Mutations of the KLF1 gene detected in Japanese with the In(Lu) phenotype.

BACKGROUND: In(Lu) is characterized by a reduced expression of antigens in the Lutheran blood group system as well as other blood group antigens. Mutations of the erythroid transcription factor, KLF1, have been reported to cause the In(Lu) phenotype, and we investigated Japanese In(Lu) to estimate the prevalence of the phenotype and KLF1 polymorphism. STUDY DESIGN AND METHODS: Blood samples were screened by monoclonal anti-CD44 and the In(Lu) phenotype was confirmed by tube tests including adsorption and elution tests using anti-Lua and anti-Lub . KLF1, LU, and A4GALT genes were analyzed by polymerase chain reaction and sequencing. RESULTS: We identified 100 of 481,322 blood donors (0.02%), and the previously characterized 20 donors, who had the In(Lu) phenotype with the LUB/LUB genotype. A total of 100 of the 120 In(Lu) individuals had mutant KLF1 alleles, and we identified 13 known and 21 novel alleles. The mutant KLF1 alleles with c.947G>A (p.Cys316Tyr), c.862A>G (p.Lys288Glu), or c.968C>G (p.Ser323Trp) were major in the In(Lu) individuals. The P1 antigen of 29 In(Lu) (two P1 /P1 , 27 P1 /P2 ) showed significantly weakened expression by hemagglutination. CONCLUSIONS: The prevalence of the In(Lu) phenotype in the Japanese population was 0.02%, and we identified 13 known and 21 novel KLF1 alleles. The KLF1 mutations cause the reduced expression of the P1 antigen.

A new ABCG2 null allele with a 27-kb deletion including the promoter region causing the Jr(a-) phenotype.

BACKGROUND: The high-prevalence antigen Jr(a) is carried on the ATP-binding cassette transporter ABCG2. The ABCG2 gene consists of 16 exons and its translation start codon is located on the second exon. Although the occurrence of the Jr(a-) phenotype is rare, several ABCG2 null alleles have been reported. We report a new ABCG2 null allele having a large deletion in this study. STUDY DESIGN AND METHODS: The Jr(a) status was determined by standard serologic tests and genomic DNA was isolated from whole blood. Exons 1 to 16 and the 5'-untranslated region of the ABCG2 gene were analyzed by polymerase chain reaction and sequencing. Expression of the ABCG2 protein on red blood cells was examined by immunoblotting. RESULTS: A Jr(a-) blood donor had a novel allele having a 27-kb deletion including noncoding Exon 1 and the promoter region of ABCG2, and the donor was apparently homozygous for the allele. In addition, we found three more individuals having heterozygosity for the same allele, with ABCG2*01N.01 having c.376C>T (p.Q126X), but did not find the allele having the 27-kb deletion in 3000 Jr(a+) individuals. Immunoblotting revealed that the ABCG2 protein was not found to be expressed in the individual with homozygosity for the ABCG2 27-kb deleted and in two individuals with an ABCG2 27-kb deleted/ABCG2*01N.01 genotype, which indirectly allows to conclude that the 27-kb deletion is responsible for a null ABCG2 allele. CONCLUSION: We first identified an ABCG2 null allele (provisional ISBT allele number ABCG2*01N.23) having a large deletion including the promoter region.

Functional analysis of an established mouse vascular endothelial cell line.

BACKGROUND: In vitrostudies using cell lines are useful for the understanding of cellular mechanisms. The purpose of our study is to develop a new immortalized aortic vascular endothelial cell (EC) line that retains endothelial characteristics and can facilitate the study of ECs. METHODS: A mouse aortic vascular EC line (MAEC) was established from p53-deficient mouse aorta and cultured for over 100 passages. The expression of endothelial markers was assessed, and the function of this cell line was analyzed by tube formation and binding assays. RESULTS: MAEC retained many endothelial properties such as cobblestone appearance, contact-inhibited growth, active uptake of acetylated low-density lipoprotein, existence of Weibel-Palade bodies and several EC markers. MAECs exhibited tube formation activity both in vitro and in vivo. Furthermore, crucially, tumor necrosis factor alpha, an inflammatory cytokine, promoted lymphocyte adhesion to MAECs, suggesting that MAECs may facilitate the study of atherosclerosis and local inflammatory reactions in vitro. CONCLUSION: We describe the morphological and cell biological characteristics of MAEC, providing strong evidence that it retained endothelial properties. This novel cell line can be a useful tool for studying the biology of ECs.

Up-regulated PAR-2-mediated salivary secretion in mice deficient in muscarinic acetylcholine receptor subtypes.

Protease-activated receptor-2 (PAR-2) is expressed in the salivary glands and is expected to be a new target for the treatment of exocrine dysfunctions, such as dry mouth; however, the salivary secretory mechanism mediated by PAR-2 remains to be elucidated. Therefore, mechanism of the PAR-2-mediated salivary secretion was investigated in this study. We found that a PAR-2 agonist peptide, SLIGRL-OH, induced salivary flow in vivo and dose-dependent increase in [Ca(2+)](i) submandibular gland (SMG) acinar cells in wild-type (WT) mice and mice lacking M(3) or both M(1) and M(3) muscarinic acetylcholine receptors (mAChRs), whereas secretions in PAR-2 knockout (PAR-2KO) mice were completely abolished. The saliva composition secreted by SLIGRL-OH was similar to that secreted by mAChR stimulation. Ca(2+) imaging in WT acinar cells and beta-galactosidase staining in PAR-2KO mice, in which the beta-galactosidase gene (LacZ) was incorporated into the disrupted gene, revealed a nonubiquitous, sporadic distribution of PAR-2 in the SMG. Furthermore, compared with the secretion in WT mice, PAR-2-mediated salivary secretion and Ca(2+) response were enhanced in mice lacking M(3) or both M(1) and M(3) mAChRs, in which mAChR-stimulated secretion and Ca(2+) response in acinar cells were severely impaired. Although the mechanism underlying the enhanced PAR-2-mediated salivary secretion in M(3)-deficient mice is not clear, the result suggests the presence of some compensatory mechanism involving PAR-2 in the salivary glands deficient in cholinergic activation. These results indicate that PAR-2 present in the salivary glands mediates Ca(2+)-dependent fluid secretion, demonstrating potential usefulness of PAR-2 as a target for dry mouth treatment.

Possible involvement of oxidative stress in salivary gland of patients with Sjogren's syndrome.

OBJECTIVE: To determine the involvement of oxidative stress in the salivary gland of patients with Sjogren's syndrome (SS). METHODS: Oxidative damage to the gland was measured by 8-hydroxy-2'-deoxyguanosine (8-OHdG) and hexanoyl-lysine (HEL) using the SS saliva. In addition, lactate dehydrogenase (LDH) and mitochondrial glutamic-oxaloacetic transaminase (m-GOT), both general markers for cell damage, were also analyzed. RESULTS: Increased levels of 8-OHdG and HEL were found in the saliva of SS patients, but not in that of patients with other salivary gland dysfunction or of healthy individuals. Levels of LDH and m-GOT were significantly correlated with 8-OHdG and HEL levels, respectively. Furthermore, the increased levels of 8-OHdG and HEL were also correlated in the SS saliva. CONCLUSION: These findings suggested the involvement of oxidative stress in glandular tissue destruction in SS. It was indicated that the detection of 8-OHdG and HEL in the saliva may become a useful tool for the diagnosis of SS.

Biological and oncogenic properties of p53-deficient salivary gland epithelial cells with particular emphasis on stromal-epithelial interactions in tumorigenesis.

OBJECTIVE: To understand the salivary gland pathobiology, we established an immortalized duct/basal cell line (MSE) from the submandibular glands of p53-deficient mice. METHODS: A variety of culture assays and xenograft experiments were conducted. Cellular characteristics were analyzed using histological, immunohistochemical, ultrastructural, and molecular techniques. RESULTS: Inoculation of a mixture of MSE and Matrigel reconstructed polarized ducts whereas cotransplantation of MSE with both Matrigel and NIH3T3 (3T3) cells developed mixed tumors of adenoma and sarcoma. A daughter adenoma line (MSA) showed some transformed phenotype in vitro, but was marginally tumorigenic in vivo. Notably, pleomorphic adenoma gene 1 (PLAG1) was expressed in MSA but not in MSE. As compared with MSE, MSA showed higher levels of insulin-like growth factor-I receptor (IGF-IR). Interestingly, 3T3 sarcoma secreted insulin-like growth factor-II (IGF-II), while MSA did not. CONCLUSION: The intrinsic tumorigenic programs of p53 null salivary epithelium are promoted by 3T3 sarcoma-derived IGF-IIin a paracrine manner through overexpression of PLAG1 and IGF-IR.

Ultrastructural spectrum of solitary fibrous tumor: a unique perivascular tumor with alternative lines of differentiation.

Eight tumors diagnosed as solitary fibrous tumor (SFT) of the oral cavity were studied. Histologic spectrum was entirely comparable with the extrapleural SFT of other sites. One tumor had glomus tumor-like foci. Immunohistochemical results confirmed most of the previous observations, indicating characteristic expression of vimentin, CD34, bcl-2, and CD99. Factor XIIIa and alpha-smooth muscle actin were less commonly reactive and a very few cells were faintly positive for factor VIII-related antigen and Ulex europaeus agglutinin 1. All were essentially negative for S-100 protein, desmin, CD31, and CD68. In stark contrast to the conclusive immunoprofile, ultrastructural investigation of six tumors demonstrated considerable cellular heterogeneity. Other than fibroblasts, perivascular undifferentiated cells and pericytes predominated, but endothelial cells were regularly present. There was a distinctive proliferation of pericytic cells in four tumors, one of which had glomoid foci of myopericytes. The extreme increase in number of Weibel-Palade bodies occurred in voluminous capillary endothelium. Occasional single and clustered cells with consistent features of endothelium showed intracytoplasmic lumen formation. Such composite cells constituted an integral segment of richly vascularized SFT. Myofibroblastic form smooth muscle differentiation was present in only a minority of cells. From phenotypic analysis by electron microscopy, SFT may originate from a unique, perivascular multipotent mesenchyme sharing with its lineage with pericytes, fibroblasts, and infrequently, endothelium. Consequently, morphological features of SFT may become diversely varied by whether predominantly constituent cells are undifferentiated, pericytic or fibroblastic in nature.

Balloon cell nevus of the soft palate: an immunohistochemical and ultrastructural study.

An ultrastructural analysis of oral balloon cell nevus of intramucosal type complemented with an immunohistochemical study was performed for the first time. The lesion was composed of large balloon cells with an admixture of small nevus cells and melanophages at the periphery. Balloon cells showed cytoplasmic accumulation of vacuoles of varying sizes and the presence of microgranular and vacuolated melanosomes were found. Residual cytoplasm contained no identifiable organelles. A spectrum of transitional forms between balloon cells and conventional nevus cells with microvacuoles was readily observed. Both cells exhibited intense immunoreactivity to multiple melanocytic markers. Ballooning phenomenon was not evident in melanophages containing a large amount of melanosome complex. It can be inferred, from the present and previous observations, that progressive vacuolization of melanosomes in nevomelanocytes may be responsible for the formation of peculiar ballooning appearance, suggesting an aberrant melanogenesis.

Rare vascular proliferations of the oral mucosa.

Benign vascular lesions-malformative, reactive, and neoplastic-are fairly common in the oral soft tissues; nevertheless, extravascular papillary endothelial hyperplasia and sinusoidal hemangioma have not been reported in this location. To our knowledge, a single case of intraoral spindle cell hemangioma has appeared in the literature. This report deals with histopathological features of these rare vascular proliferations involving the oral mucosa.