Including surrounding tissue improves ultrasound-based 3D mechanical characterization of abdominal aortic aneurysms

ObjectivesIn this study the influence of surrounding tissues including the presence of the spine on wall stress analysis and mechanical characterization of abdominal aortic aneurysms using ultrasound imaging has been investigated.MethodsGeometries of 7 AAA patients and 11 healthy volunteers were acquired using 3-D ultrasound and converted to finite element based models. Model complexity of externally unsupported (aorta-only) models was complemented with inclusion of both soft tissue around the aorta and a spine support dorsal to the aorta. Computed 3-D motion of the aortic wall was verified by means of ultrasound speckle tracking. Resulting stress, strain, and estimated shear moduli were analyzed to quantify the effect of adding surrounding material supports.ResultsAn improved agreement was shown between the ultrasound measurements and the finite element tissue and spine models compared to the aorta-only models. Peak and 99-percentile Von Mises stress showed an overall decrease of 23–30%, while estimated shear modulus decreased with 12–20% after addition of the soft tissue. Shear strains in the aortic wall were higher in areas close to the spine compared to the anterior region.ConclusionsImproving model complexity with surrounding tissue and spine showed a homogenization of wall stresses, reduction in homogeneity of shear strain at the posterior side of the AAA, and a decrease in estimated aortic wall shear modulus. Future research will focus on the importance of a patient-specific spine geometry and location.     

Identification and characterization of the murine TRPM4 channel

The transient receptor potential (TRP) channels form a superfamily with six transmembrane structures, which is common in other types of voltage-dependent channels. The TRP-melastatin (TRPM) subfamily includes the putative tumor-suppressor melastatin, which was originally found as a down-regulated protein in melanoma tumor cell lines. Here, we report a novel TRP-related protein that is a murine orthologue of human TRPM4. The function of the novel murine TRPM4 was studied in HEK-293 cells using a fluorescent calcium indicator, fura-2. The removal and re-introduction of extracellular calcium triggered changes in the intracellular calcium only in cells expressing TRPM4a, which suggests that this novel channel plays a role in the calcium entry process. We also isolated a splice variant of TRPM4 that was proven to be non-functional. Both TRPM4 variants integrated into the plasma membrane. Furthermore, FRET analysis revealed that TRPM4a and TRPM4b localized close together, suggesting a multimerization of the two molecules.     

Three types of mouse peptidylarginine deiminase: characterization and tissue distribution.

Three types of mouse peptidylarginine deiminase were separated by DEAE-Sephacel ion-exchange column chromatography, and we propose designating them peptidylarginine deiminase type I, II, and III according to the order of elution. The type II enzyme was widely distributed in various tissues including the skeletal muscle, whereas the type I enzyme was localized in the epidermis and uterus, and the type III enzyme was detected in the epidermis and hair follicles. These enzymes were distinguished by their molecular weights and substrate specificity. The molecular weights were estimated to be approximately 54,000 (type I) and 100,000 (type II and III) by Sephacryl S-200 gel filtration column chromatography. On SDS-PAGE the type II and III enzymes gave Mr = 81,000 and Mr = 76,000, respectively. Among the substrates tested, the type I enzyme showed highest activity toward BZ-L-Arg-NH2, type II toward BZ-L-Arg-O-Et, and type III toward protamine. Western blot analysis showed that antibodies against the type II enzyme were immuno-crossreactive to the type III enzyme.     

Cloning and characterization of novel tumor-targeting immunocytokines based on murine IL7

We generated and characterized novel antibody-cytokine fusion proteins (“immunocytokines”) based on murine interleukin-7 (IL7), an immunomodulatory protein which has previously shown anti-cancer activity in preclinical models and whose human counterpart is currently being investigated in clinical trials. The sequential fusion of the clinical-stage antibody fragment scFv(F8), specific to a tumor-associated splice isoform of fibronectin, yielded an immunocytokine (termed “F8-mIL7”) of insufficient pharmaceutical quality and in vivo tumor targeting performance, with a striking dose dependence on tumor targeting selectivity. By contrast, a novel immunocytokine design (termed “F8-mIL7-F8”), in which two scFv moieties were fused at the N- and C-terminus of murine IL7, yielded a protein of excellent pharmaceutical quality and with improved tumor-targeting performance [tumor: blood ratio = 16:1, 24 h after injection]. Both F8-mIL7 and F8-mIL7-F8 could induce tumor growth retardation in immunocompetent mice, but were not able to eradicate F9 tumors. The combination of F8-mIL7-F8 with paclitaxel led to improved therapeutic results, which were significantly better compared to those obtained with saline treatment. The study indicates how the engineering of novel immunocytokine formats may help generate fusion proteins of acceptable pharmaceutical quality, for those immunomodulatory proteins which do not lend themselves to a direct fusion with antibody fragments.     

Isolation and characterization of a Pseudomonas strain that degrades 4-acetamidophenol and 4-aminophenol

Though many microorganisms that are capable of using phenol as sole sourceof carbon have been isolated and characterized, only a few organisms degradingsubstituted phenols have been described to date. In this study, one strain ofmicroorganism that is capable of using phenol (3000 ppm), 4-aminophenol(4000 ppm) and 4-acetamidophenol (4000 ppm) as sole source of carbon andenergy was isolated and characterized. This strain was obtained by enrichmentculture from a site contaminated with compounds like 4-acetamidophenol,4-aminophenol and phenol in Pakistan at Bhai Pheru. The contaminated siteis able to support large bacterial community as indicated by the viable cellcounts (2 × 10⁴–5 × 10⁸) per gram of soil. Detailed taxonomic studies identified the organisms as Pseudomonas species designated as strain STI. The isolate also showed growth on other organic compounds like aniline, benzene, benzyl alcohol, benzyl bromide, toluene, -cresol, trichloroethylene and o-xylene. Optimum growth temperature and pH were found to be 30 °C and 7, respectively, while growth at 4, 25 and 35 °C and at pH 8 and 9 was also observed. Non growing suspended cells of strain ST1 degraded 68, 96 and 76.8% of 4-aminophenol (1000 ppm), phenol (500 ppm) and 4-acetamidophenol (1000 ppm), respectively, in 72 hrs. The isolation and characterization of Pseudomonas speciesstrain ST1, may contribute to efforts on phenolic bioremediation, particularly in anenvironment with very high levels of 4-acetamidophenol and 4-aminophenol.     

Using averaged models from 4D ultrasound strain imaging allows to significantly differentiate local wall strains in calcified regions of abdominal aortic aneurysms

Abdominal aortic aneurysms are a degenerative disease of the aorta associated with high mortality. To date, in vivo information to characterize the individual elastic properties of the aneurysm wall in terms of rupture risk is lacking. We have used time-resolved 3D ultrasound strain imaging to calculate spatially resolved in-plane strain distributions characterized by mean and local maximum strains, as well as indices of local variations in strains. Likewise, we here present a method to generate averaged models from multiple segmentations. Strains were then calculated for single segmentations and averaged models. After registration with aneurysm geometries based on CT-A imaging, local strains were divided into two groups with and without calcifications and compared. Geometry comparison from both imaging modalities showed good agreement with a root mean squared error of 1.22 ± 0.15 mm and Hausdorff Distance of 5.45 ± 1.56 mm (mean ± sd, respectively). Using averaged models, circumferential strains in areas with calcifications were 23.2 ± 11.7% (mean ± sd) smaller and significantly distinguishable at the 5% level from areas without calcifications. For single segmentations, this was possible only in 50% of cases. The areas without calcifications showed greater heterogeneity, larger maximum strains, and smaller strain ratios when computed by use of the averaged models. Using these averaged models, reliable conclusions can be made about the local elastic properties of individual aneurysm (and long-term observations of their change), rather than just group comparisons. This is an important prerequisite for clinical application and provides qualitatively new information about the change of an abdominal aortic aneurysm in the course of disease progression compared to the diameter criterion.

     

Quantitative assessment of 4D hemodynamics in cerebral aneurysms using proper orthogonal decomposition

Background and purposeThe comparison of different time-varying three-dimensional hemodynamic data (4D) is a formidable task. The purpose of this study is to investigate the potential of the proper orthogonal decomposition (POD) for a quantitative assessment.MethodsThe complex spatial-temporal flow information was analyzed using proper orthogonal decomposition to reduce the complexity of the system. PC-MRI blood flow measurements and computational fluid dynamic simulations of two subject-specific IAs were used to compare the different flow modalities. The concept of Modal Assurance Criterion (MAC) provided a further detailed objective characterization of the most energetic individual modes.ResultsThe most energetic flow modes were qualitatively compared by visual inspection. The distribution of the kinetic energy on the modes was used to quantitatively compare pulsatile flow data, where the most energetic mode was associated to approximately 90% of the total kinetic energy. This distribution was incorporated in a single measure, termed spectral entropy, showing good agreement especially for Case 1.ConclusionThe proposed quantitative POD-based technique could be a valuable tool to reduce the complexity of the time-dependent hemodynamic data and to facilitate an easy comparison of 4D flows, e.g., for validation purposes.     

Functional expression of the murine D2, D3 and D4 dopamine receptors in Xenopus laevis oocytes

The different murine D2-type dopamine receptors (D_2L, D_2S, D_3L, D_3S, and D₄) were expressed in Xenopus laevis oocytes. The D2-type receptors were all similarly and efficiently expressed in Xenopus oocytes and were shown to bind the D2 antagonist [¹²⁵I]sulpride. They were all shown to activate Cl⁻ influx upon agonist stimulation. Using the diagnostic inhibitor bumetanide, we were able to separate the Na⁺/K⁺/2Cl⁻ cotransporter component of the Cl⁻ influx from the total unidirectional Cl⁻ influx. The D_3L subtype was found to operate exclusively through the bumetanide-insensitive Cl⁻ influx whereas the other D2-type receptors acted on the Na⁺/K⁺/2Cl⁻ cotransporter as well. The pertussis toxin sensitivity of the receptor-activated chloride influx via the Na⁺/K⁺/2Cl⁻ cotransporter varied between the various D2-type receptors showing that they may couple to different G proteins, and activate different second messenger systems.     

Molecular characterization and differential mRNA tissue distribution of rabbit apolipoprotein D

We report for the first time the quantification of relative apolipoprotein D (apoD) mRNA concentrations in a wide selection of organs and a detailed characterization of the rabbit protein. ApoD cDNA clones were isolated from a rabbit testis cDNA library by screening with a human apoD cDNA-derived RNA probe. The 912 nucleotide sequence of rabbit apoD cDNA contains a unique reading frame coding for a protein sharing 80% homology with human apoD. The two sequences have two potential asparagine-linked glycosylation sites at the same positions, almost superimposable hydrophobicity plot, and the antigenic proteins show similar charge polymorphism, Mr, and lipoprotein distribution. This high degree of similarity shows that the rabbit system can be used as a model for apoD studies. Moreover, the two consensus sequences of the hydrophobic ligand carrier (alpha 2-microglobulin) family present in human apoD are also found in the rabbit protein and these sequences coincide with the most conserved regions. The distribution of apoD mRNA among rabbit organs was determined by Northern blot and quantitative dot blot analysis. The highest levels of mRNA were found in spleen, adrenal glands, lungs, brain, testis, and kidneys. Moderate or low concentrations were detected in all the other organs tested including liver and small intestine. Thus, our results show that the apoD gene is expressed mainly in peripheral organs, with levels as high as 59-fold that of the liver, unlike other apolipoproteins. We suggest that apoD exerts its main function locally in peripheral organs.     

Design and characterization of stabilized derivatives of human CD4D12 and CD4D1

CD4 is present on the surface of T-lymphocytes and is the primary cellular receptor for HIV-1. CD4 consists of a cytoplasmic tail, one transmembrane region, and four extracellular domains, D1-D4. A construct consisting of the first two domains of CD4 (CD4D12) is folded and binds gp120 with similar affinity as soluble 4-domain CD4 (sCD4). However, the first domain alone (CD4D1) was previously shown to be largely unfolded and had 3-fold weaker affinity for gp120 when compared to sCD4 [Sharma, D.; et al. (2005) Biochemistry 44, 16192-16202]. We now report the design and characterization of three single-site mutants of CD4D12 (G6A, L51I, and V86L) and one multisite mutant of CD4D1 (G6A/L51I/L5K/F98T). G6A, L51I, and V86L are cavity-filling mutations while L5K and F98T are surface mutations which were introduced to minimize the aggregation of CD4D1 upon removal of the second domain. Two mutations, G6A and V86L in CD4D12 increased the stability and yield of the protein relative to the wild-type protein. The mutant CD4D1 (CD4D1a) with the 4 mutations was folded and more stable compared to the original CD4D1, but both bound gp120 with comparable affinity. In in vitro neutralization assays, both CD4D1a and G6A-CD4D12 were able to neutralize diverse HIV-1 viruses with similar IC(50)s as 4-domain CD4. These stabilized derivatives of human CD4 can be useful starting points for the design of other more complex viral entry inhibitors.     

Identification, characterization, and tissue distribution of apolipoprotein D in the rat

We recently described an unknown apolipoprotein that is present on the lipoprotein particles isolated from regenerating rat sciatic nerves. In the regenerating nerve, the concentration of this apolipoprotein rises 500-fold over its concentration in the normal nerve. In this report we have identified the apolipoprotein by partial amino acid sequence analysis as apolipoprotein (apo) D. Characterization of rat apoD by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed it to be composed of a series of molecular weight isoforms of between 27 kDa and 31 kDa that increase 2 kDa in apparent molecular mass upon reduction. Rat apoD has multiple isoelectric points between pH 4.05 and 4.37, apparently resulting from N-linked glycosylation. In the rat, unlike the human, little apoD is found in plasma. However, immunocytochemical localization of apoD in 12 tissues (liver, kidney, bladder, adrenal, cerebrum, duodenum, testis, lung, spleen, pancreas, heart, and skin) showed that a variety of cells contained substantial levels of apolipoprotein. The broad distribution of apoD suggests that it may play a general role in cellular metabolism. Moreover, many of the same cell types varied dramatically in their content of apoD in different tissues, suggesting that the uptake or secretion of apoD by cells is regulated.     

生物4D打印技术在心血管组织工程中的研究及应用进展

3D生物打印技术是应用包含生物材料与活细胞在内的生物墨水来构造生物医学产品的技术，近年来得到快速发展。3D打印的组织是静态的，而人体的组织则处于实时动态之中，并且随时能够发生形态及性能的变化，要提高体外环境与体内真实环境的吻合度，就需要一种能够模拟这种动态过程的体外组织构建技术。4D打印概念的提出，给实现这种复杂技术提供了一条新的思路。4D打印可理解为“3D打印+时间”，在3D打印基础上，4D打印应用一种或多种对刺激具有响应的智能材料，这种材料可以在相应的刺激下改变它们的形态、性能及功能，以满足多种需求。本文重点关注4D打印技术在心血管系统中的最新研究进展及其潜在应用领域，为该项技术的发展提供一些理论及应用参考价值。     

Up-regulation of IL-33 expression in various types of murine cells by IL-3 and IL-4

The crucial roles of the novel cytokine IL-33 in allergic, inflammatory, infectious and autoimmune diseases are becoming characterized. However, the cytokines which regulate IL-33 expression and secretion are still largely unknown. In this study, IL-3 and IL-4 were found to up-regulate IL-33 mRNA expression in mouse peritoneal exudate cells by a two-color DNA microarray and further confirmed by real time PCR and ELISA. IL-3 and IL-4 synergistically promote IL-33 mRNA expression and IL-33 intracrine in the heterogeneous cell populations as peritoneal exudates cells, bone marrow cells and splenic cells. IL-3 and IL-4 also induced IL-33 introcrine in the peritoneal exudate cells from the macrophage-deficient op/op mice, suggesting that macrophage is not the only target of IL-3 and IL-4 in the heterogeneous peritoneal exudate cells. Furthermore, IL-3 and IL-4 were verified to promote the IL-33 intracrine in the homogeneous cell population as fibroblasts and mast cells. These results indicate that up-regulation of IL-33 expression by IL-3 and IL-4 is not a feature particular to a specific type of cells. Up to 100 cytokines were screened, but none of them stimulated the secretion or release of IL-33 in the culture system. In summary, we confirm for the first time that IL-3 and IL-4 are critical for IL-33 intracrine in murine cells of various types, indicating that IL-3 and IL-4 may play an important role in the constitutive expression of IL-33 in vivo.     

Isolation and characterization of two types of MDCK epithelial cell clones based on glycosphingolipid pattern

The Madin-Darby canine kidney (MDCK) epithelial cell line was shown previously to be heterogeneous with marked differences reported between low-passage (strain I) and high-passage (strain II) cultures (Richardson, J.C.W., Scalera, V. and Simmons, N.L. (1981) Biochim. Biophys. Acta 673, 26–36). This report describes major differences in the glycolipids of the two subpopulations of cells that comprise strain I and strain II cultures. The majority of strain II cells were strongly positive for the Forssman glycolipid antigen, while strain I cells were Forssman-deficient. Upon finding that strain I cells were contaminated with mycoplasma, we rescued Forssman-deficient cells from strain II using an anti-Forssman plus complement lysis procedure. Clones of surviving cells consisted of two distinct cell types. The first were Forssman-deficient, non-ciliated, spindle-shaped cells which generated negative (apical to basolateral) transepithelial potential differences. Clones of the second type were strongly Forssman-positive, ciliated, and formed island-shaped clusters of cuboidal cells. These latter clones generated positive potential differences and grew more slowly than the spindle-shaped clones. Spindle cells were enriched in fucolipids, while cuboidal cells contained higher levels of sulfated glycolipids. These two types of clones should provide excellent model systems in which to study the processing and polarity of glycolipids in epithelial cells.     

Genetic and structural characterization ofH-2-controlled allelic forms of murine C4

The patterns of expression of several types of genetic variation in murine C4 were investigated. Dominance was assessed forS-region-determined differences in molecular weight of the C4 α-chain fromS ^w7 strains relative to all other strains examined and in C4 hemolytic titers. Codominant expression in F₁ hybrids was shown for both of the above variations. Comparison of tryptic peptide profiles of radiolabeled C4β-chains encoded by differentS regions revealed differences in their primary structures. The demonstration of codominant expression for the variant C4 traits examined and the evidence from tryptic peptide maps for haplotype differences in C4 primary structure strongly support the hypothesis that polymorphism of murine C4 is controlled by structural genes which are located in theS region.