Bcl10 expression, rearrangement and mutation in MALT lymphoma: correlation with expression of nuclear factor-kappaB.

Mucosa-associated lymphoid tissue (MALT) lymphomas usually involve extranodal sites, especially the stomach, lung and salivary glands. The Bcl10 gene was recently isolated from the breakpoint region of t(1;14) (p22;q32) in MALT lymphomas, and considered to be an apoptosis-associated gene, and involves a caspase recruitment domain (CARD)-containing protein that activates NF-kappaB. We investigated the role of Bcl10 in MALT lymphoma by analyzing its expression, rearrangement and somatic mutation, by immunostaining, reverse transcriptase-polymerase chain reaction (RT-PCR), Southern blot and PCR in 20 cases of MALT lymphoma. Expression of NF-kappaB was studied by immunostaining. Five cases of reactive lymphadenitis (RLA) were used as the control. Bcl10 rearrangement was detected in 8 of 20 (40%) MALT lymphomas, but in none of RLA. Significant Bcl10 mutation was detected only in 1 case (5%) with MALT, but not in RLA. RT-PCR showed higher density bands of Bcl10 in MALT lymphomas than in RLA. Immunostaining showed a weak Bcl10 expression in the germinal center and very weak expression in the marginal zone B-cells in RLA, which was limited to the cytoplasm. In contrast, Bcl10 was strongly expressed in MALT lymphomas, and was mainly detected in the cytoplasm, as well as in the nuclei. Bcl10 expression did not correlate with Bcl10 mutation and re-arrangements. NF-kappaB was expressed in nuclei of MALT lymphoma cells, but not in RLA. Bcl10 expression in MALT lymphoma correlated closely with NF-kappaB expression. Our results suggest that activation of Bcl10 and NF-kappaB may be important in MALT lymphomagenesis, and that nuclear localization of Bcl10 may be important in the progression of MALT.

Corneal sensation after correction of myopia by photorefractive keratectomy and laser in situ keratomileusis.

PURPOSE: To compare the effects of photorefractive keratectomy (PRK) and laser in situ keratomileusis (LASIK) on corneal sensation. SETTING: Ohshima Hospital of Ophthalmology, Fukuoka, Japan. METHODS: Corneal sensation was measured with a Cochet-Bonnet esthesiometer in 35 patients before and 3 days, 1 week, and 1 and 3 months after correction of myopia by PRK (22 patients) or LASIK (13 patients). RESULTS: After PRK, corneal sensitivity was decreased slightly at 3 days, began to recover at 1 week, and returned to preoperative values at 3 months; none of the changes was statistically significant (P >.05). After LASIK, corneal sensation was significantly decreased at 3 days, 1 week, and 1 month; it recovered slightly at 3 months, although it remained significantly less than preoperatively. CONCLUSIONS: Laser in situ keratomileusis was associated with a negative effect on corneal sensation, which was markedly greater than the effect with PRK and was evident for at least 3 months after surgery.

Cytokine expression in murine corneas during recurrent herpetic stromal keratitis.

PURPOSE: Recurrent herpetic stromal keratitis (HSK) is a potentially blinding, immune-mediated disease. To better understand the immunopathology of recurrent HSK, we examined the cytokine profile of mouse corneas with the condition. METHODS: The eyes of latently infected mice were examined for corneal pathology and cytokine content following UV-B-stimulated herpes simplex virus (HSV) reactivation. RESULTS: Peak HSV-induced corneal disease, manifested by stromal opacification, occurred 7-14 days after viral reactivation in latently infected mice. In qualitative RT-PCR analyses, IFNgamma, IL-10, IL-4, and IL-12 p40 mRNA were simultaneously expressed before and during recurrent HSK. Competitive, semi-quantitative RT-PCR evaluation of cytokine mRNA revealed highest IFNgamma expression before and during clinical disease with a decline thereafter. IL-4 levels peaked and declined before day 14, while IL-10 peaked on days 7 or 14 and paralleled IFNgamma at lower levels. Small amounts of IL-12 p40 mRNA were detected late in the disease course. ELISA evaluation of corneal extracts demonstrated similar results, featuring early expression of Th2 cytokines relative to disease. CONCLUSIONS: The presence of Th2 cytokines during early stages of recurrent herpetic corneal lesions indicate the presence of a mixed Th1 and Th2 cell infiltrate, which is likely associated with a memory response to viral antigens. These data suggest that disease resolution in corneas with recurrent HSK may depend upon the balance between destructive and protective cytokines at individual sites of viral recurrence.

Cloning of translocation breakpoints associated with Shwachman syndrome and identification of a candidate gene.

Shwachman syndrome is an autosomal-recessive disorder characterized by exocrine pancreatic insufficiency, bone-marrow dysfunction, and metaphyseal chondrodysplasia. A de novo balanced translocation was recently documented in a patient with this disease. Toward isolating the gene(s) responsible for Shwachman syndrome, we cloned and sequenced the translocation breakpoints in the DNA of this patient. The nucleotide sequences around the breakpoints contained neither repetitive elements nor motifs reported to be implicated in recombination events, although we did detect gains or losses of oligonucleotides at the translocation junctions. By large-scale genomic sequencing and in silico gene trapping, we identified two novel transcripts in the vicinity of the breakpoints that might represent candidate genes for Shwachman syndrome, one on chromosome 6 and the other on chromosome 12. The gene on chromosome 12 was actually disrupted by the translocation.

Structural organization of the 5'-end and chromosomal assignment of human placental leucine aminopeptidase/insulin-regulated membrane aminopeptidase gene.

Placental leucine aminopeptidase (P-LAP) which is identical with cystine aminopeptidase as oxytocinase was found to be a homologue of rat insulin-regulated membrane aminopeptidase (IRAP) by cDNA cloning. In this study, we confirmed 5'-end cDNA sequence of P-LAP and isolated genomic clones containing the upstream region of human P-LAP gene. The transcription initiation sites determined by primer extension located 478 and 480 bp upstream of the initiation methionine codon, 38 bp downstream of TATA box-like motif. The 5'-flanking region of human P-LAP gene contained DNA-binding motifs for several ubiquitous transcription factors such as SP1 and AP2. Chromosomal localization by fluorescence in situ hybridization showed that the gene was assigned to 5q14.2-q15 of the human chromosome. This study establishes the genetic basis for P-LAP gene research, thereby leading to better understanding of the molecular mechanism underlying the P-LAP gene.

Efficacy of all-trans retinoic acid for molecular relapse of acute promyelocytic leukemia during remission.

We analyzed the efficacy of all-trans retinoic acid (ATRA) as an early treatment for four acute promyelocytic leukemia (APL) patients in remission who were PML/RARalpha-positive by reverse transcription-polymerase chain reaction or fluorescence in situ hybridization. ATRA 45 mg/m2 was administered orally. All became negative for PML/RARalpha transcripts after 3 to 6 months of ATRA treatment. However, the PML/RARalpha transcripts subsequently reverted to positive in three cases. Although retreatment with ATRA failed to prevent hematological relapse in two patients, one case remains in hematological remission. No serious side effects were encountered during ATRA treatment. These findings suggest that early treatment of ATRA for PML/RARalpha-positive APL patients in remission may have a therapeutic benefit and prolong the duration of hematological remission without chemotherapy.

UVA-induced immune suppression through an oxidative pathway.

Although ultraviolet B (UVB) irradiation induces local immune or systemic immune suppression, depending on the dose, the immune suppression by ultraviolet A (UVA) has not been fully investigated. In this study, we investigated the effect of UVA on the immune response in vitro and in vivo. The effect of UVA on the antigen-presenting function of epidermal cells was measured in terms of antigen-specific T cell proliferation. A murine epidermal cell suspension was exposed to UVA in vitro, pulsed with trinitrobenzenesulfonic acid, and cultured with T cells prepared from syngeneic mice previously sensitized with trinitrochlorobenzene. UVA (5-20 J per cm2) suppressed the antigen-presenting function of epidermal cells in a dose-dependent manner, accompanied with suppression of the expression of costimulatory molecules on Langerhans cells. In order to investigate the effect of an antioxidant on the immune suppression, an epidermal cell suspension was irradiated with UVA in the presence or absence of glutathione. The suppressions of antigen-presenting function and ICAM-1 expression were significantly prevented by glutathione in a dose-dependent manner. Further, the effect of UVA on the immune response at the induction phase of contact hypersensitivity was evaluated in terms of lymph node cell proliferation ex vivo. UVA irradiation suppressed the endogenous proliferation of lymph node cells in trinitrochlorobenzene-painted mice, and this suppression was significantly reversed by the application of glutathione to the skin during irradiation. These results suggest that UVA-induced immune suppression may be mediated by reactive oxygen species, at least in part.

Oxidative effects of cigarette smoke on the human skin.

The oxidative effects of cigarette smoke on the human skin were investigated. A remarkable increase in the conversion ratio of squalene (SQ) to squalene monohydroperoxide (SQHPO) due to exposure to cigarette smoke was observed using a CL-HPLC (high performance liquid chromatography with a chemiluminescence detector) system. The results showed that cigarette smoke caused lipid peroxidation. We also found that the addition of chain-breaking-type antioxidants, such as oolong tea extract, inhibited the peroxidation. When cultured human skin fibroblasts were exposed to cigarette smoke, this increased the intensity of ultraweak chemiluminescence (CL), leading us to assume that cigarette smoke caused oxidation in cultured human skin fibroblasts. When the cultured human skin fibroblasts were treated with antioxidants such as glutathione, thiotaurine, hypotaurine and ascorbic acid there was little increase in CL, meaning that oxidation had been prevented in the human skin fibroblasts. We also exposed the human forearm to cigarette smoke and obtained sebum using cotton immersed in acetone in order to measure hydroperoxide levels by means of a CL-HPLC system. The exposure of skin to the smoke caused a dose-dependent increase in hydroperoxides derived from cigarette smoke. Further exposure of the forearm to cigarette smoke increased the intensity of CL, but pretreating the skin with antioxidants such as glutathione, thiotaurine and hypotaurine inhibited this increase. From these results, we concluded that cigarette smoke had an oxidative effect on SQ, cultured human skin fibroblasts and the surface of the human skin. The application of antioxidants prevented the cigarette smoke-induced oxidation. We consider that these oxidative effects on the skin could be a cause of skin disorders and skin aging.

Human cytomegalovirus infection in a retinoblastoma cell line in vitro.

BACKGROUND: The focus of these studies was to determine whether the Y79 human retinoblastoma cell line could function as a good in vitro model system for studying human cytomegalovirus (HCMV) infection. METHODS: Y79 cells were exposed to an HCMV mutant carrying a LacZ gene, and the resulting beta-galactosidase expression in infected cells was assessed by flow cytometry. The extent to which the three classes of viral gene products immediate early, early, and late proteins - were expressed was analyzed by immunohistochemical staining and Western blotting. Infected Y79 cells were also co-cultivated on human foreskin fibroblast (SF cell) cultures to recover virus. RESULTS: Infection of Y79 cells with the virus resulted in beta-galactosidase expression as detected by flow-cytometric analysis. Immunohistochemical staining revealed that a portion of Y79 cells expressed antigens reactive to monoclonal antibodies against immediate early, early, and late HCMV proteins. The 43-kDa early gene product was also detected by Western blotting. Infected Y79 cells co-cultivated on SF cell cultures yielded infectious foci, which turned blue following X-gal staining, demonstrating productive HCMV infection in the Y79 cells. CONCLUSION: These results demonstrate that while HCMV can productively infect Y79 cultures, it does so in a highly inefficient manner, leading these authors to conclude that this cell line does not provide a particularly good model system to study HCMV infection.

In vitro and in vivo prolonged biological activities of novel vitamin C derivative, 2-O-alpha-D-glucopyranosyl-L-ascorbic acid (AA-2G), in cosmetic fields.

The biological activity of the novel vitamin C derivative, 2-O-alpha-D-glucopyranosyl-L-ascorbic acid (AA-2G), was evaluated in vitro and in vivo. The percutaneous absorption of AA-2G was determined in five Japanese males. The excretion of ascorbic acid (AA) in the subjects administered AA-2G was sustained for a longer period than in the subjects administered ascorbic acid 2-phosphate (AA-2P), which is a conventional vitamin C derivative. An analysis of the distribution of AA in the skin showed that small black specks assumed to be AA were observed in the epidermis even 3 d after applying AA-2G. The melanin synthesis in B16 melanoma cells was inhibited more by AA-2G than by AA-2P, and AA-2G also prevented more UV-induced damage of human skin keratinocytes and fibroblasts than AA-2P did. From these in vivo and in vitro results, it is supposed that the conversion of AA-2G to AA is sustained for a long time compared with that of AA-2P, and that AA-2G is an effective and available compound having vitamin C activity in human subjects.

Enhancing effect of 2-O-alpha-D-glucopyranosyl-L-ascorbic acid, a stable ascorbic acid derivative, on collagen synthesis.

The objective of the present study was to compare 2-O-alpha-D-glucopyranosyl-L-ascorbic acid (AA-2G) with ascorbic acid (AA) and ascorbic acid 2-phosphate (AA-2P) concerning the promotion of collagen production in human skin fibroblasts. Though AA-2G was still observed to be promoting collagen synthesis at the same level on the 8th day of the culture, collagen synthesis was seen to decrease on the fifth day of culturing with AA and AA-2P. This sustained collagen synthesis-promoting action is considered to be a major feature of the novel vitamin C derivative, AA-2G by conducting an experiment in which an alpha-glucosidase inhibitor was present, it was shown that AA-2G exerts its collagen synthesis-promoting action after being decomposed to AA by alpha-glucosidase. Further, we observed that for AA-2G, even on the 8th day of the culture, the amount of AA in the fibroblasts was virtually unchanged from the beginning of the experiment, whereas, in the case of adding AA and AA-2P, virtually no AA was detectable in the culture medium on the fifth day. These findings suggests that AA-2G is decomposed to AA by alpha-glucosidase in the cells. This AA promotes collagen synthesis, which is prolonged through AA-2G's sustained decomposition.

Reproduction of antiviral effect in an in vivo model of human cytomegalovirus retinal infection.

BACKGROUND: Cytomegalovirus retinitis remains a serious problem in AIDS patients, and the species specificity of human cytomegalovirus (HCMV) has hindered the development of animal models suitable for testing new therapeutic agents. Having previously described an in vivo model of HCMV retinal infection, we investigated its ability to reproduce the antiviral effects of the established anti-HCMV agent ganciclovir in order to determine the model's potential for evaluating novel agents. METHODS: Athymic rats had human fetal retinal tissue implanted in both anterior chambers. At 14 or 28 days post implantation, a suspension of a beta-galactosidase (lacZ+) mutant of HCMV was injected into each anterior chamber. Commencing 3 days prior to the injection of virus, rats in the treatment group received twice-daily intraperitoneal injections of ganciclovir (identical to a total of 100 mg/kg per day) for the duration of the study. The control rats received no drug. Twenty days after virus injection, the eyes of all rats were removed, sectioned and developed with X-gal substrate to detect any beta-galactosidase expression in the human tissue implants. RESULTS: Blue-staining foci of infection were detected in the implanted retinal tissue in 8 of 10 eyes from untreated control rats, but no beta-galactosidase expression was found in any of 12 eyes from animals which had received ganciclovir treatment. CONCLUSION: Intraperitoneal administration of ganciclovir successfully prevented HCMV replication in the intraocular retinal implants. This model of HCMV retinal infection is therefore suitable for preliminary evaluation of systemically administered antiviral agents.

[Evaluation of blood flows inside hepatic tumors using color Doppler echography with galactose-based intravenous contrast medium "SH/TA 508" administration].

The purpose of this study is to evaluate hemodynamic characteristics of various hepatic tumors using color Doppler echography administered galactose-based intravenous contrast medium "SH/TA 508 (Levovist)". Subject were 9 cases of hepatocellular carcinomas, 5 cases of metastatic liver tumors and a case of hemangioma. We evaluated the characteristics of blood flows inside various hepatic tumors, and also evaluated the first pass through the hepatic tumors during administration of Levovist. The strongly pulsatile branched blood flows inside tumor and the slow-increased and plateau patterns were observed in the all cases of hepatocellular carcinomas, the weakly pulsatile blood flows surrounding tumor and the rapid-increased and slow-decreased patterns were observed in the cases of metastatic liver tumors, and the continuous blood streams in the margin of tumor and the slow-increased and slow-decreased pattern were observed in the case of hemangioma. These findings were characteristic in various hepatic tumors, and color Doppler echography enhanced by Levovist was very useful to distinguish hepatic tumors.

Hyperimmunoglobulinemia D in idiopathic retinal vasculitis.

BACKGROUND: We examined four patients who exhibited both idiopathic retinal vasculitis and elevated serum IgD levels. Uveitis caused by Behçet's disease is also associated with high levels of serum IgD. Therefore, the clinical features of these patients were investigated and the possible relationship between retinal vasculitis and elevated serum IgD was examined after undertaking a study of increased IgD levels in patients diagnosed with uveitis. METHODS: The study population was composed of 110 patients: 49 with Behçet's disease, 15 with sarcoidosis, 10 with Vogt-Koyanagi-Harada disease, and 36 with other forms of uveitis. IgD measurements were performed using modifications of the latex photometric immunoassay. Surface IgD (sIgD) expression in peripheral lymphocytes was determined by immunofluorescence, and the correlation between serum IgD levels and the percentage of sIgD-positive cells was examined. RESULTS: Twelve of the 110 patients had an elevated serum IgD. Eight of the 12 had Behçet's disease, and 4 were diagnosed with idiopathic retinal vasculitis. These 4 patients were HLA-A24+ females whose ages ranged from 8 to 25 years. A linear correlation between the serum IgD levels and the percentage of sIgD-positive cells was found. CONCLUSION: Hyperimmunoglobulinemia D state was found in Behçet's disease and idiopathic retinal vasculitis. These diseases may represent a new clinical entity characterized by signs of retinal vasculitis and hyperimmunoglobulinemia D that results from abnormal B cell activation and immune complex-mediated responses.

Isolation, characterization and chromosomal assignment of the human WNT7A gene.

The Wnt genes compose a large gene family encoding a group of secreted signaling molecules that have been implicated in oncogenesis and a number of developmental processes. We have isolated a full-length human cDNA clone that we consider to be a novel member of the Wnt gene family. The gene (WNT7A) encodes a deduced 349-amino-acid peptide with 98% identity in amino acid sequence to murine Wnt7a. Expression of this gene is restricted to certain tissues: placenta, kidney, testis, uterus, fetal lung, and fetal and adult brain. Furthermore, we have isolated a genomic clone of WNT7A and mapped it to chromosome 3p25 by fluorescent in situ hybridization.

Functional analysis of mutations in the PIS gene, which encodes Saccharomyces cerevisiae phosphatidylinositol synthase.

The PIS gene for an enzyme phosphatidylinositol synthase having an increased Km for myo-inositol, was isolated from Saccharomyces cerevisiae. The mutant PIS gene contained a CAA codon at position 114 instead of the CAC codon observed in the wild-type gene, resulting in alteration of the amino acid from His to Gln. Oligonucleotide mediated site-directed mutagenesis of PIS at codon 114 revealed that mutant genes with codons for Ala, Thr and Leu could support yeast cell growth in vivo, but those for Asp, Lys and Tyr could not. All mutant enzymes when expressed in Escherichia coli showed greatly reduced in vitro activity.