured for 2 weeks in an adipogenic medium (containing 15% horse serum and 100 nM dexamethasone), osteogenic medium (containing 10% fetal calf serum [FCS] and 10 nM calcitriol), or control medium (10% FCS). The MSCs were identified by their abilities to form colonies. Total number of colonies, osteoblastic colonies stained positive for alkaline phosphatase (AP+), and adipocytic colonies containing adipocytes (Ad+) were quantitated. In addition, steady state mRNA levels of gene markers of adipocytic and osteoblastic phenotypes were determined using reverse-transcriptase polymerase chain reaction (RT-PCR). The adipogenic and osteogenic media induced cell differentiation and the expression of adipocytic and osteoblastic lineage-specific markers, respectively. We found no age-related changes in the osteoblastic or adipocytic colony formation or the steady state levels of mRNA of the adipogenic or osteogenic gene markers. Cells obtained from patients with OP showed a pattern of differentiation similar to those of age-matched controls. In conclusion, MSCs maintain their differentiation potential during aging and in patients with OP. Other mechanisms responsible for age-related decrease in bone formation need to be determined.

Debio 0932 is a novel oral heat shock protein 90 (Hsp90) inhibitor developed for anti-cancer therapy. Surprising-ly, during the first clinical trial, one psoriasis patient experienced complete remission of his skin manifestation. However, a possible therapeutic utility of Hsp90 in psoriasis has not previously been reported. The objective of the present study was to explore the ability of Debio 0932 to alleviate psoriasis in a preclinical model. A psoriasis xenograft transplantation model was employed where skin from 5 psoriasis patients was transplanted onto immunodeficient mice (8 xenografts per donor). Debio 0932 was administered perorally daily for 3 weeks and resulted in significant clinical alleviation of psoriasis by day 11 and reduced epidermal thickness evaluated post-treatment. Alleviation of psoriasis in the psoriasis xenograft transplantation model, which may be due to Hsp90's involvement in signalling pathways that are up-regulated in psoriasis, substantiates a potential role of Debio 0932 in psoriasis treatment.

Aging of the human skeleton is characterized by decreased boneformation and bone mass and these changes are more pronounced inpatients with osteoporosis. As osteoblasts and adipocytes share a commonprecursor cell in the bone marrow, we hypothesized that decreased boneformation observed during aging and in patients with osteoporosis is theresult of enhanced adipognesis versus osteoblastogenesis from precursorcells in the bone marrow. Thus, we examined iliac crest bone biopsiesobtained from 53 healthy normal individuals (age 30–100) and 26patients with osteoporosis (age 52–92). Adipose tissue volumefraction (AV), hematopoietic tissue volume fraction (HV) and trabecularbone volume fraction (BV) were quantitated as a percentage of totaltissue volume fraction (TV) (calculated as BV + AV + HV) usingthe point-counting method. We found an age-related increase in AV/TV(r = 0.53, p < 0.001, n =53) and an age-related decline in BV/TV (r =−0.46, p < 0.001, n = 53) as well asin the HV/TV (r = −0.318, p

Valrubicin is a semisynthetic anthracycline developed as an anti-cancer drug able to ameliorate psoriasis and non-melanoma skin cancer (NMSC) by topical application in animal models. Valrubicin decreases cell proliferation, and induces apoptosis; however its mode of action is still unknown. Valrubicin localizes in the cytoplasm and its valerate moiety resembles diacylglycerol, an activator of protein kinase C (PKC) α, which belongs to the PKC family of cytoplasmic serine/threonine protein kinases. PKCα is observed in the suprabasal layers of normal skin and is associated to keratinocyte growth arrest and differentiation processes. In hyper-proliferative skin diseases the presence of PKCα is altered.The aim of the present study was to investigate valrubicin's mode of action in keratinocytes by studying its possible effect on PKCα activation.PKCα's characteristic to translocate from the cytoplasm to the cellular membrane when activated was assessed by measuring the amount of PKCα in the soluble and membrane-bound protein fractions isolated from valrubicin stimulated keratinocytes and by visualizing PKCα in stimulated cells over time. Downstream signaling was investigated by measuring the amount of phosphorylated Myristoylated Alanine-rich C-kinase substrate (MARCKS) and extracellular signal-regulated kinases (ERK) 1/2 of valrubicin-stimulated keratinocytes. To investigate whether there was a direct interaction between valrubicin and PKCα, an activity assay employing purified PKCα protein was used.Valrubicin activates PKCα in vitro as shown by PKCα's translocation and phosphorylation of downstream signaling molecules.Valrubicin stimulates PKCα activity and downstream signaling which may contribute to its beneficial effect in psoriasis and NMSC.

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.

Psoriasis is a very common chronic skin disease, affecting 2-3% of the world's population or more than 125 million individuals worldwide. The characteristic lesion of psoriasis is due to rapid proliferation and shortened transition of keratinocytes through the epidermis. Proinflammatory white blood cells (WBCs) migrate into the psoriatic plaques, and the pathogenic cytokine environment causes the changes in keratinocyte proliferation and differentiation. Enhanced migration of WBCs is due to the upregulation and activation of adhesion molecules such as leukocyte function antigen-1 (LFA-1), which binds intercellular adhesion molecule-1 (ICAM-1) on endothelial cells. Targeting LFA-1 and preventing interaction with ICAM-1 has proven an effective strategy for treating psoriasis. We show here that a natural leukocyte-targeting bacterial protein (leukotoxin (LtxA)) that binds LFA-1 can inhibit proliferation of activated WBCs from psoriasis patients and demonstrates significant therapeutic efficacy in a psoriasis xenograft transplantation model. In ex vivo studies, LtxA preferentially targeted proinflammatory WBC subtypes, including activated CD25(+) T cells and CD14(+)CD16(+) monocytes. LFA-1 has been shown to have a significant role in the pathogenesis of numerous autoimmune and inflammatory diseases, and we propose that LtxA may be a highly effective agent for treating these diseases.

Psoriasis xenograft transplantation models where human skin is transplanted onto immune-deficient mice are generally accepted in psoriasis research. Over the last decade, they have been widely employed to screen for new therapeutics with a potential anti-psoriatic effect. However, experimental designs differ in several parameters. Especially, the number of donors and grafts per experimental design varies greatly; numbers that are directly related to the probability of detecting statistically significant drug effects. In this study, we performed a statistical evaluation of the effect of cyclosporine A, a recognized anti-psoriatic drug, to generate a statistical model employable to simulate different scenarios of experimental designs and to calculate the associated statistical study power, defined as the probability of detecting a statistically significant anti-psoriatic drug treatment effect. Results showed that to achieve a study power of 0.8, at least 20 grafts per treatment group and a minimum of five donors should be included in the chosen experimental setting. To our knowledge, this is the first time that study power calculations have been performed to evaluate treatment effects in a psoriasis xenograft transplantation model. This study was based on a defined experimental protocol, thus other parameters such as drug potency, treatment protocol, mouse strain and graft size should, also, be taken into account when designing an experiment. We propose that the results obtained in this study may lend a more quantitative support to the validity of results obtained when exploring new potential anti-psoriatic drug effects.

Background Psoriasis is a chronic inflammatory skin disorder that shows as erythematous and scaly lesions. The pathogenesis of psoriasis is driven by a dysregulation of the immune system which leads to an altered cytokine production. Proinflammatory cytokines that are up-regulated in psoriasis include tumor necrosis factor alpha (TNFα), interleukin-12 (IL-12), and IL-23 for which monoclonal antibodies have already been approved for clinical use. We have previously documented the therapeutic applicability of targeting TNFα mRNA for RNA interference-mediated down-regulation by anti-TNFα small hairpin RNAs (shRNAs) delivered by lentiviral vectors to xenografted psoriatic skin. The present report aims at targeting mRNA encoding the shared p40 subunit (IL-12B) of IL-12 and IL-23 by cellular transduction with lentiviral vectors encoding anti-IL12B shRNAs. Methods Effective anti-IL12B shRNAs are identified among a panel of shRNAs by potency measurements in cultured cells. The efficiency and persistency of lentiviral gene delivery to xenografted human skin are investigated by bioluminescence analysis of skin treated with lentiviral vectors encoding the luciferase gene. shRNA-expressing lentiviral vectors are intradermally injected in xenografted psoriatic skin and the effects of the treatment evaluated by clinical psoriasis scoring, by measurements of epidermal thickness, and IL-12B mRNA levels. Results Potent and persistent transgene expression following a single intradermal injection of lentiviral vectors in xenografted human skin is reported. Stable IL-12B mRNA knockdown and reduced epidermal thickness are achieved three weeks after treatment of xenografted psoriatic skin with lentivirus-encoded anti-IL12B shRNAs. These findings mimick the results obtained with anti-TNFα shRNAs but, in contrast to anti-TNFα treatment, anti-IL12B shRNAs do not ameliorate the psoriatic phenotype as evaluated by semi-quantitative clinical scoring and by immunohistological examination. Conclusions Our studies consolidate the properties of lentiviral vectors as a tool for potent gene delivery and for evaluation of mRNA targets for anti-inflammatory therapy. However, in contrast to local anti-TNFα treatment, the therapeutic potential of targeting IL-12B at the RNA level in psoriasis is questioned.

Valrubicin is a second generation anthracycline characterized by an excellent safety profile presenting no skin toxicity or necrosis upon contact. In its current liquid formulation (Valstar; Indevus Pharmaceuticals, Lexington, MA), it is approved solely for the treatment of bladder cancer. Recently, valrubicin was incorporated in a cream formulation rendering this drug available for topical application. The cytostatic property of valrubicin can, thus, be employed for treating hyperproliferative skin diseases as was recently described for psoriasis. In the present study, the effect of topical application of valrubicin was investigated in skin tumor development; we hypothesized that valrubicin may be employed in treating actinic keratosis, a hyperproliferative skin condition that may transform into malignancy. A two-stage chemical mouse skin carcinogenesis model that represents the multistage etiology of human skin cancer-from developing papillomas to squamous cell carcinoma (SCC) was used. Moreover, two human skin SCC cell lines: DJM-1 and HSC-1 were cultured, to further investigate the effect of valrubicin in vitro. Cell viability was assessed by adenosine triphosphate presence, proliferation as proliferative cell nuclear antigen expression and apoptosis as cytokeratin 18 cleavage, caspase activation, poly-adenosine diphosphate-ribose-polymerase cleavage and bax and bcl-2 regulation. Valrubicin significantly inhibited tumor formation in the mouse skin carcinogenesis model and significantly decreased cell viability of the cultured human skin SCC cells. In both mouse skin and SCC cells, proliferation was significantly decreased. Apoptosis was significantly increased in SCC cells but unchanged in the treated mouse skin at study completion. This study demonstrated that topical application of valrubicin has a beneficial effect in treating developing skin tumors.

Valrubicin is a cytostatic drug currently approved by the American Federal Drug Administration as a trademarked Valstar sterile solution for the treatment of bladder cancer. Valrubicin has shown an excellent therapeutic potential with minimal toxicity. This study investigated the effect in vivo of treating psoriasis with a daily topical application of valrubicin cream in a psoriasis xenograft transplantation model. Psoriasis is characterized by an accelerated keratinocyte proliferation, resulting in increased epidermal thickness. We thus studied the cytostatic potential of valrubicin on epidermal keratinocytes. In vivo, valrubicin treatment resulted in a normalization of epidermal morphology and a reduction in epidermal thickness after 12 days. In addition, the dermal vessel pattern was reduced and the stratum granulosum was regained. Staining for a regenerative proliferation marker showed a decrease in keratinocyte proliferation, and scattered epidermal cells showed apoptosis. In vitro, valrubicin was shown to localize solely to the cell cytoplasm in cultured keratinocytes and to reduce keratinocyte proliferation as well as increase apoptosis by activation of caspases 3, 7, and 9. Our results indicated that valrubicin successfully treats psoriasis in a xenograft transplantation model, suggesting that topical valrubicin may become an upcoming treatment for psoriasis.

Stereology is a set of mathematical and statistical tools to estimate three-dimensional (3-D) characteristics of objects from regular two-dimensional (2-D) sections. In medicine and biology, it can be used to estimate features such as cell volume, cell membrane surface area, total length of blood vessels per volume tissue and total number of cells. The unbiased quantification of these 3-D features allows for a better understanding of morphology in vivo compared with 2-D methods. This review provides an introduction to the field of stereology with specific emphasis on the application of stereology to dermatological research by supplying a short insight into the theoretical basis behind the technique and presenting previous dermatological studies in which stereology was an integral part. Both the theory supporting stereology and a practical approach in a dermatological setting are reviewed with the aim to provide the reader with the capability to better assess papers employing stereological estimators and to design stereological studies independently.

Udgivelsesdato: 2009-Feb BACKGROUND: Interleukin (IL)-20 is a recently discovered cytokine displaying increased levels in psoriatic lesions. Interestingly, IL-20 levels decrease with antipsoriatic treatment, correlating with clinical improvement. However, the role of IL-20 in the aetiology of psoriasis is unknown. OBJECTIVES: In this study, we investigate the effects both of blocking IL-20 signalling in psoriatic plaques and of adding IL-20 to nonlesional psoriasis skin. METHODS: We employed the human skin xenograft transplantation model in which psoriatic plaques and nonlesional keratome skin biopsies obtained from donors with moderate to severe plaque psoriasis were transplanted on to immuno-deficient mice. The transplanted mice were treated with anti-IL-20 antibodies or recombinant human IL-20. RESULTS: We demonstrate that blocking IL-20 signalling with anti-IL-20 antibodies induces psoriasis resolution and inhibits psoriasis induction. We also demonstrate that continuous IL-20 infusion, together with injection of additional nonactivated leucocytes, promotes induction of psoriasis in nonlesional skin from patients with psoriasis. CONCLUSIONS: The results suggest that IL-20 plays a critical role in the induction and maintenance of psoriasis, and IL-20 is suggested as a new possible specific target in psoriasis treatment.

Interleukin-20 (IL-20) is suggested as a new target in psoriasis treatment. It was first described in 2001, and the potential role of this cytokine in psoriasis was suggested because mice which were over-expressing IL-20 developed a psoriasis-like phenotype of the skin. Subsequently, IL-20 expression levels were found to be increased in psoriasis skin, and it was observed that these levels normalized upon psoriasis treatment. In the psoriasis xenograft transplantation model, administration of IL-20 to non-lesional psoriasis skin transplanted onto immune-deficient mice demonstrated that IL-20 was involved in the psoriasis induction. More interestingly, improvement of psoriasis was induced by blocking IL-20 signaling.

The proinflammatory cytokine Tumor Necrosis Factor alpha (TNF-) is upregulated in inflammatory psoriatic skin. The increased level of TNF- protein is thought to cause keratinocyte hyperproliferation, leukocyte infiltration as well as growth and dilation of superficial blood vessels, which are all characteristics of human psoriasis skin. Blockade of TNF- function with specific inhibitors at the protein level has resulted in a rapid clinical improvement in psoriasis patients, demonstrating that TNF- inhibition offers a promising therapy of psoriasis. Whether TNF--encoding RNA is a valid therapeutic target, however, is still a matter of speculation, as recent findings have suggested that the level of TNF- is not increased in psoriatic skin. To test the hypothesis that TNF- in skin can be stably down-regulated by RNA interference (RNAi), we designed a panel of short hairpin RNAs (shRNAs) targeting human TNF-. Their efficiency in down-regulating TNF- protein expression was evaluated using a Renilla luciferase screening-assay based upon targeting of luc-TNF- fusion RNAs and a transient co-transfection assay. The three most potent shRNAs, which at most reduced the expression from target templates to 15% of the control samples treated with irrelevant shRNAs, were selected and cloned into lentiviral vectors. The lentiviral vectors expressing TNF- shRNAs were used to transduce HEK-293 cells and verify vector-derived knock-down of stable TNF- expression in vitro. The most efficient TNF--directed shRNA, which in cell lines reduced the amount of released TNF- more than 50% upon viral transduction, was selected for in vivo studies. In vivo studies were carried out in a xenograft mouse model in which human psoriatic plaques keratome skin biopsies were transplanted onto SCID mice. Initial studies using eGFP-encoding lentiviral vectors demonstrated efficient transduction of human psoriatic skin. Grafted psoriatic skin was exposed to viral vector-encoded TNF- shRNAs by a single intradermal injection of purified VSV-G-pseudotyped lentiviral vectors (150l containing 46.4 ng p24/l was injected at a single site). Biopsies were taken three weeks after injection. qPCR-based measurements of TNF- mRNA in skin treated with lentiviral vector-encoded TNF- shRNA demonstrated a 50% reduction in the level of TNF- mRNA. Most interestingly, the epidermal thickness of the human psoriatic plaques was reduced relative to mice treated with lentiviral vectors encoding an irrelevant shRNA. In conclusion, our results demonstrate that lentiviral vector-encoded TNF- shRNAs have the potential to down-regulate TNF- production both in vitro and in vivo. Phenotypic changes in shRNA-treated psoriatic skin suggest that TNF--encoding RNA is a valid therapeutic target in psoriasis treatment.

Interleukin-20 (IL-20) is a new member of the IL-10 cytokine family discovered by a structural algorithm. IL-20 transgenic mice displayed skin abnormalities reminiscent of psoriasis, a finding that has prompted the investigation of this new interleukin in relation to this disease. This article reviews the role of IL-20 and its implication in psoriasis. It is shown that IL-20 and its receptors are found in human skin and that IL-20 is involved in proliferation, angiogenesis, and chemotaxis, all characteristics of psoriasis. We demonstrated that IL-20 induced the thickening of human epidermis in vivo; however, this thickening does not seem to be related to a direct effect of IL-20 on hyperproliferation since the growth of normal human epidermal keratinocytes (NHEKs) cultured in vitro was not affected by IL-20. On the other hand, in vitro, IL-20 stimulated human peripheral blood mononuclear cells (PBMCs) to produce proinflammatory cytokines and, in vivo, IL-20 in combination with PBMCs induced psoriasis. This may suggest that IL-20 indirectly exerts its proliferative effects on keratinocytes via immune cells present in the skin. Finally, we found that blocking IL-20 signaling in psoriasis improves psoriasis, suggesting that IL-20 is a potential target in psoriasis treatment.

Udgivelsesdato: 2005-May-13 Engraftment of mesenchymal stem cells (MSC) in peripheral tissues for replenishing of local stem cell function has been proposed as a therapeutic approach to degenerative diseases. We have previously reported the development of an immortalized human telomerase reverse transcriptase transduced MSC line (hMSC-TERT). In the present study, we co-transduced hMSC-TERT with enhanced green fluorescent protein gene, and studied tissue distribution, engraftment, and cell survival after intracardiac and intravenous injections in immunodeficient mice. The pattern of organ distribution suggested that infused cells were efficiently arrested in microvasculature during first-pass, but only for a fraction of the infused cells was arrest followed by vascular emigration and tissue engraftment. Few engrafted cells in lungs, heart, and kidney glomeruli remained after 4 weeks. These observations are consistent with several reports on limited systemic transplantability of primary MSC. HMSC-TERT may constitute a valuable tool for mechanistic studies on how to control MSC homing and engraftment.

Interconversion of bone marrow osteoblasts and adipocytes has been reported previously. However, the osteogenic potential of extramedullary adipocytes is not known. Thus, we incubated a pure culture of human subcutaneous adipocytes in control medium for 1-2 weeks. Afterward, the cells were incubated in either osteoblast medium (OB medium) containing various combinations of calcitriol, dexamethasone, ascorbic acid, and beta-glycerophosphate or in adipocyte medium (AD medium) containing HEPES, biotin, pantothenate, insulin, triiodothyronine, dexamethasone, and isobutylmethylxanthine for 4 weeks. Expression of osteoblastic and adipocytic phenotypes was examined by determination of lineage-specific mRNA markers and in vitro adipocyte and osteoblast formation. Cells were also implanted, mixed with hydroxyapatite-tricalcium phosphate powder, in the subcutaneous tissue of immunodeficient mice in order to assess in vivo bone formation potential. One week after incubation in control medium, cells formed fusiform elongated fibroblast-like cells. In OB medium, cells stained positive for alkaline phosphatase (AP) and expressed mRNAs encoding Cbfa1/Runx2, AP, and osteocalcin. In AD medium cells reacquired adipocyte morphology with multilocular lipid-filled cells. Also, the cells expressed adipocyte-specific mRNA markers: lipoprotein lipase and peroxisome proliferator-activated receptor gamma2. Bone was formed only in the in vivo implants of cells incubated in OB medium. In conclusion, extramedullary adipocytes can transdifferentiate to bone-forming cells. Because of their ease of isolation, adipocytes may be good candidates for tissue-engineering protocols aimed at creating bone tissue for the repair of nonunion fractures and large bone defects.

Age-related decreased osteoblast function is a well-known but poorly understood phenomenon. Previous studies that examined the effects of donor age on osteoblast functions employed in vitro assays that may not reflect the true osteoblast capacity for bone formation. Thus, we have developed an in vivo assay for quantifying the bone forming capacity (BFC) and we compared the BFC of osteoblastic cells obtained from young and old donors. Osteoblasts were obtained from human bone marrow stromal cell cultures and implanted subcutaneously in immuno-deficient mice (NOD/LtSz- Prkdc(scid)). After 8 weeks, the implants were removed and embedded un-decalcified in methyl methacrylate (MMA). Sections were stained histochemically with Goldner's Trichrome stain and immuno-histochemically using human-specific antibodies against known osteogenic markers. Implanted human marrow stromal cells (hMSC) were able to form bone in vivo. The donor origin of bone was verified using several human-specific antibodies. Dose-response experiments demonstrated that 5 x 10(5) hMSC per implant gave the maximal bone formation after 8 weeks. No difference in BFC was observed between cells obtained from young (24-30 years old; mean age 27 +/- 2 years, n = 5) and old (71-81 years old; mean age 75 +/- 4 years, n = 5) donors. Our study demonstrates that the capacity of hMSC to form bone in vivo is maintained with age and suggests that the observed senescence-associated decrease in bone formation is due to a defect in the bone microenvironment, the nature of which remains to be determined.

Age-related decrease in bone formation is well described. However, the cellular causes are not known. Thus, we have established cultures of bone marrow stromal cells (MSC) from young (aged 18-29 years, n = 6) and old (aged 68-81 years, n = 5) donors. MSC were serially passaged until reaching maximal life span. Cell growth, markers of cellular senescence, and osteogenic and adipogenic potential were determined in early-passage and late-passage cells established from young and old donors. MSC from old donors exhibited a decreased maximal life span compared with cells from young donors (24 +/- 11 population doublings [PD] vs 41 +/- 10 PD, P

Human aging is associated with bone loss leading to bone fragility and increased risk for fractures, a disease known as osteoporosis. Osteoporosis is one of the most prevalent and serious diseases affecting the elderly population and constitutes a major public health problem. The cellular and molecular causes of age-related bone loss are current intensive topic of investigation with the aim of identifying new approaches to abolish its negative effects on the skeleton. The aim of this chapter is to give a review on the current understanding of the contribution of aging of the osteoblasts (the bone forming cells) to the phenomenon of age-related boneloss.

Human bone marrow stromal cells (hMSCs) were stably transduced by a retroviral vector containing the gene for the catalytic subunit of human telomerase (hTERT). Transduced cells (hMSC-TERTs) had telomerase activity, and the mean telomere length was increased as compared with that of control cells. The transduced cells have now undergone more than 260 population doublings (PD) and continue to proliferate, whereas control cells underwent senescence-associated proliferation arrest after 26 PD. The cells maintained production of osteoblastic markers and differentiation potential during continuous subculturing, did not form tumors, and had a normal karyotype. When implanted subcutaneously in immunodeficient mice, the transduced cells formed more bone than did normal cells. These results suggest that ectopic expression of telomerase in hMSCs prevents senescence-associated impairment of osteoblast functions.

Mesenchymal stem cells (MSC) originate from bone marrow and give rise to various cell types, including osteoblasts, chondrocytes, adipocytes, and myocytes. Lineage-specific differentiation is dependent on activation of specific transcription factors. MSCs play an important role in bone modelling and remodelling where they give rise to the osteoblasts necessary for bone formation. Human studies have shown that the number and differentiation potential of MSC are unchanged with age and osteoporosis. In recent years, there has been an increasing interest in the clinical use of MSCs. These have been used to augment healing of bone fractures. Some animal studies have shown that MSCs infused intravenously target bone and possibly participate in bone formation. These studies represent the beginning of a new era, where transplantation with autologous MSCs or genetically-modified MSCs expanded in vitro is a potential treatment strategy to augment bone formation in patients with diverse metabolic and genetic bone diseases, including osteoporosis.

Decreased bone formation is an important pathophysiological mechanism responsible for bone loss associated with aging and osteoporosis. Osteoblasts (OBs), originate from mesenchymal stem cells (MSCs) that are present in the bone marrow and form colonies (termed colony-forming units-fibroblastic [CFU-Fs]) when cultured in vitro. To examine the effect of aging and osteoporosis on the MSC population, we quantified the number of MSCs and their proliferative capacity in vitro. Fifty-one individuals were studied: 38 normal volunteers (23 young individuals [age, 22-44 years] and 15 old individuals [age, 66-74 years]) and 13 patients with osteoporosis (age, 58-83 years). Bone marrow was aspirated from iliac crest; mononuclear cells were enriched in MSCs by magnetic activated cell sorting (MACS) using STRO-1 antibody. Total CFU-F number, size distribution, cell density per CFU-F, number of alkaline phosphatase positive (ALP+) CFU-Fs, and the total ALP+ cells were determined. In addition, matrix mineralization as estimated by alizarin red S (AR-S) staining was quantified. No significant difference in colony-forming efficiency between young individuals (mean +/- SEM; 87 +/- 12 CFU-Fs/culture), old individuals (99 +/- 19 CFU-Fs/culture), and patients with osteoporosis (129 +/- 13 CFU-Fs/culture; p = 0.20) was found. Average CFU-F size and cell density per colony were similar in the three groups. Neither the percentage of ALP+ CFU-Fs (66 +/- 6%, 65 +/- 7%, and 72 +/- 4% for young individuals, old individuals, and patients with osteoporosis, respectively) nor the percentage of ALP+ cells per culture (34 +/- 5%, 40 +/- 6%, and 41 +/- 4%) differed between groups. Finally, mineralized matrix formation was similar in young individuals, old individuals, and patients with osteoporosis. Our study shows that the number and proliferative capacity of osteoprogenitor cells are maintained during aging and in patients with osteoporosis and that other mechanisms must be responsible for the defective osteoblast (OB) functions observed in these conditions.

TNF-α is a well known target in psoriasis treatment and biological treatments targeting TNF-a are already clinically used against psoriasis and psoriasis arthritis. Attention is however given to a novel therapeutic tool: RNA interference that controls gene silencing. This study investigates the efficiency of targeting TNF-a with specific short hairpin RNA (shRNA) and explores its potential in treating psoriasis. ShRNAs targeting human TNF-α mRNA were generated. Their efficiency in down-regulating TNF-a protein expression was evaluated using a Renilla luciferase screening-assay and a transient co-transfection assay. The expression cassette encoding the most efficient TNF-a shRNA was inserted into a lentiviral vector, allowing proficient gene delivery. The lentiviral vector is integrated into the host genome and establishes life-long infection and persistent shRNA expression. The lentiviral vector expressing TNF-a shRNA was used to transduce HEK293 cells and verify vector-derived TNF-a knockdown in vitro. In vivo, psoriasis skin was exposed to lentiviral TNF-a shRNAs by a single intra-dermal injection. Psoriasis skin for the in vivo study was obtained from psoriatic plaque skin biopsies that were transplanted onto SCID mice employing the psoriasis xenograft model. Three weeks after exposure, biopsies were taken and the epidermal thickness as an endpoint for evaluating psoriasis and the levels of TNF-a mRNA were measured. The lentiviral TNF-a shRNAs down-regulated the TNF-a expression in vitro and in vivo by 50% and, most interestingly, the epidermal thickness of the psoriatic plaques was reduced. In conclusion, our results demonstrate that lentiviral TNF-a shRNAs have the potential to down-regulate TNF-a production in vitro and in vivo. The decreased epidermal thickness suggests a potential role for TNF-a shRNAs as a new therapeutic agent in psoriasis treatment.