High frequency of HTRA1 AND ABCC6 mutations in Japanese patients with adult-onset cerebral small vessel disease.

BACKGROUND: This study aimed to clarify the frequency and clinical features of monogenic cerebral small vessel disease (mgCSVD) among patients with adult-onset severe CSVD in Japan. METHODS: This study included patients with adult-onset severe CSVD with an age of onset ≤55 years (group 1) or >55 years and with a positive family history (group 2). After conducting conventional genetic tests for NOTCH3 and HTRA1, whole-exome sequencing was performed on undiagnosed patients. Patients were divided into two groups according to the results of the genetic tests: monogenic and undetermined. The clinical and imaging features were compared between the two groups. RESULTS: Group 1 and group 2 included 75 and 31 patients, respectively. In total, 30 patients had NOTCH3 mutations, 11 patients had HTRA1 mutations, 6 patients had ABCC6 mutations, 1 patient had a TREX1 mutation, 1 patient had a COL4A1 mutation and 1 patient had a COL4A2 mutation. The total frequency of mutations in NOTCH3, HTRA1 and ABCC6 was 94.0% in patients with mgCSVD. In group 1, the frequency of a family history of first relatives, hypertension and multiple lacunar infarctions (LIs) differed significantly between the two groups (monogenic vs undetermined; family history of first relatives, 61.0% vs 25.0%, p=0.0015; hypertension, 34.1% vs 63.9%, p=0.0092; multiple LIs, 87.8% vs 63.9%, p=0.0134). CONCLUSIONS: More than 90% of mgCSVDs were diagnosed by screening for NOTCH3, HTRA1 and ABCC6. The target sequences for these three genes may efficiently diagnose mgCSVD in Japanese patients.

Chemical and immunochemical detection of 8-halogenated deoxyguanosines at early stage inflammation.

Myeloperoxidase (MPO) generates reactive halogenating species that can modify DNA. The aim of this study was to investigate the formation of 8-halogenated 2'-deoxyguanosines (8- halo-dGs) during inflammatory events. 8-Bromo-2'-dG (8-BrdG) and 8-chloro-2'-dG (8-CldG) were generated by treatment of MPO with hydrogen peroxide at physiological concentrations of Cl(-) and Br(-). The formation of 8-halo-dGs with other oxidative stress biomarkers in lipopolysaccharide-treated rats was assessed by liquid chromatography tandem mass spectrometry and immunohistochemistry using a novel monoclonal antibody (mAb8B3) to 8-BrdG-conjugated keyhole limpet hemocyanin. The antibody recognized both 8-BrdG and 8-CldG. In the liver of lipopolysaccharide-treated rats, immunostaining for 8-halo-dGs, halogenated tyrosines, and MPO were increased at 8 h, whereas those of 8-oxo-2'-dG (8-OxodG) and 3-nitrotyrosine were increased at 24 h. Urinary excretion of both 8-CldG and 8-BrdG was also observed earlier than those of 8-OxodG and modified tyrosines (3-nitrotyrosine, 3-chlorotyrosine, and 3- bromotyrosine). Moreover, the levels of the 8-halo-dGs in urine from human diabetic patients were 8-fold higher than in healthy subjects (n = 10, healthy and diabetic, p < 0.0001), whereas there was a moderate difference in 8-OxodG between the two groups (p < 0.001). Interestingly, positive mAb8B3 antibody staining was observed in liver tissue from hepatocellular carcinoma patients but not in liver tissue from human cirrhosis patients. These data suggest that 8-halo-dGs may be potential biomarkers of early inflammation.

Myocarditis and myasthenia gravis by combined nivolumab and ipilimumab immunotherapy for renal cell carcinoma: A case report of successful management.

Ipilimumab plus nivolumab (Ipi/Nivo) has revolutionized advanced renal cell carcinoma (RCC) treatment. However, it encompassed fatal immune-related adverse events (irAEs). Myocarditis with concomitant myasthenia gravis (MG) has a mortality rate of 50%, and a high dose of methylprednisolone (mPSL) should be administered with careful attention to MG exacerbation. We present the case of a 59-year-old man with progressing lung metastasis of RCC. After one cycle of Ipi/Nivo, he experienced myocarditis and MG, managed by mPSL pulse therapy, plasma exchange, and high-dose intravenous immunoglobulin. We share the therapeutic course, aiming to contribute to the limited literature on rare but aggressive irAEs.

Low concentration of 4-hydroxy hexenal increases heme oxygenase-1 expression through activation of Nrf2 and antioxidative activity in vascular endothelial cells.

Large-scale clinical studies have shown that n-3 polyunsaturated fatty acids (n-3 PUFAs) such as eicosapentaenoic and docosahexaenoic acids reduce cardiovascular events without improving classical risk factors for atherosclerosis. Recent studies have proposed that direct actions of n-3 PUFAs themselves, or of their enzymatic metabolites, have antioxidative and anti-inflammatory effects on vascular cells. Although a recent study showed that plasma 4-hydroxy hexenal (4-HHE), a peroxidation product of n-3 PUFA, increased after supplementation of docosahexaenoic acid, the antiatherogenic effects of 4-HHE in vascular cells remain unclear. In the present study, we tested the hypothesis that 4-HHE induces the antioxidative enzyme heme oxygenase-1 (HO-1) through activation of nuclear factor erythroid 2-related factor 2 (Nrf2), a master regulatory transcriptional factor, and prevents oxidative stress-induced cytotoxicity in vascular endothelial cells. This mechanism could partly explain the cardioprotective effects of n-3 PUFAs. Human umbilical vein endothelial cells were stimulated with 1-10µM 4-HHE or 4-hydroxy nonenal (4-HNE), a peroxidation product of n-6 PUFAs. Both 4-HHE and 4-HNE dose-dependently increased HO-1 mRNA and protein expression, and intranuclear expression and DNA binding of Nrf2 at 5µM. Small interfering RNA for Nrf2 significantly reduced 4-HHE- or 4-HNE-induced HO-1 mRNA and protein expression. Furthermore, pretreatment with 4-HHE or 4-HNE prevented tert-butyl hydroperoxide-induced cytotoxicity. In conclusion, 4-HHE, a peroxidation product of n-3 PUFAs, stimulated expression of the antioxidant enzyme HO-1 through the activation of Nrf2 in vascular endothelial cells. This resulted in prevention of oxidative stress-induced cytotoxicity, and may represent a possible mechanism to partly explain the cardioprotective effects of n-3 PUFAs.

Galloylated catechins as potent inhibitors of hypochlorous acid-induced DNA damage.

Hypochlorous acid (HOCl), a strong oxidant derived from myeloperoxidase in neutrophils and macrophages, can chlorinate DNA bases at the site of inflammation. Because little is known about the protective role of natural antioxidants, such as polyphenols, for the myeloperoxidase-derived DNA damage, we screened the inhibitory effects of various phenolic antioxidants on the chlorination of the 2'-deoxycytidine residue by HOCl in vitro and found that green tea catechins, especially (-)-epicatechin gallate (ECg) and (-)-epigallocatechin gallate (EGCg), significantly inhibited the chlorination. These catechins also reduced nucleoside- and taurine-chloramines, which can induce secondary oxidative damage, into their native forms. Mass spectrometric and nuclear magnetic resonance analyses showed that ECg and EGCg can effectively scavenge HOCl and/or chloramine species resulting in the formation of mono- and dichlorinated ECg and EGCg. Using the HL-60 human leukemia cell line, it was found that ECg could efficiently accumulate in the cells. Immunocytometric analyses using antihalogenated 2'-deoxycytidine antibody showed that pretreatment of cells with ECg inhibited the HOCl-induced immunofluorescence. In addition, the chlorinated ECg derivatives were detected in the HOCl-treated HL-60 cells. These results showed that green tea catechins, especially 3-galloylated catechins, may be the plausible candidate for the prevention of inflammation-derived DNA damage and perhaps carcinogenesis.

Immunogenicity of a brominated protein and successive establishment of a monoclonal antibody to dihalogenated tyrosine.

During inhalation of allergens and experimental sepsis, formation of brominated tyrosine has been reported. In this study, we first examined the immunogenicity of brominated protein prepared by treatment of N-bromosuccinimide (NBS). The immunized serum obtained reacted with brominated bovine serum albumin (BSA). The NBS dose-dependent formation of immunoreactivity, which was estimated by enzyme-linked immunosorbent assay, was observed, and the increase coincided with 3,5-dibromotyrosine (DiBrY) formation in the modified BSA, which was chemically determined by liquid chromatography/quadrupole tandem mass spectrometry (LC/MS/MS). Second, by use of immunized mice, monoclonal antibodies to the brominated one were prepared. The two established novel monoclonal antibodies obtained from the immunized mice reacted with DiBrY, 3,5-dichlorotyrosine (DiClY), and 3,5-diiodotyrosine (DiIY). Moreover, 3,5-dihalo-4-hydroxybenzoic acids (3,5-dichloro-4-hydroxybenzoic acid and 3,5-dibromo-4-hydroxybenzoic acid) were recognized by these antibodies. These results suggest that dihalogenated tyrosines (DiBrY, DiClY, and DiIY) are the epitopes. Lastly, we used the antibody in an immunohistochemical study. Lipopolysaccharide (LPS) was intraperitoneally administered to mice, and livers were removed. Positive staining of LPS-treated mouse liver tissues by both the anti-dihalotyrosine antibody and anti-myeloperoxidase antibody was estimated, suggesting that inflammatory tissue damage induces the formation of dihalotyrosine in vivo.

A randomized controlled trial of consensus interferon with or without lactoferrin for chronic hepatitis C patients with genotype 1b and high viral load.

Recently, lactoferrin has been reported to have anti-HCV effects. The aim of this study was to investigate the effect of combination therapy using consensus interferon (CIFN) and lactoferrin in patients with chronic hepatitis C. Twenty-one patients with chronic HCV infection, who were positive for HCV-RNA genotype 1b with serum viral loads from 100 to 700KIU/ml, were randomly assigned to two groups; the CIFN + Lac group received CIFN with lactoferrin and the CIFN group received CIFN alone. Nine patients in each group completed this trial; the other patients dropped out because of side effects. Three, two and four patients were categorized as complete responders, relapsers and non-responders, respectively, in the CIFN + Lac group, and four, one and four in the CIFN group, respectively. There was no statistically significant difference in virologic response between the two groups. During the follow up after CIFN therapy with continued lactoferrin, there were two relapsers in the CIFN + Lac group and their HCV-RNA titers before treatment were over 400KIU/ml. In conclusion, the combination therapy of CIFN and lactoferrin did not increase the response rate or prevent relapse after discontinuation of IFN.

Comparison of Therapeutic G-CSF Cycles and Prophylactic G-CSF Cycles in Patients Receiving Paclitaxel and Carboplatin Combination Chemotherapy for Ovarian Cancer: A Retrospective Study Report.

OBJECTIVE: The aim of the present study was to investigate the differences between therapeutic granulocyte-colony stimulating factor (G-CSF) cycles and prophylactic G-CSF cycles in patients receiving paclitaxel and carboplatin combination chemotherapy for ovarian cancer. MATERIAL AND METHOD: Medical records of 15 women who received paclitaxel and carboplatin combination chemotherapy for ovarian cancer between January 2003 and December 2012 were analyzed retrospectively. All 15 patients completed 6 cycles of paclitaxel and carboplatin as the first-line chemotherapy. The complications were compared between therapeutic G-CSF cycles and prophylactic G-CSF cycles. RESULTS: The number of chemotherapy cycles correlated with the ratio of prophylactic G-CSF cycles. It was considered that earlier prophylactic G-CSF injections were chosen due to a gradual decrease in WBC and neutrophil counts. The WBC and neutrophil counts were significantly higher in prophylactic G-CSF cycles than in therapeutic G-CSF cycles. However, there were no significant differences in the intervals of chemotherapy, delay of chemotherapy, and incidence of febrile neutropenia between the therapeutic G-CSF and prophylactic G-CSF cycles. CONCLUSION: Prophylactic G-CSF injections were not effective in preventing the incidence of febrile neutropenia in patients receiving paclitaxel and carboplatin combination chemotherapy for ovarian cancer.

Characteristics of PCR fragments amplified using five microsatellite markers for identifying the Nagoya breed.

The Nagoya breed is a native chicken of Aichi Prefecture, Japan, a dual-purpose breed for eggs and meat. A method for distinguishing the Nagoya breed from Aichi Prefecture from other chickens using five microsatellite markers (ABR0015, ABR0257, ABR0417, ABR0495 and ADL0262) has already been utilized in order to check the authenticity of Nagoya breed-labeled chicken on the market. The present study was conducted to investigate nucleotide sequences and sizes of PCR fragments containing the five microsatellite regions for the Nagoya breed and to confirm that the genomic identification could continue to be applied in the future. The DNA sequencing of fragments containing the five markers showed that ABR0015, ABR0417 and ABR0495 had a single haplotype, ABR0257 had three haplotypes, and ADL0262 had two haplotypes, although all the markers exhibited one fixed fragment size each upon sequencing of the fragments and fragment analysis. The results of the fragment analysis of each marker using DNA samples of 28 Nagoya breed males (G0 generation) reared in 2000-2001 and 20 of their offspring males (G8) reared in 2008-2009 showed one fixed fragment size in both populations. Therefore, we confirmed that the five microsatellite markers are useful tools for accurately distinguishing the Nagoya breed from other chickens.

Endogenous formation of novel halogenated 2'-deoxycytidine. Hypohalous acid-mediated DNA modification at the site of inflammation.

A potential role of DNA damage by leukocyte-derived reactive species in carcinogenesis has been suggested. Leukocyte-derived peroxidases, such as myeloperoxidase and eosinophil peroxidase, use hydrogen peroxide and halides (Cl- and Br-) to generate hypohalous acids (HOCl and HOBr), halogenating intermediates. It has been suggested that these oxidants lead to the formation of halogenated products upon reaction with nucleobases. To verify the consequences of phagocyte-mediated DNA damage at the site of inflammation, we developed a novel monoclonal antibody (mAb2D3) that recognizes the hypohalous acid-modified DNA and found that the antibody most significantly recognized HOCl/HOBr-modified 2'-deoxycytidine residues. The immunoreactivity of HOCl-treated oligonucleotide was attenuated by excess methionine, suggesting that chloramine-like species may be the plausible epitopes of the antibody. On the basis of further characterization combined with mass spectrometric analysis, the epitopes of mAb2D3 were determined to be novel N4,5-dihalogenated 2'-deoxycytidine residues. The formation of the dihalogenated 2'-deoxycytidine in vivo was immunohistochemically demonstrated in the lung and liver nuclei of mice treated with lipopolysaccharides, an experimental inflammatory model. These results strongly suggest that phagocyte-derived oxidants, hypohalous acids, endogenously generate the halogenated DNA bases such as a novel dihalogenated 2'-deoxycytidine in vivo. Halogenation (chlorination and/or bromination) of DNA therefore may constitute one mechanism for oxidative DNA damage at the site of inflammation.