Oral administration of milk-derived phospholipids inhibits penetration of cutaneous nerve fibres into epidermis in a mouse model of acute dry skin.

The density of intraepidermal nerve fibres has been shown to be higher in itchy dry skin than in healthy skin, suggesting that epidermal hyperinnervation is at least partly involved in peripheral itch sensitization. We investigated whether oral administration of milk-derived phospholipids (MPLs) would inhibit epidermal hyperinnervation in a mouse model of dry skin. We found that the number of intraepidermal nerve fibres was significantly lower in the MPL group than in the control group. Expression of nerve growth factor (NGF) levels in the epidermis was significantly decreased by oral administration of MPLs, whereas expression of semaphorin (Sema)3A, a nerve repulsion factor, was increased in the MPL group. These results suggest that dietary MPLs attenuate the penetration of nerve fibres into the epidermis by reducing epidermal NGF levels and increasing Sema3A level. Thus, dietary MPLs may have beneficial effects in the prevention and/or alleviation of dry skin-induced itch by reducing intraepidermal nerve fibre density.

Antibody against chromatin assembly factor-1 is a novel autoantibody specifically recognized in systemic lupus erythematosus.

Autoantibodies to proliferating cell nuclear antigen (PCNA) are specifically, if rarely, present in systemic lupus erythematosus (SLE) patient sera. Even SLE patients lacking PCNA reactivity often show reaction to PCNA-binding protein. Here, immunoreactivity to chromatin assembly factor-1 (CAF-1), an essential molecule for DNA replication and a PCNA-binding protein, was compared for the sera of SLE patients, normal healthy controls (NHCs) and other disease controls, and in autoimmune sera reactive to standard autoantigens, by enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence, and immunoblotting. CAF1 and IRF1 expression in SLE and NHC peripheral mononuclear cells were compared by quantitative real-time polymerase chain reaction. Serum interferon-γ-inducing protein-10 and anti-double-stranded (ds)DNA antibody levels were measured by ELISA. Increased CAF-1 autoimmune reactivity was recognized in SLE or serum anti-dsDNA antibody-positive patients. Significantly greater central nervous system (CNS) involvement (aseptic meningitis) and serum anti-dsDNA antibody titers were present more often in anti-CAF-1 antibody-positive than antibody-negative SLE patients. IFN-γ positively regulated CAF-1 expression in vitro and was associated with anti-CAF-1 antibody production in SLE. Thus, a novel anti-CAF-1 autoantibody is frequently found in patients with SLE and is a useful biomarker for diagnosis, especially in cases with CNS involvement. Aberrant IFN-γ regulation appears to play an important role in anti-CAF-1 antibody production in SLE.

Neurotropin inhibits the increase in intraepidermal nerve density in the acetone-treated dry-skin mouse model.

Epidermal hyperinnervation is considered one cause of sensitization to itch, and is thought to regulated by keratinocyte-derived axonal guidance molecules, including nerve growth factor (NGF) and semaphorin (Sema)3A. Neurotropin (NTP) shows antipruritic effects in allergic disease and is also used for pain relief. Using cultured rat dorsal root ganglion neurones, we previously found that NTP inhibited NGF-induced neurite outgrowth. However, no such inhibitory effect has been shown in vivo. We therefore assessed the effects of intraperitoneal administration of NTP on nerve density and expression of NGF and Sema3A mRNAs in the epidermis of acetone-treated mice showing epidermal hyperinnervation. We found that NTP significantly reduced intraepidermal nerve growth in these acetone-treated mice. NTP significantly upregulated epidermal Sema3A mRNA, but had no effect on expression of epidermal NGF mRNA. These findings indicate that NTP may reduce intraepidermal nerve density by inducing expression of Sema3A in the epidermis.

Possible role of the JAK/STAT pathways in the regulation of T cell-interferon related genes in systemic lupus erythematosus.

Changes in gene expression in CD3+ T cells associated with disease progression in systemic lupus erythematosus (SLE) patients were determined. The genes related to SLE disease-related activities were identified and their gene regulatory networks were investigated. Analyses of gene expression were performed by both DNA microarray and real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). The expression of certain genes including interferon (IFN) regulatory factor (IRF)-related genes, such as IFN-regulated, -related, and -signature genes was increased in the active phase of SLE. Pathway network analyses suggested that these IRF-related genes are regulated through the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway. JAK/STAT pathway-mediated regulation of IRF-related genes may have an important role in the disease activity of SLE. Inhibitors of JAK/STAT cascade may be useful as therapeutic agents.

Neurotropin inhibits both capsaicin-induced substance P release and nerve growth factor-induced neurite outgrowth in cultured rat dorsal root ganglion neurones.

BACKGROUND: Neurotropin (NTP), a biological extract from rabbit skin inoculated with vaccinia virus, is an effective analgesic and anti-allergic agent, and has antipruritic effects in various dermatoses including eczema, dermatitis and urticaria. In patients receiving haemodialysis who have pruritus, NTP appears to exert its antipruritic effect by lowering the plasma levels of substance P (SP), but its underlying mechanisms are not fully understood. AIM: To investigate the antipruritic mechanisms of NTP. METHODS: The effects of NTP on capsaicin-induced SP release from neonatal rat dorsal root ganglion (DRG) neurones were assessed by measuring SP concentrations in culture media by a competitive ELISA. The effects of NTP on nerve growth factor (NGF)-induced neurite outgrowth were assessed by measuring the length of the longest process of cultured DRG neurones. The neuronal cytotoxicity of NTP was determined using a methylthiazole tetrazolium cytotoxicity assay. RESULTS: NTP dose-dependently inhibited capsaicin-induced release of SP from cultured DRG neurones, whereas NTP alone had no effect on SP release. Moreover, NTP dose-dependently inhibited NGF-induced neurite outgrowth in cultured DRG neurones. NTP had no observable cytotoxicity. CONCLUSIONS: These results suggest that NTP exerts its antipruritic effects by inhibiting both SP release and neurite outgrowth of cutaneous sensory nerves.

In vitro model for penetration of sensory nerve fibres on a Matrigel basement membrane: implications for possible application to intractable pruritus.

BACKGROUND: Epidermal hyperinnervation occurs in dermatoses with intractable pruritus, such as atopic dermatitis, suggesting that the hyperinnervation is partly responsible for abnormal itch perception. OBJECTIVES: To investigate the mechanisms of penetration of sensory nerve fibres into the basement membrane of the skin. METHODS: A rat dorsal root ganglion neurone culture system consisting of Matrigel and a Boyden chamber containing a nerve growth factor (NGF) concentration gradient was used. In some experiments, matrix metalloproteinase (MMP) blockers and semaphorin 3A (Sema3A) were added to the culture system. Matrigel-coated membranes were stained with anti-Tau antibody, and the number of nerve fibres that crossed the membrane was counted. Expression of MMPs in the cultured neurones was examined at mRNA and protein levels by quantitative reverse transcription-polymerase chain reaction and immunocytochemistry, respectively. The activity was also examined by zymography. RESULTS: Nerve fibres penetrated into Matrigel in the presence of an NGF concentration gradient, which was dose-dependently inhibited by GM6001, a broad-spectrum MMP inhibitor. Transcripts for MMP2, but not MMP9, were increased in the cultured neurones, and the penetration was dose-dependently inhibited by MMP-2 blockers. MMP-2 and its activity were partially localized on the NGF-responsive growth cones. NGF also upregulated pro-MMP-2 activation molecules in the cultured neurones. Sema3A stimulation showed the opposite effects on these NGF-dependent events. Interestingly, MMP2 expression was modulated by extracellular matrix (ECM) substrates for this enzyme. CONCLUSIONS: Membrane-associated MMP-2 contributes to penetration of nerve fibres into Matrigel through modulation by axonal guidance molecules and/or ECM. These findings provide insight for understanding the development of intractable pruritus involving epidermal nerve density.

Changes in the gene expression of peripheral blood mononuclear cells during the menstrual cycle of females is associated with a gender bias in the incidence of systemic lupus erythematosus.

OBJECTIVE: The incidence of systemic lupus erythematosus (SLE) is far higher in females than in males and the onset and/or disease activity is influenced by pregnancy and the menstrual cycle. Sex hormones seem to influence the pathogenesis of SLE, therefore, changes in gene expression in peripheral blood mononuclear cells (PBMC) were examined during the menstrual cycle in females, under the comparison of gene expression of patients with SLE. METHODS: The detection and a quantitative analysis of the gene expression was performed by DNA microarray or real-time quantitative polymerase chain reaction (RQ-PCR) method. RESULTS: There were thirteen known genes which showed significant quantitative changes during the menstrual cycles of females, but not in males. Among these genes, statistical quantitative differences between normal controls and SLE patients were observed in six genes. CONCLUSION: Based on these findings, certain genes (such as the tumor necrosis factor receptor superfamily, member 14; TNFRSF14, and signal regulatory protein, gamma; SIRPG) appear to contribute to gender difference of SLE.

Epithelial fibroblast growth factor receptor 1 regulates enamel formation.

The interaction between epithelial and mesenchymal tissues plays a critical role in the development of organs such as teeth, lungs, and hair. During tooth development, fibroblast growth factor (FGF) signaling is critical for regulating reciprocal epithelial and mesenchymal interactions. FGF signaling requires FGF ligands and their receptors (FGFRs). In this study, we investigated the role of epithelial FGF signaling in tooth development, using the Cre-loxp system to create tissue-specific inactivation of Fgfr1 in mice. In K14-Cre;Fgfr1(fl/fl) mice, the apical sides of enamel-secreting ameloblasts failed to adhere properly to each other, although ameloblast differentiation was unaffected at early stages. Prior to eruption, enamel structure was compromised in the K14-Cre;Fgfr1(fl/fl) mice and displayed severe enamel defects that mimic amelogenesis imperfecta (AI), with a rough, irregular enamel surface. These results suggest that there is a cell-autonomous requirement for FGF signaling in the dental epithelium during enamel formation. Loss of Fgfr1 affects ameloblast organization at the enamel-secretory stage and, hence, the formation of enamel.

Protein biomarker analysis by mass spectrometry in patients with rheumatoid arthritis receiving anti-tumor necrosis factor-alpha antibody therapy.

OBJECTIVE: To investigate the mechanism of action of anti-tumor necrosis factor-alpha (TNF-alpha) antibody in patients with rheumatoid arthritis (RA), we analyzed serum or plasma proteins by mass spectrometry system. METHODS: Ten RA patients who received treatment with anti-TNF-alpha antibody were studied. Samples obtained before and after therapy were analyzed by a two-dimensional liquid chromatography tandem mass spectrometry (2D LC-MS/MS) system after pretreatment by a recently developed method to remove high molecular weight proteins. RESULTS: Using this system, certain proteins were identified after treatment with anti-TNF-alpha antibody, including proteins related to the TNF-alpha-mediated pathway for nuclear factor kappa B (NF-kappaB) activation and/or to the metabolism (including regeneration) of articular cartilage. CONCLUSION: Our mass spectrometry system appears to be useful for proteomic analysis. The efficacy of anti-TNF-alpha antibody therapy for RA may be related to various consequence of the inhibition of TNF-alpha activity.

Skin sulfhydryl oxidase. Purification and some properties.

A sulfhydryl-oxidizing enzyme has been found in skin of young rats and a method for purifying the enzyme over 600-fold has been developed. Enzymatic activity was assayed either by its ability to oxidize dithiothreitol of by measuring its ability to renature reductively denatured ribonuclease A. Skin sulfhydryl oxidase catalyzed the oxidation of various thiols: dithiothreitol, dithioerythritol, D-penicillamine, and L-cysteine. Glutathione and 2-mercaptoethanol were very poor substrates for the enzyme. The enzyme also reactivated reductively denatured ribonuclease A, with neither the presence of a thiol nor prior reduction of the enzyme being necessary. The molecular weight of the enzyme was estimated to be 66 000 +/- 2000, and the isoelectric point was determined to be at pH 4.65. Alkylating reagents alone had some inhibiting effect on skin sulfhydryl oxidase; when the enzyme was preincubated with thiols which were substrates, inhibition by alkylating reagents was greatly increased. After preincubation with dithiothreitol, treatment of the enzyme with alkylating reagents or N-ethylmaleimide caused significant inhibition; preincubation with a poor substrate, reduced glutathione, did not enhance inhibition by alkylating reagents or N-ethylmaleimide.

Skin proteasomes (high-molecular-weight protease): purification, enzymologic properties, gross structure, and tissue distribution.

Proteasomes (high-molecular-weight protease) were purified from rat skin, and their enzymologic properties, gross structure, and tissue distribution were investigated. Skin proteasomes were purified by successive (NH4)2SO4 fractionation and by phenyl Sepharose CL-4B and HPLC gel filtration chromatography. The molecular weights of the proteasomes were estimated from gel filtration to be 750 kD. On sodium dodecylsulfate-polyacrylamide gel electrophoresis, the purified enzymes dissociated into several bands, the majority falling into the range of 36-20 kD. Two-dimensional electrophoretic analysis demonstrated approximately 10-15 separate protein spots with pl values varying between 3 and 10. As analyzed by electron microscopy, the gross structure of the enzymes showed an almost symmetrical ring-shaped particle with a small hole in the center. Succinyl-leucyl-leucyl-valyl-tyrosine-4-methylcoumaryl-7-amide, a fluorogenic substrate for serine proteinases, demonstrated the highest activity in terms of substrate specificity. Sodium dodecylsulfate, Ca++, and some free fatty acids activated enzyme activity. Activity was inhibited by diisopropylfluorophosphate, leupeptin, N-ethylmaleimide, iodoacetamide, and chymostatin. These results show that both serine and cysteine residues are related to the enzyme activity of proteasomes. Total and specific enzyme activities in the epidermis were, respectively, 10 and 20 times higher than in the dermis. Immunohistochemical studies utilizing the avidin-biotin complex method with monoclonal antibody revealed that the enzyme is distributed throughout the epidermis. These findings indicate the epidermal localization of proteasomes.

Local injection of hepatocyte growth factor/scatter factor (HGF/SF) alters cyclic growth of murine hair follicles.

Hepatocyte growth factor/scatter factor (HGF/SF) has recently been shown to stimulate the hair follicle growth of mouse vibrissae in vitro. In this study, we analyzed the effect of cutaneous injections of recombinant human HGF/SF on hair follicle growth using mice in different hair cycle stages. Five male newborn mice, five male mice in second anagen, and five male mice in second telogen were administered a dorsal intradermal injection of 1 microg HGF/SF dissolved in 0.1% albumin-phosphate-buffered saline once daily for five or seven consecutive days, and then sacrificed on days 7 or 10. Hair follicle growth was evaluated photometrically and histologically using three parameters: the skin color of the reverse side of the resected skin, the skin thickness, and the area occupied by hair follicle tissue. The HGF/SF injected skin of newborn mice had hair follicles that were histologically longer and larger than those of the 0.1% albumin-phosphate-buffered saline injected skin. Mice that had received HGF/SF injection in second anagen, retained anagen hair follicles after 10 d only at the injection site, suggesting that HGF/SF delayed the transition from anagen to telogen. The HGF/SF injected skin of telogen mice had a significant increase in hair follicle tissue in the dermis, suggesting a mild anagen inducible activity by HGF/SF. Furthermore, precise measurements of the 20 hairs plucked from the HGF/SF injection sites revealed mild hair elongation in all the aforementioned experiments. These results imply that HGF/SF acts as a paracrine factor that alters cyclic hair growth of mice.

Production of blister in normal human skin in vitro by blister fluids from epidermolysis bullosa.

Pathogenic mechanisms involved in the blister formation of epidermolysis bullosa (EB) are not clearly understood at present. In this paper, we attempted to produce experimental blistering in vitro similar to the histologic picture of EB simplex (EBS) and recessive dystrophic EB (RDEB). It was demonstrated by light and electron microscopy that the medium containing fresh blister fluids from the patients with EBS or RDEB could produce similar histologic features in normal human skin (in vitro) to those of the skin lesions of patients (in vivo). This observation may open new avenues of approach to studying these diseases.

Hepatocyte growth factor/scatter factor stimulates hair growth of mouse vibrissae in organ culture.

Hepatocyte growth factor/scatter factor (HGF/SF) is a multifunctional polypeptide that acts as a mitogen, motogen, or morphogen, depending on the biologic context. In this study, we examined the effect of HGF/SF on hair growth using a serum-free organ culture system. Vibrissal hair follicles isolated from newborn mice were cultured at 31 degrees C in 95% O2/5% CO2 for 72 h in the presence of various cytokines or growth factors, and elongation of hair shaft, DNA, and protein synthesis in hair follicles were measured. Among the agents tested, only HGF/SF significantly increased hair follicle length (p < 0.001), 3H-thymidine (p < 0.001), and 35S-cysteine (p < 0.05) incorporation. The effect of HGF/SF was dose dependent, with maximal stimulation obtained at 10 ng/ml. The increase in hair follicle length and thymidine incorporation were specifically inhibited by a neutralizing antibody against HGF/SF. These results indicate that HGF/SF can promote hair growth and may have clinical utility in this regard.

Efficient gene activation system on mammalian cell chromosomes using recombinant adenovirus producing Cre recombinase.

To develop a method for activating genes located on cell chromosomes, an on/off switching unit regulated by the site-specific recombinase Cre was constructed. The switching unit was designed to express firstly the neo gene and secondly the reporter lacZ gene by Cre-mediated excisional deletion of the neo gene. CV1 cell lines bearing the switching unit on a cell chromosome were isolated and activation of the lacZ gene was examined after infection with a Cre-producing recombinant adenovirus. In one cell line virtually 100% of the cells stably expressed the lacZ gene, whereas in another cell line lacZ-expressing cell populations reached only to about 90% and decreased after cell divisions. The Southern blot analyses showed that the latter type of cells contained a head-to-tail array of the switching units, and that consequently the lacZ-expressing units were excised from a cell chromosome and present as extrachromosomal circular DNAs. These results showed that the system offers efficient activation of genes introduced into cell chromosomes and that the organization of the reporter units are important for efficiency and duration of the activated gene expression.

The effect of various cytokines on hair growth of mouse vibrissae in organ culture.

Hepatocyte growth factor/scatter factor (HGF) is a multifunctional polypeptide which acts as a mitogen, motogen or morphogen depending on the biological context. In this study, we examined the effect of HGF on hair growth using a serum-free organ culture system. Vibrissal hair follicles isolated from newborn mice were cultured at 31 degrees C in 95% O2-5%CO2 for 72 h in the presence of various cytokines or growth factors. DNA, protein synthesis and elongation of the hair shaft in the hair follicles were measured. Among the agents tested, only HGF significantly increased hair follicle length (P < 0.001) and 3H-thymidine (P < 0.001) incorporation. The effect of HGF was dose-dependent, with maximal stimulation obtained at 10 ng/ml. The increase in hair follicle length and thymidine incorporation were specifically inhibited by a neutralizing antibody against HGF. These results indicate that HGF is able to promote hair growth and may have clinical utility in this regard.

Organ culture conditions of human hair follicles.

Experimental results revealed that although [3H]thymidine uptake in the hair bulb increased time dependently for 12 days under normal culture conditions (95% air-5% CO2 at 37 degrees C), striking morphological changes occurred in the hair bulb cells as demonstrated by histological findings. As such, organ culture conditions applicable to human hair follicles were studied utilizing observations from both histology and DNA synthesis. We found that culture conditions of 95% O2-5% CO2 at 31 degrees C were superior when compared to normal culture conditions for cultures of human hair follicles when attempting to maintain the normal morphology of hair germinative cells. The hair bulb and the germinative cells successfully maintained their normal morphology throughout the 96 and 48 h culture period, respectively. Autoradiographs of [3H]thymidine-labeled follicles showed localization in the germinative cells below Auber's critical line. Hair bulb DNA synthesis increased time dependently for 96 h after culture initiation. Under conditions of 95% O2-5% CO2 at 31 degrees C, the synthesis of DNA in hair germinative cells was observed. Such an organ culture method may prove useful for studies on the human hair growth mechanism.

An attempt to treat intractable skin ulcers with autologous dermal graft.

Five patients with a total of seven chronic intractable skin ulcers and impaired peripheral blood circulation were treated with autologous dermal grafts which did not contain any epidermal component. These ulcers were refractory to conventional therapy. Results from the dermal grafting were variable, with four of the ulcers (in two patients) healing 1-8 months after 1-5 graftings. These findings suggest the possibility that dermal grafting may be a simple yet effective treatment for deep, super-infected skin ulcers in which tendons and bones have become exposed. Dermal grafting may also provide an additional method by which to treat chronic skin ulcers which do not respond to conventional surgical therapy and where amputation of affected extremities is being considered.