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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.

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.

Defensins are potent antimicrobial and proinflammatory peptides. The human neutrophil defensins human neutrophil peptide (HNP)-1–3 are synthesized as 94 amino acide (aa) preproHNPs, which are converted to 75 aa proHNPs by cotranslational removal of a 19 aa endoplasmic reticulum signal peptide. At the promyelocytic stage of myelopoiesis, proHNPs are further proteolytically modified and accumulate in azurophil granules as 29–30 aa HNPs. In contrast, proHNPs produced by more mature myeloid cells are not subjected to proteolytic cleavage and undergo a high degree of constitutive exocytosis. The proHNPs are devoid of antimicrobial potential, and the significance of their secretion is unknown. To investigate whether mature neutrophils contain proHNPs, we developed antibodies against proHNP-1 by DNA immunization of rabbits. In addition, antibodies against the 45 aa proHNP pro-piece were raised by conventional immunization procedures. These antibodies allowed detection of proHNPs in homogenates of peripheral blood neutrophils. The proHNPs were isolated by affinity chromatography, and their identity was confirmed by mass spectrometry and N-terminal aa sequence analysis. Finally, the neutrophil proHNPs were identified as novel matrix proteins of specific granules by subcellular fractionation experiments, release studies, and immunoelectron microscopy. Thus, human neutrophils not only store large amounts of mature defensins in azurophil granules but also contain a more easily mobilized reservoir of unprocessed prodefensins in specific granules.

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.

Prodefensins (proHNPs) are the 74 aa antimicrobially inactive precursor peptides of defensins (HNPs). ProHNPs are synthesized in neutrophil precursors from the late promyelocytic stage and until the band cell stage. In promyelocytes, proHNP is proteolytically cleaved to yield 29–30 aa HNPs that are efficiently stored in primary (azurophil) granules. Beyond the promyelocytic stage, proHNPs are no longer proteolytically processed and significant amounts of proHNP escape the cell through constitutive exocytosis. The fate and biological implications of proHNP constitutively secreted from myeloid cells is unclear. We have raised antibodies against a synthetic propiece of proHNP (45 N-terminal amino acids) and developed an accurate, sensitive and specific ELISA for proHNP using these antibodies, with a detection limit of 4.0 ng/ml. We found levels of pro HNP in bone marrow plasma of 9 μg/ml and levels in blood plasma of 1.7 μg/ml. This indicates that the bone marrow is an important organ for generation of this pro-antibiotic molecule giving rise to plasma concentrations in the same order of magnitude as lysozyme and hCAP-18. Studies of plasma from patients with ph- chronic myeloproliferative disorders showed a mean concentration of 3.5 μg/ml indicating that proHNP is a parameter of myelopoietic activity. ProHNP in plasma was apparently not complexed to higher molecular weight structures, yet it was not present in urine. This indicates that proHNP from bone marrow is processed in circulation.