Genetics in pulmonary arterial hypertension in a large homogeneous Japanese population.

Pulmonary arterial hypertension (PAH) is a rare but serious disease with a grave prognosis. Bone morphogenetic protein type 2 receptor (BMPR2) gene is a strong pathogenic factor for PAH. As a collaborative team from Kyorin University and Keio University in Japan, we have analyzed the BMPR2 gene in 356 probands and more than 50 family members, including secondary patients. Importantly, the study population is a racially, ethnically, and socially homogeneous population. In PAH patients, there is a high incidence of unique mutations in BMPR2, and several mutations are frequently observed in the Japanese population, suggesting that these common and recurring mutations may be highly pathogenic or have high penetrance, explaining why they are found frequently throughout the world. We have also mapped each breakpoint of exonic deletions/duplications and found that most break and rejoining points are in the Alu elements. Reviewing the distribution of the reported mutations on each exon of BMPR2 revealed that the number and frequency of mutations are imbalanced among exons. The penetrance of BMPR2 gene mutations was 3-fold higher in females than males. Full elucidation of BMPR2-mediated pathogenic mechanisms in PAH requires persistent efforts to achieve precision or individualized medicine as a therapeutic strategy for PAH.

Uptake and retention of radio-caesium in earthworms cultured in soil contaminated by the Fukushima nuclear power plant accident.

To understand the effects of radionuclides on non-human biota and the environment, it is essential to study the intake and metabolism of radio-isotopes in earthworms which are among the most important soil organisms, and Eisenia fetida, which were used in this study, are known to be sufficiently sensitive to chemicals and representative of common earthworms. In this study, we assessed the concentration ratios, uptake and retention, absorbed dose rate, and distribution of radio-caesium in earthworms. The concentration ratios of (137)Cs (i.e., the concentrations of radio-caesium in earthworms relative to those in dry soil) were higher early in the culturing period and decreased gradually over the experimental period. (137)Cs taken up by E. fetida was cleared rapidly after the worms were cultured in radio-caesium-free soil, suggesting that the metabolism of radio-caesium in earthworms is very rapid. Autoradiography demonstrated that the concentration of radio-caesium within the digestive tract was as high as that in the soil, while radio-caesium in the body tissue was lower than radio-caesium in the soil and was almost uniformly distributed among earthworm tissues. The highest absorbed dose rate of total exposure to radio-caesium ((137)Cs + (134)Cs) was calculated to be 1.9 × 10(3) (µGy/day) in the earthworms.

Interruption of NFkappaB-STAT1 signaling mediates EGF-induced cell-cycle arrest.

It is known that EGF induces the cell-cycle arrest in A431 cells that possess high numbers of EGF receptors and it was previously suggested that p21/WAF1 protein was a major effector molecule of the EGF-mediated cell-cycle arrest of A431 cells. Here, we further investigate this phenomenon using the decoy double-strand oligonucleotides for STAT-binding sequence (STAT decoy) and IkappaB, an inhibitor of the nuclear factor kappa B (NFkappaB). Addition of STAT decoy restored EGF-induced A431 cell-growth arrest. Interestingly, infection of adenovirus vectors to express IkappaB (AxIkappaBalphaDeltaN) as the inhibitor of NFkappaB also reversed the A431 cell-growth inhibition. The individual treatment of two inhibitors partially inhibited the WAF1 gene expression, whereas simultaneous treatment of two inhibitors exhibited more efficient inhibition. These observations suggest the activation of NFkappaB via IkappaB degradation and STAT1 via specific receptor kinase activity synergistically induce WAF1 gene expression in A431 cells. Thus, NFkappaB and STAT1 pathways mutually interact to play an important role in the EGF-induced intracellular reaction.

Development of chimeric molecules for recognition and targeting of antigen-specific B cells in pemphigus vulgaris.

Pemphigus vulgaris (PV) is an autoimmune blistering disease characterized by circulating pathogenic IgG antibodies against desmoglein 3 (Dsg3). The purpose of this study was to develop chimeric molecules for specific recognition and elimination of autoimmune B cells in PV. Mouse hybridoma cell lines producing anti-Dsg3 antibody (5H10, 12A2) were developed as an in vitro model system for targeting B cells. Dsg3-GFP, a baculoprotein containing the entire extracellular domain of Dsg3 fused with green fluorescence protein, recognized and targeted the hybridoma cells through their surface immunoglobulin receptors in an antigen-specific way. The epitopes of these monoclonal antibodies were mapped on the amino terminal EC1 and part of EC2, a region considered functionally important in cadherins. Chimeric toxin molecules containing the mapped region (Dsg3deltaN1) and modified Pseudomonas exotoxin were produced in bacteria (Dsg3deltaN1-PE40-KDEL, PE3 7-Dsg3deltaN1-KDEL) and tested in vitro on hybridoma cell lines. The chimeric toxins, but not Dsg3deltaN1 alone, showed dose-dependent toxic activity with a reduction in hybridoma cell number to 40-60% of toxin-negative control cultures, compared with little or no effect on anti-Dsg3-negative hybridoma cells. Furthermore, these toxins showed toxic effects on anti-Dsg3 IgG-producing B cells from Dsg3deltaN1-immunized mice, with a 60% reduction in cell number compared with Dsg3deltaN1 alone. Thus, specific recognition and targeting of antigen-specific B cells in PV was demonstrated; this strategy may hold promise as a future therapeutic option for PV and other autoimmune diseases.

Ex vivo delivery of suicide genes into melanoma cells using epidermal growth factor receptor-specific Fab immunogene.

The Fab fragment of monoclonal antibody B4G7 against human epidermal growth factor (EGF) receptor was conjugated with cationic poly-L-lysine and the resulting conjugate was further complexed with reporter genes or therapeutic genes. This Fab/DNA complex was designated as "Fab immunogene." The Fab immunogene transfer in vitro was mediated through the EGF receptors in two melanoma cell lines. The frequency of cells expressing beta-galactosidase (beta-Gal) reporter gene was approximately 1%. The induction of suicide effects after Fab immunogene transfer of herpes simplex virus thymidine kinase (TK) or Escherichia coli cytosine deaminase (CD) gene was quite remarkable, and the growth of melanoma cells was inhibited for over 7 days in the presence of ganciclovir (GCV) or 5-fluorocytosine (5-FC). Similarly, when melanoma cells treated in vitro with the Fab immunogene carrying TK or CD were transplanted into the back of nude mouse, subsequent systemic administration of GCV or 5-FC effectively suppressed the growth of tumors, indicating the occurrence of in vivo suicide effects.

[Targeting delivery of therapeutic genes using monoclonal antibody; immunogene approach].

We are developing the "immunogene" system for the targeted delivery of therapeutic genes. The immunogene system utilizes the EGF receptor-mediated endocytosis. The Fab fragment of monoclonal antibody B4G7 against human EGF receptor was conjugated with polylysine to form an "Fab immunoporter", which forms an affinity complex with DNA. The transfection efficiency of Fab immunogene was approximately 10-fold higher than the Lipofectin. Gene transfer of HSV-tk gene into A431 tumor cells with Fab immunoporter was successful and the subsequent treatment with ganciclovir induced remarkable suicide effects conferring 1000-fold higher drug sensitivity. Thus, the immunogene system could be useful as a gene transfer vehicle targeting the EGF receptor-hyperproducing tumor cells.

Antisense oligonucleotide of WAF1 gene prevents EGF-induced cell-cycle arrest in A431 cells.

A431 cells hyperproduce EGF receptors and possess inactive p53 proteins. It has been suggested that a cyclin-dependent kinase (CDK) inhibitor p21/WAF1 plays a crucial role in the EGF-induced cell-cycle arrest of A431 cells. Here, we investigated the role of WAF1 gene transcription in the EGF-induced cell-cycle arrest by transfecting the 18-mer antisense oligonucleotide which corresponds to the 5' region of WAF1 gene (AS/WAF1). When A431 cells were treated with EGF, a cascade of responses were observed, including immediate hyperphosphorylation of EGF receptor on tyrosine residues, accumulation of WAF1 mRNA and p21/WAF1 protein, dephosphorylation of RB protein which is a substrate of CDK-cyclin, and cell-cycle arrest. In the presence of AS/WAF1, EGF induced the tyrosine-phosphorylation of EGF receptor, but WAF1 mRNA was reduced to a half; accumulation of p21/WAF1 protein and its downstream responses were no longer observed; A431 cells grew continuously. Thus, the transfection of antisense efficiently prevented A431 cells from the EGF-induced arrest. These observations suggest that p21/WAF1 protein is a major effector molecule of the EGF-mediated cell-cycle arrest of A431 cells.

Targeted in vivo delivery of therapeutic gene into experimental squamous cell carcinomas using anti-epidermal growth factor receptor antibody: immunogene approach.

The "Fab immunogene" is a novel gene transfer vehicle in which the Fab fragment of anti-human epidermal growth factor (EGF) receptor antibody B4G7 is conjugated with poly-L-lysine to form an affinity complex with DNA. It was developed to target delivery of therapeutic genes into EGF receptor-hyperproducing tumor cells. Various characteristic features of the immunogene have been documented (Chen et al., 1998). Here we add further evidence to prove that in vitro transfer of beta-galactosidase/Fab immunogene is exclusively to EGF receptor-positive cells and that the herpes simplex virus thymidine kinase (TK)/Fab immunogene induces substantial suicide effects on A431 tumor cells when treated together with ganciclovir. The in vivo specificity of the immunogene transfer was examined using A431 tumor-bearing nude mice. When these nude mice were injected intraperitoneally with the chloramphenicol acetyltransferase (CAT)/Fab immunogene, CAT DNA was detected in the tumors as well as in liver and kidney but not brain, whereas CAT mRNA and enzyme activity were detected only in the tumors. Local and intraperitoneal injection of the TK/Fab immunogene and subsequent administration of ganciclovir effectively suppressed the growth of A431 tumors transplanted on the backs of nude mice. These observations suggest a possible application of the Fab immunogene system in cancer gene therapy.

Receptor-mediated gene delivery using the Fab fragments of anti-epidermal growth factor receptor antibodies: improved immunogene approach.

We previously developed the "immunogene" approach toward cancer gene therapy using epidermal growth factor receptor (EGFR)-mediated endocytosis. Here, we describe an improved immunogene system, in which the antigen-binding (Fab) fragments of the monoclonal antibody (Ab) B4G7 against the human EGFR were conjugated with poly-L-lysine to form a gene delivery vehicle (designated Fab "immunoporter"). Within 12 hours, the beta-galactosidase beta-gal) gene was transferred via the Fab immunoporter to virtually all of the nuclei of human squamous carcinoma A431 cells that overproduce the EGFR, and the beta-gal enzyme activity was detected within 24 hours and retained for more than 3 days. The beta-gal gene was not transferred into human and mouse cells that were deficient in EGFRs, but it was delivered if those mouse cells were transformed with human EGFR genes. Beta-gal gene transfer via the Fab immunoporter was inhibited by pretreatment with excess amounts of the Fab fragment. The transfer efficiency of the beta-gal gene to A431 cells via the Fab immunoporter was approximately 2%, which is as high as the lipofection method and 20- to 100-fold higher than the whole Ab immunoporter. The transfer of the herpes simplex virus thymidine kinase gene into A431 tumor cells as a form of the thymidine kinase/Fab immunogene was successful, and subsequent treatment with ganciclovir induced remarkable suicide effects which conferred 1000-fold higher drug sensitivity. Thus, the Fab immunogene was substantially improved with regard to the whole Ab immunogene and could be used as a potent gene transfer vehicle for the in vivo targeting of EGFR-hyperproducing tumor cells.

Characterization of autoantibodies in pemphigus using antigen-specific enzyme-linked immunosorbent assays with baculovirus-expressed recombinant desmogleins.

Pemphigus vulgaris (PV) and pemphigus foliaceus (PF) are autoimmune skin diseases caused by autoantibodies against desmoglein (Dsg) 3 and Dsg1, respectively. Routine immunofluorescence testing of skin and serum from patients cannot distinguish between these two severe diseases since both have IgG Abs directed against keratinocyte cell surfaces. In this study, recombinant Dsg3 and Dsg1, produced as secreted proteins by baculovirus expression, have been utilized to develop ELISAs for the specific characterization of their autoantibodies. Of 49 PV sera, 46 were positive in the Dsg3 ELISA and 44 of 46 PF sera were positive in the Dsg1 ELISA, compared with only 3 of 23 sera of bullous pemphigoid, and none of 53 normal control sera in both ELISAs. Both the Dsg3 and Dsg1 ELISAs were more specific and sensitive than conventional immunofluorescence staining. These Ag-specific ELISAs revealed that more than one-half of PV sera (26 of 49) had anti-Dsg1 Abs in addition to anti-Dsg3 Abs. PV patients who had not only oral mucous lesions but also significant skin involvement tended to have higher titers of anti-Dsg1 Abs. Furthermore, the ELISA reactivity correlated well with clinical disease activity in 5 of 6 PV and 5 of 5 PF patients. This ELISA provides a sensitive and highly specific assay for the diagnosis of patients with PV and PF, the correlation of disease activity with serum Ab levels, and a novel tool for investigating the immunopathogenesis of pemphigus.

[Expression of EGF receptor subfamily in tumor].

The members of EGF receptor subfamily are transmembrane glycoproteins with tyrosine kinase activity. Once EGF or EGF-related peptides binds to the receptors, they undergo autophosphorylation and binding to the SH2 protein, which in turn activates the intracellular signaling cascade. In the squamous carcinoma of head and neck, esophagus and lung, the EGF receptor gene amplification and EGF receptor hyperproduction are frequently observed. In the adenocarcinoma of pancreas, breast and stomach, hyperproduction of the EGF receptor subfamily is common, suggesting the involvement of these growth factor receptors in the tumorigenesis. The prognostic value of EGF receptor hyperproduction appears considerable when combined with other factors. EGF receptor subfamily members are useful targets for the immunotoxin therapy and immunogene therapy.

Immunogene approach toward cancer therapy using erythrocyte growth factor receptor-mediated gene delivery.

In this article we describe an improved method to produce a conjugate of anti-erythrocyte growth factor (EGF) receptor monoclonal antibody with polylysine via thio-ether bonds. The resulting antibody/polylysine conjugate was found to be a much more stable DNA (gene) carrier than the previous conjugate formed via disulfide bonds. We designated the conjugate as an "immunoporter" and the immunoporter/DNA (gene) complex as an "immunogene." The fluorescent microscopic observation showed that the immunoporter as well as immunogene bound specifically to the EGF receptors on the cell surface, and the loaded reporter gene, such as beta-galactosidase (beta-GAL), was detected in the cell nucleus at 2 hours after transfection. The enzyme activity from the beta-GAL gene was detected at 12 hours and increased for 3 to 5 days. Similar kinetics were obtained for another reporter gene, chloramphenicol acetyltransferase. Furthermore, the immunoporter delivered the herpes simplex virus thymidine kinase gene and induced substantial suicide effects on tumor cells when gancyclovir or acyclovir was added. Thus, the immunogene approach was successful in delivering therapeutic genes to EGF receptor overexpressing tumor cells. Further technical refinement may prove useful as a supplementary treatment of patients with squamous cell carcinomas.

Conformational epitopes of pemphigus antigens (Dsg1 and Dsg3) are calcium dependent and glycosylation independent.

The target molecule of pemphigus autoantibodies is a transmembrane desmosomal component, desmoglein 3 (Dsg3) in pemphigus vulgaris (PV) and Dsg1 in pemphigus foliaceus (PF). In this study, we examined the effects of calcium and glycosylation on the anti-genicity of the pemphigus antigens and on the generation of conformational epitopes. We used recombinant baculovirus proteins, PVIg and PFIg, which are considered to reflect accurately the native conformation of the extracellular domain of their respective proteins Dsg3 and Dsg1. These baculoproteins could immunoadsorb heterogeneous autoantibodies from the corresponding sera of PV and PF patients, completely blocking indirect immunofluorescence staining of normal human skin. Chelating calcium from the solution containing the baculoproteins using ethylenediaminetetraacetic acid (EDTA) or ethyleneglycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) abolished immunoadsorption by both PVIg and PFIg; however, immunoadsorption by the baculoproteins was restored after dialysis against 1 mM calcium. Nonglycosylated forms of both baculoproteins produced in the presence of tunicamycin retained their immunoadsorptive ability. Furthermore, immunoadsorption by the baculo-proteins was prevented irreversibly by treatment with low pH, high pH, and boiling, but not with the non-ionic detergent Nonidet P-40. These findings indicate that formation of the conformational epitopes on the pemphigus antigens is dependent on calcium but independent of glycosylation, and provide direct evidence that calcium plays an important role in determining the antigenic properties of the pemphigus antigens.

Characterization of paraneoplastic pemphigus autoantigens by immunoblot analysis.

We investigated the antigen molecules for six clinically typical cases of paraneoplastic pemphigus (PNP) using immunofluorescence, immunoprecipitation, and immunoblotting. All the PNP sera showed a clear reactivity with transitional epithelia of rat urinary bladder and immunoprecipitated the 250-kD, 230-kD, 210-kD, 190-kD, and 170-kD proteins in various combinations, confirming the diagnosis of PNP. Immunoblot analysis demonstrated slightly different reactivity from that of immunoprecipitation. With immunoblotting of normal human epidermal extract, bovine desmosome preparation, and extract of cultured squamous cell carcinoma cells, all the PNP sera reacted with a characteristic doublet of the 210-kD and 190-kD proteins. However, immunoblotting detected the 250-kD desmoplakin I and the 230-kD bullous pemphigoid antigen less frequently and did not detect the 170-kD protein. Further immunoblot studies indicated that the 210-kD protein is different from desmoplakin II and that the 190-kD protein is most frequently detected by PNP sera. Two of the six PNP sera specifically reacted with the extracellular domain of recombinant pemphigus vulgaris antigen protein, indicating that pemphigus vulgaris antigen may be involved in PNP. In future studies to unravel the complex mechanisms of the PNP antigens, the immunoblot technique may be a useful tool.

Regulation of translocation of the desmoyokin/AHNAK protein to the plasma membrane in keratinocytes by protein kinase C.

Desmoyokin was identified as a desmosomal plaque protein. We previously demonstrated that desmoyokin is identical to a protein encoded by a human gene, AHNAK, whose expression is suppressed in neuroblastoma cells. Although this protein is distributed in the cytoplasm and the nucleus in various cells, it is associated closely with the plasma membrane in keratinocytes. In keratinocytes, desmoplakin translocates from the cytoplasm to the plasma membrane following both high calcium switch and protein kinase C (PKC) activation by 12-O-tetradecanoylphorbol-13-acetate (TPA). In the low calcium medium, the desmoyokin/AHNAK protein resides diffusely in the cytoplasm and the nucleus. However, 2 h after shift to the high calcium medium, the desmoyokin/AHNAK protein localized to the cell boundary in all cells in a pattern similar to that of desmoplakin. Selective PKC inhibitors completely inhibited the calcium-induced translocation of the desmoyokin/AHNAK protein, but the inhibition of desmoplakin translocation by these inhibitors was only partial. TPA also induced translocation of both the desmoyokin/AHNAK protein and desmoplakin, which was completely inhibited by PKC inhibitors. The calcium-induced phosphorylation of the desmoyokin/AHNAK protein was confirmed by immunoprecipitation using [32P]orthophosphate-labeled keratinocytes. Furthermore, the study of extractability with non-ionic detergent indicated that desmoplakin, but not the desmoyokin/AHNAK protein, is associated with the cytoskeleton. These results suggested an involvement of PKC in the translocation of the desmoyokin/AHNAK protein in keratinocytes. It was, however, also suggested that different mechanisms are likely involved in the translocation of the desmoyokin/AHNAK protein and desmoplakin.

Hydrogen peroxide preferentially enhances the tyrosine phosphorylation of epidermal growth factor receptor.

We found that hydrogen peroxide (H2O2) enhances EGF receptor tyrosine phosphorylation in intact cells as well as solubilized membrane of an EGF receptor hyperproducing cell line NA. An antioxidant MnCl2 effectively inhibited this enhancement. Interestingly, overall phosphorylation of the EGF receptor enhanced by H2O2 was half that of the EGF-enhanced phosphorylation when the receptor immunoprecipitated from [32P]orthophosphate-labeled cells was examined. Tryptic phospho-peptide mapping of these receptors revealed that EGF enhanced the phosphorylation on five specific residues including serine 671, 1,046 and 1,047, threonine 669 and tyrosine 1,173, whereas H2O2 enhanced the phosphorylation remarkably on tyrosine 1,173 and three other residues and only moderately on serine 1,046 and 1,047 and threonine 669. Thus, H2O2 preferentially enhances the tyrosine phosphorylation of EGF receptor through oxidant stress.

Response of atherosclerotic intimal smooth muscle cells to epidermal growth factor in vitro.

Increased proliferation of intimal smooth muscle cells (SMCs) plays an important role in the early phase of atherogenesis. To investigate growth mechanisms of these cells, we used intimal SMCs from rabbits fed an atherogenic diet and examined the sequential events that may facilitate induction of intimal SMC proliferation as well as the possible effects of growth-promoting factors secreted by these cells. In serum-free medium, epidermal growth factor (EGF) stimulated [3H]thymidine uptake by quiescent intimal SMCs at a rate six times higher than quiescent medial SMCs. There was no significant difference between the two cell types in terms of the number of specific EGF receptor per cell, the dissociation constant of EGF, and the time course of EGF binding and internalization. Furthermore, in both types of cells, c-fos, c-jun, and c-myc mRNAs were induced after 1, 1, and 4 hours of EGF treatment, respectively, whereas they required 8 hours of contact with EGF to induce proliferation. Growth response of medical SMCs to EGF was greatly enhanced when rabbit serum, deficient in lipoproteins and free of platelet-derived growth factor, was added to the medium. Moreover, EGF induced a twofold to fourfold increase in DNA synthesis in medial SMCs cocultured with intimal SMCs compared with medial SMCs incubated alone. Likewise, DNA synthesis of medial SMCs grown in medium conditioned by intimal SMCs was six times higher than that observed in medium conditioned by medical SMCs. Adding EGF to the medium conditioned by intimal SMCs increased their DNA synthesis even further.(ABSTRACT TRUNCATED AT 250 WORDS)

Light-dependent induction of early-response gene expression by calphostin-C.

Calphostin-C is a compound possessing the ability to inhibit protein kinase C (PKC) by oxidative modification in vitro and to enhance the epidermal growth factor (EGF) receptor phosphorylation in vivo in a light-dependent manner. Here, we found that calphostin-C induced c-fos and c-jun mRNA accumulation in the lung adenocarcinoma cell line A549 in a light-dependent manner. Nuclear run-on assay revealed that this mRNA accumulation took place at the transcription level. However, unlike in vitro, calphostin-C did not inhibit cytosolic PKC activity in vivo, and the gene expression induced by calphostin-C was inhibited by another PKC inhibitor, staurosporine. Thus, it was suggested that calphostin-C activates cytosolic PKC-dependent signaling pathway to the induction of "early-response gene" expression in a light-dependent manner.

A novel gene delivery system using EGF receptor-mediated endocytosis.

A monoclonal antibody to the human epidermal growth factor (EGF) receptor was conjugated with polylysine and the resulting conjugate was affinity-linked with DNA (gene). This novel gene delivery system utilizes receptor-mediated endocytosis and would be especially suitable for gene therapy for EGF receptor-overproducing squamous cell carcinomas.