Evaluation of immunohistochemical markers to detect the genotoxic mode of action of fine and ultrafine dusts in rat lungs

Data on local genotoxicity after particle exposure are crucial to resolve mechanistic aspects such as the impact of chronic inflammation, types of DNA damage, and their role in lung carcinogenesis. We established immunohistochemical methods to quantify the DNA damage markers poly(ADP-ribose) (PAR), phosphorylated H2AX (γ-H2AX), 8-hydroxyguanosine (8-OH-dG), and 8-oxoguanine DNA glycosylase (OGG1) in paraffin-embedded tissue from particle-exposed rats. The study was based on lungs from a subchronic study that was part of an already published carcinogenicity study where rats had been intratracheally instilled with saline, quartz DQ12, amorphous silica (Aerosil^® 150), or carbon black (Printex^® 90) at monthly intervals for 3 months. Lung sections were stained immunohistochemically and markers were quantified in alveolar lining cells. Local genotoxicity was then correlated with already defined endpoints, i.e. mean inflammation score, bronchoalveolar lavage parameters, and carcinogenicity. Genotoxicity was most pronounced in quartz DQ12-treated rats, where all genotoxicity markers gave statistically significant positive results, indicating considerable genotoxic stress such as occurrence of DNA double-strand breaks (DSB), and oxidative damage with subsequent repair activity. Genotoxicity was less pronounced for Printex^® 90, but significant increases in γ-H2AX- and 8-OH-dG-positive nuclei and OGG1-positive cytoplasm were nevertheless detected. In contrast, Aerosil^® 150 significantly enhanced only 8-OH-dG-positive nuclei and oxidative damage-related repair activity (OGG1) in cytoplasm. In the present study, γ-H2AX was the most sensitive genotoxicity marker, differentiating best between the three types of particles. The mean number of 8-OH-dG-positive nuclei, however, correlated best with the mean inflammation score at the same time point. This methodological approach enables integration of local genotoxicity testing in subchronic inhalation studies and makes immunohistochemical detection, in particular of γ-H2AX and 8-hydroxyguanine, a very promising approach for local genotoxicity testing in lungs, with prognostic value for the long-term outcome of particle exposure.     

Rapid induction of mild therapeutic hypothermia by extracorporeal veno-venous blood cooling in humans

Aim

Mild therapeutic hypothermia is beneficial in patients successfully resuscitated from non-traumatic out-of-hospital cardiac arrest. The effect of fast induction of hypothermia in these patients remains to be investigated. The aim of this study was to evaluate the efficacy and safety of extracorporeal veno-venous blood cooling in humans successfully resuscitated from cardiac arrest.

Methods

We performed an interventional study in patients after successful resuscitation from cardiac arrest admitted to the emergency department of a tertiary care centre. The extracorporeal veno-venous circulation was established via a percutaneously introduced double lumen dialysis catheter in the femoral vein, and a tubing circuit and heat exchanger. A paediatric cardiopulmonary bypass roller pump and a heater-cooler system were used to circulate the blood. Main outcome measures were feasibility, efficacy, and safety.

Results

We included eight consecutive cardiac arrest patients with a median oesophageal temperature of 35.9 °C (interquartile range 34.9–37.0). A median time of 8 min elapsed (interquartile range 5–15 min) to reach oesophageal temperatures below 34 °C, which reflects a cooling rate of 12.2 °C/h (interquartile range 10.8 °C/h to 14.1 °C/h). The predefined target temperature of 33.0 °C was reached after 14 min (interquartile range 8–21 min). No device or method related adverse events were reported.

Conclusion

Extracorporeal veno-venous blood cooling is a feasible, safe, and very fast approach for induction of mild therapeutic hypothermia in patients successfully resuscitated from cardiac arrest.     

Interleukin-6 at discharge predicts all-cause mortality in patients with sepsis

Purpose

Interleukin-6 (IL-6) is a proinflammatory cytokine that plays a central role in the pathogenesis of sepsis. We aim to investigate the association between IL-6 and all-cause mortality in patients with sepsis.

Methods

A cohort of 40 elderly patients with sepsis was identified between March 2009 and June 2010 at Rambam Health Medical Campus, Haifa, Israel. The cohort was followed up for all-cause mortality occurring during the 6 months after hospital discharge. Cox proportional hazard model was used to assess the association between IL-6 and all-cause mortality.

Results

Iinterleukin-6 at discharge had a higher predictive accuracy for all-cause mortality when compared with IL-6 at admission. The area under the curve was 0.752 (P = .015) and 0.545 (P = .661), respectively. Eleven (27.5%) patients died during follow-up; the subjects who died have higher IL-6 levels at discharge (median, 50.6 pg/mL [interquartile range, 39.6-105.9]) compared with survivors at the end of follow-up (median, 35.4 [interquartile range, 15.8-49]; P = .014). The risk of all-cause mortality was higher in subjects with IL-6 levels above the median compared with subjects with lower IL-6 levels (log-rank P = .017). On multivariate Cox proportional analysis, adjusting for the potential confounders, IL-6 at discharge remained an independent predictor for 6 month all-cause mortality (hazard ratio, 6.05 [1.24-24.20]) for levels above the median compared with lower levels.

Conclusions

Iinterleukin-6 at discharge is an independent predictor of all-cause mortality in patients with sepsis. Compared with IL-6 at admission, IL-6 at discharge better predicts all-cause mortality.     

Development and Characterization of An Unshielded PatLoc Gradient Coil for Human Head Imaging

A cylindrical head gradient insert for human imaging with non‐linear spatial encoding magnetic fields (SEMs) has been designed, optimized and successfully integrated with a modified 3T clinical MR system. This PatLoc (parallel acquisition technique using localized gradients) SEM coil uses SEMs that resemble second‐order magnetic shim fields, but with much higher amplitude as well as the possibility for rapid switching. This work describes the optimization of a coil design and measurement methods to characterize its SEMs, induced self‐eddy currents and concomitant fields. Magnetic field maps of the SEMs are measured and it is demonstrated that the induced self‐eddy current magnetic fields are small and can be compensated. A method to measure concomitant fields is presented and those fields are compared to simulated data. Finally, in vivo human images acquired using the PatLoc system are presented and discussed. © 2013 Wiley Periodicals, Inc. Concepts Magn Reson Part B (Magn Reson Engineering) 43B: 111–125, 2013     

Mapping translocation breakpoints by next-generation sequencing

Balanced chromosome rearrangements (BCRs) can cause genetic diseases by disrupting or inactivating specific genes, and the characterization of breakpoints in disease-associated BCRs has been instrumental in the molecular elucidation of a wide variety of genetic disorders. However, mapping chromosome breakpoints using traditional methods, such as in situ hybridization with fluorescent dye-labeled bacterial artificial chromosome clones (BAC-FISH), is rather laborious and time-consuming. In addition, the resolution of BAC-FISH is often insufficient to unequivocally identify the disrupted gene. To overcome these limitations, we have performed shotgun sequencing of flow-sorted derivative chromosomes using "next-generation" (Illumina/Solexa) multiplex sequencing-by-synthesis technology. As shown here for three different disease-associated BCRs, the coverage attained by this platform is sufficient to bridge the breakpoints by PCR amplification, and this procedure allows the determination of their exact nucleotide positions within a few weeks. Its implementation will greatly facilitate large-scale breakpoint mapping and gene finding in patients with disease-associated balanced translocations.     

Meta-analysis of determinants for pet ownership in 12 European birth cohorts on asthma and allergies: a GA2LEN initiative

Background: Studies on pet ownership as a risk or protective factor for asthma and allergy show inconsistent results. This may be on account of insufficient adjustment of confounding factors. Aim: The objective of this study was to describe determinants of cat and dog ownership in European families with and without allergies. Methods: Within the EU‐funded network of excellence GA²LEN, we performed meta‐analyses with data from 12 ongoing European birth cohort studies on asthma and allergy. Each of the birth cohort studies enrolled between 485 and 4089 children. Pet ownership, allergic status (asthma, allergic rhinitis, eczema) of parents and siblings, parental education, access to ground floor, and number of people living at home were assessed by questionnaires. Results: Among the 25 056 families from seven European countries cats (14.9%) were more common than dogs (12.0%). Allergic family history significantly reduced the odds to own a cat (adjusted combined random‐effect OR 0.91; 95% CI 0.85–0.99), or dog (0.90; 0.86–0.94). A higher parental educational level had even more pronounced effects on cat (0.84; 0.71–0.98), and dog ownership (0.61; 0.54–0.70). Elder siblings reduced the odds to own cats, but not dogs. Convenient ground access significantly increased the odds, whereas crowding at home was not associated with cat or dog ownership. Conclusions: The chances to own a cat or dog were significantly reduced in allergic families, in parents with a higher educational level, and in homes without convenient ground access. In addition to parental allergies, social and housing factors should be considered as potential confounders in studies on pet exposure and allergic diseases.     

Predictivity of allergic sensitization (RAST) for the onset of allergic diseases in adults

Background:  Specific IgE antibodies are often detected without any clinical manifestation of allergies. We aimed to analyse the predictivity of allergic sensitization for incident symptoms of allergic diseases in adults during a 10-year follow-up .
Methods:  In 1994/95 specific IgE antibodies against five common inhalant allergens (grass pollen, birch pollen, house dust mite, cat dander and Cladosporium ) were diagnosed by radioallergosorbent test in 4178 adults aged 25–74 years. A subset of 2656 participants could be re-evaluated in 2004/05. Information on socio-economic factors and medical history, including data on atopic diseases, was assessed by a combination of a personal interview and a self-administered questionnaire. Logistic regression models were applied to study associations between allergic sensitization and incident allergic diseases.
Results:  Allergic sensitization was an important predictor for incident hay fever (OR 7.95, CI 95% 4.64–13.62) and asthma (OR 1.82, CI 95% 1.29–2.57). Specific IgE antibodies were mainly related to outdoor allergens (grass and birch pollen) for hay fever and indoor allergens (mite and cat dander) for asthma, while for atopic dermatitis no specific IgE antibodies were identified as major predictors.
Conclusions:  Allergic sensitization not only covers clinically apparent allergies, but indicates a prognostic factor for later allergies, even in adulthood.     

Correlations between reduced expression of the metastasis suppressor gene KAI-1 and accumulation of p53 in uterine carcinomas and sarcomas

Kangai (KAI)-1 (CD82) is a metastasis suppressor gene, which belongs to the family of tetraspanin proteins. A loss of KAI-1 expression is associated with the advanced stages of many human malignancies. The present study was designed to investigate the expression pattern of KAI-1 in the normal endometrium and uterine tumors and to correlate it with the expression of tumor suppressor protein p53. KAI-1 could be found in the normal endometrium throughout the menstrual cycle. Thirteen of 42 endometrial carcinomas demonstrated moderate KAI-1 expression, but low expression of p53. Twenty-nine of 42 endometrial carcinomas showed reduced or absent KAI-1 expression, which correlated with strong expression of p53 (p < 0.001). There were significant correlations between KAI-1 expression and histological type, e.g., 93% of endometrioid carcinomas displayed a low or moderate immunostaining for KAI-1, whereas nearly all of the serous/clear cell carcinomas were KAI-1 negative (p < 0.001); tumor grading, e.g., 73% of high grade tumors showed no KAI-1 expression (p < 0.001). Most of the investigated uterine sarcomas were negative for KAI-1, whereas they displayed a strong immunostaining for p53. In conclusion, KAI-1 and p53 show inverse expression. The reduced KAI-1 expression may be the result of dysregulated p53 function and could be an important step in the endometrial carcinogenesis.     

Mutations in CYP1B1 cause primary congenital glaucoma by reduction of either activity or abundance of the enzyme

Primary congenital glaucoma (PCG) is an autosomal recessive disorder caused predominantly by mutations in the CYP1B1 gene. A total of five frequent single nucleotide polymorphisms (SNPs) have been identified in the coding sequence of CYP1B1: rs10012C>G (p.R48G), rs1056827G>T (p.A119S), rs1056836C>G (p.V432L), rs1056837C>T (p.D449D), and rs1800440A>G (p.N453S). We performed a functional characterization of four common CYP1B1 variants presenting different coding SNP haplotypes (RAVDN, GSLDN, RALDS, and RALDN) and five CYP1B1 mutations reported for PCG patients: c.182G>A (p.G61E), c.608A>G (p.N203S), c.1033_1035del (p.L343del), c.241 T>A (p.Y81N), and c.685G>A (p.E229 K). Each mutation was embedded in its corresponding background SNP haplotype. The common variants revealed variation in enzymatic activity; among them, RAVDN showed the highest activity. Mutants p.G61E, p.N203S, and p.L343del each revealed a residual activity (<10%) of their respective haplotype. The microsomal CYP1B1 abundance relative to total protein also showed variation in common variants and a significant reduction in p.L343del, p.Y81N, and p.E229 K. The free energy of folding (DeltaDeltaG) values suggest that the lower stability of the mutants is one key property leading to the experimentally observed lower protein abundance. Our new measure of relative enzymatic activity (U/mg total protein), which combines activity and abundance values, was significantly lower for all five mutations compared to the corresponding background haplotype. We classified p.Y81N and p.E229 K not as mutations but as hypomorphic alleles, since their relative activity values are intermediate between bona fide mutations and the common variant with the lowest activity (RALDS). We propose that CYP1B1 mutations can act by either reducing enzymatic activity (p.G61E and p.N203S), reducing the abundance of the enzyme (p.Y81N and p.E229 K), or both (p.L343del).