Radiation-Induced Lens Opacity and Cataractogenesis: A Lifetime Study Using Mice of Varying Genetic Backgrounds.

Recent epidemiological findings and reanalysis of historical data suggest lens opacities resulting from ionizing radiation exposures are likely induced at lower doses than previously thought. These observations have led to ICRP recommendations for a reduction in the occupational dose limits for the eye lens, as well as subsequent implementation in EU member states. The EU CONCERT LDLensRad project was initiated to further understand the effects of ionizing radiation on the lens and identify the mechanism(s) involved in radiation-induced cataract, as well as the impact of dose and dose-rate. Here, we present the results of a long-term study of changes to lens opacity in male and female adult mice from a variety of different genetic (radiosensitive or radioresistant) backgrounds, including mutant strains Ercc2 and Ptch1, which were assumed to be susceptible to radiation-induced lens opacities. Mice received 0.5, 1 and 2 Gy 60Co gamma-ray irradiation at dose rates of 0.063 and 0.3 Gy min-1. Scheimpflug imaging was used to quantify lens opacification as an early indicator of cataract, with monthly observations taken postirradiation for an 18-month period in all strains apart from 129S2, which were observed for 12 months. Opacification of the lens was found to increase with time postirradiation (with age) for most mouse models, with ionizing radiation exposure increasing opacities further. Sex, dose, dose rate and genetic background were all found to be significant contributors to opacification; however, significant interactions were identified, which meant that the impact of these factors was strain dependent. Mean lens density increased with higher dose and dose rate in the presence of Ercc2 and Ptch1 mutations. This project was the first to focus on low (<1 Gy) dose, multiple dose rate, sex and strain effects in lens opacification, and clearly demonstrates the importance of these experimental factors in radiobiological investigations on the lens. The results provide insight into the effects of ionizing radiation on the lens as well as the need for further work in this area to underpin appropriate radiation protection legislation and guidance.

Lower versus higher hemoglobin threshold for transfusion in ARDS patients with and without ECMO.

BACKGROUND: Efficacy and safety of different hemoglobin thresholds for transfusion of red blood cells (RBCs) in adults with an acute respiratory distress syndrome (ARDS) are unknown. We therefore assessed the effect of two transfusion thresholds on short-term outcome in patients with ARDS. METHODS: Patients who received transfusions of RBCs were identified from a cohort of 1044 ARDS patients. After propensity score matching, patients transfused at a hemoglobin concentration of 8 g/dl or less (lower-threshold) were compared to patients transfused at a hemoglobin concentration of 10 g/dl or less (higher-threshold). The primary endpoint was 28-day mortality. Secondary endpoints included ECMO-free, ventilator-free, sedation-free, and organ dysfunction-free composites. MEASUREMENTS AND MAIN RESULTS: One hundred ninety-two patients were eligible for analysis of the matched cohort. Patients in the lower-threshold group had similar baseline characteristics and hemoglobin levels at ARDS onset but received fewer RBC units and had lower hemoglobin levels compared with the higher-threshold group during the course on the ICU (9.1 [IQR, 8.7-9.7] vs. 10.4 [10-11] g/dl, P < 0.001). There was no difference in 28-day mortality between the lower-threshold group compared with the higher-threshold group (hazard ratio, 0.94 [95%-CI, 0.59-1.48], P = 0.78). Within 28 days, 36.5% (95%-CI, 27.0-46.9) of the patients in the lower-threshold group compared with 39.5% (29.9-50.1) of the patients in the higher-threshold group had died. While there were no differences in ECMO-free, sedation-free, and organ dysfunction-free composites, the chance for successful weaning from mechanical ventilation within 28 days after ARDS onset was lower in the lower-threshold group (subdistribution hazard ratio, 0.36 [95%-CI, 0.15-0.86], P = 0.02). CONCLUSIONS: Transfusion at a hemoglobin concentration of 8 g/dl, as compared with a hemoglobin concentration of 10 g/dl, was not associated with an increase in 28-day mortality in adults with ARDS. However, a transfusion at a hemoglobin concentration of 8 g/dl was associated with a lower chance for successful weaning from the ventilator during the first 28 days after ARDS onset. TRIAL REGISTRATION: ClinicalTrials.gov NCT03871166.

Neurotransmitter receptor density changes in Pitx3ak mice--a model relevant to Parkinson's disease.

Parkinson's disease (PD) is the second most common neurodegenerative disorder, characterized by alterations of nigrostriatal dopaminergic neurotransmission. Compared to the wealth of data on the impairment of the dopamine system, relatively limited evidence is available concerning the role of major non-dopaminergic neurotransmitter systems in PD. Therefore, we comprehensively investigated the density and distribution of neurotransmitter receptors for glutamate, GABA, acetylcholine, adrenaline, serotonin, dopamine and adenosine in brains of homozygous aphakia mice being characterized by mutations affecting the Pitx3 gene. This genetic model exhibits crucial hallmarks of PD on the neuropathological, symptomatic and pharmacological level. Quantitative receptor autoradiography was used to characterize 19 different receptor binding sites in eleven brain regions in order to understand receptor changes on a systemic level. We demonstrated striking differential changes of neurotransmitter receptor densities for numerous receptor types and brain regions, respectively. Most prominent, a strong up-regulation of GABA receptors and associated benzodiazepine binding sites in different brain regions and concomitant down-regulations of striatal nicotinic acetylcholine and serotonergic receptor densities were found. Furthermore, the densities of glutamatergic kainate, muscarinic acetylcholine, adrenergic α1 and dopaminergic D2/D3 receptors were differentially altered. These results present novel insights into the expression of neurotransmitter receptors in Pitx3(ak) mice supporting findings on PD pathology in patients and indicating on the possible underlying mechanisms. The data suggest Pitx3(ak) mice as an appropriate new model to investigate the role of neurotransmitter receptors in PD. Our study highlights the relevance of non-dopaminergic systems in PD and for the understanding of its molecular pathology.

Radiation cataractogenesis: a review of recent studies.

The lens of the eye is recognized as one of the most radiosensitive tissues in the human body, and it is known that cataracts can be induced by acute doses of less than 2 Gy of low-LET ionizing radiation and less than 5 Gy of protracted radiation. Although much work has been carried out in this area, the exact mechanisms of radiation cataractogenesis are still not fully understood. In particular, the question of the threshold dose for cataract development is not resolved. Cataracts have been classified as a deterministic effect of radiation exposure with a threshold of approximately 2 Gy. Here we review the combined results of recent mechanistic and human studies regarding induction of cataracts by ionizing radiation. These studies indicate that the threshold for cataract development is certainly less than was previously estimated, of the order of 0.5 Gy, or that radiation cataractogenesis may in fact be more accurately described by a linear, no-threshold model.

The German Mouse Clinic: a platform for systemic phenotype analysis of mouse models.

The German Mouse Clinic (GMC) is a large scale phenotyping center where mouse mutant lines are analyzed in a standardized and comprehensive way. The result is an almost complete picture of the phenotype of a mouse mutant line--a systemic view. At the GMC, expert scientists from various fields of mouse research work in close cooperation with clinicians side by side at one location. The phenotype screens comprise the following areas: allergy, behavior, clinical chemistry, cardiovascular analyses, dysmorphology, bone and cartilage, energy metabolism, eye and vision, host-pathogen interactions, immunology, lung function, molecular phenotyping, neurology, nociception, steroid metabolism, and pathology. The German Mouse Clinic is an open access platform that offers a collaboration-based phenotyping to the scientific community (www.mouseclinic.de). More than 80 mutant lines have been analyzed in a primary screen for 320 parameters, and for 95% of the mutant lines we have found new or additional phenotypes that were not associated with the mouse line before. Our data contributed to the association of mutant mouse lines to the corresponding human disease. In addition, the systemic phenotype analysis accounts for pleiotropic gene functions and refines previous phenotypic characterizations. This is an important basis for the analysis of underlying disease mechanisms. We are currently setting up a platform that will include environmental challenge tests to decipher genome-environmental interactions in the areas nutrition, exercise, air, stress and infection with different standardized experiments. This will help us to identify genetic predispositions as susceptibility factors for environmental influences.

[Perioperative discontinuation of antiplatelet therapy of patients with coronary stents. Reevaluating the risks]. / Perioperatives Pausieren der antithrombozytären Therapie bei Patienten mit koronaren Stents : Ein neu zu bewertendes Risiko?

According to the present guidelines, patients with coronary stents are to be treated with dual antiplatelet therapy. In case surgery is needed, the risk of a fatal stent thrombosis by withdrawing antithrombotics needs to be balanced in each individual case against the risk of haemorrhagic complications on continued antiplatelet medication. We present a case of fatal stent thrombosis and discuss the current evidence regarding perioperative continuation and interruption of antiplatelet therapy for this patient population. In summary the haemorrhagic risk with acetylsalicylic acid for secondary prevention seems very low, and it should be discontinued only in selected cases. Continued dual anticoagulation concepts are also discussed.

Sequencing of the factor 8(F8) coding regions in 10 Turkish hemophilia A patients reveals three novel pathological mutations, and one rediagnosis of von Willebrand's disease type 2N.

The most common cause for severe cases of hemophilia A is the homologous recombination involving intron 22 and related sequences outside the F8 gene. F8 coding regions of the gene including the exon/intron junctions were sequenced in 10 Turkish hemophilia A patients all of whom have been typed negative for intron 22 inversion and who did not have a detectable change by DGGE analysis. Pathological changes including two novel deletions (c. 205del CT and c. 3699del ACAT), one novel missense mutation (9546A) and two recurrent missense mutations were observed in five patients. The c. 2110C > T is another novel pathological change affecting exonic splicing enhancer site in two patients. One of the remaining three patients had a recurrent vWD type 2N mutation in the F8 binding site of the vWF (C788R). The S1269S polymorphism (c. 3864A > C) detected phenotype. Conclusively, sequencing of the promoter and the coding regions of 10 hemophilia A patients contributes four novel pathological mutations to the F8 mutations list and reveals a rediagnosis of hemophilia A but is still not sufficient to confirm hemophilia A phenotype in two patients.

Analysis of mRNA in hemophilia A patients with undetectable mutations reveals normal splicing in the factor VIII gene.

BACKGROUND: haemophilia A (HA) is characterized by partial or total deficiency of factor VIII (FVIII) protein activity. It is caused by a broad spectrum of mutations in the FVIII gene. Despite tremendous improvements in mutation screening methods, in about 2% of HA patients no DNA change could be found, even after sequencing the whole coding part of the FVIII gene including the flanking splice sites, as well as the promotor and the 3' UTR regions. OBJECTIVES, PATIENTS AND METHODS: In the present study we performed a detailed RNA analysis of three groups of patients. The first included control patients with known splicing defects, the second included two patients with already identified nucleotide changes close to splicing sites, that could potentially alter the normal splicing process, and a third group of 11 unrelated patients whose genomic DNA have already been screened for mutations by DHPLC and direct sequencing with no mutation being identified. RESULTS: Both candidate splice site mutations were shown to result in either skipping or alternative splicing of at least one exon, therefore these DNA changes must be considered as causal for the patients' HA phenotype. In contrast, no abnormalities on the RNA level were observed in any of 11 unrelated patients without mutations in the FVIII gene. CONCLUSIONS: These findings exclude mutations that could be located deep in the introns and affecting either normal splicing or lead to mechanisms causing some unknown rearrangements of the FVIII gene. In fact, our results point to the presence of still unknown factor(s) causing HA, which might be either allelic or in the close proximity of the FVIII gene or non-allelic associated with other genetic loci that are involved in the processing of the FVIII protein.

[2% Haemophilia A patients without mutation in the FVIII gene]. / 2% Hämophilie-A-Patienten ohne Mutation im FVIII-Gen.

In Germany, approximately 6,000 patients are suffering from haemophilia A. Screening methods cover 97% of the mutations. For the other patients the coding sequences of the FVIII gene have to be sequenced in total. Out of 1,350 patients, no mutation was observed in 80 patients. In 5 patients, we observed an inversion in intron 1. Known mutations were detected in 16 patients, and in 19 cases novel mutations were characterized (14 in coding regions and 5 in flanking introns). The mutations are mainly base pair substitutions, small deletions or insertions (max. 4 bp) and predicted to cause amino acid exchanges or frameshifts leading to premature stop codons. Moreover, 5 polymorphisms were identified in exons 14 and 26 as well as in introns 7 and 19. Further studies are necessary to identify their causative effects. Surprisingly, in 23 patients out of this subgroup of 80, no mutation was identified in the FVIII gene. Therefore, mutations in non-coding areas or even in other genes have to be considered responsible for the haemophilia A like phenotype. One of them codes for the von Willebrand factor (vWF). We confirmed in two of our cases mutations in the vWF gene.

[Significance of mutation analysis in patients with haemophilia A]. / Bedeutung der Mutationsdiagnostik bei Patienten mit Hämophilie A.

Haemophilia A represents the most frequent hereditary bleeding disorder in humans. The disease is caused by mutations within the factor VIII gene leading to decreased or absent factor VIII activities with a bleeding tendency depending on the degree of factor VIII deficiency. Nowadays, the causative mutations can be routinely detected and have substantially improved diagnostic and understanding of the pathophysiology of haemophilia A. Identification of the gene defects in haemophilic families have enabled fast and save carrier diagnosis. The correlation of the genetic defects with the clinical course revealed that the type of mutation represents the most important genetic predisposing factor for inhibitor formation, the most severe complication of treatment with factor VIII concentrates. Mitigated clinical courses of haemophilia A were shown to be due to special types of mutations or the presence of concomitant thrombophilic mutations. Molecular models of the factor VIII protein allowed to investigate the effects of specific mutations thus giving new insights in the structure/function relationship of the factor VIII molecule. These findings might promote the development of novel recombinant factor VIII concentrates with higher efficacy, longer half life and reduced immunogenicity.

[Characterization of factor VIII antibody epitopes from haemophilia A patients using cellulose bound FVIII peptide libraries]. / Charakterisierung von Faktor-VIII-Antikörperepitopen mit Faktor-VIII-Peptid-Bibliotheken.

Approximately 30% of patients suffering from severe haemophilia A develop antibodies against factor VIII (FVIII) neutralizing the effect of the pro-coagulant activity of intravenously injected FVIII as a complication of replacement therapy. Generally, various epitopes on the FVIII molecule are bound by these antibodies. The detailed structure of such epitopes is unknown. In this study epitopes on the FVIII molecule are identified using solid phase bound peptide arrays carrying the whole amino acid sequence of FVIII as small oligopeptides. The binding of FVIII antibodies by specific peptide sequences on the array indicates potential epitopes. FVIII antibodies of inhibitor patients and healthy blood donors are currently investigated by this method. Identified epitopes may lead to new concepts in therapy aiming at avoidance of inhibitor formation or improvement of inhibitor eradication. As participant of the 'haemophilia A' consortium dealing with genotype/phenotype correlation in haemophilia A we investigate, if the site or type of the mutation correlates with the epitopes, and if there is any relation between epitopes and clinical course. Furthermore, the influence of epitopes on therapeutical effects and the outcome of immune tolerance induction is under scrutiny.

[The national GTH haemophilia registry as database within the scope of the German human genome project]. / Das bundesweite GTH-Hämophilie-Register als Datenbasis im Rahmen des Deutschen Human-Genom-Projekts.

The Committee of Haemophilia of the GTH has established a central registry for all German centers treating patients with haemophilia. The intention was to establish a suitable system for collecting and analyzing epidemiological data relevant to bleeding disorders. The registry provides the database within the scope of the German Human-Genome-Project. The set goal is the complete molecular characterization of the genetic mutations on chromosome X of haemophilia A patients in Germany and subsequent correlation with the phenotype. An electronic network is applied for communication. A Java-application was developed for online electronic data acquirement by the participating centers. Offline data entry and sending encrypted data carriers is possible, too. A high level of security is assured by personalized access. Data are anonymized and scrambled by secure encoding. The concept was confirmed by the official data security offices. A considerable improvement for the epidemiological sciences and a better basis on therapy for patients with bleeding disorders is expected. Furthermore the registry is available for other scientific projects.

[Gathering and evaluation of phenotype data of haemophilia A patients for correlation with genotype data]. / Erfassung und Bewertung phänotypischer Parameter von Hämophilie-A-Patienten und Korrelation mit dem Genotyp.

Haemophilia A is caused by a genetic defect of the factor VIII gene resulting in complete or considerable functional loss of factor VIII molecule within blood. The high bleeding risk of patients can be prevented by intravenous injections of factor VIII protein. However, 25% of patients affected with severe haemophilia, develop factor VIII antibodies against the concentrate substituted. Within this study we try to comprise the phenotypic parameters (e. g. detailed documentation of disease course, basic laboratory values) and the therapy-associated data (e. g. applicated type and amount of factor VIII, number of substitutions, factor VIII recovery, inhibitor development and inhibitor elimination). We hope to identify differences of variable therapeutic treatments on course of disease as already identified for the factor VIII gene defects. At least we expect that certain mutations and mutation types, respectively, can be referred to typical phenotypes and similar course of treatment protocols.

Characterization of a new, dominant V124E mutation in the mouse alphaA-crystallin-encoding gene.

PURPOSE: During an ethylnitrosourea (ENU) mutagenesis screening, mice were tested for the occurrence of dominant cataracts. The purpose of the study was morphologic description, mapping of the mutant gene, and characterization of the underlying molecular lesion in a particular mutant, Aey7. METHODS: Isolated lenses were photographed and histologic sections of the eye were analyzed according to standard procedures. Linkage analysis was performed with a set of microsatellite markers covering all autosomal chromosomes. cDNA was amplified after reverse transcription of lens mRNA. For PCR, cDNA or genomic DNA was used as a template. RESULTS: Nuclear opacity and posterior suture anomaly were visible at eye opening and progressed to a nuclear and zonular cataract at 2 months of age. The opacity as well as the microphthalmia was more pronounced in the homozygotes than in the heterozygotes. The mutation was mapped to chromosome 17 between the markers D17Mit133 and D17Mit180. This position made the alphaA-crystallin-encoding gene (Cryaa) an excellent candidate gene. Sequence analysis revealed a mutation of a T to an A at position 371 in the Cryaa cDNA. The mutation was confirmed by an additional MnlI restriction site in the genomic DNA of homozygous mutants leading to replacement of Val with Glu at codon 124 affecting the C-terminal region of the alphaA-crystallin. CONCLUSIONS: The Aey7 mutant represents the first dominant mouse cataract mutation affecting the Cryaa gene. The mutation leads to progressive opacification of the lens. Compared with the beta- and gamma-crystallin-encoding genes, mutations in the alpha-crystallin-encoding genes are rare.

Regulation of the human SIX3 gene promoter.

A 2-kb promoter fragment of SIX3, a human transcription factor essential for vertebrate eye development, has been characterized in a gene reporter assay system. The peak of activity implies the 2-kb sequence of SIX3, whereas 5'-deletion constructs of the promoter decreases successively to 60% of the activity starting from the entire promoter. In contrast, cutting off 300 bp of the 3' promoter extinguishes its activity completely. Coexpression experiments of different other transcription factors illuminate the regulation of SIX3 during eye development: Pax6 activates the -703/-349 SIX3 promoter threefold, and PROX1 even eightfold. In contrast, Msx2 represses the entire SIX3 promoter. Furthermore, Six3 is regulated by its own negative feedback loop. In conclusion, SIX3 expression underlies a complex regulation, which is an important part to understand the network of transcription factors during eye development.

Aey2, a new mutation in the betaB2-crystallin-encoding gene of the mouse.

PURPOSE: During an ethylnitrosourea (ENU) mutagenesis screen, mice were tested for the occurrence of dominant cataracts. One particular mutant was found that caused progressive opacity and was referred to as Aey2. The purpose of the study was to provide a morphologic description, to map the mutant gene, and to characterize the underlying molecular lesion. METHODS: Isolated lenses were photographed, and histologic sections of the eye were analyzed according to standard procedures. Linkage analysis was performed using a set of microsatellite markers covering all autosomal chromosomes. cDNA from candidate genes was amplified after reverse transcription of lens mRNA. RESULTS: The cortical opacification visible at eye opening progressed to an anterior suture cataract and reached its final phenotype as total opacity at 8 weeks of age. There was no obvious difference between heterozygous and homozygous mutants. The mutation was mapped to chromosome 5 proximal to the marker D5Mit138 (8.7 +/- 4.2 centimorgan [cM]) and distal to D5Mit15 (12.8 +/- 5.4 cM). No recombinations were observed to the markers D5Mit10 and D5Mit25. This position makes the genes within the betaA4/betaB-crystallin gene cluster excellent candidate genes. Sequence analysis revealed a mutation of T-->A at position 553 in the Crybb2 gene, leading to an exchange of Val for GLU: It affects the same region of the Crybb2 gene as in the Philly mouse. Correspondingly, the loss of the fourth Greek key motif is to be expected. CONCLUSIONS: The Aey2 mutant represents the second allele of Crybb2 in mice. Because an increasing number of beta- and gamma-crystallin mutations have been reported, a detailed phenotype-genotype correlation will allow a clearer functional understanding of beta- and gamma-crystallins.

Ethylnitrosourea-induced mutation in mice leads to the expression of a novel protein in the eye and to dominant cataracts.

A novel ENU-induced mutation in the mouse leading to a nuclear and zonular opacity of the eye lens (Aey1) was mapped to chromosome 1 between the markers D1Mit303 and D1Mit332. On the basis of the chromosomal position, the gamma-crystallin encoding gene cluster (Cryg) and the betaA2-crystallin encoding gene Cryba2 were tested as candidate genes. An A --> T mutation destroys the start codon of the Cryge gene in the mutants; this mutation was confirmed by the absence of a restriction site for NcoI in the corresponding genomic fragment of homozygous mutants. The next in-frame start codon is 129 bp downstream; this predicted truncated gammaE-crystallin consists of 131 amino acids, resulting in a molecular mass of 14 kD. However, another open reading frame was observed just 19 bp downstream of the regular Cryge start codon, resulting in a protein of 119 amino acids and a calculated molecular weight of 13 kD. Western blot analysis using polyclonal antibodies against gamma-crystallins or the novel Aey1-specific protein demonstrated the specific expression of the Aey1 protein in the cataractous lenses only; the truncated form of the gammaE-crystallin could not be detected. Therefore, it is concluded that the novel protein destroys the sensitive cellular structure of the eye lens.