Karin Stenderup

Telomerase expression extends the proliferative life-span and maintains the osteogenic potential of human bone marrow stromal cells.

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.

Number and Proliferative Capacity of Osteogenic Stem Cells Are Maintained During Aging and in Patients with 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.

Hidradenitis suppurativa: a disease of the absent sebaceous gland? Sebaceous gland number and volume are significantly reduced in uninvolved hair follicles from patients with hidradenitis suppurativa

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.

Amelioration of Psoriasis by Anti-TNF-α RNAi in the Xenograft Transplantation Model

Tumor necrosis factor-α (TNF-α) is upregulated in psoriatic skin and represents a prominent target in psoriasis treatment. The level of TNF-α-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-α mRNA and, next, the transfer of the most potent TNF-α shRNA variant, as assessed in vitro, to human skin in the psoriasis xenograft transplantation model by the use of lentiviral vectors. TNF-α 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-α mRNA as detected in skin biopsies 3 weeks after a single vector injection of lentiviral vectors encoding TNF-α shRNA. Our data show efficient lentiviral gene delivery to psoriatic skin and therapeutic applicability of anti-TNF-α shRNAs in human skin. These findings validate TNF-α 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.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.

Interleukin-20 as a target in psoriasis treatment

Abstract:  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.

Resolution of Psoriasis by a Leukocyte-Targeting Bacterial Protein in a Humanized Mouse Model

Psoriasis is a very common chronic skin disease, affecting 2-3% of the worlds 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.

Targeting of human interleukin-12B by small hairpin RNAs in xenografted psoriatic skin.

BackgroundPsoriasis 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.MethodsEffective 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.ResultsPotent 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.ConclusionsOur 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 in a Topical Formulation Treats Psoriasis in a Xenograft Transplantation Model

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.

Application of stereology to dermatological research

Abstract:  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.

Topical application of valrubicin has a beneficial effect on developing skin tumors

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.