Aberrant maspin expression in gallbladder epithelium is associated with intestinal metaplasia in patients with cholelithiasis.

OBJECTIVE: Aberrant expression of maspin protein related to DNA hypomethylation in the promoter region is frequently observed in gallbladder carcinomas, whereas the non-tumorous gallbladder epithelium is maspin negative. We investigated maspin expression in non-tumorous gallbladder epithelium in patients with cholelithiasis. METHODS: An immunohistochemical study of maspin expression was performed in 69 patients with cholelithiasis and 30 patients with gastric cancer without cholelithiasis. RESULTS: Immunoreactivity for maspin was observed in focal and patchy regions of the gallbladder epithelium. Positive immunoreactivity for maspin was significantly associated with the presence of intestinal metaplasia in patients with cholelithiasis (p<0.05). CONCLUSION: The high incidence of aberrant maspin expression in both intestinal metaplasia and carcinoma of the gallbladder supports the assumption that intestinal metaplasia of the gallbladder may predispose to gallbladder carcinoma.

Homozygosity and linkage disequilibrium mapping of autosomal recessive distal myopathy (Nonaka distal myopathy).

Autosomal recessive distal myopathy or Nonaka distal myopathy (NM) is characterized by its unique distribution of muscular weakness and wasting. The patients present with spared quadriceps muscles even in a late stage of the disease. The hamstring and tibialis anterior muscles are affected severely in early adulthood. We have localized the NM gene to the region between markers D9S319 and D9S276 on chromosome 9 by linkage analysis. To further refine the localization of the NM gene, we conducted homozygosity and linkage disequilibrium analysis for 14 patients from 11 NM families using 18 polymorphic markers. All of the patients from consanguineous NM families were found to be homozygous for six markers located within the region between markers D9S2178 and D9S1859. We also provided evidence for significant allelic associations between the NM region and five marker loci. Examination of the haplotype analysis identified a predominant ancestral haplotype comprising the associated alleles 199-160-154-109 (marker order: D9S2179-D9S2180-D9S2181-D9S1804), present in 60% of NM chromosomes and in 0% of parent chromosomes. On the basis of the data obtained in this study, the majority of NM chromosomes were derived from a single ancestral founder, and the NM gene is probably located within the 1.5-Mb region between markers D9S2178 and D9S1791.

Oxidative stress abolishes leptomycin B-sensitive nuclear export of transcription repressor Bach2 that counteracts activation of Maf recognition element.

The mammalian transcription activator Nrf2 plays critical roles in executing oxidative stress response by binding to the regulatory DNA sequence Maf recognition element. Bach2 is an Nrf2-related transcription repressor and a tissue-specific partner of the Maf oncoprotein family. We show here how Bach2 is regulated by an oxidative stress-sensitive conditional nuclear export. In cultured cells, Bach2 was localized in cytoplasm through its C-terminal evolutionarily conserved cytoplasmic localization signal (CLS). The CLS directed leptomycin B-sensitive nuclear export of reporter proteins, suggesting its dependence on the nuclear exporter Crm1/exportin 1. However, the CLS sequence does not bear a resemblance to the leucine-rich class of nuclear export signal, and mutagenesis analysis indicated that a stretch of nonhydrophobic amino acids is essential for its activity. Oxidative stressors aborted the CLS activity and induced nuclear accumulation of Bach2. Whereas oxidative stress is known to activate MARE-dependent transcription, overexpression of Bach2 in cultured cells silenced the inducibility of MARE. The results suggest that Bach2 mediates nucleocytoplasmic communication to couple oxidative stress and transcription repression in mammalian cells.

Role of peroxynitrite in airway microvascular hyperpermeability during late allergic phase in guinea pigs.

We investigated the role of peroxynitrite, which is formed by a rapid reaction between nitric oxide (NO) and superoxide anion (O(2)(-)), in the airway microvascular hyperpermeability during the late allergic response (LAR) in sensitized guinea pigs in vivo. The occurrence of LAR was assessed as a 100% increase in the transpulmonary pressure, which was monitored by the esophageal catheter technique. Airway microvascular permeability was assessed by Monastral blue dye trapping between the endothelium using an image analyzer. In the LAR phase (4 to 6 h after antigen inhalation), microvascular hyperpermeability and eosinophil infiltration within the airway wall were observed. NO production and xanthine oxidase (XO)/xanthine dehydrogenase activity, which are responsible for O(2)(-) production, were enhanced during the LAR. Peroxynitrite formation assessed by nitrotyrosine immunostaining was also exaggerated at that time. The microvascular hyperpermeability during the LAR was largely reduced by NO synthase inhibitor (L-NAME, 72.7% inhibition; p < 0.05), XO inhibitor (AHPP, 60.8% inhibition; p < 0. 05) and peroxynitrite scavenger (ebselen, 81.0% inhibition; p < 0. 05). L-NAME had a small but significant inhibitory effect on airway eosinophil accumulation, but AHPP and ebselen had no effect. These results suggest that excessive production of O(2)(-) and NO occurs in the LAR. These two molecules appear to cause airway microvascular hyperpermeability via peroxynitrite formation.

[Blast crisis of chronic myelocytic leukemia that was difficult to differentiate from Ph+ acute lymphoblastic leukemia].

We encountered a 44-year-old woman with suspected chronic myelocytic leukemia (CML) in the acute phase that was difficult to be differentiate from Philadelphia chromosome (Ph)-positive acute lymphoblastic leukemia (ALL). At disease onset, her bone marrow showed an increase in blasts that were negative for myeloperoxydase (MPO) and Positive for CD10, 19, 34, and HLA.DR. Standard type Ph was detected by chromosome analysis, and both major and minor BCR/ABL m-RNA were detected by reverse-transcriptase polymerase chain reaction (RT-PCR) methods. Neutrophil alkaliphosphatase (NAP) score was normal, and neither eosinophilia nor basophilia was observed in peripheral blood. Under a presumptive diagnosis of Ph-positive ALL (L2), the patient was given AdVP (doxorubicin, vincristine, and prednisolone) therapy followed by a regimen of LMVP (L-asparaginase, mitoxantrone, and VP), and obtained a complete remission 2 months later. At that time, FISH analyses of her bone marrow and blood cells no longer detected bone marrow Ph or BCR/ABL fusion gene. A month later, however, the leukemia relapsed with an increase in MPO-positive blasts in bone marrow, and the patient died soon thereafter. We finally concluded that her leukemia was not Ph-positive ALL, but CML in the acute phase at disease onset.

[Hemophagocytic syndrome due to miliary tuberculosis in the course of aplastic anemia].

We report a 63 year-old female with aplastic anemia (AA) who was complicated with hemophagocytic syndrome induced by systemic miliary tuberculosis. Two years before admission to our hospital, she was diagnosed as AA and had been treated with granulocyte colony-stimulating factor, erythropoietin and methenolone acetate. In May, 1996, She was transferred to our hospital because of high fever and exacervation of pancytopenia. She showed severe pancytopenia, and an increase in macrophages showing remarkable erythrophagocytosis and decrease in hemopoietic cells in the bone marrow. In initial examination, high titer of IgM antibody to herpes simplex virus type I was identified and methylprednisolone pulse therapy was started under the diagnosis of virus associated hemophagocytic syndrome. Ten days later, however, she died for intestinal hemorrhage followed by multiorgan failure. In autopsy, multiple epitheloid cell granulomas with acid-fast bacilli were found in bone marrow, lungs, liver, spleen and kidneys.

An Nrf2/small Maf heterodimer mediates the induction of phase II detoxifying enzyme genes through antioxidant response elements.

The induction of phase II detoxifying enzymes is an important defense mechanism against intake of xenobiotics. While this group of enzymes is believed to be under the transcriptional control of antioxidant response elements (AREs), this contention is experimentally unconfirmed. Since the ARE resembles the binding sequence of erythroid transcription factor NF-E2, we investigated the possibility that the phase II enzyme genes might be regulated by transcription factors that also bind to the NF-E2 sequence. The expression profiles of a number of transcription factors suggest that an Nrf2/small Maf heterodimer is the most likely candidate to fulfill this role in vivo. To directly test these questions, we disrupted the murine nrf2 gene in vivo. While the expression of phase II enzymes (e.g., glutathione S-transferase and NAD(P)H: quinone oxidoreductase) was markedly induced by a phenolic antioxidant in vivo in both wild type and heterozygous mutant mice, the induction was largely eliminated in the liver and intestine of homozygous nrf2-mutant mice. Nrf2 was found to bind to the ARE with high affinity only as a heterodimer with a small Maf protein, suggesting that Nrf2/small Maf activates gene expression directly through the ARE. These results demonstrate that Nrf2 is essential for the transcriptional induction of phase II enzymes and the presence of a coordinate transcriptional regulatory mechanism for phase II enzyme genes. The nrf2-deficient mice may prove to be a very useful model for the in vivo analysis of chemical carcinogenesis and resistance to anti-cancer drugs.

Bach proteins belong to a novel family of BTB-basic leucine zipper transcription factors that interact with MafK and regulate transcription through the NF-E2 site.

Members of the small Maf family (MafK, MafF, and MafG) are basic region leucine zipper (bZip) proteins that can function as transcriptional activators or repressors. The dimer compositions of their DNA binding forms determine whether the small Maf family proteins activate or repress transcription. Using a yeast two-hybrid screen with a GAL4-MafK fusion protein, we have identified two novel bZip transcription factors, Bach1 and Bach2, as heterodimerization partners of MafK. In addition to a Cap'n'collar-type bZip domain, these Bach proteins possess a BTB domain which is a protein interaction motif; Bach1 and Bach2 show significant similarity to each other in these regions but are otherwise divergent. Whereas expression of Bach1 appears ubiquitous, that of Bach2 is restricted to monocytes and neuronal cells. Bach proteins bind in vitro to NF-E2 binding sites, recognition elements for the hematopoietic transcription factor NF-E2, by forming heterodimers with MafK. Furthermore, a DNA binding complex that contained MafK as well as Bach2 or a protein related closely to Bach2 was found to be present in mouse brain cells. Bach1 and Bach2 function as transcription repressors in transfection assays using fibroblast cells, but they function as a transcriptional activator and repressor, respectively, in cultured erythroid cells. The results suggest that members of the Bach family play important roles in coordinating transcription activation and repression by MafK.

Incubation with IgE increases cholinergic neurotransmission in human airways in vitro.

Airway cholinergic hyperresponsiveness is frequently observed in asthmatic patients. Recent reports suggest the possible involvement of IgE in hyperresponsiveness, although the exact mechanism is still uncertain. In this study, we examined whether incubation with IgE could facilitate the cholinergic function in human airways. Bronchi were obtained from 20 patients undergoing lung resection. Cholinergic contractile responses were induced by electrical field stimulation (EFS) or exogenous acetylcholine (ACh), and they were assessed by isometric tension measurement. EFS-induced ACh release from cholinergic nerves was also measured by high performance liquid chromatography. Incubation with IgE significantly enhanced EFS-induced bronchial contraction and ACh release as compared with the values of the bronchi incubated with heat inactivated IgE (control) (p < 0.05, respectively), but it did not alter the contractile responses induced by exogenous ACh. Pretreatment with the muscarinic M2-receptor agonist pilocarpine reduced the EFS-induced ACh release in the control tissues (p < 0.05), but not in the tissues incubated with IgE. The M2-receptor antagonist methoctramine significantly enhanced the EFS-induced contraction in control bronchi (p < 0.05), but this augmentation was not observed in the tissues incubated with IgE. These results suggest that IgE itself can enhance cholinergic bronchial contraction via facilitation of ACh release from cholinergic nerves and that this augmentation is related to autoreceptor M2 dysfunction at nerve endings.

Endogenous nitric oxide modifies antigen-induced microvascular leakage in sensitized guinea pig airways.

To examine the role of endogenous nitric oxide in allergic airway inflammation, we investigated the effect of a nitric oxide synthase inhibitor, N omega-nitro-L-arginine methyl ester (l-NAME), on antigen-induced airway microvascular leakage in actively sensitized guinea pigs by using Evans blue dye. Three weeks after sensitization with ovalbumin (10 micrograms), the tracheas were cannulated, and lungs were artificially ventilated. Animals were pretreated with atropine and propranolol (both 1 mg/kg, intravenously) to avoid neural modification. Ovalbumin inhalation (3 mg/ml, 1 minute) challenge caused significant microvascular leakage in all airways portions, which was significantly suppressed in a dose-dependent manner by pretreatment with intravenous injection of L-NAME (1 and 10 mg/kg) but not with the inactive enantiomer D-NAME (10 mg/kg). This inhibition by L-NAME was significantly reversed by co-administration of L-arginine (100 mg/kg, intravenously). Pretreatment with a vasoconstrictor, phenylephrine (20 micrograms/kg, intravenously), had no inhibitory effects on antigen-induced airway microvascular leakage despite increasing systemic blood pressure. Inhalation of representative mast cell-derived mediators, histamine (2 mg/ml, 1 minute) or leukotriene D4 (5 micrograms/ml, 1 minute), produced significant microvascular leakage in all airways. L-NAME (10 mg/kg, intravenously) partially but significantly inhibited leukotriene D4-induced leakage, whereas histamine-induced leakage was not affected. These results suggest that endogenous nitric oxide acts to increase airway microvascular leakage after airway allergic reaction.

A neurokinin 1-receptor antagonist improves exercise-induced airway narrowing in asthmatic patients.

Recent reports suggest the involvement of vascular phenomena in exercise-induced asthma. Sensory neuropeptides, such as substance P (SP), which causes airway vascular dilatation and plasma leakage, have been demonstrated to play a role in hyperpnea-induced airway narrowing in animal studies. The purpose of this study was to investigate the importance of tachykinins in exercise-induced airway narrowing in patients with asthma using a selective neurokinin 1-receptor (NK1-receptor) antagonist, FK-888. In a double-blind, placebo-controlled, crossover trial, nine subjects with stable asthma were given FK-888 (2.5 mg) or placebo by inhalation 20 min before each exercise at a level previously demonstrated to cause a fall of at least 40% in specific airway conduction (SGaw). Inhalation of FK-888 had no significant effect on baseline SGaw. While the recovery from exercise-induced airway narrowing was significantly faster after treatment with FK-888 the area under the curve for SGaw during the 50 min after exercise was significantly reduced (p<0.05) and the time taken for the SGaw to recover to within 65% of baseline after exercise was also significantly shorter with FK-888 than the placebo (p<0.05). However, treatment with FK-888 did not significantly attenuate the maximal fall in SGaw. These results suggest that NK1-receptor-mediated mechanisms are involved in the recovery phase of exercise-induced airway narrowing. The possible mechanisms of these phenomena are discussed.

Possible role of Streptococcus pyogenes in mucocutaneous lymph node syndrome. XII. Variable responses of platelets in MCLS seem to be explainable by streptococcal pyrogenic exotoxin.

As a model system for mucocutaneous lymph node syndrome (MCLS), we have advocated and used mice which had been rendered tolerant to Streptococcus pyogenes-associated antigens by neonatal infection with group A beta-hemolytic streptococci, because these mice have shown a variety of peculiar bioimmunological characteristics bearing a striking resemblance to those of MCLS patients. The results of our current investigations reaffirmed the reliability of the animal model by indicating that mice subjected to neonatal infection with S. pyogenes, or inoculation with streptococcal pyrogenic exotoxin (SPE) in Freund's adjuvant, were perfect counterparts of patients with MCLS on account of their platelet activation and hyperaggregability in response to provocative treatment, which are familiar findings in this disease.