[Case of retroperitoneal fibrosis exhibiting a variety of auto- antibodies and complicated by Sjogren's syndrome].
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Autoantibodies↗
Biomedical subjects
N Harada
Publications and source records attributed to N Harada.
Identification of the critical portions of the human IL-4 receptor alpha chain for activation of STAT6.
Interleukin-4 (IL-4) has been shown to activate Janus kinase (Jak)-1 and Jak-3, followed by activation of STAT (signal transducers and activators of transcription) 6. This Jak-STAT pathway is central to the initiation of IL-4 activities. In this study, we identified the essential region for the proliferation signal and activation of Jak-1, Jak-3, and STAT6 in the cytoplasmic domain of the human IL-4 receptor alpha chain (hIL-4R alpha) using a mouse T cell line CTLL-2. We found that the region between amino acid 353 and 393 is critical for the proliferation signal and activation of STAT6, but not for tyrosine phosphorylation of Jaks. These results suggest that in addition to the "Box-1" portion, which is known to be essential for Jak-1 activation, the more membrane-distal region of hIL-4R alpha is also necessary for activation of STAT6.
Animals↗
Activated protein C reduces the severity of compression-induced spinal cord injury in rats by inhibiting activation of leukocytes.
Activated protein C (APC), an important inhibitor of the coagulation system, has recently been shown to prevent tissue injury by blocking the activation of leukocytes. To determine whether APC can also prevent post-traumatic spinal cord injury (SCI), a condition in which leukocytes play an important role, we tested the effects of APC on SCI induced in rats by compression trauma. Administration of APC, either before or after the induction of SCI, markedly reduced the motor disturbances in these animals. In contrast, neither an inactive derivative of activated factor X (DEGR-Xa), a selective inhibitor of thrombin generation, nor active site-blocked APC (DIP-APC) reduced the motor disturbances. Histological examination revealed that intramedullary hemorrhages, observed 24 hr after trauma, were significantly reduced in the animals administered APC. The increase in the tissue level of tumor necrosis factor-alpha (TNF-alpha) and the accumulation of neutrophils in the damaged segment of the spinal cord were significantly inhibited in the animals that had received APC, but these were not inhibited in those administered DIP-APC or DEGR-Xa. The induction of leukocytopenia had the same effect as APC, in that it significantly reduced motor disturbances, tissue levels of TNF-alpha, and neutrophil accumulation in the animals subjected to compressive SCI. These findings suggest that in SCI, APC reduces motor disturbances primarily by reducing the amount of TNF-alpha at the site of injury, thus inhibiting neutrophil accumulation and the resultant damage to the endothelial cells.
Animals↗
Anatomical relationships between aromatase and tyrosine hydroxylase in the quail brain: double-label immunocytochemical studies.
The activation of male sexual behavior in Japanese quail (Coturnix japonica) requires the transformation of testosterone to 17beta-estradiol by the enzyme aromatase (estrogen synthetase). There are prominent sex differences in aromatase activity that may be regulated in part by sex differences in catecholaminergic activity. In this study, we investigate, with double-label immunocytochemistry methods, the anatomical relationship between the catecholamine synthesizing enzyme, tyrosine hydroxylase (TH) and aromatase (ARO) in the quail brain. The immunoreactivity observed for each antigen generally matched the previously described distribution. One exception is the observation that cells weakly labeled for aromatase were found widely distributed throughout the telencephalon. The presence of telencephalic aromatase was confirmed independently by radioenzymatic assays. There was an extensive overlap between the distribution of the two antigens in many brain areas. In all densely labeled aromatase-immunoreactive (ARO-ir) cell groups, including the preoptic medial nucleus, nucleus of the stria terminalis, mediobasal hypothalamus, and paleostriatum ventrale, ARO-ir cells were found in close association with TH-ir fibers. These TH-ir fibers often converged on an ARO-ir cell, and one or more TH-ir punctate structure(s) were found in close contact with nearly every densely labeled ARO-ir cell. In the telencephalon (mostly the neostriatum), all TH-ir fibers were found to be part of fiber groups that surrounded weakly immunoreactive aromatase cells. The few cells exhibiting an intracellular colocalization were detected in the anteroventral periventricular nucleus. These results are consistent with the hypothesis that catecholaminergic inputs regulate brain aromatase.
Animals↗
Origin and mechanism of formation of 45,X/47,XX,+21 mosaicism in a fetus.
Chromosome analysis of amniotic fluid cells from a 17-week-old fetus with a nuchal cystic hygroma showed a 45,X/47,XX,+21 karyotype. Analyses of cord blood lymphocytes, skin fibroblasts, amniotic membrane, and chorionic villi demonstrated both cell lines in various proportions. We studied the origin and mechanism of formation of the double mosaic aneuploid using Q-banded chromosomal heteromorphisms, and one RFLP, two VNTRs, one tetranucleotide repeat, 28 CA repeat markers, mapped to every member of chromosomes. The heteromorphic markers examined showed no discordant patterns in parent-to-child transmission or between the two cell lines except for those in chromosomes 21 and X. Fetal DNA was extracted from its established monoclonal fibroblast cell lines with 45,X or 47,XX,+21 karyotypes. Genotyping with the DNA markers showed that each cell line was identical at every locus, except for chromosome 21 or X loci, indicating that the fetus was not a chimera but a mosaic. The 21-trisomic cells had one paternal allele and two maternal heterozygous alleles at the D21S270 locus, and the 45,X (21-disomic) cells had two biparental alleles. Alleles at two X chromosomal loci, DXS991 and DXS8057, were biparental in the 47,XX,+21 cells, whereas only the paternal allele was retained in the 45,X cells. Based on these findings, we concluded that the fetus started as a 47,XX,+21 zygote that had resulted from nondisjunction at the maternal first meiotic division and that one each of the maternally derived chromosomes 21 and X was lost during an early mitotic division, leading to the mosaicism.
Alleles↗
Neuroanatomical distribution and variations across the reproductive cycle of aromatase activity and aromatase-immunoreactive cells in the pied flycatcher (Ficedula hypoleuca).
The anatomical distribution and seasonal variations in aromatase activity and in the number of aromatase-immunoreactive cells were studied in the brain of free-living male pied flycatchers (Ficedula hypoleuca). A high aromatase activity was detected in the telencephalon and diencephalon but low to negligible levels were present in the optic lobes, cerebellum, and brain stem. In the diencephalon, most aromatase-immunoreactive cells were confined to three nuclei implicated in the control of reproductive behaviors: the medial preoptic nucleus, the nucleus of the stria terminalis, and the ventromedial nucleus of the hypothalamus. In the telencephalon, the immunopositive cells were clustered in the medial part of the neostriatum and in the hippocampus as previously described in another songbird species, the zebra finch. No immunoreactive cells could be observed in the song control nuclei. A marked drop in aromatase activity was detected in the anterior and posterior diencephalon in the early summer when the behavior of the birds had switched from defending a territory to helping the female in feeding the nestlings. This enzymatic change is presumably controlled by the drop in plasma testosterone levels observed at that stage of the reproductive cycle. No change in enzyme activity, however, was seen at that time in other brain areas. The number of aromatase-immunoreactive cells also decreased at that time in the caudal part of the medial preoptic nucleus but not in the ventromedial nucleus of the hypothalamus (an increase was even observed), suggesting that differential mechanisms control the enzyme concentration and enzyme activity in the hypothalamus. Taken together, these data suggest that changes in diencephalic aromatase activity contribute to the control of seasonal variations in reproductive behavior of male pied flycatchers but the role of the telencephalic aromatase in the control of behavior remains unclear at present.
Animals↗
Establishment of a monoclonal antibody to human myeloma cell: relation to chemotherapy and extramedullar infiltration.
Resistance of myeloma cells to melphalan (L-PAM) is a serious problem. To investigate mechanisms of drug resistance, we generated a monoclonal antibody, clone O3, to melphalan-resistant myeloma cells, KHM-11R. Western blot analysis showed that molecular weight of O3 antigen was approximately 90 kDa. Expression of O3 antigen was approximately two times higher in KHM-11R than in parental melphalan sensitive cell line, KHM-11. O3 was preferentially expressed in plasma cell, B-cell, and monocytic cell lines, but not in T-cell lines. Analysis of bone marrow samples from myeloma patients revealed that 13 of 23 samples expressed O3 antigen at various levels, and that O3 antigen expression in patients correlate with preceding chemotherapy, advanced clinical stage and extramedullar invasion of myeloma cells. Furthermore, patients expressing O3 antigen at the time of diagnosis tended to have poor prognosis. The investigation of O3 antigen in myeloma cells will be useful to reveal the pathophysiology of extramedullar invasion and the mechanism of cell killing by melphalan.
Adult↗
Dietary Mg and/or K restriction enhances paraquat toxicity in rats.
Effect of mineral restriction was studied to clarify which mineral in the diet is most indispensable in preventing paraquat (PQ) toxicosis. ODS rats were chosen as the experimental animal owing to the inability to synthesize vitamin C similarly to humans. Rats were fed with either mineral-adequate or restricted diets dosed with 125 ppm PQ. The mineral-adequate diet was based on the American Institute of Nutrition-76, and the restricted diet was one-half the amounts. Measurements were made on the onset day of PQ toxicosis, body weight changes during the feeding experiment, and changes of two acute phase reactant proteins cysteine proteinase inhibitor and alpha1-proteinase inhibitor. The minerals tested were divided into three classes: I, largely needed, Ca, K, Na, and Mg; II, moderately needed, Mn, Fe, Zn, and Cu; and III, minutely needed, Cr and Se, respectively. Rats fed with a Mg-restricted diet showed a severe toxicosis but those with a K-restricted diet, a mild toxicosis. No appreciable effect was observed by restriction of other minerals. A synergistic effect was observed in the restriction of Mg and K.
Animals↗
Role of neutrophil elastase in stress-induced gastric mucosal injury in rats.
Activated neutrophils play an important role in tissue injury by releasing various inflammatory mediators capable of damaging endothelial cells. To investigate whether neutrophil elastase (NE) is involved in stress-induced gastric mucosal injury, we examined the effects of 2 NE inhibitors (ONO-5046 and L-658 758) as well as nitrogen mustard-induced leukocytopenia on the formation of gastric mucosal lesions, gastric mucosal blood flow, gastric mucosal microvascular permeability, and gastric neutrophil accumulation in rats subjected to water immersion-restraint stress (WIR). Gastric mucosal injury peaked 8 hours after WIR. Gastric mucosal blood flow, as measured by laser-Doppler flow cytometry, decreased to 45% of its initial level 8 hours after WIR. Gastric mucosal microvascular permeability, evaluated by Evans blue dye leakage to the gastric mucosa, showed an increase, peaking 8 hours after WIR. Gastric accumulation of neutrophils, determined by measuring gastric myeloperoxidase activity and by histologic examination, was also significantly increased 8 hours after WIR. Both of the NE inhibitors markedly prevented the formation of gastric mucosal lesions. They also decreased the reduction in gastric mucosal blood flow seen in animals subjected to WIR while preventing increases in gastric mucosal microvascular permeability. Gastric neutrophil accumulation was significantly reduced in animals given either inhibitor 8 hours after WIR. Leukocytopenia produced effects similar to those produced by the inhibitors. Taken together, these observations strongly suggest that NE promotes stress-induced gastric mucosal injury in rats by reducing gastric mucosal blood flow and increasing neutrophil accumulation.
Animals↗
Distribution and effects of testosterone on aromatase mRNA in the quail forebrain: a non-radioactive in situ hybridization study.
A number of studies have been devoted to the analysis of the anatomical distribution, control by steroids and functional significance of aromatase (the enzyme metabolizing testosterone into 17beta-estradiol) in the quail brain. In particular, the sexually dimorphic nucleus preopticus medialis has been the main focus of investigation because testosterone aromatization in this structure mediates the activation of male sexual behavior and aromatase activity is itself testosterone-dependent in this nucleus. No information on the anatomical distribution of aromatase gene expression is, however, available so far in this avian species. In the present study we applied a non-radioactive in situ hybridization technique to describe the distribution of aromatase mRNA containing neurons in the quail prosencephalon. We also analyzed, at a neuronal level of resolution, the induction by testosterone of this mRNA in the medial preoptic nucleus. Dense clusters of aromatase gene expressing neurons were observed within the medial preoptic nucleus, the nucleus of the stria terminalis, the ventro-medial hypothalamus and the tuberal region. Scattered neurons expressing lower levels of aromatase mRNA were also found in the dorsal thalamic area and central gray. The specificity of the staining was confirmed by demonstrating the absence of signal in sections that had been hybridized with a sense probe. Moreover, the distribution of the aromatase mRNA containing cells completely overlapped with the distribution of the aromatase-immunoreactive cells. Aromatase-mRNA expression was controlled by testosterone (or its metabolites) in the entire medial preoptic nucleus. Castration resulted in a decrease in the number of aromatase mRNA-containing cells and this effect was totally reversed by testosterone treatment. These data further support the idea that testosterone regulates the rate of its own aromatization by modulating the expression of aromatase rather than by acting at a post transcriptional level.
Animals↗
Interferon-alpha treatment of acute lymphoblastic leukemia relapse after unrelated bone marrow transplantation.
A 32-year-old Japanese male in his second remission of acute lymphoblastic leukemia (ALL) received a matched unrelated donor bone marrow transplant (BMT) from the Japan Marrow Donor Program. On day +83, a bone marrow examination revealed 5.2% leukemic cells. Despite the cessation of cyclosporine, leukemic cells in the bone marrow increased to 18.4% on day +91. Treatment was started with interferon (IFN)-alpha-2b 3 x 10(6) U/body s.c. daily on day +92 and leukemic cells in the bone marrow disappeared completely. The toxicity of IFN-alpha treatment included leukoencephalopathy consisting of somnolence, disorientation, short-term memory loss, lack of coordination and ataxia, myelotoxicity requiring multiple platelet transfusions and exacerbation of graft-versus-host disease (GVHD) of oral cavity, skin and lung. Because of progressive GVHD, IFN-alpha was discontinued on day +124. On day +132, a bone marrow aspirate showed 6.4% leukemic cells. The patient died of progressive ALL on day +178. IFN-alpha may be useful for the treatment of leukemic relapse following BMT, although its toxicity is marked.
Adult↗
Macrophages, estrogen and the microenvironment of breast cancer.
Estrogen is a major mitogenic stimulus to established breast cancer. Estrogen sources include ovarian, extraglandular sites and breast tissue. Which source primarily maintains benign and breast cancer tissue estrogen concentrations remains unclear. While macrophages may comprise up to 50% of the mass of breast carcinomas, previous studies neglected to study them as possible sources of estrogen. We present evidence that breast macrophages constitute an in situ source of estradiol and that the amount produced is sufficient to mediate cellular proliferation. We utilized immunohistochemistry and RT-PCR to study cell-specific aromatase expression in (i) 29 breast biopsies, (ii) human monocytes/macrophages and (iii) a myeloid cell line (THP-1) capable of differentiating into macrophages. Use of a breast cancer cell line (MCF-7) provided biologic confirmation of the role of aromatization in cell proliferation. We demonstrated considerable amounts of immunoreactive-aromatase (irARO) in breast tissue macrophages and a positive correlation between the proportion of irARO present in macrophages and lesion severity. Using in vitro techniques, we demonstrated that monocytes and THP-1 cells require differentiation into macrophages to produce aromatase in amounts approaching placental levels. The amount of estrogen produced by THP-1 cells stimulated MCF-7 cells to proliferate, an effect blocked by aromatase inhibitors. Estrogen production by macrophages in breast tissue appears sufficient to stimulate the proliferation of adjacent epithelial cells and to autoregulate cytokine production. These findings represent a new dimension of cellular regulation in breast tissue with major biologic implications, amenable to pharmacological manipulation.
Antineoplastic Agents↗
Molecular analysis of aberrant expression of aromatase in breast cancer tissues.
Aromatase mRNA in non-malignant breast tissues was mainly transcribed form skin fibroblast/fetal liver-specific exon 1 (exon 1b) of the aromatase gene. However, in half the cases of breast cancers, switching of the alternative exons 1 from exon 1b to ovary-specific exon 1 (exon 1c) was observed, and expression levels of aromatase mRNA in breast cancer tissues were significantly higher than those in the distal regions to tumors or in non-malignant breast tissues. Co-culture or addition of the conditioned medium of breast cancer cells, MCF-7 caused increase of aromatase mRNA in cultured adipose stromal cells from breast tissues together with switching from exon 1b to exon 1c. Removal of fetal calf serum (FCS) from the culture medium or addition of forskolin or phorbol ester (TPA) also induced rapid elevation of aromatase mRNA and switching to exon 1c, whereas TGFbeta almost abolished the expression, suggesting that cancer cells might secret forskolin- or TPA-like stimulatory factors, or consume TGFbeta-like inhibitory factors in serum for expression of aromatase mRNA. The promoter region responsible for transcription from exon 1b and the switching was investigated using a newly developed reporter carrying 4 major alternative exons 1 and promoters. Transcriptional elements responsible for preferential utilization of exon 1b were identified on the promoter region between -300 and -400 of exon 1b. Gel shift assay showed a unique site for a FCS-dependent DNA binding factor just downstream of GRE (glucocorticoid responsive element) in this promoter region.
Adipose Tissue↗
Demonstration of aromatase activity and its regulation in breast tumor and benign breast fibroblasts.
Breast tumors from post-menopausal women contain higher amounts of estradiol than would be predicted from levels circulating in plasma. This observation raised the hypothesis that tumors may synthesize estradiol in situ and increase their tissue estradiol levels via this mechanism. The key enzyme involved in tissue estrogen synthesis, aromatase, is present in breast tumors but, according to some investigators, not in sufficient concentration to be biologically meaningful. We postulated that foci of cells in breast tumors might contain high amounts of aromatase and this locally produced estrogen might act in a paracrine or autocrine fashion. To test this hypothesis, we utilized immunohistochemistry to localize the aromatase enzyme, an histological scoring system to quantitate it, and culture of isolated breast cells to demonstrate its potential regulation. In 26 archival breast tumors, 16 (62%) contained aromatase by radiometric assay. With the immunohistochemical method, we detected areas with staining in the stroma as well as tumor epithelial cells. Staining ranged from the intensity approaching that seen in placenta to levels just distinguishable from background. We adopted an histological scoring system (H-score) from that used to quantitate progesterone receptor levels in tissue and used it to quantitate aromatase activity. A higher histologic score was found in stromal spindle cells (13) than in tumor epithelial cells (4.8). The biochemical aromatase results correlated with the H-score of stromal but not epithelial cells. To further study stromal cells from tumors, we isolated stromal cells from breast tumors and the benign areas of breast distal to the tumor and grew them in culture. Addition of dexamethasone, phorbol esters, and cyclic AMP analogues stimulated aromatase enzyme and messenger RNA levels substantially. Use of aromatase enzyme inhibitors such as letrozole blocked estrogen production but did not alter aromatase message levels. Epithelial cells, whether nonmalignant or cancer derived, exhibited no regulation by dexamethasone, phorbol esters, or cAMP analogues. These data, taken together, suggest that stromal cells may be more important than epithelial cancer cells for estrogen production in breast tumors. The ability to stimulate aromatase activity substantially with various enhancers of aromatase provides further credence for an important biologic role of estrogen production in tumor tissue.
Aromatase↗
Inhibitors of aromatase prevent degradation of the enzyme in cultured human tumour cells.
The effects of two steroidal (4-hydroxyandrostenedione and atamestane) and three non-steroidal (fadrozole, vorozole and pentrozole) aromatase inhibitors on the levels of aromatase mRNA and protein were examined using cultured JEG-3 and HepG2 cells. Immunocytochemical studies demonstrated increased quantities of immunoreactive aromatase in both cell types as a result of these treatments. To clarify this effect in detail, quantitation of aromatase protein in JEG-3 cells was performed after various treatments using an enzyme-linked immunosorbent assay. Time-dependent increase was observed with all the aromatase inhibitors except 4-hydroxyandrostenedione. The three non-steroidal agents caused an approximately fourfold elevation in the cells 24 h after the treatment compared with untreated controls. The inhibitors also appeared to block the rapid degradation observed in JEG-3 cells after induction with forskolin. However, aromatase mRNA levels in JEG-3 cells remained unchanged. Furthermore, the increase in aromatase protein in JEG-3 cells due to the inhibitor action was not blocked by treatment with cycloheximide, an inhibitor of protein synthesis. These results thus suggest that aromatase inhibitors increase aromatase protein through stabilization and reduced protein turnover as a side-effect of their binding.
Androstenedione↗
Expression of Bcl-2 family of proteins in fresh myeloma cells.
Members of the Bcl-2 family of proteins, Bcl-2, Bcl-X(L), Bcl-Xs and Bax, are considered to play important roles in the regulation of apoptosis and drug resistance. To understand the significance of these proteins in fresh human myeloma cells, expression of Bcl-2 family of proteins was analyzed by Western blotting in 17 cases with multiple myeloma (MM) and three cases with plasma cell leukemia (PCL). Bcl-2 and Bcl-X(L) were found in 12 and nine samples, respectively. All PCL cases showed co-expression of Bcl-2 and Bcl-X(L). Analysis of MM cases showed that Bcl-2 was preferentially expressed in samples from cases with early clinical stage while Bcl-X(L) tended to be expressed in samples from cases at advanced clinical stage. Bcl-X(L) was significantly expressed in tumor cells from cases with extramedullar lesions. There was no correlation between the expression levels of Bcl-2 or Bcl-X(L) and preceding chemotherapy. Expression of Bax was found in only one patient who had pleural effusion caused by invasion of myeloma cells and a high serum LDH level. Survival analysis revealed that there was no statistical significance in expression of Bcl-2 or Bcl-X(L) although Bcl-X(L) tended to be expressed in cases with poor prognosis. These findings indicate that expression of Bcl-2 family of proteins is heterogeneously regulated in fresh myeloma cells. Expression of Bcl-X(L) and Bcl-2 may correlate with extramedullar invasion and early stage of the disease, respectively. Absence of Bax in myeloma cells may contribute to low sensitivity of myeloma cells to anti-cancer agents since Bax is reported to mediate cytotoxicity of some anti-cancer drugs.
Adult↗
Aromatase in the human central nervous system.
OBJECTIVE: Oestrogen produced locally by aromatase is thought to participate in numerous biological functions in the adult central nervous system (CNS). However, little is known about aromatase expression in the human CNS. DESIGN: We examined aromatase expression in human brain regions, (4 men, 2 women) obtained from autopsy, by reverse transcriptase (RT)-polymerase chain reaction (PCR) and also studied alternative use of multiple exons 1 of its gene, which is involved in tissue specific expression of aromatase in human. RESULTS: The amount of aromatase mRNA determine by RT-PCR assay in 6 cases tended to be highest in pons, thalamus, hypothalamus and hippocampus. Analysis of multiple exons 1 revealed that 1f, considered specific for brain, as well as 1b (fibrolast type) and 1d (gonadal type), were expressed. 1d and 1f tended to be utilized in hypothalamus, thalamus and amygdala. The amount of overall mRNA expression was also higher in hypothalamus, thalamus and amygdala than in other regions of the brain. There were no differences of utilization of exons 1 and mRNA expression of aromatase between female and male brain. CONCLUSIONS: These results demonstrate that aromatase is expressed widely in various regions of human brain tissues in both men and women.
Adult↗
Thimerosal-induced Ca2+ mobilization in isolated guinea pig cochlear outer hair cells.
Intracellular calcium mobilization of isolated guinea pig cochlear outer hair cells (OHCs) was investigated using thimerosal, a -SH group oxidizing agent, and fura-2 fluorescence ratio imaging microscopy. In the presence of thimerosal, intracellular Ca2+ concentrations ([Ca2+]i) of OHCs were elevated in a dose-dependent manner. Even in Ca(2+)-free medium, Ca2+ response was still induced. The effects of thimerosal on [Ca2+]i were completely blocked and reversed by dithiothreiotol (DTT). Neither 1-100 microM ryanodine nor 5-20 mM caffeine altered the effects of thimerosal. Pretreatment with pertussis toxin (PTX) for 30 min did not affect the thimerosal-induced increase in [Ca2+]i. The increase in [Ca2+]i when Ca2+ was added during thimerosal application in Ca(2+)-free medium was almost completely blocked by 500 microM LaCl3, while nifedipine did not inhibit further increase in [Ca2+]i caused by thimerosal. Thus, oxidation of the -SH group of the OHC membrane can induce a Ca2+ release from intracellular Ca2+ stores, which are ryanodine- and caffeine-insensitive, and Ca2+ influx through non-specific Ca2+ channels, but not the nifedipine-sensitive Ca2+ channels. The possible oxidation of -SH group gated Ca2+ channels in OHCs is worthy of further study.
Animals↗