Factor VII Deficiency Due to Compound Heterozygosity for the p.Leu13Pro Mutation and a Novel Mutation in the HNF4 Binding Region (-58G>C) in the F7 Promoter.

We investigated the molecular basis of factor VII (FVII) deficiency in a Japanese patient and identified compound heterozygous mutations. Factor VII activity and antigen levels in the patient were less than 5.0% and 6.5% of controls, respectively. All exons, exon-intron boundaries, and the 5' promoter region of F7 from genomic DNA were amplified using polymerase chain reaction (PCR). Sequencing analysis of PCR fragments revealed that the patient was heterozygous for a known T to C substitution at nucleotide position 38, which resulted in the p.Leu13Pro missense mutation (Factor VII Morioka) in the signal peptide region, and a novel mutation in the 5' promoter region (-58G>C). An electrophoretic mobility shift assay showed that the mutation in the promoter region reduced the binding of hepatocyte nuclear factor (HNF). It is presumed that the reduced binding of HNF-4 to the F7 promoter region reduces F7 transcription and thus reduces the synthesis and expression of FVII.

Depletion of Epidermal Langerhans Cells in the Skin Lesions of Pellagra Patients.

Pellagra is a nutrient deficiency disease caused by insufficient niacin levels. Recent studies have shown that numbers of epidermal Langerhans cells decreased in other diseases caused by nutritional deficiencies, including necrolytic migratory erythema and acrodermatitis enteropathica. Epidermal Langerhans cells are capable of modulating or even halting the inflammatory reaction. The aim of this study was to examine changes in the number of Langerhans cells and other dendritic cells, and maturation of epidermal Langerhans cells in the lesional and adjacent non-lesional skin in pellagra patients. Seven pellagra patients and 10 healthy individuals who served as controls were included. The number and distribution of dendritic cells and other cutaneous cells were examined by immunohistochemistry. Epidermal Langerhans cells decreased considerably in the skin lesions of pellagra patients, whereas other dendritic cells did not change. The decrease in the number of Langerhans cells was positively correlated with the histological severity of skin lesions. As the number of Langerhans cells was not reduced in the undisturbed neighboring skin, the depletion of epidermal Langerhans cells did not precede skin damage but was a cause of prolonged severe inflammation.

Factor X Deficiency with Heterozygous Mutations of Novel p.G435S and Known p.G244R in a Patient Presenting with Severe Umbilical Hemorrhage.

A 10-day-old male patient was referred to our hospital with severe umbilical bleeding. Prothrombin time (PT) and activated partial thromboplastin time (APTT) were prominently prolonged. Plasma coagulation factor X (FX) activity and antigen levels were 1% and 0.6%, respectively. A DNA sequence analysis of his leukocytes revealed a compound heterozygous state; known Gly244 to Arg (p.G244R) in exon 6 and a novel mutation of Gly 435 to Ser (p.G435S) in exon 8. A pedigree analysis showed that p.G244R originated from the paternal side, while p.G435S was from the maternal side. A p.G244R mutation was reported previously as FXDebrecen and this mutated protein was synthesized as a non-secretable protein. The glycine at amino acid position 435 in the C-terminal region is completely conserved in the trypsin-like serine protease family, including thrombin, FVII, protein C, plasmin, trypsin, and chymotrypsin. In a three-dimensional structural model of FX, Gly 435 was located within the 11th ß-strand and buried in the back of the catalytic pocket. Therefore, the substitution to serine was expected to disrupt this structure. p.G435S FX was also predicted to be synthesized and exist in the cytoplasm, but not to be secreted into culture media by a cDNA expression assay. These two mutations may be responsible for the type 1 (null levels of both activity and antigen in plasma) FX deficiency with severe bleeding phenotype.

Clinical analysis of leg ulcers and gangrene in rheumatoid arthritis.

Leg ulcers are often complicated in patients with rheumatoid arthritis (RA), however, the etiology is multifactorial. We examined the cases of leg ulceration or gangrene in seven RA patients who were hospitalized over the past 3 years. One patient was diagnosed as having pyoderma gangrenosum. Although vasculitis was suspected in three patients, no histological evidence was obtained from the skin specimens. In these patients, angiography revealed the stenosis or occlusion of digital arteries. In the remaining three patients, leg ulcers were considered to be due to venous insufficiency. Treatment should be chosen depending on the causes of leg ulcers.

Efficacy of high-concentration tacalcitol ointment in psoriasis vulgaris after changing from other high-concentration vitamin D3 ointments.

Three high-concentration vitamin D3 ointments are currently available in Japan for the treatment of psoriasis. The aim of the present study is to investigate the efficacy of high-concentration tacalcitol in patients with psoriasis vulgaris who have already been treated with another high-concentration vitamin D3 ointment, calcipotriol or maxacalcitol. The psoriasis area and severity index score was improved in more than half the patients after changing to the tacalcitol ointment. Many patients treated with maxacalcitol once a day achieved greater clinical improvement by changing to high-concentration tacalcitol. In contrast, some patients who had responded to a high-concentration tacalcitol ointment showed exacerbation after changing to maxacalcitol once a day. Interviews with 50 patients (including the 34 patients enrolled in the present study) indicated that high-concentration tacalcitol ointment was an acceptable therapy in terms of the number of daily applications and drug cost. The results of this clinical study suggest that high-concentration tacalcitol ointment meets the preference of many patients who wish to use an ointment once a day.

Evidence that PI3K, Rac, Rho, and Rho kinase are involved in basic fibroblast growth factor-stimulated fibroblast-Collagen matrix contraction.

Fibroblast-collagen matrix contraction has been used as a model system to study how cells organize connective tissue. Previous work showed that lysophosphatidic acid (LPA)-stimulated floating collagen matrix contraction is independent of Rho kinase while platelet-derived growth factor (PDGF)-stimulated contraction is Rho kinase-dependent. The current studies were carried out to determine the signaling mechanisms of basic fibroblast growth factor (bFGF)-stimulated fibroblast-collagen matrix contraction. Both bFGF and LPA promoted equally collagen matrix contraction well. Three different inhibitors, LY294002 for phosphatidylinositol-3-kinase (PI3K), C3 exotransferase for Rho and Y27632 for Rho kinase, suppressed the bFGF-stimulated fibroblast-collagen matrix contraction. With bFGF stimulation, fibroblasts spread with prominent stress fiber network formation and focal adhesions. In the presence of Rho kinase inhibitor, focal adhesions and stress fibers were mostly lost. We demonstrated that bFGF stimulation for fibroblast caused transient Rac and Rho activation but did not activate Cdc42. In addition, bFGF enhanced fibroblast migration in wound healing assay. The present study implicates PI3K, Rac, Rho, and Rho kinase as being involved in bFGF-stimulated collagen matrix contraction. The elucidation of bFGF-triggered signal transduction may be an important clue to understand the roles of bFGF in wound healing.

Clinical usefulness and patient satisfaction for treatment with low-dose cyclosporin administration in patients with moderate psoriasis vulgaris.

The Japanese guidelines for psoriasis therapy with cyclosporin microemulsion preconcentrate (CyA MEPC) has been revised, and the clinical application of CyA MEPC is being expanded to include mild to moderate psoriasis. In this study, we aimed to confirm the clinical efficiency of low-dose cyclosporin therapy in patients with moderate psoriasis vulgaris. After informed consent was obtained, 19 patients with psoriasis vulgaris were enrolled in this study. Each patient basically administrated CyA MEPC, 2.5 mg/kg/day, orally over 12 weeks. When the psoriasis area and severity index (PASI) score showed a 75% reduction from the initial value, the dosage of CyA MEPC was reduced to 1.5 mg/kg/day and added a topical application of active vitamin D3 ointment. We interviewed the patients as to their satisfaction for the usefulness and cost of the treatment. All patients obtained improvement within 12 weeks. In 10 patients whose PASI score reduced over 75%, we could reduce CyA MEPC dosage. No adverse effects were noted in any patients during the treatment. It is of note that the cost for 1.5 mg/kg/day administration of CyA MEPC was accepted by all the patients. In conclusion, this preliminary study suggests that the CyA MEPC is effective, safe and would provide patients with acceptable costs.

Successful treatment with cyclosporin administration for persistent benign migratory glossitis.

We herein describe a 54 year-old female patient with a 5-year history of persistent and painful benign migratory glossitis (BMG), which was remarkably improved by systemic administration of cyclosporin. She had noted some white patches leaving smooth denuded red areas with whitish elevated borders on the dorsum of her tongue, and finally felt strong pain. The lesion was refractory to the previous treatment with topical corticosteroid treatment for the last 2 years. Because clinicopathological findings were compatible with BMG, systemic administration of 20 mg/day prednisolone and topical 0.1% dexamethasone application were started, however, she suffered a severe relapse after tapering the dosage of prednisolone to 10 mg/day. Because some investigations have suggested that BMG is an oral manifestation of psoriasis, we introduced cyclosporin administration. The systemic treatment of cyclosporin microemulsion pre-concentrate, 3 mg/kg/day, resulted in a satisfactory improvement. Two months later, we could reduce cyclosporin microemulsion pre-concentrate dosage to 1.5 mg/kg/day for maintenance therapy, and the disease has been well controlled so far.

Harlequin sign (hemifacial flushing and contralateral hypohidrosis) in a 4-year-old girl with Horner syndrome.

We report a Japanese infant with Horner syndrome whose clinical examination and testing suggested the location of the causative lesion. A 4-year-old Japanese girl had an acquired right ocular ptosis and unequal pupils presenting shortly after birth. She also exhibited left hemifacial flushing and loss of sweating on the contralateral side (harlequin sign). Physical examination demonstrated 2.0 mm of ptosis of the right upper lid with normal elevator function. The diameters of the pupils were 4 mm on the left and 2.5 mm on the right. No sweating was induced in the right frontal region at 40 degrees C for 15 minutes of sweat challenge test. Otherwise, no abnormalities were found by the neurophysiologic examinations or magnetic resonance imaging of the brain. Based on the clinical examination, we speculated that the responsible lesion might be in the preganglionic areas. Harlequin sign was informative for making the diagnosis of Horner syndrome.

Basic fibroblast growth factor stimulates human keratinocyte motility by Rac activation.

Topical application of human recombinant basic fibroblast growth factor (bFGF) promotes wound healing. bFGF, however, has been reported to have little in vitro effects on keratinocyte compared with other cell types such as endothelial cells or fibroblasts. The aim of this study was to investigate the mechanism(s) of bFGF-stimulated keratinocyte migration. Normal human keratinocytes, seeded on coverslips that were noncoated or coated with type I collagen or fibronectin, were stimulated with bFGF to evaluate their ability to spread. Keratinocyte migration was measured using a Boyden chamber assay. The lysates of keratinocytes, which were plated on noncoated, type I collagen-coated or fibronectin-coated plastic dishes and stimulated with bFGF, were subjected to pulldown assays to detect guanine triphosphate-loaded Rac. Morphologically, keratinocytes formed lamellipodia only when they were stimulated with bFGF on the collagen-coated coverslips. Keratinocyte migration was significantly enhanced by bFGF. Guanine triphosphate-loaded Rac was detected only in the lysate of bFGF-stimulated keratinocytes on collagen-coated dishes. This in vitro study shows that bFGF exerts a stimulatory effect on keratinocyte migration in the presence of type I collagen as a scaffold, and, at least, Rac activation is involved.

Daily versus intermittent application of high-concentration tacalcitol ointment in combination with low-dose cyclosporin for psoriasis vulgaris.

In this study, we aimed to compare the clinical effectiveness of highly-concentrated tacalcitol ointment daily versus intermittent application in patients with psoriasis vulgaris who simultaneously took a low dose of cyclosporin. All the patients in both groups showed significant improvements, and the patients in the intermittent application group obtained more patient satisfaction in cost performance. The treatment cost of low-dose cyclosporin and intermittent application of highly-concentrated tacalcitol ointment was less than half of that of high-dose cyclosporin and daily application of highly-concentrated tacalcitol ointment. This preliminary study suggests that the combination therapy with low-dose cyclosporin administration and intermittent application of highly-concentrated tacalcitol is effective, safe and provides acceptable costs for the treatment.

Genetic analyses of two cases of Werner's syndrome.

We report two cases of Werner's syndrome (WS). First, a 42-year-old Japanese man was referred on suspicion of systemic sclerosis (SSc) because of scleroderma-like skin atrophy and foot ulcers. Second, a 51-year-old woman with malignant fibrous histiocytoma was referred on suspicion of premature aging syndrome. Because both patients had many typical manifestations compatible with WS, we made a clinical diagnosis of WS. Genetic analyses revealed a homozygous mutation, an A deletion at nucleotide 3677 of WS gene (WRN) in the first case and a homozygous mutation, a G to C substitution at one base upstream of exon 26 of WRN in the second case. Both mutations were consistent with those previously reported in Japanese WS patients.

Psychrobacter okhotskensis sp. nov., a lipase-producing facultative psychrophile isolated from the coast of the Okhotsk Sea.

A facultatively psychrophilic bacterium, strain MD17(T), which hydrolyses lipids at 5 degrees C, was isolated from the Monbetsu coast of the Okhotsk Sea in Hokkaido, Japan, when ice carried by the cold current came to the area. The isolate is an aerobic, non-motile coccobacillus that reduces nitrate to nitrite and hydrolyses Tweens 20, 40, 60 and 80, but not gelatin, DNA or alginic acid. The isolate grows at 0 degrees C, but not at temperatures higher than 36 degrees C; its optimum growth temperature is 25 degrees C. It grows in the presence of 0-10 % NaCl. Its major isoprenoid quinone is ubiquinone-8 (Q-8) and its DNA G+C content is 46.7 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain MD17(T) is closely related to Psychrobacter glacincola DSM 12194(T) (99.0 % similarity) and Psychrobacter immobilis DSM 7229(T) (98.7 % similarity). DNA-DNA hybridization revealed 45.9 % relatedness between strain MD17(T) and P. immobilis ATCC 43116(T) and 33.4 % between strain MD17(T) and P. glacincola ATCC 700754(T). Based on physiological and biochemical characteristics, phylogenetic position (as determined by 16S rRNA gene sequence analysis) and DNA-DNA relatedness, it is concluded that the isolate should be designated as a novel species, for which the name Psychrobacter okhotskensis sp. nov. is proposed. The type strain is MD17(T) (=NCIMB 13931(T)=JCM 11840(T)).

Bacillus krulwichiae sp. nov., a halotolerant obligate alkaliphile that utilizes benzoate and m-hydroxybenzoate.

Obligate alkaliphilic strains, AM31D(T) and AM11D, that utilize benzoate and m-hydroxybenzoate were isolated from soil obtained from Tsukuba, Ibaraki, Japan. The isolates grew at pH 8-10, but not at neutral pH. They were Gram-positive, facultatively anaerobic, straight rods with peritrichous flagella and produced ellipsoidal spores. The isolates reduced nitrate to nitrite and grew in 0-14 % NaCl, but not in higher concentrations. The major isoprenoid quinones were menaquinone-5, -6 and -7, and the cellular fatty acid profile consisted of significant amounts of 15-C branched-chain acids, isoC(15 : 0) and anteisoC(15 : 0). Phylogenetic analysis based on 16S rRNA gene sequencing indicated that strain AM31D(T) was a member of group 6 (alkaliphiles) in the genus Bacillus. DNA-DNA hybridization revealed a low relatedness of the isolates with several phylogenetically close neighbours, including Bacillus alcalophilus and Bacillus pseudalcaliphilus (less than 19.3 %). Based on phenotypic characteristics, phylogenetic data and DNA-DNA relatedness data, it was concluded that these isolates merited classification as a new species, for which the name Bacillus krulwichiae is proposed. The type strain of this species is AM31D(T) (=NCIMB 13904(T)=JCM 11691(T)=IAM 15000(T)).

Functions of the stratum corneum in systemic sclerosis as distinct from hypertrophic scar and keloid functions.

Both transepidermal water loss (TEWL) and skin surface high-frequency conductance are functions of the skin barrier. Systemic sclerosis (SSc) and hypertrophic scar (HS)/keloid are characterized by abnormal fibrotic changes in the dermis. Since the close interrelationship between the epidermis and the dermis has been well established, we analyzed the stratum corneum functions of forearm skin in 39 SSc patients after assessing the degree of the skin thickening and compared those functions with 10 age-matched normal controls. We also analyzed the stratum corneum functions of HS or keloid lesions in seven patients using the same methods, and compared those functions to adjacent or contralateral normal skin in identical patients. Neither the TEWL, nor high-frequency conductance of forearm skin in SSc patients were significantly different from those in normal controls. There was no correlation between the levels of TEWL or high-frequency conductance and the degree of skin thickening in SSc. In HS or keloid conditions, high-frequency conductance was significantly elevated (42.5+/-8.9 vs. 26.4+/-5.7, P<0.001). Although TEWL was elevated, there was no statistical significance (48.6+/-39.7 vs. 25.1+/-10.1). Our results revealed that stratum corneum functions are distinct between SSc and HS or keloid. This may reflect the various natures of dermal changes, which in turn differentiate the functions of the stratum corneum in the diseases.

A case of polypoid dermatofibroma.

Dermatofibroma is a common benign cutaneous tumor that usually appears as a slowly growing firm nodule. Polypoid nodular dermatofibroma is a variant type that is rarely encountered. We reported a case of polypoid dermatofibroma with a review of the previously reported cases. Polypoid dermatofibroma tends to arise on the leg, especially below the knee. Its size is often larger than that of common dermatofibroma. It is speculated that both the underlying firm tissue and long-term development may lead the tumor to form a polypoid appearance.