Associação entre a expressão imuno-histoquímica de 8-OHdG e parâmetros clínico-patológicos em uma série de casos de queilites actínicas / Association between immunohistochemical expression of 8-OHdG and clinicopathological parameters in a series of actinic cheilitis cases

A queilite actínica (QA) é uma desordem potencialmente maligna que pode evoluir para o carcinoma de células escamosas de lábio inferior (CCELI). Os fatores associados à sua probabilidade de transformação maligna ainda não estão bem estabelecidos, no entanto, a exposição à radiação ultravioleta (RUV) representa o principal fator de risco tanto para ocorrência da QA como do CCELI. A RUV causa danos oxidativos ao DNA, que levam à formação de 8-hidroxi-2'desoxiguanosina (8-OHdG). Esta pesquisa objetivou avaliar a associação entre a expressão da 8-OHdG e parâmetros clínico-patológicos, em uma série de casos de QA. A amostra foi constituída de 57 casos de QA, analisadas morfologicamente e classificadas de acordo com a gradação histopatológica de displasia epitelial (OMS). Foram observados casos sem displasia (7), com displasia epitelial leve (22), displasia epitelial moderada (17) e displasia epitelial severa (11). A expressão da 8- OHdG foi analisada de forma semiquantitativa por dois examinadores calibrados (Kappa=0,79), atribuindo um escore de imunoreatividade (EIR) que variava de 0 a 6 para os casos analisados. Os dados foram analisados através dos testes não paramétricos de Mann-Withney e de Kruskall-Wallis, com nível de significância de 5% (p<0,05). Observou-se que a maioria dos casos mostrou imunorreatividade forte (64,9%). Não foram constatadas diferenças estatisticamente significativas entre a expressão de 8-OHdG e as características clínicas da QA, nem entre a expressão de 8-OHdG e a gradação da displasia epitelial (p=0,350). A forte imunoexpressão de 8-OHdG na maioria dos casos de QA, independente das alterações morfológicas presentes, sugere que o dano oxidativo ao DNA está envolvido na patogênese desta desordem (AU).

Actinic cheilitis (AC) is a potentially malignant disorder that can progress to lower lip squamous cell carcinoma (LLSCC). It is not yet well-established which factors may be associated with the probability of malignant transformation of this condition, but the exposure to ultraviolet radiation (UVR) represents the main risk factor for both AC and LLSCC. RUV causes oxidative damage to DNA, which leads to the formation of 8-hydroxy-2'deoxyguanosine (8-OHdG), This research aimed to evaluate the expression of 8-OHdG, in a series of cases of AC and to associate its expression with clinical-pathological parameters. The sample consisted of 57 cases of AC, analyzed morphologically and classified according to the histopathological gradation of epithelial dysplasia (WHO). Cases without dysplasia (7), with mild epithelial dysplasia (22), moderate epithelial dysplasia (17) and severe epithelial dysplasia (11) were observed. The analysis of the expression of 8-OHdG was performed in a semi-quantitative way by two calibrated examiners (Kappa = 0.79), assigning an immunoreactivity score for each evaluated case, which could vary from 0 to 6. The data were analyzed using Mann-Withney and KruskallWallis non-parametric tests, with a significance level of 5% (p <0.05). It was observed that most cases showed strong immunoreactivity (64.9%). There were no statistically significant differences between the expression of 8-OHdG and the clinical characteristics of QA, neither between the expression of 8-OHdG and the gradation of epithelial dysplasia present in the evaluated cases (p = 0.350). The strong immunoexpression of 8-OHdG in most cases of QA, regardless of the morphological changes present, suggests that oxidative damage to DNA is involved in the pathogenesis of this disorder (AU).

Deoxyguanosine is a TLR7 agonist.

Toll-like receptor 7 (TLR7) is an innate immune sensor for single-strand RNA (ssRNA). Recent structural analysis revealed that TLR7 has an additional binding site for nucleosides such as guanosine, and is activated when both guanosine and ssRNA bind. The nucleoside binding site also accommodates imidazoquinoline derivatives such as R848, which activate TLR7 in the absence of ssRNA. Here, we report that deoxyguanosine (dG) triggered cytokine production in murine bone marrow derived macrophages and plasmacytoid dendritic cells, as well as in human peripheral blood mononuclear cells, including type I interferons and pro-inflammatory factors such as TNF and IL-6. This signalling activity of dG was dependent on TLR7 and its adaptor MyD88 and did not require amplification via the type I interferon receptor. dG-triggered cytokine production required endosomal maturation but did not depend on the concurrent provision of RNA. We conclude that dG induces an inflammatory response through TLR7 and propose that dG is an RNA-independent TLR7 agonist.

Unexpected role of the human cytomegalovirus contribute to essential hypertension in the Kazakh Chinese population of Xinjiang.

Human cytomegalovirus (HCMV) infection, chronic inflammation and oxidative stress, the renin-angiotensin system (RAS), endothelial function, and DNA methylation play roles in the pathogenesis of essential hypertension (EH); however, the mechanism by which HCMV predisposes patients to hypertension remain unclear. Our group previously demonstrated an association between EH and HCMV infection in Kazakh Chinese. Here, we investigated the relationship between HCMV infection and other clinicopathological features in 720 Kazakh individuals with or without hypertension (n=360 each; age: 18-80). Multiple linear and logistic regression analyses were used to determine the associations between HCMV infection, clinical characteristics, and EH. Notably, patients with EH, particularly those with HCMV infection, exhibited a marked increase in tumor necrosis factor-α (TNF-α) and 8-hydroxy-2-deoxyguanosine (8-OHDG) levels, but a decrease in endothelial nitric oxide synthase (eNOS) and renin levels. Similarly, elevated TNF-α and 8-OHDG levels were independent predictors of increased HCMV antibody titers, whereas eNOS and renin were negatively correlated with the latter. Moreover, serum angiotensin-converting enzyme (sACE, ACE) methylation was increased, whereas 11-ß hydroxysteroid dehydrogenase 2 (HSD11ß2; HSD3B2) methylation was decreased in patients with EH who were also infected with HCMV. A positive correlation between HSD3B2 methylation and HCMV IgG titer and blood pressure was additionally observed, whereas angiotensin-converting enzyme (ACE) methylation was inversely correlated with blood pressure. Collectively, these data indicate that HCMV may contribute to EH development in the Kazakh Chinese by increasing TNF-α and 8-OHDG levels, suppressing eNOS and renin, and manipulating HSD3B2 and ACE methylation.

An Investigation of the Effects of Curcumin on the Changes in the Central Nervous System of Rats Exposed to Aroclor 1254 in the Prenatal Period.

BACKGROUND & OBJECTIVE: Aroclor 1254 is a widespread toxic compound of Polychlorinated Biphenyls (PCBs), which can create significant nervous problems. No remedies have been found to date. The aim of this study was to reveal the damage that occurs in the central nervous system of rat pups exposed to Aroclor 1254 in the prenatal period and to show the inhibiting effect of curcumin, which is a strong anti-oxidant and neuroprotective substance. METHOD: The study established 3 groups of adult female and male Wistar albino rats. The rats were mated within these groups and the offspring rats were evaluated within the group given Aroclor 1254 only (n=10) and the group was given both Aroclor 1254 and curcumin (n=10) and the control group (n=10). The groups were compared in respect of pathomorphological damage. The immunohistochemical evaluation was made of 8-hydroxdeoxyguanosine (8-OHdG), 4-hydroxynoneal (4HNE), myelin basic protein (MBP) expressions and TUNEL reaction. The biochemical evaluation was made of the changes in the TAS-TOS and Neuron Specific Enolase (NSE) levels. Damage was seen to have been reduced with curcumin in the 8OHdG and TUNEL reactions, especially in the forebrain and the midbrain, although the dosage applied did not significantly change TAS and TOS levels. Consequently, it was understood that Aroclor 1254 caused damage in the central nervous system of the pup in the prenatal period, and curcumin reduced these negative effects, particularly in the forebrain and the midbrain. CONCLUSION: It was concluded that curcumin could be a potential neuroprotective agent and would be more effective at higher doses.

8-hydroxy-2'-deoxyguanosine (8-OHdG) biomarker detection down to picoMolar level on a plastic antibody film.

An innovative biosensor assembly relying on a simple and straightforward in-situ construction is presented to monitor urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG) down to the pmol/L level. The sensing film of the biosensor consisted of a molecularly imprinted polymer (MIP) layer for 8-OHdG assembled on a gold electrode through electropolymerization of monomer combined with the template. The analytical features of the resulting biosensor were assessed by Cyclic Voltammetry (CV) and Electrochemical Impedance Spectroscopy (EIS). Some experimental parameters such as the initial concentration of the monomer and the ratio template-monomer were investigated and optimized in order to finely tune the performance of the MIP-based sensor. Under optimal conditions, the developed biosensor was able to rebind 8-OHdG with a linear response against EIS from 0.1 to 100pg/ml 3.5-3500 pM. The interference of coexisting species was tested, also with calibrations on urine samples, and good selectivity towards 8-OHdG was obtained. RAMAN spectroscopy, FTIR and SEM evaluations of the prepared films confirmed the formation of a polyphenol thin-film on the electrode surface. The presence and distribution of the imprinted cavities on the MIP layer was confirmed by confocal microscopy imaging of the film, after a post-treatment with Fluorescein Isothiocyanate (FITC) labeled 8-OHdG antibody. Overall, this label-free biosensor for urinary 8-OHdG detection constitutes a promising low-cost alternative to the conventional immunoassay approaches, due to its simplicity, stability, high sensitivity and selectivity for biological sample assays, opening new doors for other applications.

Advancing age increases sperm chromatin damage and impairs fertility in peroxiredoxin 6 null mice.

Due to socioeconomic factors, more couples are choosing to delay conception than ever. Increasing average maternal and paternal age in developed countries over the past 40 years has raised the question of how aging affects reproductive success of males and females. Since oxidative stress in the male reproductive tract increases with age, we investigated the impact of advanced paternal age on the integrity of sperm nucleus and reproductive success of males by using a Prdx6(-/-) mouse model. We compared sperm motility, cytoplasmic droplet retention sperm chromatin quality and reproductive outcomes of young (2-month-old), adult (8-month-old), and old (20-month-old) Prdx6(-/-) males with their age-matched wild type (WT) controls. Absence of PRDX6 caused age-dependent impairment of sperm motility and sperm maturation and increased sperm DNA fragmentation and oxidation as well as decreased sperm DNA compaction and protamination. Litter size, total number of litters and total number of pups per male were significantly lower in Prdx6(-/-) males compared to WT controls. These abnormal reproductive outcomes were severely affected by age in Prdx6(-/-) males. In conclusion, the advanced paternal age affects sperm chromatin integrity and fertility more severely in the absence of PRDX6, suggesting a protective role of PRDX6 in age-associated decline in the sperm quality and fertility in mice.

Glycoxidative damage to human DNA: Neo-antigenic epitopes on DNA molecule could be a possible reason for autoimmune response in type 1 diabetes.

Advanced glycation end-products (AGEs) are known to be mutagenic, diabetogenic and vascular disease risk factors. Methylglyoxal (MG) is a dicarbonyl species that reacts with biological macromolecule (proteins, DNA and lipids) to give AGEs. Nonenzymatic glycation of MG with lysine (Lys) in the presence of copper (Cu(2+)) is reported to generate reactive oxygen species (ROS) capable of causing DNA damage. We show that DNA modification in MG-Lys-Cu(2+) system results in the generation of strand breaks, base modification, hyperchromicity and increased fluorescence intensity. Superoxide generation in the MG-Lys system was found to be significantly higher when compared with that in the MG and Lys alone. Moreover, d-penicillamine and pyridoxal phosphate significantly inhibited the formation of glycation products. The presence of a major DNA glycation adduct, N(2)-carboxyethyl-2'-deoxyguanosine (CEdG), was detected by high performance liquid chromatography (HPLC) and confirmed by nuclear magnetic resonance (NMR). As reported earlier, modified DNA (MG-Lys-Cu(2+)-DNA) was highly immunogenic in experimental animals. Furthermore, induced anti-MG-Lys-Cu(2+)-DNA antibodies were effective probe for detecting glycoxidative lesions in human genomic DNA of type I diabetes patients. Our results clearly imply that interaction of MG-Lys and Cu(2+) leads to the formation of AGEs and also the production of potent ROS, capable of causing DNA damage, thereby playing an important role in diabetes mellitus.

Disposable immunosensor using a simple method for oriented antibody immobilization for label-free real-time detection of an oxidative stress biomarker implicated in cancer diseases.

This work proposes a novel approach for a suitable orientation of antibodies (Ab) on an immunosensing platform, applied here to the determination of 8-hydroxy-2'-deoxyguanosine (8OHdG), a biomarker of oxidative stress that has been associated to chronic diseases, such as cancer. The anti-8OHdG was bound to an amine modified gold support through its Fc region after activation of its carboxylic functions. Non-oriented approaches of Ab binding to the platform were tested in parallel, in order to show that the presented methodology favored Ab/Ag affinity and immunodetection of the antigen. The immunosensor design was evaluated by quartz-crystal microbalance with dissipation, atomic force microscopy, electrochemical impedance spectroscopy (EIS) and square-wave voltammetry. EIS was also a suitable technique to follow the analytical behavior of the device against 8OHdG. The affinity binding between 8OHdG and the antibody immobilized in the gold modified platform increased the charge transfer resistance across the electrochemical set-up. The observed behavior was linear from 0.02 to 7.0 ng/mL of 8OHdG concentrations. The interference from glucose, urea and creatinine was found negligible. An attempt of application to synthetic samples was also successfully conducted. Overall, the presented approach enabled the production of suitably oriented Abs over a gold platform by means of a much simpler process than other oriented-Ab binding approaches described in the literature, as far as we know, and was successful in terms of analytical features and sample application.

Selection of monoclonal antibodies against 6-oxo-M(1)dG and their use in an LC-MS/MS assay for the presence of 6-oxo-M(1)dG in vivo.

Oxidative stress triggers DNA and lipid peroxidation, leading to the formation of electrophiles that react with DNA to form adducts. A product of this pathway, (3-(2'-deoxy-ß-d-erythro-pentofuranosyl)-pyrimido[1,2-α]purine-10(3H)-one), or M(1)dG, is mutagenic in bacterial and mammalian cells and is repaired by the nucleotide excision repair pathway. In vivo, M(1)dG is oxidized to a primary metabolite, (3-(2-deoxy-ß-d-erythro-pentofuranosyl)-pyrimido[1,2-α]purine-6,10(3H,5H)-dione, or 6-oxo-M(1)dG, which is excreted in urine, bile, and feces. We have developed a specific monoclonal antibody against 6-oxo-M(1)dG and have incorporated this antibody into a procedure for the immunoaffinity isolation of 6-oxo-M(1)dG from biological matrices. The purified analyte is quantified by LC-MS/MS using a stable isotope-labeled analogue ([(15)N(5)]-6-oxo-M(1)dG) as an internal standard. Healthy male Sprague-Dawley rats excreted 6-oxo-M(1)dG at a rate of 350-1893 fmol/kg·d in feces. This is the first report of the presence of the major metabolite of M(1)dG in rodents without exogenous introduction of M(1)dG.

Effects of purine nucleoside phosphorylase deficiency on thymocyte development.

BACKGROUND: Inherited or acquired defects in purine nucleoside phosphorylase (PNP) impair purine metabolism, as well as the survival and function of T lymphocytes. However, the effects of PNP deficiency on thymocyte development are not well known. OBJECTIVES: We sought to study thymocyte development in PNP-deficient (PNP-KO) mice. METHODS: Maturation, proliferation, and apoptosis were determined in thymocytes from PNP-KO mice and hematopoietic stem cells from these mice grown ex vivo into thymocyte-like cells. RESULTS: Reduced percentages of CD4(+)CD8(+) double-positive (DP) thymocytes with normal percentages of CD4(-)CD8(+) and CD4(+)CD8(-) single-positive thymocytes were found in the thymi of PNP-KO mice. Similarly, reduced DP-like thymocytes grew ex vivo from hematopoietic stem cells of PNP-KO mice. Thymi of PNP-KO mice contained increased apoptotic DP thymocytes. Increased apoptosis of PNP-deficient DP thymocytes occurred after exposure to deoxyguanosine (dGuo), although not after Fas ligation, and could be prevented by restoring PNP activity within the cells. In DP thymocytes from PNP-KO mice, dGuo caused mitochondrial membrane potential dissipation and induced release of cytochrome c from the mitochondria followed by nuclear DNA fragmentation. Inhibition of the caspase pathway prevented dGuo-induced nuclear DNA fragmentation but not mitochondrial membrane potential dissipation, indicating that PNP deficiency induces apoptosis that is initiated in the mitochondria of DP thymocytes. 5-Bromo-2-deoxyuridine incorporation demonstrated that PNP deficiency does not interfere with DP or single-positive thymocyte proliferation. CONCLUSIONS: PNP is important for the survival of DP thymocytes. Accumulation of dGuo in cases of PNP deficiency leads to mitochondria-initiated apoptosis of DP thymocytes, which can be prevented by restoring PNP activity in the cells.

Optimizing immunoslot blot assays and application to low DNA adduct levels using an amplification approach.

Immunoslot blot assays have been used for the analysis of many DNA adducts, but problems are frequently encountered in achieving reproducible results. Each step of the assay was examined systematically, and it was found that the major problems are in the DNA fragmentation step and the use of the manifold apparatus. Optimization was performed on both the malondialdehyde-deoxyguanosine (M(1)dG) adduct and the O(6)-carboxymethyl-deoxyguanosine (O(6)CMdG) adduct to demonstrate the applicability to other DNA adducts. Blood samples from the European Prospective Investigation on Cancer (EPIC) study (n = 162) were analyzed for M(1)dG adducts, and the data showed no correlation with adduct levels in other tissues, indicating that the EPIC blood samples were not useful for studying M(1)dG adducts. Blood samples from a processed meat versus vegetarian diet intervention (n = 6) were analyzed for O(6)CMdG, and many were below the limit of detection. The reduction of background adduct levels in standard DNA was investigated using chemical and whole genome amplification approaches. The latter gave a sensitivity improvement of 2.6 adducts per 10(7) nucleotides for the analysis of O(6)CMdG. Subsequent reanalysis for O(6)CMdG showed a weakly significant increase in O(6)CMdG on the processed meat diet compared with the vegetarian diet, demonstrating that further studies are warranted.

Interpretation of urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine is adversely affected by methodological inaccuracies when using a commercial ELISA.

The DNA lesion 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), a urinary marker of oxidative stress, is produced from reactions of reactive oxygen species with host DNA 2'-deoxyribonucleotides. The current gold-standard assessment is by complex chromatographic methods using HPLC or LC-MS/MS. Several studies have reported that commercial 8-oxodG ELISA kits correlate sufficiently with chromatographic techniques to be an easier alternative for laboratories without access to gold-standard techniques. However, the assumption that significant correlation translates into a similar ability to differentiate disease categories or treatment groups is yet to be tested. Using LC-MS/MS and two variants of a commercial ELISA, we measured urinary 8-oxodG and creatinine concentrations in young children with cystic fibrosis, a disease associated with oxidative stress, and age-matched controls. We show that, despite significant correlation, both ELISAs overestimate the levels of 8-oxodG, and neither ELISA accurately depicted the difference in group means that was observed by gold-standard LC-MS/MS. The implications of these findings for study outcomes add further support for chromatographic techniques, despite their cost and complexity, to remain the gold standard in urinary 8-oxodG assessment.

Analysis of 7,8-dihydro-8-oxo-2'-deoxyguanosine in cellular DNA during oxidative stress.

Analysis of cellular 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxo-dGuo) as a biomarker of oxidative DNA damage has been fraught with numerous methodological problems. This is primarily due to artifactual oxidation of dGuo that occurs during DNA isolation and hydrolysis. Therefore, it has become necessary to rely on using the comet assay, which is not necessarily specific for 8-oxo-dGuo. A highly specific and sensitive method based on immunoaffinity purification and stable isotope dilution liquid chromatography (LC)-multiple reaction monitoring (MRM)/mass spectrometry (MS) that avoids artifact formation has now been developed. Cellular DNA was isolated using cold DNAzol (a proprietary product that contains guanidine thiocyanate) instead of chaotropic- or phenol-based methodology. Chelex-treated buffers were used to prevent Fenton chemistry-mediated generation of reactive oxygen species (ROS) and artifactual oxidation of DNA bases. Deferoxamine was also added to all buffers in order to complex any residual transition metal ions remaining after Chelex treatment. The LC-MRM/MS method was used to determine that the basal 8-oxo-dGuo level in DNA from human bronchoalveolar H358 cells was 2.2 +/- 0.4 8-oxo-dGuo/10(7) dGuo (mean +/- standard deviation) or 5.5 +/- 1.0 8-oxo-dGuo/10(8) nucleotides. Similar levels were observed in human lung adenocarcinoma A549 cells, mouse hepatoma Hepa-1c1c7 cells, and human HeLa cervical epithelial adenocarcinoma cells. These values are an order of magnitude lower than is typically reported for basal 8-oxo-dGuo levels in DNA as determined by other MS- or chromatography-based assays. H358 cells were treated with increasing concentrations of potassium bromate (KBrO3) as a positive control or with the methylating agent methyl methanesulfonate (MMS) as a negative control. A linear dose-response for 8-oxo-dGuo formation (r(2) = 0.962) was obtained with increasing concentrations of KBrO3 in the range of 0.05 mM to 2.50 mM. In contrast, no 8-oxo-dGuo was observed in H358 cell DNA after treatment with MMS. At low levels of oxidative DNA damage, there was an excellent correlation between a comet assay that measured DNA single strand breaks (SSBs) after treatment with human 8-oxo-guanine glycosylase-1 (hOGG1) when compared with 8-oxo-dGuo in the DNA as measured by the stable isotope dilution LC-MRM/MS method. Availability of the new LC-MRM/MS assay made it possible to show that the benzo[a]pyrene (B[a]P)-derived quinone, B[a]P-7,8-dione, could induce 8-oxo-dGuo formation in H358 cells. This most likely occurred through redox cycling between B[a]P-7,8-dione and B[a]P-7,8-catechol with concomitant generation of DNA damaging ROS. In keeping with this concept, inhibition of catechol-O-methyl transferase (COMT)-mediated detoxification of B[a]P-7,8-catechol with Ro 410961 caused increased 8-oxo-dGuo formation in the H358 cell DNA.

Urea, the most abundant component in urine, cross-reacts with a commercial 8-OH-dG ELISA kit and contributes to overestimation of urinary 8-OH-dG.

Urinary 8-OH-dG is commonly analyzed as a marker of oxidative stress. For its analysis, ELISA and HPLC methods are generally used, although discrepancies in the data obtained by these methods have often been discussed. To clarify this problem, we fractionated human urine by reverse-phase HPLC and assayed each fraction by the ELISA method. In addition to the 8-OH-dG fraction, a positive reaction was observed in the first eluted fraction. The components in this fraction were examined by the ELISA. Urea was found to be the responsible component in this fraction. Urea is present in high concentrations in the urine of mice, rats, and humans, and its level is influenced by many factors. Therefore, certain improvements, such as a correction based on urea content or urease treatment, are required for the accurate analysis of urinary 8-OH-dG by the ELISA method. In addition, performance of the ELISA at 4 degrees C reduced the recognition of urea considerably and improved the 8-OH-dG analysis.

Immunolocalization of 8-OHdG and OGG1 in pancreatic islets of streptozotocin-induced diabetic rats.

This study examined whether oxidative DNA damage and its repair system contribute to the occurrence of diabetes in an experimental rat model. The changed morphological findings of the 8-hydroxydeoxyguanosine (8-OHdG) and 8-oxoG-DNA glycosylase (OGG1) were examined in the pancreatic islets in streptozotocin-induced diabetic rats (60 mg/kg, i.p.). The patterns of immunolocalization were mainly observed in the periphery of the normal pancreatic islet: 8-OHdG in the nucleus and OGG1 in the cytoplasm. The altered immunolocalization of 8-OHdG and OGG1 were greatest in the first hours after streptozotocin injection, and then declined in parallel with the morphological observations of pancreatic beta cell destruction. These results suggested that increased oxidative DNA damage might play a role as the inducer of diabetes and that OGG1 may not successfully mediate DNA repair in streptozotocin-induced diabetic rat pancreas.

Pancreatic islets are very poor in rectifying oxidative DNA damage.

OBJECTIVE: Free radicals that escape scavenging by antioxidant defense damage lipids, proteins, and DNA. Damage to DNA can be repaired. Therefore, both cells' antioxidant defense and their ability to repair oxidatively damaged DNA decide its fate to survive oxidative stress. Pancreatic islets cells with poor antioxidant defense were checked for their ability to remove oxidative damage form DNA. METHODS: For ex vivo DNA repair, assay-cultured pancreatic islets and liver slices were treated with 1 and 10 mM H2O2, respectively, for 30 minutes. After incubation for different time intervals, 8-hydroxy-2'-deoxyguanosine (8-OHdG) in DNA of these cells was estimated using monoclonal antibody raised against 8-OHdG by competitive enzyme-linked immunosorbent assay. For in vitro DNA repair assay, oxidatively damaged pBR322 was incubated with nuclear extracts of islet and liver cells, and 8-OHdG retained in the plasmid was quantitated. RESULTS: Oxidative damage induced by H2O2 was removed quickly and efficiently from DNA by liver cells compared with islet cells. The repair of oxidatively damaged plasmid DNA in vitro was also performed more efficiently (P < 0.05) by nuclear extracts from liver cells compared with islet cell. CONCLUSIONS: We clearly demonstrate that in addition to their low antioxidant defense, islets are very poor in rectifying the oxidative DNA damage.

Activation of ATM signaling pathway is involved in oxidative stress-induced expression of mito-inhibitory p21WAF1/Cip1 in chronic non-suppurative destructive cholangitis in primary biliary cirrhosis: an immunohistochemical study.

Biliary epithelial cells (BECs) of chronic non-suppurative destructive cholangitis (CNSDC) in primary biliary cirrhosis (PBC) reportedly express p21(WAF1/Cip1) and p16(INK4a), which may induce cell cycle arrest and are related to progressive loss of BECs of PBC. Given that the ATM pathway plays a role in the induction of p21(WAF1/Cip1), we examined its possible involvement in bile duct damage of PBC. The expression of phosphorylated-ATM (p-ATM) reflecting the activation of ATM, p21(WAF1/Cip1) and 8-hydroxy-deoxyguanosine (8-OHdG), an oxidative stress marker, was examined immunohistochemically in the liver tissues of 20 cases of stage 1/2 PBC, 9 extrahepatic biliary obstruction (EBO), 35 chronic viral hepatitis (CVH), 17 nonalcoholic steatohepatitis (NASH), and 18 histologically normal liver. p21(WAF1/Cip1), p-ATM and 8-OHdG were frequently and extensively co-expressed in the nuclei of CNSDC in PBC, and their expressions were correlated. In contrast, the expression of these three molecules was absent or faint in small bile ducts in normal livers, CVH, and EBO, and these molecules were clearly expressed in the nuclei of hepatocytes of NASH, in which oxidative stress is involved in hepatocellular damage. In conclusion, oxidative stress-induced p21(WAF1/Cip1) expression in BECs in PBC is closely associated with activation of the ATM pathway and the resultant reduced regeneration or cell cycle arrest of BECs may be related to the progressive loss of small bile ducts of PBC.

Oral administration of a synthetic agonist of Toll-like receptor 9 potently modulates peanut-induced allergy in mice.

BACKGROUND: Agonists of Toll-like receptor 9 have been shown to induce potent T(H)1-type immune responses and prevent and reverse ovalbumin-induced T(H)2-dominant allergic asthma in mice. OBJECTIVE: We examined oral administration of a synthetic agonist of Toll-like receptor 9 (immune modulatory oligonucleotide [IMO]) to modulate peanut-induced allergy in mice. METHODS: In the prevention model mice were sensitized 3 times by means of oral administration of peanut in the presence or absence of IMO. In a treatment protocol mice were sensitized orally with peanut on days 0 and 14 and treated 4 times with oral administration of IMO starting on day 21. RESULTS: In the prevention study mice that received the combination of IMO/peanut showed decreased IgE and increased IgG2a levels in the serum and intestinal tissue compared with mice sensitized with peanut only. In spleen cell recall experiments, production of IL-5 and IL-13 was inhibited and production of IFN-gamma was increased in mice immunized with the peanut/IMO combination compared with those sensitized with peanut only. In the treatment model IMO-treated mice showed decreased IgE, IL-5, and IL-13 levels and increased IgG2a and IFN-gamma levels in the serum, intestines, and spleen cells compared with PBS-treated mice. A reduction in local inflammation and restoration of normal structure in the intestines was found in the mice that received IMO in both models. CONCLUSION: These results indicate that IMOs can switch peanut-induced T(H)2 immune responses toward T(H)1 responses accompanied by reduced inflammation in the gastrointestinal tract and anaphylaxis in both the prevention and treatment models. CLINICAL IMPLICATIONS: IMOs might be suitable candidates for the management of peanut-induced allergy.

Osteonecrosis in stroke-prone spontaneously hypertensive rats: effect of glucocorticoid.

BACKGROUND: High-dose administration of a steroid hormone has been associated with a major risk of osteonecrosis. In this study we investigated the effects of a steroid hormone on the incidence of osteonecrosis of the femoral head in stroke-prone spontaneously hypertensive rats/Nagasaki (SHRSP/Ngsks). METHODS: A total of 71 SHRSP/Ngsks were divided into two groups: a control group (C group, n = 40) and a steroid hormone group (S group, n = 31) given 5 mg (about 20 mg/kg) of methylprednisolone acetate during the 17th week of age. We compared the groups' laboratory data, histological appearance, incidence of osteonecrosis, and expression of oxidative stress on immunohistochemical analysis using the monoclonal antibodies anti-4HNE and anti-8OHdG. RESULTS: The S group showed an increase in total cholesterol, with the amounts of high-density lipoprotein, low-density lipoprotein, and triglycerides all significantly higher than in the C group. Histological examination showed that the frequency of necrosis of the femoral head was significantly higher in the S group (95.2%) than in the C group (51.2%). Most of the histological features of the osteonecrosis demonstrated typical features of a similar sort in the two groups. However, the S group showed bone marrow spaces in the femoral head that were occupied by an increased number of adipocytes and that were swollen, partially degenerative, and necrotic. On immunohistochemical analysis, the stains of anti-4HNE and anti-8OHdG antibody were stronger in the S group than in the C group. CONCLUSIONS: This study confirmed, to a remarkable degree, the suspicion that the administration of steroid hormone increases the number of adipocytes in marrow. Fat degeneration and necrosis, considered early signs of osteonecrosis, were also observed. It has been hypothesized that osteonecrosis is produced by the ischemic change accompanying compartment pressure load in marrow, where fat degeneration, necrosis, and endothelial cell injury might occur together with oxidative stress.