Gastrointestinal symptoms in idiopathic pulmonary fibrosis patients treated with pirfenidone and herbal medicine.

Pirfenidone is an antifibrotic agent for patients with pulmonary fibrosis, but this drug has adverse gastrointestinal (GI) effects. The first aim of this study was to assess GI symptoms due to pirfenidone by using a new questionnaire for reflux symptoms and dismotility symptoms. Whether adding herbal medicine of rikkunshi-to improved GI symptoms due to pirfenidone therapy was also investigated. This was a randomized controlled trial performed on 17 IPF patients. The patients were assigned to two groups, and the study period was 8 weeks. The pirfenidone group received pirfenidone therapy for 8 weeks with add-on rikkunshi-to from 4 weeks, while the control group did not receive either of these agents. To assess the effects of RK, plasma levels of acyl-ghrelin and des-acyl-ghrelin, serum KL-6 and surfactant protein-D, and pulmonary function tests were monitored. GI symptoms were most severe during the initial 2 weeks of pirfenidone therapy at a dose of 600 mg/day. Both reflux symptoms and dismotility symptoms deteriorated. Rikkunshi-to improved GI symptoms to the level prior to pirfenidone therapy. Plasma levels of des-acyl-ghrelin and acyl-/des-acyl-ghrelin ratio changed significantly at 8 weeks compared to 2 weeks. GI adverse events due to PFD were most severe in the first 2 weeks of treatment at a dose of 600 mg/day, and both reflux and dismotility symptoms deteriorated, but the drug was well tolerated at 1200 mg/day. Rikkunshi-to contributed to improvement of GI symptoms, but plasma ghrelin levels did not reflect the improvement of GI symptoms.

Successful treatment of lichen amyloidosus associated with atopic dermatitis using a combination of narrowband ultraviolet B phototherapy, topical corticosteroids and an antihistamine.

Lichen amyloidosus (LA) is a type of primary localized cutaneous amyloidosis characterized by multiple pruritic discrete hyperkeratotic papules with amyloid deposition in the papillary dermis. Clinical regression is usually difficult to achieve, even after treatment. In this study, we report a case of an adult man with LA associated with atopic dermatitis (AD) which was successfully treated with narrowband ultraviolet B (NB-UVB) phototherapy, topical corticosteroids and an oral antihistamine. This case suggests that NB-UVB phototherapy may be a useful adjuvant for LA associated with AD.

Pigmented spots as a sign of mammary Paget's disease.

Pigmented mammary Paget's disease is a rare variant of mammary Paget's disease. The clinical appearance mimics malignant melanoma. This paper describes a case of asymptomatic, slightly pigmented spots on the right mammary nipple. The pigmented nipple was histopathologically diagnosed as mammary Paget's disease with an underlying intraductal carcinoma. This case suggests the importance of conducting skin biopsies of developing pigmented spots on the nipples in elderly people.

Inhibition of cytokine-induced expression of T-cell cytokines by antihistamines.

OBJECTIVE: To investigate immunomodulatory properties of 4 antihistamines available in Japan. METHOD: Isolated peripheral blood T cells from healthy volunteers were preincubated with cetirizine, loratadine, olopatadine, or fexofenadine for 30 minutes and then stimulated with interleukin (IL)-1 2 or IL-4 to skew immune response towards type 1 or type 2 helper T cells. RNA was extracted 6 hours later and semiquantitative reverse transcription polymerase chain reaction (RT-PCR) was performed using primers for IL-5 and interferon (IFN) gamma. Supernatants were collected 24 hours after stimulation, and cytokine production was quantified by enzyme-linked immunosorbent assay (ELISA). RESULTS: RT-PCR revealed that IL-12-induced expression of IFN-gamma was partially suppressed by loratadine and fexofenadine and that all 4 agents tested inhibited IL-4-induced expression of IL-5. ELISA demonstrated that IL-12-induced IFN-gamma production was significantly suppressed by cetirizine and fexofenadine and IL-4-induced IL-5 production was downregulated by three agents with the exception of cetirizine. This study demonstrates that antihistamines have varying immunomodulatory properties, suggesting treatment choice for atopic dermatitis can be directed by disease signs and symptoms.

Niemann-Pick disease: coupling and uncoupling of inhibited sphingomyelinase activity and exogenous cholesterol esterification in fibroblasts by ionophore treatment.

In order to elucidate a biochemical relationship between sphingomyelin and cholesterol metabolisms, we examined the effects of several ionophores (monensin, nigericin, A23187, ionomycin, lasalocid) on sphingomyelinase activity and cholesterol esterification in cultured human fibroblasts. Phase-contrast microscopy showed the presence of foamy cells with monensin and nigericin treatments only. Electron microscopic examination revealed lamellated membranous bodies and cytoplasmic vacuoles in cells treated with monensin and nigericin. Monensin and nigericin treatments led to reduction of acid sphingomyelinase activity and disturbance of the esterification of lipoprotein-derived cholesterol in cultured fibroblasts, which is compatible with the biochemical changes of Niemann-Pick disease, type C. A23187, ionomycin, and lasalocid treatments showed only sphingomyelinase reduction in treated fibroblasts. Experimental models in this culture system could be produced in these ways, mimicking subtypes of Niemann-Pick disease, type A, B and type C.

Variant parameter values-as defined by the Chicago Criteria-produced by ManoScan and a new system with Unisensor catheter.

BACKGROUND: Recently reported normal values for esophageal motility obtained by high-resolution manometry (HRM) using a system with a Unisensor catheter were significantly different from those obtained by the ManoScan(®) , which could result in a wrong diagnosis. To clarify whether these differences were due to system or subject differences, we compared the manometric parameter values between ManoScan and a new system with a Unisensor catheter (Starlet) in the same subjects. METHODS: A total of 103 volunteers without any symptoms related to esophageal motility disorders were recruited. Esophageal HRM was performed using both the ManoScan and the Starlet in all subjects. Data from the ManoScan were analyzed using ManoView, and data from the Starlet were analyzed by a program with e-sleeve function. Integrated relaxation pressure, distal contractile integral, contractile front velocity (CFV), intrabolus pressure, and distal latency were calculated by both analyzing programs, and the values of these parameters were compared between the two systems by a signed rank test. KEY RESULTS: Data from a total of 97 participants were analyzed. The values of all parameters, except CFV, measured by the Starlet were significantly higher than those obtained by the ManoScan (p < 0.01). CONCLUSIONS & INFERENCES: Both systems can measure esophageal motility appropriately; nevertheless, we confirmed that the two systems showed different values of the parameters defined by the Chicago criteria. These differences should be recognized to evaluate esophageal motility precisely.

Assignment of three patients with xeroderma pigmentosum to complementation group E and their characteristics.

Three cases belonging to xeroderma pigmentosum (XP) complementation group E were analyzed clinically and photobiologically. The three Japanese patients were a 50-yr-old female (XP80TO), a 42-yr-old female (XP81TO), and a 41-yr-old female (XP82TO). They were assigned to complementation group E by the cell hybridization study. All showed lowered minimal erythema doses between those of normal Japanese and XP group A subjects at wavelengths of 280, 290, and 300 nm of monochromatic ultraviolet (UV) light. Patients XP80TO and XP81TO, but not patient XP82TO, showed a delayed peak reaction at 48 h to UV erythema. All fibroblast strains from these patients had a reduced level of 40%-44% unscheduled DNA synthesis (UDS) after irradiation with 10 J/m2 of 254 nm UV. Primary cultured epidermal cells from these patients exhibited a relatively low level of UDS (ie, 38%-51% of normal epidermal cells). All of the group E fibroblast strains were twice as sensitive to 254 nm UV killing [n (extrapolation number) = 1.3-1.8, Do (mean lethal dose) = 2.2-2.8 J/m2)] as normal fibroblasts (n = 1.5, Do = 5.0 J/m2). All of the above group E patients had mild XP symptoms, but not neurological abnormalities, at the fifth decade of age. Patients XP80TO and XP81TO had developed skin malignancies (patients XP80TO developed three basaliomas; patient XP81TO developed two basaliomas) at the ages of 46 and 41 yr, respectively.

Human peptidylarginine deiminase type III: molecular cloning and nucleotide sequence of the cDNA, properties of the recombinant enzyme, and immunohistochemical localization in human skin.

Peptidylarginine deiminase catalyzes the post-translational modification of proteins through the conversion of arginine to citrulline in the presence of calcium ions. In rodents, peptidylarginine deiminase has been classified into four isoforms, types I, II, III, and IV, which are distinct in their molecular weights, substrate specificities, and tissue localization. Of these isoforms, only type III was detected in epidermis and hair follicles. Although the role of this enzyme in these tissues is not yet clear, indirect data have shown that several structural proteins such as filaggrin, trichohyalin, and keratin are substrates for peptidylarginine deiminase. In this study, we cloned the full-length cDNA of human peptidylarginine deiminase type III (3142 bp) from cultured human keratinocytes by reverse transcription-polymerase chain reaction and by rapid amplification of cDNA ends methods. This cDNA contained a 1995 bp open reading frame encoding 664 amino acids (Mr = 74 770). To explore the physicochemical and enzymatic properties of human peptidylarginine deiminase type III, we constructed a plasmid for producing a recombinant human peptidylarginine deiminase type III in bacteria. The enzymatic characteristics of the recombinant enzyme were very similar to those of the rodent peptidylarginine deiminase type III. The recombinant enzyme showed the catalytic activities toward structural proteins of epidermis and hair follicle, filaggrin and trichohyalin, in which the deiminations maxima of about 60% and 13% arginine residues were observed in filaggrin and trichohyalin, respectively. An immunohistochemical study of human scalp skin with a monospecific anti-peptidyl-arginine deiminase type III antibody revealed that the type III enzyme was localized to the inner root sheath and outer root sheath of hair follicles. Peptidylarginine deiminase type III in the inner root sheath was notable between supramatrix and keratogenous zone and was scarcely detected in cornified hair zone. The enzyme was also expressed in the cuticle layer of hair. On the other hand, expression of the enzyme in the epidermis was very low. These data imply that human peptidylarginine deiminase type III is the predominant isoform in hair follicles and may function as a modulator of hair structural proteins, including trichohyalin during hair and hair follicle formation.

Rat epidermal cathepsin B: purification and characterization of proteolytic properties toward filaggrin and synthetic substrates.

The aim of this study was to purify epidermal cathepsin B from rat skin and investigate its proteolytic activities on filaggrin and several synthetic substrates. The molecular weight of purified monomeric cathepsin B was estimated to be 30 kDa by SDS-polyacrylamide gel electrophoresis. The amino acid composition, similar to that of liver cathepsin B, indicated the enzyme to be an acidic protease. The enzyme had strong hydrolytic activity toward N-benzyloxy-carbonyl-L-arginyl-L-arginine-7-amido-4-methylcoumarin (Z-Arg-Arg-MCA) (152 mU/mg) and N-benzyloxycarbonyl-L-phenylalanyl-L-arginine-7-amido-4-methylcoumarin (424 mU/mg), but had no proteolytic activity toward L-arginine-7-amido-4-methylcoumarin. The Km value for Z-Arg-Arg MCA was 0.34 mM and pH optimum was 5.5. Cathepsin B degraded rat epidermal filaggrin into small fragments at pH 4.0 and 5.5., and was inhibited by a specific cysteine proteinase inhibitor, N-[N-(L-3-trans-carboxyoxirane-2-carbonyl)L-leucyl]- agmatin. This study demonstrated that filaggrin was susceptible to degradation by cathepsin B. Such an action may have relevance to skin differentiation in which acid proteases are thought to participate.

Stimulation of human keratinocyte growth by alginate oligosaccharides, a possible co-factor for epidermal growth factor in cell culture.

Oligosaccharides, involved in regulation of plant developmental and defensive processes, were tested to determine their ability to enhance proliferation of human keratinocytes. A mixture of alginate oligosaccharides remarkably stimulated keratinocyte growth and [3H]thymidine uptake in the presence of epidermal growth factor (EGF). The activity was comparable to bovine pituitary extract, a common complement in keratinocyte culture, and additive on BPE-induced stimulation. The most effective oligosaccharide in the mixture was identified and its chemical structure was determined. These findings demonstrate a novel activity of alginate oligosaccharide(s) in keratinocyte growth and suggest a possible co-factor for EGF-dependent stimulation in medium for keratinocytes.

Expression of type XVI collagen in cultured skin fibroblasts is related to cell growth arrest.

The expression of type XVI collagen in various phases of cell growth in cultured skin fibroblasts was studied. A marked increase in type XVI collagen mRNA level was found in stationary phases of cell growth (non-adherent and confluent phases), whereas the expression of type I and III collagens was undetectable in the non-adherent phase but became greater in the confluent phase. When suspended cells were further cultured over 72 h (suspension arrest), mRNA level and gene transcription of type XVI collagen were time-dependently increased whereas those of type I collagen remained undetectable. When the confluent cells were further cultured for 72 h under the condition of serum deprivation (serum deprivation arrest), mRNA levels of both type XVI collagen and type I collagen were elevated. The level of type XVI collagen polypeptide in the culture media of suspension-arrested and serum deprivation-arrested cells paralleled the mRNA level of type XVI collagen. The results indicate that expression of type XVI collagen (a member of the fibril-associated collagens with interrupted triple helices), unlike interstitial collagens (type I collagen), is related to cell growth arrest brought about by two different growth inhibiting systems, suspension arrest and serum deprivation arrest.

Risk and preventive factors for skin phototype.

Risk and preventive factors for skin phototype are discussed. Skin phototype was firstly proposed by Fitzpatrick based on skin response of the Caucasian, whereas the Japanese skin type was proposed for Japanese skin by Satoh and Kawada. Since skin phototype is determined by a personal interview of sunburn and suntan experience, it has a limitation in accuracy for personal interview and racial difference. The skin phototype concept is practicable and useful for predicting individual's sensitivity to UV, risk and preventive factors, and choosing sunscreens even with the limitation.

Precursor of rat epidermal cathepsin L: purification and immunohistochemical localization.

Immunoblot study using anti-rat cathepsin L antibody revealed almost only a precursor present in a crude extract of homogenized lower epidermis of rat skin, while the precursor and mature form were detected in the upper epidermis. To elucidate mechanisms of synthesis of cathepsin L, we have purified a precursor of cathepsin L from rat epidermis and investigated its localization in the skin. The precursor was purified after separation from the mature form in the final purification step of active fraction for N-benzyloxycarbonyl-L-phenylalanyl-L-arginine-7-amido-4-methylcoumari n. The precursor showed a single protein band with Mr 39 kDa on SDS/polyacrylamide gel electrophoresis and was immunoreacted with the anti-rat cathepsin L antibody. Two types of NH(2)-terminal sequences obtained were identical to the amino acid residues from -96 to -86 and those from -93 to -87 deduced from cDNA of the precursor of cathepsin L. The precursor was processed to mature form of the enzyme and the enzyme activity remarkably increased after 48-h incubation of the whole epidermis in 1 M acetate buffer (pH 3. 5) at 20 degrees C. In histological sections of the skin, a thin and diffuse staining pattern was present in the spinous layer and a dense and linear staining in the granular layer of the epidermis. In contrast, rat liver showed more mature form than the precursor by immunohistological findings. These results suggest that cathepsin L may have some roles in the terminal differentiation.

Isolation and molecular cloning of epidermal- and hair follicle-specific peptidylarginine deiminase (type III) from rat.

Peptidylarginine deiminase (PAD) is a post-translational modification enzyme that catalyzes deimination of arginine residues of proteins in the presence of calcium ions. There are three types of PAD in rodent tissues: PAD types I, II, and III [Terakawa et al. (1991) J. Biochem. 110, 661-666]. Type III enzyme was detected only in the epidermis and in hair follicles. In this study, we have purified PAD type III from 2-day-old rat epidermis by a four-step procedure that included soybean trypsin inhibitor-affinity chromatography. The enzyme was purified about 600-fold from the crude extract and the recovery was 23%. The final preparation of the enzyme gave only a single protein band on SDS-PAGE and showed an apparent molecular weight of 76,000. Subsequently, we cloned and sequenced the full-length cDNA encoding rat PAD type III by reverse transcription-polymerase chain reaction (RT-PCR) using degenerate oligonucleotide primers designed from the internal amino acid sequences and by the rapid amplification of the cDNA ends method. The composite cDNA sequence contained a 5' untranslated region of 42 bp, an open reading frame of 1,995 bases that encoded 664 amino acids (Mr=75,036), a 3' untranslated region of 1,063 bp, and part of a poly(A)+ tail. The entire reading frame sequence of rat PAD type III showed 51% homology with that of rat PAD type II, and the C-terminal region is highly conserved between the two types. The cloned gene was expressed in Escherichia coli cells to produce PAD type III, which had not only enzymatic activity, but also immunoreactivity against specific antibodies toward PAD type II. Furthermore, the specific expression of the enzyme in the epidermis and hair follicles was confirmed by RT-PCR assays of mRNAs from several tissues.

Rat epidermal cathepsin L-like proteinase: purification and some hydrolytic properties toward filaggrin and synthetic substrates.

We have purified cathepsin L-like proteinase from rat epidermis, determined its NH2-terminal amino acid sequence, and investigated its proteolytic activities on an intermediate filament-associated protein filaggrin and several synthetic substrates. The amino acid sequence of its NH2-terminus was determined to be Val-Pro-Asn-Ser-Leu-Asp-Trp-Arg-Glu-Lys-Gly-Tyr-Val-Thr-Pro-, which differed from that of rat cathepsin L and was not found in the amino acid sequence data bank. The enzyme consisted of a single-chain form with M(r) 30,000. Its hydrolytic properties toward synthetic substrates were similar to those of cathepsin L in other tissues. The enzyme effectively proteolyzed rat epidermal filaggrin into small fragments at pH 4.0-6.0 and was inhibited by a specific cysteine proteinase inhibitor, N-[N-(L-3-trans-carboxyoxirane-2-carbonyl)L-leucyl]-agmatin. However, cathepsins D and E from rat epidermis did not hydrolyze filaggrin. This study demonstrated that filaggrin was susceptible to degradation by rat epidermal cathepsin L-like proteinase, suggesting that this proteolytic activity may have relevance to skin differentiation, in which acid proteases are thought to participate.