Nitrosative stress in the bronchial mucosa of severe chronic obstructive pulmonary disease

BACKGROUND: Reactive nitrogen species, formed via the reaction of nitric oxide (NO) with superoxide anion and via (myelo)peroxidase-dependent oxidation of NO(2)(-), have potent proinflammatory and oxidizing actions. Reactive nitrogen species formation and nitrosative stress are potentially involved in chronic obstructive pulmonary disease (COPD) pathogenesis. OBJECTIVES: To investigate the expression of markers of nitrosative stress, including nitrotyrosine (NT), inducible NO synthase (iNOS), endothelial NO synthase (eNOS), myeloperoxidase (MPO), and xanthine oxidase (XO) in bronchial biopsies and bronchoalveolar lavage from patients with mild to severe stable COPD compared with control groups (smokers with normal lung function and nonsmokers). METHODS: The expression of NT, iNOS, eNOS, MPO and XO in the bronchial mucosa and bronchoalveolar lavage of patients was measured by using immunohistochemistry, Western blotting, and ELISA and correlated with the inflammatory cell profile. RESULTS: Patients with severe COPD in stable phase had higher numbers of NT(+) and MPO(+) cells in their bronchial submucosa compared with mild/moderate COPD, smokers with normal lung function, and nonsmokers (P < .01). iNOS(+) and eNOS(+) but not XO(+) cells were significantly increased in smokers with COPD or normal lung function compared with nonsmokers (P < .05 and P < .01, respectively). In patients with COPD, the number of MPO(+) cells was significantly correlated with the number of neutrophils (r = +0.61; P < .0025) in the bronchial submucosa. Furthermore, the number of NT(+) and MPO(+) cells was negatively correlated with postbronchodilator FEV(1). CONCLUSION: These data suggest that nitrosative stress, mainly mediated by MPO and neutrophilic inflammation, may contribute to the pathogenesis of severe COPD.     

Evaluation of the proficiency of trained non-laboratory health staffs and laboratory technicians using a rapid and simple HIV antibody test

In Cambodia, nearly half of pregnant women attend antenatal care (ANC), which is an entry point of services for prevention of mother-to-child transmission of HIV (PMTCT). However, most of ANC services are provided in health centres or fields, where laboratory services by technicians are not available. In this study, those voluntary confidential counselling and testing (VCCT) counsellors involved in PMTCT were trained by experienced laboratory technicians in our centre on HIV testing using Determine (Abbot Laboratories) HIV1/2 test kits through a half-day training course, which consisted of use of a pipette, how to process whole blood samples, and how to read test result. The trained counsellors were midwives working for ANC and delivery ward in our centre without any experience on laboratory works. The objective of this study was to assess the feasibility of the training by evaluating the proficiency of the trained non-laboratory staffs. The trained counsellors withdrew blood sample after pre-test counselling following ANC, and performed the rapid test. Laboratory technicians routinely did the same test and returned reports of the test results to counsellors. Reports by the counsellors and the laboratory technicians were compared, and discordant reports in two groups were re-tested with the same rapid test kit using the same blood sample. Cause of discordance was detected in discussion with both groups. Of 563 blood samples tested by six trained VCCT counsellors and three laboratory technicians, 11 samples (2.0%) were reported positive in each group, however four discordant reports (0.7%) between the groups were observed, in which two positive reports and two negative reports by the counsellors were negative and positive by the laboratory technicians, respectively. Further investigation confirmed that all the reports by the counsellors were correct, and that human error in writing reports in the laboratory was a cause of these discordant reports. These findings lead us the conclusion that the half-day training using the rapid and simple test was feasible for non-laboratory staffs to attain enough proficiency to implement VCCT services for PMTCT in resource-limited settings, and that human error was more likely to occur in laboratory before giving reports to counsellors.     

Isolation and radiation hybrid mapping of a dinucleotide repeat polymorphism at the human calcium-sensing receptor (CASR) locus

Calcium-sensing receptor (CASR) in parathyroid gland regulates calcium homeostasis by sensing decreases in extracellular calcium levels and effecting an increase in secretion of parathyroid hormone. A polymorphic dinucleotide (CA) sequence was isolated from a genomic clone containing the human CASR gene and was mapped to 3q13.3–q21. This polymorphism will be useful in the genetic study of disorders affecting calcium metabolism, such as hypercalcemia, hypocalcemia, osteoporosis, hyperparathyroidism, and hypoparathyroidism. Received: June 2, 1998 / Accepted: June 24, 1998     

Sarcomatoid carcinoma of the urinary tract

Sarcomatoid carcinoma is a rare variant of malignant tumor arising from the urinary tract. This tumor had been termed carcinosarcoma because of its carcinomatous and sarcomatous components. There is still some confusion in the terminology between true carcinosarcoma and sarcomatoid carcinoma; however, the latter is now regarded as primarily a malignant epithelial tumor with pseudosarcomatous transformation.
Four cases of sarcomatoid carcinoma arising from the urinary tract are reported. The patients were a 77 year old female, and three males aged 62, 69 and 80 years. All but the eldest patient complained of gross hematurla. Surgical removal was performed in the younger three cases, and an autopsy was done in the remaining case. All the tumors were macroscopically polypoid. Histopathologic examination revealed fasciculated spindle-cell tumors with myxold stroma or malignant fibrous histiocytoma-like spindle cell tumors. The epithelial nature was proven in these sarcomatous cells by immunohistochemical andlor electron-microscopic examinations. Only a small amount of squamous cell carcinoma components was also evident in the latter three cases. Although the younger three patients were alive at 44, 23 and 39 months'follow-up, respectively, constant careful monitoring Is recommended.     

Liver tumor-promoting effect of beta-naphthoflavone, a strong CYP 1A1/2 inducer, and the relationship between CYP 1A1/2 induction and Cx32 decrease in its hepatocarcinogenesis in the rat

Interrelationships among induction of cytochrome P-450 (CYP) 1A1/2, decrease in connexin 32 (Cx32), and liver tumor-promoting activity by beta-naphthoflavone (BNF) in the promotion stage were examined in a 2-stage liver carcinogenesis model. A total of 20 male Fischer 344 rats were initiated with a single intraperitoneal injection of 150 mg/kg of diethylnitrosamine (DEN) or were given the saline vehicle alone. Starting 2 weeks later, they were fed a diet containing 2%, 1%, or 0% BNF for 6 weeks. All animals were subjected to a two-thirds partial hepatectomy at week 3 and were sacrificed at week 8. Absolute and relative liver weights were significantly increased in the DEN+BNF groups as compared to the DEN-alone group. Diffuse hepatocellular hypertrophy with cytoplasmic eosinophilia, sometimes accompanied by development of adenoma-like hepatic foci, was observed in the BNF-treated rats. Remarkable induction of cytochrome CYP 1A1/2 and significant increase in CYP 2E1 were noted in the DEN+BNF groups, and positive immunohistochemical staining for both was observed diffusely. The areas of Cx32-positive spots per hepatocyte in the centrilobular areas of livers of the BNF-treated rats were significantly decreased, but no changes were observed in periportal areas. The numbers and areas of foci positive for glutathione S-transferase placental form were increased in the BNF-treated groups. These results suggest that BNF is a liver tumor promoter that, unlike phenobarbital, does not induce CYP 2B1/2 isozymes, and there seems to be no direct relationship between CYP 1A1/2 induction and Cx32 reduction in BNF hepatocarcinogenesis.     

Arg-Gly-Asp (RGD) peptide ameliorates carbon tetrachloride-induced liver fibrosis via inhibition of collagen production and acceleration of collagenase activity

Liver cirrhosis is caused by a relative imbalance between synthesis and degradation of collagens. Arg-Gly-Asp (RGD) peptide is a major adhesive domain of several extracellular matrix (ECM) components, such as that involved in the binding of fibronectin to the alpha5beta1 integrin receptor. We previously reported that RGD peptide increased the expression of matrix metalloproteinase in hepatic stellate cells (HSCs) which play a major role in hepatic fibrosis. We evaluated whether RGD-peptides inhibit the progression of liver fibrosis in an animal model of carbon tetrachloride-induced hepatotoxicity. RGD peptide (GRGDS) (1 mg/kg body weight) was injected intraperitoneally (i.p.) 3 times a week for one month. The group treated with control peptide (GRGES) showed pathologically typical hepatic fibrosis, while the RGD-treated group showed minimal fibrotic changes. The liver contents of collagen and hydroxyproline in the RGD-treated group was significantly lower than that of the control group. Collagenase activity measured in liver homogenates was significantly higher in the treated group than in the control group. In an in vitro study using TWNT-4 cells derived from human HSCs, RGD peptide (100 mug/ml) reduced the expression of type I collagen and tissue inhibitor of matrix metalloproteinase-1, and increased that of matrix metalloproteinase-1. These results indicated that RGD peptides inhibited liver fibrosis associated with both decreased collagen production and increased collagenase acitivity, and suggested that RGD peptide might be useful for the therapy of hepatic fibrosis.     

Immunohistochemistry and angiography in adenomatous hyperplasia and small hepatocellular carcinomas

The sinusoidal structure and blood supply of 38 liver nodules less than 2 cm In diameter were Investigated. There were 18 cases of adenomatous hyperplasia (AH) and 20 cases of hepatocetlular carcinoma (HCC). Growth pattern, encapsulation and vascularity were examined, and Immunohistochemistry performed for factor VIII related antigen (factor VIII), type IV collagen (collagen IV), lamlnln and CD68. There were significant differences between AH and small HCC, except for the expression of CD68. There were differences In tumor size, vasculartty and the components of the basement membrane between AH and small, well differentiated HCC. The cases of AH were supplied by the portal system and maintained the sinusoidal structure, but small well-differentiated HCC were supplied by a mixture of portal and arterial vessels. In spite of their small size, moderately and poorly differentiated HCC had capillary and were supplied by branches of the hepatic artery.     

Studies of bovine enterovirus structure by ultraviolet resonance Raman spectroscopy

The structural comparison of bovine enterovirus MZ468 strain before and after the heat treatment was studied by ultraviolet resonance Raman (UVRR) spectra excited at both 235 and 251 nm. The difference between full, heated full and purified empty particles, which were expected as an in vitro model of uncoating, were demonstrated. At 235 nm excitation, the Raman bands of the capsid protein dominated in all the UVRR spectra. The UVRR spectra of the empty particles exhibited non-homogenious broadening for tryptophan W3 band and W7 Fermi doublet bands, which were characteristics of hydrophobic environment, when compared with those of the full particles. The results indicates that some Trp indole rings of the full particles were packaged inside the viral capsids and not strained by virion assembly. On the other hand, the Raman bands assigned to guanine residues of the single stranded-RNA genome were enhanced strongly in the 251-nm excited UVRR spectrum. The spectral differences between the packaged (full particles) and the unpackaged virions (heated full particles) indicates that some guanine residues had strong hydrogen bonds in the full particles.     

The inhibitory NK cell receptor CD94/NKG2A and the activating receptor CD94/NKG2C bind the top of HLA-E through mostly shared but partly distinct sets of HLA-E residues

The human non-classical MHC class I molecule HLA-E is a ligand for both an inhibitory NK cell receptor (CD94/NKG2A) and an activating receptor (CD94/NKG2C). To identify HLA-E surface recognized by both receptors, especially to determine if both receptors recognize the same epitope, we made a series of individually Ala-substituted HLA-E proteins and analyzed their binding to CD94/NKG2A orCD94/NKG2C. Eight HLA-E mutations that significantly impaired HLA-E binding to CD94/NKG2A are all found in the top of alpha1/alpha2 domain of HLA-E. These results suggest that CD94/NKG2A binds a HLA-E surface equivalent to a NKG2D binding site on MICA. Of the eight mutations that impaired HLA-E binding to CD94/NKG2A, six significantly impaired HLA-E binding to CD94/NKG2C suggesting that CD94/NKG2C also binds a similar surface of HLA-E. Unexpectedly, the two HLA-E mutations (D69A and H155A) selectively abrogated HLA-E binding to CD94/NKG2A, not largely affected CD94/NKG2C. These results indicate that a mostly shared, but partly distinct set of HLA-E residues is discriminated by the two receptors.