First left-right comparative study of topical rapamycin vs. vehicle for facial angiofibromas in patients with tuberous sclerosis complex.

BACKGROUND: Tuberous sclerosis complex (TSC) is an autosomal dominant disorder causing multiple hamartomas. Treatment of TSC lesions with mammalian target of rapamycin inhibitors is effective. Recently, several reports have shown the efficacy of topical rapamycin (sirolimus) for angiofibromas. However, almost all studies have been case studies and the 0·1% solution caused skin irritation. A comparative study of topical rapamycin and a vehicle has not yet been reported. OBJECTIVES: To compare the efficacy of topical rapamycin formulation with that of vehicle for angiofibromas. METHODS: A left-right comparative study between rapamycin 0·2% topical formulation and vehicle was conducted in 11 patients with TSC. Two formulations, an ointment and a gel, were prepared and in vitro percutaneous absorption of rapamycin was determined. RESULTS: In vitro percutaneous absorption of rapamycin was significantly greater with the gel compared with the ointment. In the clinical study, the rapamycin-treated cheek showed significant improvements relative to the vehicle-treated cheek in all outcome measures after 12 weeks of treatment. The improvement was particularly remarkable in children aged ≤ 10 years. No side-effects were noted, and rapamycin was not detected in the blood of the patients. CONCLUSIONS: Topical rapamycin was significantly effective against angiofibromas. Both formulations used were effective and safe. The 0·2% gel is especially useful because of its better skin penetration and low irritancy. Initiation of topical rapamycin therapy in early childhood would be beneficial for patients with TSC.

A topical combination of rapamycin and tacrolimus for the treatment of angiofibroma due to tuberous sclerosis complex (TSC): a pilot study of nine Japanese patients with TSC of different disease severity.

BACKGROUND: Dysregulation of mTOR signalling by mutations in tuberin and/or hamartin leads to the formation of tuberous sclerosis complex (TSC). Trials to treat TSC using mTOR inhibitors, including rapamycin, have been performed. Although rapamycin improves many TSC lesions, significant side-effects appear after systemic administration. Topical administration has been recommended. OBJECTIVES: The efficacy of rapamycin-tacrolimus ointment was examined for TSC-related angiofibroma. METHODS: Left-right comparisons of the tacrolimus ointments with/without 0·2% rapamycin was conducted in symmetrical facial angiofibromas in nine patients with definitive TSC. After the 3-month treatment, a cumulative score for redness, flatness and papule size was used to evaluate the efficacy of the treatment. Blood rapamycin levels were analysed by liquid chromatography-electrospray mass spectrometry (LC-ESI/MS). RESULTS: At the end of the treatment, all of the scores significantly improved for rapamycin-tacrolimus treatment compared with tacrolimus alone. No adverse reactions were noted and blood levels of rapamycin were below the detection limit in all cases. CONCLUSIONS: Topical application of rapamycin-tacrolimus ointment is a safe and useful treatment for TSC-related angiofibroma.

Clinical and genetic features of 20 Japanese patients with vascular-type Ehlers-Danlos syndrome.

BACKGROUND: Vascular-type Ehlers-Danlos syndrome (vEDS) is a severe autosomal dominant inherited disorder resulting from mutations within the α1 type III collagen gene (COL3A1). The majority of published mutations are base changes leading to the substitution of single glycine residues within the triple-helical domain of type III collagen. Although clinical characteristics and mutations in the COL3A1 gene have been analysed for some patients from Europe and America, similar analyses have not yet been performed for Japanese patients with vEDS. OBJECTIVES: To analyse the genetic and phenotypic findings in Japanese patients with vEDS. METHODS: We analysed the clinical features of 20 unrelated individuals with vEDS. To quantify type III collagen production, the fibroblasts were cultured with (3) H-proline, and the radiolabelled collagenous proteins were analysed using sodium dodecyl sulphate-polyacrylamide gel electrophoresis and fluorography. Mutations in COL3A1 were detected by sequence analysis of cDNA from patients' fibroblasts and subsequently by a genomic DNA sequence analysis. RESULTS: Thin and translucent skin with extensive bruising and hypermobility of the small joints were observed in about 90% of the patients, whereas the prevalence of serious clinical findings such as rupture/dissection/aneurysm of the arteries (30%) or rupture of the gastrointestinal tract (25%) was relatively low. Sequence analyses of the COL3A1 gene demonstrated heterozygous point mutations leading to glycine substitution in only nine patients (45%), while heterozygous splice-site mutations at the junction of the triple-helical exons were observed in the remaining 11 patients (55%). The average type III collagen production level in the cultured dermal fibroblasts was 14·6% of the normal value. The types of complication were not associated with specific mutations in COL3A1. CONCLUSION: The analysis in the present series revealed a low frequency of patients presenting with serious clinical findings such as arterial rupture/arterial dissection/aneurysm and perforation or rupture of the gastrointestinal tract, and revealed a higher prevalence of splice-site mutations at the junction of the triple-helical exons than of glycine substitution mutations in COL3A1.

Cells derived from tuberous sclerosis show a prolonged S phase of the cell cycle and increased apoptosis.

Tuberous sclerosis complex (TSC) is a multisystemic disorder characterized by systemic hamartomas. Although the disease-determining genes TSC1 and TSC2 have been isolated, the molecular pathogenesis of the disease is not understood. We examined cell cycle abnormalities in skin specimens and cultured cells derived from specific lesions of TSC patients with confirmed TSC1 or TSC2 mutations. None of the specimens used in this study showed loss of heterozygosity (LOH). We detected more cells positive for PCNA and fewer cells positive for MPP2 in the epidermis of TSC patients than in the epidermis of control patients without TSC. Incorporation of 5-bromo-2-deoxyuridine (BrdU) was similar in fibroblasts derived from TSC lesions and in normal human fibroblasts. These results suggest that the cell cycle of TSC cells shows a prolonged S phase. Flow cytometric analysis confirmed S phase prolongation in TSC cells. Many apoptotic cells were detected by a nick end labeling assay in both skin tissue and cultured fibroblasts derived from specific TSC lesions. Examination of cyclin levels showed increased nuclear cyclin A and cytoplasmic cyclin B and decreased nuclear cdc2 levels. We conclude that suppression of either TSC1 or TSC2 may change cyclin levels, prolong S phase and induce apoptotic cell death.

Analysis of all exons of TSC1 and TSC2 genes for germline mutations in Japanese patients with tuberous sclerosis: report of 10 mutations.

Twenty-seven Japanese patients with the tuberous sclerosis complex (TSC), consisting of 23 sporadic and 4 familial cases, were tested for mutations in the TSC1 and TSC2 genes, using single-strand conformational polymorphism analysis and direct sequencing. Four possible pathogenic mutations were found in the TSC1 gene, including three frame shifts and a nonsense mutation in a familial case. All mutations were expected to result in a truncated hamartin gene product. The TSC2 gene analysis identified six possible pathogenic mutations only in the sporadic cases, including two frame shifts, one in-frame deletion, and three missense mutations. Two of the TSC2 mutations were expected to result in a truncated tuberin gene product. These results of the Japanese TSC patients were compatible with the reports from Europe and the United States, i.e., (1) TSC1 mutations are rarer in sporadic cases than in familial cases, (2) substantial numbers of sporadic cases arise from mutations in the TSC2 gene, and (3) mutations of the TSC1 gene may cause premature truncation of hamartin.

LBP-p40 binds DNA tightly through associations with histones H2A, H2B, and H4.

Laminin binding protein precursor p40 (LBP-p40) was long believed to be located exclusively in the cytoplasm. We recently reported localization of epitope-tagged LBP-p40 to the nucleus tightly associated with nuclear structure as well as on ribosomes. In this paper, we analyze the interaction of LBP-p40 with DNA and nuclear proteins in vitro. LBP-p40 was found to bind to a double-stranded DNA cellulose column at moderate salt. However, when mixed with a high salt nuclear extract, LBP-p40 was eluted from the DNA cellulose column only at higher salt. An LBP-p40 affinity column indicated that both histone H1 and in particular the core histones associate with LBP-p40. Using recombinant core histone molecules fused with glutathione S-transferase (GST), we demonstrate that histones H2A, H2B, and H4 are capable of interacting with LBP-p40, whereas H3 is not. These results suggest that association of LBP-p40 with histones H2A, H2B, and H4 confers tight binding of LBP-p40 to chromatin DNA in the nucleus.

The induction of apoptosis in HeLa cells by the loss of LBP-p40.

To analyze the function of the laminin-binding protein precursor p40 (LBP-p40) in higher eukaryotic cells, plasmid DNA expressing antisense or sense cDNA for p40 under the control of the LacSwitch system was introduced into HeLa cells. Stable transformants were isolated, and the expression of p40 was assayed by Western and Northern blotting. The expression level of p40 was not affected in HeLa cell transformants cultured in 10% serum-supplemented media with the induction of antisense (AS)-p40 with 5 mM IPTG. However, both the protein and message for endogenous p40 in serum-depleted media with 5 mM IPTG were reduced to about 30 - 10% of the expression level in serum-free media without 5 mM IPTG. Colony formation was inhibited with the suppression of p40. AS-p40 clones died in 7 days when cultured in serum-depleted media with 5 mM IPTG, while clones without 5 mM IPTG AS-p40 clones never died, even in serum-depleted media. Additionally, sense (S)-p40 clones and control CAT clones survived more than 2 weeks in serum-free media with 5 mM IPTG. DNA fragmentation assay revealed that cell death induced by the reduction of AS-p40 resulted from apoptosis. Both the inhibition of cell growth and apoptotic cell death were partially rescued by the transfer of the p40 cDNA expression vector to AS-p40 clones. Moreover, the introduction of a synthetic hammerhead ribozyme for LBP-p40 using a fusigenic viral liposome suppressed the message for LBP-p40 even in the presence of 10% serum, and it also induced apoptosis.

Sustained transgene expression in vitro and in vivo using an Epstein-Barr virus replicon vector system combined with HVJ liposomes.

For long-term gene expression in tissues, we constructed an Epstein-Barr virus (EBV) replicon-based plasmid, pEB, containing the latent viral DNA replication origin (oriP) and EBV nuclear antigen-1 (EBNA-1). When pEB was transferred to human cells (HeLa-S3, HEK 293 and FS 3) and rodent cells (BHK-21) using HVJ-cationic liposomes, luciferase expression was observed in those cells for at least 10 days. Luciferase activity was two to 10 times higher in those cell lines on and after day 3 post-transfection of pEBActLuc compared with plasmids without the EBV replicon sequence. Southern blot analysis showed that the pEB vector luciferase gene was maintained extrachromosomally in BHK-21 cells. In human cells, transformation was five to 20 times more efficient with pEBc than with pcDNA3, and 18-35% of the introduced EBV replicon plasmid was replicated autonomously. The luciferase gene or lacZ gene was introduced into mouse liver using HVJ-AVE liposomes. Luciferase gene expression was observed for at least 35 days in cells transfected with pEBActLuc, whereas it was not detected on day 14 in cells transfected with pActLuc, which lacks the EBV sequence. By the transfer of pEBActNlacF, the lacZ gene expression rate in hepatocytes was approximately 35 and 12% on days 7 and 35, respectively.

OK-432 therapy for recalcitrant warts.

Twelve patients with warts recalcitrant to various treatment, including cryotherapy, were treated with OK-432 injection therapy. Six patients received only subcutaneous injection, three received only intralesional injection, and the other three received both subcutaneous and intralesional injections. Complete clearing of the warts occurred in nine (75%) of 12 patients, while the other three patients were not cured. Grouping the patients by the method of injection, the success rate of subcutaneous injection was 5/9 (55.6%), and that of intralesional injection was 4/6 (66.7%). Although five patients complained of mild fever and malaise, these side effects gradually disappeared during the repeated injections. OK-432 injection is considered as a hopeful therapy for recalcitrant warts.

A case of tuberous sclerosis with a giant angiofibroma.

This is a case study of tuberous sclerosis with unusually large facial angiofibromas. The patient had other intense skin manifestations. Histological findings in the angiofibroma included large and glial-appearing cells specific for this disease. Karyotype analysis revealed a translocation of chromosome (12q-, 15q+).

Differential localization of TGF-beta-precursor isotypes in psoriatic human skin.

Transforming growth factor-beta (TGF-beta) can act as a multi-functional regulator of both cell growth and differentiation. Three isoforms of TGF-betas, namely TGF-beta 1 TGF-beta 2 and TGF-beta 3, have been identified in human tissues. Previously we reported the expression of TGF-beta isoforms in normal human skin. However little is known about the role of TGF-beta isoforms in the pathogenesis of psoriasis. Using the TGF-beta precursor-specific antibodies to strengthen the specificity, we studied the immunohistochemical distribution of TGF-betas 1-3 in psoriatic skin. TGF-beta 2, which was found in the intercellular space of all the layers of the epidermis in normal human skin, was decreased in the psoriatic epidermis. The intensity of immunoreactivity has the tendency to decrease in the lower epidermis rather than in the upper epidermis of the transitional lesion. In contrast, TGF-beta 3 was present in the subepidermal area of the psoriatic skin as in the normal human skin. TGF-beta 1 was observed in neither epidermis nor dermis in both normal and psoriatic skin. Since TGF-beta is a potent growth inhibitor for human keratinocytes, the decrease of TGF-beta 2 in the epidermis of psoriatic skin may contribute to epidermal hyperplasia, a hallmark of psoriasis.

The loss of 40-kDa chromosomal protein in tuberous sclerosis lesions.

Tuberous sclerosis is an genetic disease characterized by systemic hamartomas. Some of the cultured cells derived from tuberous sclerosis show the distinct chromosomal disarrangement and abnormal cell division. To detect the proteins responsible for this phenomenon, we investigated many nuclear components related to cell division. 40-kDa protein (p40) recognized by one monoclonal antibody M108, which was present in nucleus, chromosomes and cytoplasm of many vertebrate culture cells, significantly decreased in the tuberous sclerosis cells. Immunoblot analysis revealed that in all the fractions the quantity of p40 of severe tuberous sclerosis cells was approximately 4 times less than p40 in normal fibroblasts.

Differential localization of TGF-beta-precursor isotypes in normal human skin.

Transforming growth factor-beta (TGF-beta) can act as a multi-functional regulator of both cell growth and differentiation. Three isotypes of TGF-beta s namely TGF-beta 1, TGF-beta 2 and TGF-beta 3, have been found in human tissues. Up to now, little is known about the distribution patterns of the TGF-beta isotypes in human skin. Using the TGF-beta-precursor (latency-associated peptides) specific antibodies to confirm the specificity, we studied the immunohistochemical distribution of TGF-beta 1-3 in human skin. TGF-beta 2 was found mainly in the intercellular space of all the layers of the epidermis as well as in the cytoplasm with a weak staining. In contrast, TGF-beta 3 was present in the subepidermal area of the dermis. TGF-beta 1 was observed obviously in neither epidermis nor dermis. These results showed the differential localization of TGF-beta isotypes in human skin, suggesting that the TGF-beta 2 and TGF-beta 3 may regulate the human skin function in an epithelial autocrine or mesenchymal-epithelial interaction manner.

A monoclonal antibody against the nucleus reveals the presence of a common protein in the nuclear envelope, the perichromosomal region, and cytoplasmic vesicles.

A monoclonal antibody that recognizes antigenic determinants on the nucleus of cultured mammalian cells was isolated. Immunofluorescence studies using this antibody showed that the recognized antigen was present not only on the nucleus but also in cytoplasmic vesicles of interphase cells and in the perichromosomal region of mitotic cells. Premature chromosome condensation analysis showed that the reactive site for this monoclonal antibody could be detected in the perichromosomal region during the G2 and M phases, but not during the G1 and S phases. Finally, immunoblot analysis showed that this monoclonal antibody prepared against the nucleus recognized a protein of approximately 40 kD both in the cytoplasm and in the perichromosomal regions.