Photon-counting CT-angiography in pre-TAVR aortic annulus assessment: effects of retrospective vs. prospective ECG-synchronization on prosthesis valve selection.

To compare the diagnostic value of ultrahigh-resolution CT-angiography (UHR-CTA) compared with high-pitch spiral CTA (HPS-CTA) using a first-generation, dual-source photon-counting CT (PCD-CT) scanner for preprocedural planning of transcatheter aortic valve replacement (TAVR). Clinically referred patients with severe aortic valve stenosis underwent both, retrospective ECG-gated cardiac UHR-CTA (collimation: 120 × 0.2 mm) and prospective ECG-triggered aortoiliac HPS-CTA (collimation: 144 × 0.4 mm, full spectral capabilities) for TAVR planning from August 2022 to March 2023. Radiation dose was extracted from the CT reports, and the effective dose was calculated. Two radiologists analyzed UHR-CTA and HPS-CTA datasets, assessing the image quality of the aortic annulus, with regard to the lumen visibility and margin delineation using a 4-point visual-grading scale (ranges: 4 = "excellent" to 1 = "poor"). Aortic annulus area (AAA) measurements were taken for valve prosthesis sizing, with retrospective UHR-CTA serving as reference standard. A total of 64 patients were included (mean age, 81 years ± 7 SD; 28 women) in this retrospective study. HPS-CTA showed a lower radiation dose, 4.1 mSv vs. 12.6 mSv (p < 0.001). UHR-CTA demonstrated higher image quality to HPS-CTA (median score, 4 [IQR, 3-4] vs. 3 [IQR, 2-3]; p < 0.001). Quantitative assessments of AAA from both CTA datasets were strongly positively correlated (mean 477.4 ± 91.1 mm2 on UHR-CTA and mean 476.5 ± 90.4 mm2 on HPS-CTA, Pearson r2 = 0.857, p < 0.001) with a mean error of 22.3 ± 24.6 mm2 and resulted in identical valve prosthesis sizing in the majority of patients (91%). Patients with lower image quality on HPS-CTA (score value 1 or 2, n = 28) were more likely to receive different sizing recommendations (82%). Both UHR-CTA and HPS-CTA acquisitions using photon-counting CT technology provided reliable aortic annular assessments for TAVR planning. While UHR-CTA offers superior image quality, HPS-CTA is associated with lower radiation exposure. However, severely impaired image quality on HPS-CTA may impact on prosthesis sizing, suggesting that immediate post-scan image evaluations may require complementary UHR-CTA scanning.

Pre-Procedural Assessment of the Femoral Access Route for Transcatheter Aortic Valve Implantation: Comparison of a Non-Contrast Time-of-Flight Magnetic Resonance Angiography Protocol with Contrast-Enhanced Dual-Source Computed Tomography Angiography.

Background: We aimed to evaluate the feasibility of a non-contrast time-of-flight magnetic resonance angiography (TOF-MRA) protocol for the pre-procedural access route assessment of transcatheter aortic valve implantation (TAVI) in comparison with contrast-enhanced cardiac dual-source computed tomography angiography (CTA). Methods and Results: In total, 51 consecutive patients (mean age: 82.69 ± 5.69 years) who had undergone a pre-TAVI cardiac CTA received TOF-MRA for a pre-procedural access route assessment. The MRA image quality was rated as very good (median of 5 [IQR 4-5] on a five-point Likert scale), with only four examinations rated as non-diagnostic. The TOF-MRA systematically underestimated the minimal effective vessel diameter in comparison with CTA (for the effective vessel diameter in mm, the right common iliac artery (CIA)/external iliac artery (EIA)/common femoral artery (CFA) MRA vs. CTA was 8.04 ± 1.46 vs. 8.37 ± 1.54 (p < 0.0001) and the left CIA/EIA/CFA MRA vs. CTA was 8.07 ± 1.32 vs. 8.28 ± 1.34 (p < 0.0001)). The absolute difference between the MRA and CTA was small (for the Bland-Altman analyses in mm, the right CIA/EIA/CFA was -0.36 ± 0.77 and the left CIA/EIA/CFA was -0.25 ± 0.61). The overall correlation between the MRA and CTA measurements was very good (with a Pearson correlation coefficient of 0.87 (p < 0.0001) for the right CIA/EIA/CFA and a Pearson correlation coefficient of 0.9 (p < 0.0001) for the left CIA/EIA/CFA). The feasibility agreement between the MRA and CTA for transfemoral access was good (the right CIA/EIA/CFA agreement was 97.9% and the left CIA/EIA/CFA agreement was 95.7%, Kohen's kappa: 0.477 (p = 0.001)). Conclusions: The TOF-MRA protocol was feasible for the assessment of the access route in an all-comer pre-TAVI population. This protocol might be a reliable technique for patients at an increased risk of contrast-induced nephropathy.

Proteome effects of genome-wide single gene perturbations.

Protein abundance is controlled at the transcriptional, translational and post-translational levels, and its regulatory principles are starting to emerge. Investigating these principles requires large-scale proteomics data and cannot just be done with transcriptional outcomes that are commonly used as a proxy for protein abundance. Here, we determine proteome changes resulting from the individual knockout of 3308 nonessential genes in the yeast Schizosaccharomyces pombe. We use similarity clustering of global proteome changes to infer gene functionality that can be extended to other species, such as humans or baker's yeast. Furthermore, we analyze a selected set of deletion mutants by paired transcriptome and proteome measurements and show that upregulation of proteins under stable transcript expression utilizes optimal codons.

Matrix Metalloproteinase-2 Isoforms Differ within the Aortic Wall of Ascending Aortic Aneurysms Associated with Bicuspid Aortic Valve.

The pathogenesis of ascending thoracic aortic aneurysm (aTAA) is thought to differ between patients with bicuspid aortic valve (BAV) and tricuspid aortic valve (TAV), and one of the causes is different hemodynamics. Influenced by hemodynamics, the tissue levels of proteins associated with aTAA might differ between aTAAs with BAV and TAV and between different localities within the aortic wall. We therefore analyzed aTAA tissue levels of MMP-2 (matrix metalloproteinase-2) isoforms (Pro-MMP-2, active MMP-2, and total MMP-2) and tissue levels of MMP-14, TIMP-2 (tissue inhibitor of metalloproteinase-2), MMP-9, and TIMP-1 in 19 patients with BAV and 23 patients with TAV via gelatin zymography and enzyme-linked immunosorbent assay (ELISA), respectively. TAV and BAV groups' protein levels did not differ significantly. Whereas the TAV group exhibited no significant differences in protein levels between the aneurysm's anterior and posterior parts, the BAV group revealed significantly higher levels of Pro-MMP-2, total MMP-2, and TIMP-2 in the aneurysm's posterior parts (mean Pro-MMP-2 200.52 arbitrary units (AU) versus 161.12 AU, p=0.007; mean total MMP-2 235.22 AU versus 193.68 AU, p=0.002; mean TIMP-2 26.90 ng/ml versus 25.36 ng/ml, p=0.009), whereas the other proteins did not differ significantly within the aortic wall. Thus, MMPs are distributed more heterogeneously within the aortic wall of aTAAs associated with BAV than in those associated with TAV, which is a new aspect for understanding the underlying pathogenesis. This heterogeneous protein level distribution might be attributable to differences in the underlying pathogenesis, especially hemodynamics. This result is important for further studies as it will be essential to specify the location of samples to ensure data comparability regarding the main goals of understanding the pathogenesis of aTAA, optimizing treatments, and establishing a screening method for its potentially deadly complications.

A potential key mechanism in ascending aortic aneurysm development: Detection of a linear relationship between MMP-14/TIMP-2 ratio and active MMP-2.

OBJECTIVES: Elevated matrix metalloproteinase-2 (MMP-2) tissue levels have been associated with ascending thoracic aortic aneurysm (aTAA). As MMP-2 activation is controlled by interactions among matrix metalloproteinase-14 (MMP-14), a tissue inhibitor of metalloproteinases-2 (TIMP-2) and Pro-MMP-2 in cell culture, this activation process might also play a role in aTAA. METHODS: Via gelatin zymography we analyzed tissue levels of MMP-2 isoforms (Pro-MMP-2, active MMP-2, total MMP-2) and via enzyme-linked immunosorbent assay (ELISA,) MMP-14,TIMP-2 and total MMP-2 tissue levels in N = 42 patients with aTAA. As controls, MMP-14 and TIMP-2 aortic tissue levels in N = 9 patients undergoing coronary artery bypass surgery were measured via ELISA, and levels of MMP-2 isoforms in N = 11 patients via gelatin zymography. RESULTS: Active MMP-2 was significantly higher in aTAA than in controls. Patients with aTAA exhibited significantly lower Pro-MMP-2 and TIMP-2 levels. Total MMP-2 and MMP-14 did not differ significantly between groups. Regression analysis revealed a linear relationship between TIMP-2 and the MMP-14/TIMP-2 ratio, as well as active MMP-2 in aTAA. Aneurysmatic tissue can be accurately distinguished from control aortic tissue (AUC = 1) by analyzing the active MMP-2/Pro-MMP-2 ratio with a cutoff value of 0.11, whereas MMP-14 and TIMP-2 roles are negligible in ROC analysis. CONCLUSION: A larger amount of MMP-2 is activated in aTAA than in control aortic tissue-a factor that seems to be a central process in aneurysm development. When active MMP-2 exceeds 10% compared to Pro-MMP-2, we conclude that it originates from aneurysmatic tissue, which we regard as a starting point for further studies of aTAA biomarkers. The tissue's MMP-14/TIMP-2 ratio may regulate the degree of Pro-MMP-2 activation as a determining factor, while the enzymatic activities of MMP-14 and TIMP-2 do not seem to play a key role in aneurysm development.

Chicken immunoregulatory Ig-like receptor families: an overview and expression details on ggTREM-A1.

Paired immunoregulatory receptors facilitate the coordination of the immune response at the cellular level. In recent years, our group characterized chicken homologues to mammalian immunoregulatory Ig-like receptor families. The first part of this review focuses on the current progress on chicken immunoregulatory Ig-like receptor families. One of these receptors is gallus gallus TREM-A1, which was described as the only member of the chicken TREM family with activating potential. The second part of this review presents a study initiated to further characterize ggTREM-A1 expression. For this purpose we established real-time RT-PCR and generated a specific mab to analyze the expression profile of ggTREM-A1 on mRNA and protein level, respectively. GgTREM-A1 mRNA was predominantly expressed in macrophages, but was also detected in brain, bone marrow, bursa, thymus, spleen and PBMC. Analyzing ggTREM-A1 surface expression by mab staining validated the expression on macrophages. Additionally, we showed high expression on blood monocytes, heterophils and NK cells and on monocytes isolated from bone marrow. Moreover, we detected ggTREM-A1 protein also on thrombocytes, B and T cell subsets, but antigen expression seemed to be lower and more variable in these cells. Immunohistochemistry of chicken brain tissue, combining ggTREM-A1 mab and various markers specific for various brain cell subsets showed expression of ggTREM-A1 on microglial cells, but also on neurons, astrocytes and oligodendrocytes. In conclusion, ggTREM-A1 is expressed on a variety of cells, relevant for the immune system, possibly combining physiological function of different mammalian TREM.

Complexity of expressed CHIR genes.

The chicken leukocyte receptor complex (LRC) encodes an unprecedented number of chicken Ig-like receptor (CHIR) genes compared to the mammalian LRC. Although there are at least 100 CHIR genes in the LRC, only little information is available about the number and variability of expressed CHIR. Recently, we showed that CHIR with one Ig domain encode a variety of different affinity IgY receptors, which are highly variable in different chicken strains. The current report focused on expressed CHIR with two Ig domains. Oligonucleotides specific for conserved regions at the 5' end of Ig1 and 3' end of Ig2 were used on PBMC mRNA obtained from two individual chickens with different MHC haplotypes (M11, R11). Sequencing of 142 colonies of M11 and 117 of R11 yielded 98 and 70 different CHIR2D amino acid sequences, respectively. Comparing a total of 219 CHIR sequences, including also a genomic dataset from an LSL chicken, revealed a single amino acid sequence identical between all three chicken strains, and four sequence pairs either shared between M11 and R11 or between M11 and LSL. Calculating Wu-Kabat variability revealed three amino acid positions, which were highly variable and the analysis of synonymous/non-synonymous ratio indicated positive selection. This analysis of expressed CHIR genes in different chickens demonstrates an unusual polymorphism of expressed receptors, where only few are conserved between chickens.

Chicken IgY binds its receptor at the CH3/CH4 interface similarly as the human IgA: Fc alpha RI interaction.

Chicken IgY, the ancestral form of mammalian IgE and IgG, is recognized by the high-affinity FcY receptor CHIR-AB1, a member of the leukocyte receptor family. In this study, we have characterized the receptor ligand interaction site by consecutive truncations of the Fcv IgY domains and mutational analyses of selected residues. Using several fusion proteins that linked the human Cgamma2 and Cgamma3 domains with the Fcv IgY domains, a binding assay revealed that both the Fcv3 and Fcv4 domains were essential for the IgY CHIR-AB1 interaction. Sequence comparisons of chicken IgY with human IgA demonstrated that 11 of the 19 contact residues important for the IgA FcalphaRI interaction have been conserved in chicken IgY, although the overall amino acid identity is only 34%. Among the 19 amino acids at respective positions in IgY, the mutation of two residues in the Fcv3 and two in the Fcv4 domain completely abolished the IgY to CHIR-AB1 binding revealed by two independent assays. Three further mutations substantially altered the interaction. Molecular modeling on the Cv3 to Cv4 crystal structure revealed that all critical residues, although on two domains, are in close proximity. The importance of N-linked carbohydrates was demonstrated by the failure of the CHIR-AB1 interaction after mutation of the glycosylation site. The identification of the IgY Cv3/Cv4 interdomain region as critical for binding to CHIR-AB1 significantly enhances our understanding of the IgY receptor interaction and allows further conclusions regarding the FcR phylogeny.

The chicken leukocyte receptor complex encodes a family of different affinity FcY receptors.

Chicken Ig-like receptors (CHIR) form a large family in the leukocyte receptor complex on microchromosome 31 with inhibitory, activating, and bifunctional receptors. Recently, we characterized CHIR-AB1 as a high-affinity, primordial FcY receptor. Given that the CHIR family represents a multigene family, it is plausible that more than a single receptor binds to IgY. Therefore, after comparing CHIR-AB1-like sequences in databases, we cloned CHIR-AB1 homologues from two individual chickens representing the lines M11 and R11 with primers binding to highly conserved regions. In both lines this approach yielded 18 different CHIR-AB amino acid versions, with one sequence out of each line that was identical with the previously characterized B19 CHIR-AB1 Ig domain and two additional R11-M11 identical sequence pairs. All M11-derived CHIR-AB homologues were then expressed as soluble human Ig fusion proteins. Following standardization of the fusion protein concentration with an ELISA, the IgY, IgM, and IgA binding activities were determined by ELISA. Six fusion proteins recognized IgY, whereas none bound to IgM and IgA. The affinities of selected fusion proteins were determined using surface plasmon resonance yielding an equilibrium binding constant between 25 nM for high binders and 260 nM for low binders. Sequence comparisons and subsequent mutational analysis of selected residues identified five amino acids that are potentially involved in IgY binding. These results imply that multiple FcY receptors of variable affinity are encoded by the CHIR locus and that different chicken lines may express both unique as well as highly conserved FcY receptors.

A novel activating chicken IgY FcR is related to leukocyte receptor complex (LRC) genes but is located on a chromosomal region distinct from the LRC and FcR gene clusters.

FcRs have multifaceted roles in the immune system. Chicken FcRs were demonstrated on macrophages decades ago; however, only recently the chicken Ig-like receptor AB1, encoded in the leukocyte receptor complex, was molecularly identified as a high-affinity FcR. The present study was initiated to identify additional receptors with the capability to bind chicken immunoglobulins. Based on database searches, we cloned a novel chicken FcR, designated gallus gallus FcR (ggFcR), which was shown to bind selectively chicken IgY. The receptor consists of four extracellular C2-set Ig domains, followed by a transmembrane region containing arginine as a positively charged amino acid and a short cytoplasmic tail. ggFcR associates with the common gamma-chain, indicative for an activating receptor, and real-time RT-PCR revealed high expression on PBMC, thrombocytes, and macrophages. The genomic organization is similar to most Ig-like receptor genes, where each Ig domain is encoded by a separate exon. Additionally, the ggFcR signal peptide is encoded by two exons, the second of which is 36 bp, a hallmark for genes encoded in the leukocyte receptor complex. Phylogenetic analysis also showed a relationship to genes encoded in the leukocyte receptor complex. Surprisingly, ggFcR is not encoded in the leukocyte receptor complex, but it is located as a single isolated gene at the extremity of chicken chromosome 20.

Characterization of the chicken CD200 receptor family.

The modulation of myeloid cells via inhibitory and activating immunoglobulin superfamily members has been a subject of intense study in mammals. One such example is the inhibitory receptor for CD200, which is shown to regulate the activation threshold of myeloid cells by interaction with the broadly distributed CD200 molecule. By looking at sequence homology and synteny conservation in the chicken genome, we identified two members of the CD200 receptor family in chicken on chromosome one. Cloning and further characterization of the protein sequence yielded a potentially inhibitory ggCD200R-B1 with a splice variant lacking a transmembrane region and a potentially soluble ggCD200R-S1. Both showed a typical V/C2-set Ig domain arrangement and we present evidence that these two genes have evolved by gene duplication. The inhibitory receptor displayed an uncharged transmembrane region and a long cytoplasmic tail encoding four tyrosine residues, one of them embedded in a motif similar to the mammalian NPxY motif. Further characterization of ggCD200R-B1 showed that it is expressed as a highly glycosylated protein and that its cytoplasmic tyrosine residues can be phosphorylated. Real-time RT-PCR analysis of various tissues and primary cells showed that ggCD200R-B1 is predominantly expressed in macrophages, whereas ggCD200R-S1 is highly expressed in peripheral blood mononuclear cells, but not macrophages. In summary, we showed that there is a homologue of mammalian CD200R conserved in chicken suggesting a similar function in avian species. Furthermore, the presence of potentially soluble CD200R molecules implies an important role for these in the regulation of myeloid cells in chicken.

Chicken C-type lectin-like receptor B-NK, expressed on NK and T cell subsets, binds to a ligand on activated splenocytes.

B-NK is a C-type lectin with an immunorecptor tyrosine-based inhibition motif, that is located in the vicinity of the chicken MHC and that has been described as a potential chicken NK cell receptor. We have generated an epitope tagged B-NK version for immunization and for biochemical studies. B-NK was expressed as a heavily glycosylated, homodimeric, type II transmembrane protein. With the help of a newly developed B-NK specific mab, the tissue distribution of B-NK has been analyzed. In the blood, B-NK was mainly present on a small population of gammadelta T cells, whereas in spleen it was expressed by alphabeta T cells. Moreover, B-NK was also found on CD3(-)CD8(+) sorted splenocytes that were in vitro expanded by IL-2 and on embryonic splenocytes, both of which resemble chicken NK cells. In order to characterize cells expressing B-NK ligands, a BWZ.36 based reporter system was employed, that induced beta-galactosidase activity upon ligand binding. While potential B-NK ligands such as MHC class I or the C-type lectin B-lec did not induce any signal, a trypsin sensitive B-NK ligand was expressed on phorbol myristate or concanavalin A activated splenocytes, but not unstimulated splenocytes. In summary, B-NK is expressed by NK cells and T cell subsets, and it binds to a ligand on activated cells.

The chicken leukocyte receptor complex encodes a primordial, activating, high-affinity IgY Fc receptor.

Fc receptors are key players of the immune system that link the fine specificity of immunoglobulins and innate effector responses. Here, we describe a nonmammalian Fcgamma receptor, CHIR-AB1, a member of the leukocyte receptor complex, that binds IgY with high affinity with its single Ig domain. It is expressed on immature and mature B lymphocytes, monocytes, macrophages, and natural killer cells and harbors motifs of activating and inhibitory Fc receptors. In the absence of FcepsilonRIgamma, CHIR-AB1 can be expressed on B cells but cross-linking does not induce intracellular calcium release. In contrast, cells expressing CHIR-AB1 and FcepsilonRIgamma are triggered to release intracellular calcium upon stimulation with heat-aggregated IgY. CHIR-AB1 thus represents a primordial Fc receptor that combines features of different mammalian counterparts.

The chicken immunoregulatory receptor families SIRP, TREM, and CMRF35/CD300L.

Mammalian immunoregulatory families of genes encoding activating and inhibitory Ig-like receptor pairs have been located on distinct chromosomes. In chicken, a single Ig-like receptor family with many members had been described so far. By looking at sequence similarity and synteny conservations in the chicken genome, the signal-regulatory protein (SIRP), triggering receptor expressed on myeloid cells (TREM), and CMRF35/CD300L Ig-like gene families were identified on chromosomes 20, 26, and 3, respectively. Further analysis of the three corresponding genomic regions and partial bacterial artificial chromosome sequencing were used to identify more members and to realign several contigs. All putative genomic sequences were monitored by investigating existing expressed sequence tag and cloning cDNA. This approach yielded a single pair of activating and inhibitory SIRP, two inhibitory, and one activating TREM as well as one inhibitory CMRF35/CD300L with a potentially soluble variant and an additional member lacking categorizing motifs. The CMRF35/CD300L and TREM receptors were composed of one or two V-set Ig domains, whereas in SIRP, either a single Ig V domain was present or a combination of a V and C1 domains. Like in many Ig superfamily members, separate exons encode individual Ig domains. However, in two CMRF35/CD300L genes, the signal peptide and the distal Ig domain were encoded by a single exon. In conclusion, the mammalian diversity of immunoregulatory molecules is present the chicken suggesting an important role for TREM, SIRP, and CMRF35/CD300L in a functionally conserved network.

The chicken leukocyte receptor complex: a highly diverse multigene family encoding at least six structurally distinct receptor types.

The chicken Ig-like receptors (CHIR) have been described as two Ig domain molecules with long cytoplasmic tails containing inhibitory motifs. In this study, we demonstrate that CHIR form a large family, with multiple members showing great sequence variability among members as well as a great diversity in domain organization and properties of the transmembrane and cytoplasmic segments. We characterize various novel receptor types with motifs indicative of inhibitory, activating, or both functions. In addition to the inhibitory receptors with two ITIM, receptors with a single immunoreceptor tyrosine-based switch motif or receptors lacking a cytoplasmic domain were isolated. Activating receptors with a short cytoplasmic domain and a transmembrane arginine assembled with the newly identified chicken FcepsilonRIgamma chain. Three bifunctional receptor types were characterized composed of one or two C2-type Ig-like domains, a transmembrane region with a positively charged residue and combinations of cytoplasmic motifs such as ITIM, immunoreceptor tyrosine-based switch motif, and YXXM. RT-PCR revealed distinct expression patterns of individual CHIR. All receptor types shared a conserved genomic architecture, and in single Ig domain receptors a pseudoexon replaced the second Ig exon. Southern blot analyses with probes specific for the Ig1 domain were indicative of a large multigene family. Of 103 sequences from the Ig1 domain of a single animal, 41 unique sequences were obtained that displayed extensive variability within restricted Ig regions. Fluorescence in situ hybridization localized the CHIR gene cluster to microchromosome 31 and identified this region as orthologous to the human leukocyte receptor complex.