Strontium hexaferrite nanomagnets suspended in a cosmetic preparation: a convenient tool to evaluate the biological effects of surface magnetism on human skin.

BACKGROUND/PURPOSE: Magnetic therapy has been popular for ages, but its therapeutic abilities remain to be demonstrated. We aimed to develop a homogeneous, stable dispersion of magnetic nanoparticles in a skin-care preparation, as a tool to analyze the biological and physiological effects of superficial magnetism in skin. METHODS: SrFe(12)O(19) nanoparticles were generated by ultrasound, dispersed in glycerol, stabilized in Dermud cream and permanently magnetized. The magnetic cream was applied on the epidermis of human skin organ cultures. The effects on UV-induced cell toxicity, apoptosis and inflammatory cytokine expression were analyzed. A clinical test was performed to check skin moisturization. RESULTS: Nanomagnets were found to be homogenously and stably dispersed. After magnetization, the preparation generated a magnetic field of 1-2 G. Upon cream application, no cytotoxicity and no impairment of cellular vitality were found after 24 and 48 h, respectively. The anti-apoptotic and anti-inflammatory properties of Dermud were not modified, but its long-term effect on moisturization in vivo was slightly increased. CONCLUSION: Nanomagnetic Dermud cream can be used as a tool to analyze the biological effects of nanomagnets dispersed on the skin surface at the cellular and molecular levels, thus allowing to explore the possible therapeutic uses of superficial magnetism for skin care.

Inhibition of cholesterol transport into skin cells in cultures by phytosterol-loaded microemulsion.

Cholesterol and plant phytosterols are lipophilic compounds solubilized by intestinal micelles in a competitive manner. In this work, we used radioactive cholesterol- and phytosterol-loaded oil-in-water microemulsions to follow their incorporation and mutual competition in HaCaT keratinocytes, SZ95 sebocytes, and skin pieces in cultures. Dynamic light scattering showed homogenous nanostructures of 10.5+/-1.5 nm diameter and cryo-transmission electron microscopy confirmed the presence of uniform spherical droplets of 7.0+/-1.0 nm diameter. Up to 320 nmol/ml of cholesterol can be solubilized and transported into cells with minimal toxic effect by 0.5 wt% nanodroplets in a cell medium. Phytosterols inhibit incorporation of cholesterol into cells, in vitro, at molar ratios (phytosterols/cholesterol) of 4 and above. The loaded nanodroplets accumulate in intracellular vesicles (presumably endosomes). No metabolic conversion of cholesterol or phytosterols was found in these cells, in vitro, after 24 h, at 37 degrees C.

Are desmoglein autoantibodies essential for the immunopathogenesis of pemphigus vulgaris, or just "witnesses of disease"?

Pemphigus vulgaris (PV) is fascinating to dermatologists, epithelial biologists and immunologists alike, as its pathogenesis has been clarified to a much greater extent than that of most other organ-specific autoimmune diseases, and as it has provided abundant novel insights into desmoglein biology and pathology along the way. Historically, the most influential PV pathogenesis concept is that of Stanley and Amagai. This concept holds that autoantibodies against desmogleins are both essential and sufficient for epidermal blister formation (acantholysis) by impeding the normal functioning of these major adhesion proteins. However, as with most good theories, this landmark concept has left a number of intriguing and important questions open (or at least has not managed to answer these to everyone's satisfaction). Moreover, selected dissenting voices in the literature have increasingly called attention to what may or may not be construed as inconsistencies in this dominant PV pathogenesis paradigm of the recent past. The present debate feature therefore bravely rises to the challenge of re-examining the entire currently available evidence, as rationally and as undogmatically as possible, by provocatively asking a carefully selected congregation of experts (who have never before jointly published on this controversial topic!) to discuss how essential anti-desmoglein autoantibodies really are in the immunopathogenesis of PV. Not surprisingly, some of our expert "witnesses" in this animated debate propose diametrically opposed answers to this question. While doing so, incisive additional questions are raised that relate to the central one posed, and our attention is called to facts that may deserve more careful consideration than they have received so far. Together with the intriguing (often still very speculative) complementary or alternative pathogenesis scenarios proposed in the following pages, this offers welcome "food for thought" as well as very specific suggestions for important future research directions--within and beyond the camp of PV aficionados. The editors trust that this attempt at a rational public debate of the full evidence that is currently at hand will constructively contribute to further dissecting the exciting--and clinically very relevant!--immunopathogenesis of PV in all its complexity.

Protective effect of intravenous immunoglobulin (IVIG) in an experimental model of pemphigus vulgaris.

Uncontrolled studies have found intravenous immunoglobulin (IVIG) to be effective in the treatment of pemphigus vulgaris (PV). The aim of this study was to evaluate the role of IVIG in preventing IgG autoantibodies binding to desmoglein-3 and blister formation using a controlled experimental design. The ability of IVIG to affect the binding of IgG affinity purified from two patients with PV to desmoglein-3 in comparison to IgG from one donor, was conducted by enzyme-linked immunosorbent assay (ELISA). The specificity was confirmed by competition assay. We assessed the effect of IVIG on the induction of experimental-PV in CD1 newborn mice by subcutaneous subjection of IgG affinity purified from two patients with PV. The treatment was conducted by subcutaneous administration of IVIG together with IgG from the pemphigus patients or appropriate control. The skin of the newborns was examined 24-48 h later for blisters, and samples of the affected areas were analysed by immunohistochemistry. IVIG as a whole molecule and its F(ab)(2) portion inhibited the binding of anti-desmoglein-3 antibody to recombinant desmoglein-3 in a dose-dependent manner. The specificity was confirmed by competition assays. In-vivo, IVIG and its F(ab)(2) portion prevented blister formation in the newborn mice. Cutaneous lesions were noted only in the groups of newborn mice who were injected with IgG fractions from the PV patients. Immunopathological evaluation revealed that IVIG prevented the formation of acanthylosis with IgG deposition in the intercellular spaces. These results point to the efficacy of IVIG in the prevention of blister formation in an experimental PV model.

Possible apoptotic mechanism in epidermal cell acantholysis induced by pemphigus vulgaris autoimmunoglobulins.

Through a still unclear mechanism, pemphigus vulgaris autoantibodies (PV-IgG) induce intra-epidermal acantholytic lesions responsible for severe to fatal skin wounding. We present evidence that PV lesions contain apoptotic keratinocytes, and that cell death is induced in the lesional tissue apparently before cell separation. These data suggest that apoptosis could be the cause of the acantholytic phenomenon. We show that PV-IgG and an antibody against Fas receptor (anti-FasR) induce lesions in vitro in a similar way, causing: (1) secretion of soluble FasL; (2) elevated cellular amounts of FasR, FasL (soluble and membranal), Bax and p53 proteins; (3) reduction in levels of cellular Bcl-2; (4) enrichment in caspase 8, and activation of caspases 1 and 3; (5) co-aggregation of FasL and FasR with caspase 8 in membranal death-inducing signaling complex (DISC). Hence, the Fas-mediated death signaling pathway seems to be involved in lesion formation. Moreover, we have shown that in skin organ cultures and in keratinocyte cultures, PV-IgG can induce caspase activation and DNA fragmentation, and caspase inhibitors can prevent the formation of PV-IgG-induced epidermal lesions. Altogether, these results suggest that PV-IgG-induced acantholysis may proceed through the death-signaling pathway. They highlight new perspectives on mechanisms of tissue damage in autoimmune diseases.

The distribution of pemphigus vulgaris-IgG subclasses and their reactivity with desmoglein 3 and 1 in pemphigus patients and their first-degree relatives.

BACKGROUND: Pemphigus vulgaris (PV) autoantibodies (PV-IgG) have been found in 40-70% of sera of first-degree relatives of pemphigus patients. OBJECTIVES: To determine the possible role of PV-IgG subclasses in the pathogenesis of the disease. PATIENTS AND METHODS: Study groups comprised 25 PV patients, 55 unaffected family members and 56 sera of healthy individuals. Indirect immunofluorescence (IIF) staining and Western immunoblotting (WB) techniques were used to determine total PV-IgG and PV-IgG subclasses and their reactivity to desmoglein (Dsg) 1 and 3. RESULTS: By IIF staining, circulating PV-IgG were found in 64% of the patients, in 15% of the relatives and in none of the controls (P < or = 0.001); by WB the results were 91%, 49% and 12%, respectively (P < or = 0.001). The distribution of PV-IgG subclasses 1-3 was similar among patients and their relatives. PV-IgG4 was found in 62% of the patients but in only one relative and was absent in the controls (P < or = 0.001). PV-IgG1, 2 and 4 were found to react mainly with Dsg3 and PV-IgG3 mainly with Dsg1 and 3. CONCLUSIONS: These results support the concept of a genetic predisposition in pemphigus. The non-complement-fixing PV-IgG4 and at least one complement-fixing PV-IgG subclass appear to be involved in the pathogenesis of the disease. The absence of PV-IgG4 among relatives who were PV-IgG carriers seems to be linked to the fact that they do not develop pemphigus. The exact nature of this linkage is still unclear.

Changes in protein kinase C during vitellogenesis in the crayfish Cherax quadricarinatus--possible activation by methyl farnesoate.

During ovarian maturation in the crayfish Cherax quadricarinatus, changes in ovarian protein kinase C (PKC) isoenzymes take place in parallel to yolk accumulation (as shown by immunoblot analysis). Significant changes were recorded in the amounts of specific isoenzymes and in their distribution between the cytosol and the membranes. Ovarian maturation was accompanied by the appearance of high- and low-molecular-weight immunoreactive PKC isoenzyme species. Among the isoenzymes tested, PKC alpha was the most clearly activated during ovarian maturation, as shown by significant translocation from the cytosol to the particulate fraction and the appearance of high-molecular-weight species. Moreover, a similar picture was obtained in the ovaries of intersex individuals upon induction of secondary vitellogenesis by androgenic gland ablation. Immunohistological staining showed PKC alpha to be localized mainly in the cytosol of premature oocytes, whereas in later maturation stages, it was concentrated around the nucleus in a vesicular structure and in the oocyte membrane. In secondary vitellogenic stages, PKC was localized in the plasma membrane and apparently in follicular cells. In addition, its activity was demonstrated by in vitro phosphorylation assays of a crayfish ovarian homogenate. Activation of total PKC phosphorylation of histone, an external substrate, was induced by phosphatidylserine plus 12-O-tetradecanoylphorbol-13-acetate (TPA) or methyl farnesoate. Both TPA and methyl farnesoate stimulated activation of PKC alpha in organ culture, causing its translocation from the cytosol to the membranes and inducing autophosphorylation of threonine residues. The changes in PKC isoenzymes during ovarian maturation in the crayfish suggest their involvement in this process as well as a possible regulatory role for methyl farnesoate through a direct effect on some PKC isoenzymes.

Increase of oxidatively modified protein is associated with a decrease of proteasome activity and content in aging epidermal cells.

For the process of aging in epidermal cells to be characterized, the status of oxidized and damaged protein accumulation and removal by the proteasome has been investigated. Modified protein content and proteasome activity were assayed in lysates of epidermal cells from donors of different ages. Increased levels of oxidized proteins, glycated proteins, and proteins modified by the lipid peroxidation product 4-hydroxy-2-nonenal were observed in cells from old donors. At the same time, a decline of chymotrypsin-like and peptidylglutamyl-peptide hydrolase activities of the proteasome was found in aging keratinocytes. This age-related decline of the proteasome peptidase activities can be explained, at least in part, by a decreased proteasome content as observed by immunoblotting and enzyme-linked immunosorbent assay. In keratinocyte cultures, a decrease of proteasome activity and content was observed upon serial passaging. In cultures, as well as in skin, an inverse relationship was found between the aging marker 1-galactosidase and the proteasome content. These results suggest that proteasome is downregulated during replicative senescence as well as in aged cells in vivo, possibly resulting in the accumulation of modified proteins.

Identification of the gregarization-associated dark-pigmentotropin in locusts through an albino mutant.

In response to crowding, locusts develop characteristic black patterns that are well discernible in the gregarious phase at outbreaks. We report here a dark-color-inducing neuropeptide (dark-pigmentotropin) from the corpora cardiaca of two plague locusts, Schistocerca gregaria and Locusta migratoria. The chromatographic isolation of this neuropeptide was monitored by using a bioassay with an albino mutant of L. migratoria. The neurohormone, consisting of 11 amino acids, is identical to [His7] corazonin, previously isolated from corpora cardiaca of another acridid without known function. The present results show that even in isolated (solitary) nymphs, [His7] corazonin induces gregarious black patterns. Its primary structure shows some similarity with the vertebrate melanophore stimulating hormone.