Immunodetection of 3-nitrotyrosine in the liver of zymosan-treated rats with a new monoclonal antibody: comparison to analysis by HPLC

Zymosan-induced peritonitis is associated with an increased production of reactive nitrogen oxides that may contribute to the often-observed failure of multiple organ systems in this model of acute inflammation. Quantitative biochemical evidence is provided for a marked 13-fold increase in protein-bound 3-nitrotyrosine (NTyr), a biomarker of reactive nitrogen oxides, in liver tissue of zymosan-treated rats. In order to investigate the localization of NTyr in this affected tissue, a monoclonal antibody, designated 39B6, was raised against 3-(4-hydroxy-3-nitrophenylacetamido) propionic acid-bovine serum albumin conjugate and its performance characterized. 39B6 was judged by competition ELISA to be approximately 2 orders of magnitude more sensitive than a commercial anti-NTyr monoclonal antibody. Binding characteristics of 39B6 were similar, but not identical, to that of a commercial affinity-purified polyclonal antibody in ELISA and immunohistochemical analyses. Western blot experiments revealed high specificity of 39B6 against NTyr and increased immunoreactivity of specific proteins from liver tissue homogenates of zymosan-treated rats. Immunohistochemical analysis of liver sections indicated a marked zymosan-induced increase in immunofluorescent staining, which was particularly intense in or adjacent to nonparenchymal cells, but not in the parenchymal cells of this tissue. Quantitative analysis of fractions enriched in these cell populations corroborated the immunofluorescent data, although the relative amounts detected in response to zymosan treatment was greatly reduced compared to whole liver tissue. These results demonstrate the high specificity of the newly developed antibody and its usefulness in Western blot and immunohistochemical analysis for NTyr, confirm the presence of NTyr by complementary methods, and suggest the possible involvement of reactive nitrogen oxides in hepatic vascular dysfunction.

Oxidative stress in brain during experimental bacterial meningitis: differential effects of alpha-phenyl-tert-butyl nitrone and N-acetylcysteine treatment

Antioxidant treatment has previously been shown to be neuroprotective in experimental bacterial meningitis. To obtain quantitative evidence for oxidative stress in this disease, we measured the major brain antioxidants ascorbate and reduced glutathione, and the lipid peroxidation endproduct malondialdehyde in the cortex of infant rats infected with Streptococcus pneumoniae. Cortical levels of the two antioxidants were markedly decreased 22 h after infection, when animals were severely ill. Total pyridine nucleotide levels in the cortex were unaltered, suggesting that the loss of the two antioxidants was not due to cell necrosis. Bacterial meningitis was accompanied by a moderate, significant increase in cortical malondialdehyde. While treatment with either of the antioxidants alpha-phenyl-tert-butyl nitrone or N-acetylcysteine significantly inhibited this increase, only the former attenuated the loss of endogenous antioxidants. Cerebrospinal fluid bacterial titer, nitrite and nitrate levels, and myeloperoxidase activity at 18 h after infection were unaffected by antioxidant treatment, suggesting that they acted by mechanisms other than modulation of inflammation. The results demonstrate that bacterial meningitis is accompanied by oxidative stress in the brain parenchyma. Furthermore, increased cortical lipid peroxidation does not appear to be the result of parenchymal oxidative stress, because it was prevented by NAC, which had no effect on the loss of brain antioxidants.

Simultaneous determination of 3-hydroxyanthranilic and cinnabarinic acid by high-performance liquid chromatography with photometric or electrochemical detection

A convenient and rapid method for the simultaneous determination by HPLC of 3-hydroxyanthranilic acid and the dimer derived by its oxidation, cinnabarinic acid, is described. Buffers or biological samples containing these two Trp metabolites were acidified to pH 2.0 and extracted with ethyl acetate with recoveries of 96.5 +/- 0.5 and 93.4 +/- 3.7% for 3-hydroxyanthranilic and cinnabarinic acid, respectively. The two compounds were separated on a reversed-phase (C18) column combined with ion-pair chromatography and detected photometrically or electrochemically. The method was applied successfully to biological systems in which formation of either 3-hydroxyanthranilic or cinnabarinic acid had been described previously. Thus, interferon-gamma-treated human peripheral blood mononuclear cells formed and released significant amounts of 3-hydroxyanthranilic acid into the culture medium and mouse liver nuclear fraction possessed high "cinnabarinic acid synthase" activity. In contrast, addition of 3-hydroxyanthranilic acid to human erythrocytes resulted in only marginal formation of cinnabarinic acid. We conclude that the method described is specific, sensitive, and suitable for the detection of the two Trp metabolites in biological systems.

Radiofrequency electromagnetic field exposure and non-specific symptoms of ill health: A systematic review

This article is a systematic review of whether everyday exposure to radiofrequency electromagnetic field (RF-EMF) causes symptoms, and whether some individuals are able to detect low-level RF-EMF (below the ICNIRP [International Commission on Non-Ionizing Radiation Protection] guidelines). Peer-reviewed articles published before August 2007 were identified by means of a systematic literature search. Meta-analytic techniques were used to pool the results from studies investigating the ability to discriminate active from sham RF-EMF exposure. RF-EMF discrimination was investigated in seven studies including a total of 182 self-declared electromagnetic hypersensitive (EHS) individuals and 332 non-EHS individuals. The pooled correct field detection rate was 4.2% better than expected by chance (95% CI: -2.1 to 10.5). There was no evidence that EHS individuals could detect presence or absence of RF-EMF better than other persons. There was little evidence that short-term exposure to a mobile phone or base station causes symptoms based on the results of eight randomized trials investigating 194 EHS and 346 non-EHS individuals in a laboratory. Some of the trials provided evidence for the occurrence of nocebo effects. In population based studies an association between symptoms and exposure to RF-EMF in the everyday environment was repeatedly observed. This review showed that the large majority of individuals who claims to be able to detect low level RF-EMF are not able to do so under double-blind conditions. If such individuals exist, they represent a small minority and have not been identified yet. The available observational studies do not allow differentiating between biophysical from EMF and nocebo effects.

Assessment of recent HIV-1 infection by a line immunoassay for HIV-1/2 confirmation

BACKGROUND: Knowledge of the number of recent HIV infections is important for epidemiologic surveillance. Over the past decade approaches have been developed to estimate this number by testing HIV-seropositive specimens with assays that discriminate the lower concentration and avidity of HIV antibodies in early infection. We have investigated whether this "recency" information can also be gained from an HIV confirmatory assay. METHODS AND FINDINGS: The ability of a line immunoassay (INNO-LIA HIV I/II Score, Innogenetics) to distinguish recent from older HIV-1 infection was evaluated in comparison with the Calypte HIV-1 BED Incidence enzyme immunoassay (BED-EIA). Both tests were conducted prospectively in all HIV infections newly diagnosed in Switzerland from July 2005 to June 2006. Clinical and laboratory information indicative of recent or older infection was obtained from physicians at the time of HIV diagnosis and used as the reference standard. BED-EIA and various recency algorithms utilizing the antibody reaction to INNO-LIA's five HIV-1 antigen bands were evaluated by logistic regression analysis. A total of 765 HIV-1 infections, 748 (97.8%) with complete test results, were newly diagnosed during the study. A negative or indeterminate HIV antibody assay at diagnosis, symptoms of primary HIV infection, or a negative HIV test during the past 12 mo classified 195 infections (26.1%) as recent (< or = 12 mo). Symptoms of CDC stages B or C classified 161 infections as older (21.5%), and 392 patients with no symptoms remained unclassified. BED-EIA ruled 65% of the 195 recent infections as recent and 80% of the 161 older infections as older. Two INNO-LIA algorithms showed 50% and 40% sensitivity combined with 95% and 99% specificity, respectively. Estimation of recent infection in the entire study population, based on actual results of the three tests and adjusted for a test's sensitivity and specificity, yielded 37% for BED-EIA compared to 35% and 33% for the two INNO-LIA algorithms. Window-based estimation with BED-EIA yielded 41% (95% confidence interval 36%-46%). CONCLUSIONS: Recency information can be extracted from INNO-LIA-based confirmatory testing at no additional costs. This method should improve epidemiologic surveillance in countries that routinely use INNO-LIA for HIV confirmation.

Relation of depression to various markers of coagulation and fibrinolysis in patients with and without coronary artery disease

BACKGROUND: It has been suggested that changes in blood coagulation and fibrinolysis might explain the observed association between depression and coronary artery disease (CAD). So far, only a few coagulation factors have been investigated in this regard, and the results were not consistent. DESIGN: The aim of our study was to analyse a broad range of coagulation and fibrinolytic factors, with emphasis on factors directly involved in clot formation and degradation or reflecting coagulation activation, in patients with CAD and controls without CAD, as assessed by coronary angiography, who also underwent a diagnostic procedure for depression. METHODS: We screened 306 patients with CAD and controls without CAD for depression using the Hospital Anxiety and Depression Scale and Allgemeine Depressions Skala-L questionnaires. In participants with positive screening result, diagnosis of major depression was confirmed or excluded by a structured interview. We analysed the following coagulation and fibrinolytic factors: fibrinogen, prothrombin fragment F1+2, factor XIII A-subunit, factor XIII B-subunit, tissue plasminogen activator, plasminogen activator inhibitor-1, thrombin-activable fibrinolysis inhibitor, and D-dimer. RESULTS: We did not observe significant associations between depression and CAD, nor between depression and cardiovascular risk factors. Coagulation and fibrinolytic factors showed no differences between patients with CAD and controls, but they were associated with several cardiovascular risk factors. Depression was not associated with coagulation and fibrinolytic factors. No associations were found either when both CAD and depression were taken into account. CONCLUSION: Our study gives no evidence that there is a significant relation among depression, CAD, and blood coagulation and fibrinolysis.

Supramalleolar lateral closing wedge osteotomy for the treatment of varus ankle arthrosis

BACKGROUND: Medial ankle joint pain with localized cartilage degeneration due to medial joint overload in varus malalignment of the hindfoot lends itself to treatment by lateral closing wedge supramalleolar osteotomy. METHODS: From 1998 to 2003, nine patients between the ages of 21 to 59 years were operated. The etiology of the malalignment and degeneration was posttraumatic in eight and childhood osteomyelitis in one. Preoperative and postoperative standing radiographs were analyzed to determine the correction of the deformity and the grade of degeneration. Function and pain were assessed using the American Orthopaedic Foot and Ankle Society (AOFAS) Ankle-Hindfoot Scale. The average followup was 56 (range 15 to 88) months. RESULTS: The average time to osseous union was 10 +/- 3.31 weeks. There were no operative or postoperative complications. The average AOFAS score improved from 48 +/- 16.0 preoperatively to 74 +/- 11.7 postoperatively (p<0.004). The average pain subscore improved from 16 +/- 8.8 to 30 +/- 7.1 (p<0.008). The average tibial-ankle surface angle improved from 6.9 +/- 3.8 degrees of varus preoperatively to 0.6 +/- 1.9 degrees of valgus postoperatively (p<0.004). In the sagittal plane, the tibial-lateral-surface angle remained unchanged. At the final followup, two patients showed progression of radiographic ankle arthrosis grades. In one patient, it rose from grade 0 to I. In the other patient it advanced from grade II to III, with subsequent ankle arthrodesis required 16 months after the index procedure. Seven patients returned to their previous work. CONCLUSIONS: Lateral supramalleolar closing wedge osteotomy was an easy and safe procedure, effectively correcting hindfoot malalignment, relieving pain, restoring function, and halting progression of the degeneration in the short-term to mid-term in seven of nine patients.

Intrasexual competition among females and the stabilization of a conspicuous colour polymorphism in a Lake Victoria cichlid fish

The maintenance of colour polymorphisms within populations has been a long-standing interest in evolutionary ecology. African cichlid fish contain some of the most striking known cases of this phenomenon. Intrasexual selection can be negative frequency dependent when males bias aggression towards phenotypically similar rivals, stabilizing male colour polymorphisms. We propose that where females are territorial and competitive, aggression biases in females may also promote coexistence of female morphs. We studied a polymorphic population of the cichlid fish Neochromis omnicaeruleus from Lake Victoria, in which three distinct female colour morphs coexist: one plain brown and two blotched morphs. Using simulated intruder choice tests in the laboratory, we show that wild-caught females of each morph bias aggression towards females of their own morph, suggesting that females of all three morphs may have an advantage when their morph is locally the least abundant. This mechanism may contribute to the establishment and stabilization of colour polymorphisms. Next, by crossing the morphs, we generated sisters belonging to different colour morphs. We find no sign of aggression bias in these sisters, making pleiotropy unlikely to explain the association between colour and aggression bias in wild fish, which is maintained in the face of gene flow. We conclude that female-female aggression may be one important force for stabilizing colour polymorphism in cichlid fish.

Parallel evolution of facial stripe patterns in the Neolamprologus brichardilpulcher species complex endemic to Lake Tanganyika

Colour pattern diversity can be due to random processes or to natural or sexual selection. Consequently, similarities in colour patterns are not always correlated with common ancestry, but may result from convergent evolution under shared selection pressures or drift. Neolamprologus brichardi and Neolamprologus pulcher have been described as two distinct species based on differences in the arrangement of two dark bars on the operculum. Our study uses DNA sequences of the mitochondrial control region to show that relatedness of haplotypes disagrees with species assignment based on head colour pattern. This suggests repeated parallel evolution of particular stripe patterns. The complete lack of shared haplotypes between populations of the same or different phenotypes reflects strong philopatric behaviour, possibly induced by the cooperative breeding mode in which offspring remain in their natal territory and serve as helpers until they disperse to nearby territories or take over a breeding position. Concordant phylogeographic patterns between N. brichardi/N. pulcher populations and other rock-dwelling cichlids suggest that the same colonization routes have been taken by sympatric species and that these routes were affected by lake level fluctuations in the past. (C) 2007 Elsevier Inc. All rights reserved.

Inhibition of ErbB2/neuregulin signaling augments paclitaxel-induced cardiotoxicity in adult ventricular myocytes

Paclitaxel (Taxol) has been successfully combined with the monoclonal antibody trastuzumab (Herceptin) in the treatment of ErbB2 overexpressing cancers. However, this combination therapy showed an unexpected synergistic increase in cardiac dysfunction. We have studied the mechanisms of paclitaxel/anti-ErbB2 cardiotoxicity in adult rat ventricular myocytes (ARVM). Myofibrillar organization was assessed by immunofluorescence microscopy and cell viability was tested by the TUNEL-, LDH- and MTT-assay. Oxidative stress was measured by DCF-fluorescence and myocyte contractile function by video edge-detection and fura-2 fluorescence. Treatment of ARVM with paclitaxel or antibodies to ErbB2 caused a significant increase in myofilament degradation, similarly as observed with an inhibitor of MAPK-signaling, but not apoptosis, necrosis or changes in mitochondrial activity. Paclitaxel-treatment and anti-ErbB2 reduced Erk1/2 phosphorylation. Paclitaxel increased diastolic calcium, shortened relaxation time and reduced fractional shortening in combination with anti-ErbB2. A minor increase in oxidative stress by paclitaxel or anti-ErbB2 was found. We conclude, that concomitant inhibition of ErbB2 receptors and paclitaxel treatment has an additive worsening effect on adult cardiomyocytes, mainly discernible in changes of myofibrillar structure and function, but in the absence of cell death. A potential mechanism is the modulation of the MAPK/Erk1/2 signaling by both drugs.

A pedigree with autosomal dominant thrombocytopenia, red cell macrocytosis, and an occurrence of t(12:21) positive pre-B acute lymphoblastic leukemia

Sampling and analyzing new families with inherited blood disorders are major steps contributing to the identification of gene(s) responsible for normal and pathologic hematopoiesis. Familial occurrences of hematological disorders alone, or as part of a syndromic disease, have been reported, and for some the underlying genetic mutation has been identified. Here we describe a new autosomal dominant inherited phenotype of thrombocytopenia and red cell macrocytosis in a four-generation pedigree. Interestingly, in the youngest generation, a 2-year-old boy presenting with these familial features has developed acute lymphoblastic leukemia characterized by a t(12;21) translocation. Tri-lineage involvement of platelets, red cells and white cells may suggest a genetic defect in an early multiliear progenitor or a stem cell. Functional assays in EBV-transformed cell lines revealed a defect in cell proliferation and tubulin dynamics. Two candidate genes, RUNX1 and FOG1, were sequenced but no pathogenic mutation was found. Identification of the underlying genetic defect(s) in this family may help in understanding the complex process of hematopoiesis.

BOLD correlates of continuously fluctuating epileptic activity isolated by independent component analysis

Combined EEG/fMRI recordings offer a promising opportunity to detect brain areas with altered BOLD signal during interictal epileptic discharges (IEDs). These areas are likely to represent the irritative zone, which is itself a reflection of the epileptogenic zone. This paper reports on the imaging findings using independent component analysis (ICA) to continuously quantify epileptiform activity in simultaneously acquired EEG and fMRI. Using ICA derived factors coding for the epileptic activity takes into account that epileptic activity is continuously fluctuating with each spike differing in amplitude, duration and maybe topography, including subthreshold epileptic activity besides clear IEDs and may thus increase the sensitivity and statistical power of combined EEG/fMRI in epilepsy. Twenty patients with different types of focal and generalized epilepsy syndromes were investigated. ICA separated epileptiform activity from normal physiological brain activity and artifacts. In 16/20 patients, BOLD correlates of epileptic activity matched the EEG sources, the clinical semiology, and, if present, the structural lesions. In clinically equivocal cases, the BOLD correlates aided to attribute proper diagnosis of the underlying epilepsy syndrome. Furthermore, in one patient with temporal lobe epilepsy, BOLD correlates of rhythmic delta activity could be employed to delineate the affected hippocampus. Compared to BOLD correlates of manually identified IEDs, the sensitivity was improved from 50% (10/20) to 80%. The ICA EEG/fMRI approach is a safe, non-invasive and easily applicable technique, which can be used to identify regions with altered hemodynamic effects related to IEDs as well as intermittent rhythmic discharges in different types of epilepsy.