Neuromusculature of Gyrodactylus rysavyi, a monogenean gill and skin parasite of the catfish Clarias gariepinus

Phalloidin fluorescence technique, enzyme cytochemistry and immunocytochemistry in conjunction with confocal scanning laser microscopy were used for the first time to describe the nervous and muscle systems of the viviparous monogenean parasite, Gyrodactylus rysavyi inhabiting the gills and skin of the Nile catfish Clarias gariepinus. The body wall muscles are composed of an outer layer of circular fibres, an intermediate layer of paired longitudinal fibres and an inner layer of well-spaced bands of diagonal fibres arranged in two crossed directions. The musculature of the pharynx, intestine, reproductive tract and the most prominent muscles of the haptor were also described. Two characteristic muscular pads were found lying in the anterior region of the haptor in close contact with the hamuli. To each one of these pads, a group of ventral extrinsic muscles was connected. The role of this ventral extrinsic muscle in the body movement was discussed. The mechanism operating the marginal hooklets was also discussed. The central nervous system (CNS) consists of paired cerebral ganglia from which three pairs of longitudinal ventral, lateral and dorsal nerve cords arise. The nerve cords are connected at intervals by many transverse connectives. The CNS is better developed ventrally than dorsally or laterally and it has the highest reactivity for all neuroactive substances examined. Both the central and the peripheral nervous system (PNS) are bilaterally symmetrical. Structural and functional correlates of the neuromusculature of the pharynx, haptor and reproductive tracts were explained. The results implicated acetylcholine, FMRFamide-related peptides (FaRPs) and serotonin in sensory and motor function. The results were compared with those of the monogeneans Macrogyrodactylus clarii and M. congolensis inhabiting the gills and skin respectively of the same host fish C. gariepinus.

Acute skin GVHD following syngeneic BMT for CLL

A 41-year-old woman received a syngeneic BMT for CLL and subsequently developed acute skin GVHD. Transfusion-related allogeneic GVHD was excluded on the basis of an unchanged HLA type in circulating lymphocytes. Short tandem repeat PCR was used to confirm syngeneicity between donor and recipient. The patient had a personal and family history of autoimmune disease which may have made her particularly susceptible to development of syngeneic GVHD. The distinction between allogeneic and syngeneic or autologous GVHD is important because of therapeutic implications.

Cytosolic DNA triggers inflammasome activation in keratinocytes in psoriatic lesions

The proinflammatory cytokine interleukin-1β (IL-1β) plays a central role in the pathogenesis and the course of inflammatory skin diseases, including psoriasis. Posttranscriptional activation of IL-1β is mediated by inflammasomes; however, the mechanisms triggering IL-1β processing remain unknown. Recently, cytosolic DNA has been identified as a danger signal that activates inflammasomes containing the DNA sensor AIM2. In this study, we detected abundant cytosolic DNA and increased AIM2 expression in keratinocytes in psoriatic lesions but not in healthy skin. In cultured keratinocytes, interferon-γ induced AIM2, and cytosolic DNA triggered the release of IL-1β via the AIM2 inflammasome. Moreover, the antimicrobial cathelicidin peptide LL-37, which can interact with DNA in psoriatic skin, neutralized cytosolic DNA in keratinocytes and blocked AIM2 inflammasome activation. Together, these data suggest that cytosolic DNA is an important disease-associated molecular pattern that can trigger AIM2 inflammasome and IL-1β activation in psoriasis. Furthermore, cathelicidin LL-37 interfered with DNA-sensing inflammasomes, which thereby suggests an anti-inflammatory function for this peptide. Thus, our data reveal a link between the AIM2 inflammasome, cathelicidin LL-37, and autoinflammation in psoriasis, providing new potential targets for the treatment of this chronic skin disease.

Impact of vitamin D3 on cutaneous immunity and antimicrobial peptide expression

Antimicrobial peptides (AMPs) are effectors of cutaneous innate immunity and protect primarily against microbial infections. An array of AMPs can be found in and on the skin. Those include peptides that were first discovered for their antimicrobial properties but also proteins with antimicrobial activity first characterized for their activity as chemokines, enzymes, enzyme inhibitors and neuropeptides. Cathelicidins were among the first families of AMPs discovered in skin. They are now known to exert a dual role in innate immune defense: they have direct antimicrobial activity and will also initiate a host cellular response resulting in cytokine release, inflammation and angiogenesis. Altered cathelicidin expression and function was observed in several common inflammatory skin diseases such as atopic dermatitis, rosacea and psoriasis. Until recently the molecular mechanisms underlying cathelicidin regulation were not known. Lately, vitamin D3 was identified as the major regulator of cathelicidin expression and entered the spotlight as an immune modulator with impact on both, innate and adaptive immunity. Therapies targeting vitamin D3 signalling may provide novel approaches for the treatment of infectious and inflammatory skin diseases by affecting both innate and adaptive immune functions through AMP regulation.

Control of cutaneous antimicrobial peptides by vitamin D3

Constant exposure to a wide variety of microbial pathogens represents a major challenge for our skin. Antimicrobial peptides (AMPs) are mediators of cutaneous innate immunity and protect primarily against microbial infections. Cathelicidins were among the first AMPs identified in human skin and recent evidence suggests that they exert a dual role in innate immune defense: At first, due to their antimicrobial activity they kill pathogens directly. In addition, these peptides initiate a potent host response to infection resulting in cytokine release, inflammation and a cellular response. Disturbed cathelicidin expression and function was observed in several common inflammatory skin diseases, such as psoriasis where cathelicidin peptide converts inert self-DNA and self-RNA into an autoimmune stimulus. In atopic dermatitis decreased levels of cathelicidin facilitating microbial superinfections have been discussed. Furthermore, abnormally processed cathelicidin peptides induce inflammation and a vascular response in rosacea. Until recently, the molecular mechanisms underlying cathelicidin regulation were unknown. Recently, the vitamin D3 pathway was identified as the major regulator of cathelicidin expression. Consequently, vitamin D3 entered the spotlight as an immune modulator with impact on both innate and adaptive immunity. Therapies targeting vitamin D3 signaling may provide new approaches for infectious and inflammatory skin diseases by affecting both innate and adaptive immune functions.

VDR and MEK-ERK dependent induction of the antimicrobial peptide cathelicidin in keratinocytes by lithocholic acid

Cathelicidin is an antimicrobial peptide (AMP) and signaling molecule in innate immunity and a direct target of 1,25-dihydroxyvitamin D3 (1,25D3) in primary human keratinocytes (NHEK). The expression of cathelicidin is dysregulated in various skin diseases and its regulation differs depending on the epithelial cell type. The secondary bile acid lithocholic acid (LCA) is a ligand of the vitamin D receptor (VDR) and can carry out in vivo functions of vitamin D3. Therefore we analyzed cathelicidin mRNA- and peptide expression levels in NHEK and colonic epithelial cells (Caco-2) after stimulation with LCA. We found increased expression of cathelicidin mRNA and peptide in NHEK, in Caco-2 colon cells no effect was observed after LCA stimulation. The VDR as well as MEK-ERK signaled the upregulation of cathelicidin in NHEK induced by LCA. Collectively, our data indicate that cathelicidin induction upon LCA treatment differs in keratinocytes and colonic epithelial cells. Based on these observations LCA-like molecules targeting cathelicidin could be designed for the treatment of cutaneous diseases that are characterized by disturbed cathelicidin expression.

Vitamin D analogs differentially control antimicrobial peptide/"alarmin" expression in psoriasis

Antimicrobial peptides (AMPs) are strongly expressed in lesional skin in psoriasis and play an important role as proinflammatory "alarmins" in this chronic skin disease. Vitamin D analogs like calcipotriol have antipsoriatic effects and might mediate this effect by changing AMP expression. In this study, keratinocytes in lesional psoriatic plaques showed decreased expression of the AMPs beta-defensin (HBD) 2 and HBD3 after topical treatment with calcipotriol. At the same time, calcipotriol normalized the proinflammatory cytokine milieu and decreased interleukin (IL)-17A, IL-17F and IL-8 transcript abundance in lesional psoriatic skin. In contrast, cathelicidin antimicrobial peptide expression was increased by calcipotriol while psoriasin expression remained unchanged. In cultured human epidermal keratinocytes the effect of different vitamin D analogs on the expression of AMPs was further analyzed. All vitamin D analogs tested blocked IL-17A induced HBD2 expression by increasing IkappaB-alpha protein and inhibition of NF-kappaB signaling. At the same time vitamin D analogs induced cathelicidin through activation of the vitamin D receptor and MEK/ERK signaling. These studies suggest that vitamin D analogs differentially alter AMP expression in lesional psoriatic skin and cultured keratinocytes. Balancing AMP "alarmin" expression might be a novel goal in treatment of chronic inflammatory skin diseases.

Exposure to anthrax toxin alters human leucocyte expression of anthrax toxin receptor 1

Anthrax is a toxin-mediated disease, the lethal effects of which are initiated by the binding of protective antigen (PA) with one of three reported cell surface toxin receptors (ANTXR). Receptor binding has been shown to influence host susceptibility to the toxins. Despite this crucial role for ANTXR in the outcome of disease, and the reported immunomodulatory consequence of the anthrax toxins during infection, little is known about ANTXR expression on human leucocytes. We characterized the expression levels of ANTXR1 (TEM8) on human leucocytes using flow cytometry. In order to assess the effect of prior toxin exposure on ANTXR1 expression levels, leucocytes from individuals with no known exposure, those exposed to toxin through vaccination and convalescent individuals were analysed. Donors could be defined as either 'low' or 'high' expressers based on the percentage of ANTXR1-positive monocytes detected. Previous exposure to toxins appears to modulate ANTXR1 expression, exposure through active infection being associated with lower receptor expression. A significant correlation between low receptor expression and high anthrax toxin-specific interferon (IFN)-γ responses was observed in previously infected individuals. We propose that there is an attenuation of ANTXR1 expression post-infection which may be a protective mechanism that has evolved to prevent reinfection.

Mycosis fungoides shows concurrent deregulation of multiple genes involved in the TNF signaling pathway:an expression profile study

Mycosis fungoides (MF) is the most frequent type of cutaneous T-cell lymphoma, whose diagnosis and study is hampered by its morphologic similarity to inflammatory dermatoses (ID) and the low proportion of tumoral cells, which often account for only 5% to 10% of the total tissue cells. cDNA microarray studies using the CNIO OncoChip of 29 MF and 11 ID cases revealed a signature of 27 genes implicated in the tumorigenesis of MF, including tumor necrosis factor receptor (TNFR)-dependent apoptosis regulators, STAT4, CD40L, and other oncogenes and apoptosis inhibitors. Subsequently a 6-gene prediction model was constructed that is capable of distinguishing MF and ID cases with unprecedented accuracy. This model correctly predicted the class of 97% of cases in a blind test validation using 24 MF patients with low clinical stages. Unsupervised hierarchic clustering has revealed 2 major subclasses of MF, one of which tends to include more aggressive-type MF cases including tumoral MF forms. Furthermore, signatures associated with abnormal immunophenotype (11 genes) and tumor stage disease (5 genes) were identified.

Glucocorticoid receptor and Klf4 co-regulate anti-inflammatory genes in keratinocytes

The glucocorticoid (GC) receptor (GR) and Kruppel-like factor Klf4 are transcription factors that play major roles in skin homeostasis. However, whether these transcription factors cooperate in binding genomic regulatory regions in epidermal keratinocytes was not known. Here, we show that in dexamethasone-treated keratinocytes GR and Klf4 are recruited to genomic regions containing adjacent GR and KLF binding motifs to control transcription of the anti-inflammatory genes Tsc22d3 and Zfp36. GR- and Klf4 loss of function experiments showed total GR but partial Klf4 requirement for full gene induction in response to dexamethasone. In wild type keratinocytes induced to differentiate, GR and Klf4 protein expression increased concomitant with Tsc22d3 and Zfp36 up-regulation. In contrast, GR-deficient cells failed to differentiate or fully induce Klf4, Tsc22d3 and Zfp36 correlating with increased expression of the epithelium-specific Trp63, a known transcriptional repressor of Klf4. The identified transcriptional cooperation between GR and Klf4 may determine cell-type specific regulation and have implications for developing therapies for skin diseases.

Schistosoma mansoni cercariae experience influx of macromolecules during skin penetration

We have observed that when cercariae penetrate the skin of mice, there is influx into their tissues of Lucifer Yellow and certain labelled molecules of up to 20 kDa molecular weight. This observation was made using a variety of fluorescent membrane-impermeant compounds injected into the skin before the application of cercariae. This unexpected phenomenon was investigated further by transforming cercariae in vitro in the presence of the membrane-impermeant compounds and examining the distribution by microscopy. In schistosomula derived from this procedure, the nephridiopore and surface membrane were labelled while the pre- and post-acetabular glands were not labelled. The region associated with the oesophagus within the pharyngeal muscle clearly contained the fluorescent molecules, as did the region adjacent to the excretory tubules and the germinal mass. We used cercariae stained with carmine to aid identification of regions labelled with Lucifer Yellow. Although the mechanism of this influx is unclear, the observation is significant. From it, we can suggest an hypothesis that, during skin penetration, exposure of internal tissues of the parasite to external macromolecules represents a novel host-parasite interface.

A novel and potent trypsin inhibitor peptide, AC-17, from the skin secretion of the edible frog, Rana esculenta

Protease inhibitors are found in many venoms and evidence suggests that they occur widely in amphibian skin secretions. Kunitz inhibitors have been found in the skin secretions of bombinid toads and ranid frogs, Kazal inhibitors in phyllomedusine frogs and Bowman–Birk inhibitors in ranid frogs. Selective protease inhibitors could have important applications as therapeutics in the treatment of diseases in which discrete proteases play an aetiologcal role. Here we have examined the skin secretion of the edible frog, Rana esculenta, for protease inhibitors using trypsin as a model. HPLC fractions of secretions were screened for inhibitory activity using a chromogenic substrate as reporter. Three major peptides were resolved with trypsin inhibitory activity in HPLC fractions — one was a Kunitz-type inhibitor, a second was a Bowman–Birk inhibitor but the third represented a novel class of trypsin inhibitor in European frog skin. Analysis of the peptide established the structure of a 17-mer with an N-terminal Ala (A) residue and a C-terminal Cys (C) residue with a single disulphide bridge between Cys 12 and 17. Peptide AC-17 resembled a typical “Rana box” antimicrobial peptide but while it was active against Escherichia coli (MIC 30 µM) it was devoid of activity against Staphylococcus aureus and of haemolytic activity. In contrast, the peptide was a potent inhibitor of trypsin with a Ki of 5.56 µM. AC-17 represents the prototype of a novel trypsin inhibitor from the skin secretion of a European ranid frog that may target a trypsin-like protease present on the surface of Gram-negative bacteria.

Determining the role of mononuclear phagocytes in prion neuroinvasion from the skin

Many prion diseases are acquired by peripheral exposure, and skin lesions are an effective route of transmission. Following exposure, early prion replication, upon FDCs in the draining LN is obligatory for the spread of disease to the brain. However, the mechanism by which prions are conveyed to the draining LN is uncertain. Here, transgenic mice were used, in which langerin(+) cells, including epidermal LCs and langerin(+) classical DCs, were specifically depleted. These were used in parallel with transgenic mice, in which nonepidermal CD11c(+) cells were specifically depleted. Our data show that prion pathogenesis, following exposure via skin scarification, occurred independently of LC and other langerin(+) cells. However, the depletion of nonepidermal CD11c(+) cells impaired the early accumulation of prions in the draining LN, implying a role for these cells in the propagation of prions from the skin. Therefore, together, these data suggest that the propagation of prions from the skin to the draining LN occurs via dermal classical DCs, independently of langerin(+) cells.

Non-enzymatic glycosylation of skin collagen in patients with type 1 (insulin-dependent) diabetes mellitus and limited joint mobility

Forearm skin biopsies were obtained from diabetic subjects with and without limited joint mobility, and from non-diabetic control subjects. Collagen purified from these samples was assayed for non-enzymatic glycosylation. The level in all diabetic patients was significantly greater than that in control subjects (p less than 0.001), but those diabetic patients with limited joint mobility had a level of collagen glycosylation similar to that in those with normal joints (15.3 +/- 1.3 and 16.5 +/- 1.3 nmol fructose/10 mg protein, respectively; mean +/- SEM). Glycosylation of collagen in the diabetic patients correlated with glycosylated haemoglobin measured at the time of skin biopsy (r = 0.60). These results do not support the hypothesis that non-enzymatic glycosylation of collagen, as reflected by the ketoamine link, plays an important role in the development of limited joint mobility in diabetes.