A new method to improve RF safety of implantable medical devices using inductive coupling at 3.0 T MRI.

OBJECTIVE: To enhance RF safety when implantable medical devices are located within the body coil but outside the imaging region by using a secondary resonator (SR) to reduce electric fields, the corresponding specific absorption rate (SAR), and temperature change during MRI. MATERIALS AND METHODS: This study was conducted using numerical simulations with an American Society for Testing and Materials (ASTM) phantom and adult human models of Ella and Duke from Virtual Family Models, along with corresponding experimental results of temperature change obtained using the ASTM phantom. The circular SR was designed with an inner diameter of 150 mm and a width of 6 mm. Experimental measurements were carried out using a 3 T Medical Implant Test System (MITS) body coil, electromagnetic (EM) field mapping probes, and an ASTM phantom. RESULTS: The magnitudes of B1+ (|B1+|) and SAR1g were reduced by 15.2% and 5.85% within the volume of interest (VoI) of an ASTM phantom, when a SR that generates opposing electromagnetic fields was utilized. Likewise, the Δ|B1+| and ΔSAR1g were reduced by up to 56.7% and 57.5% within the VoI of an Ella model containing a copper rod when an opposing SR was used. CONCLUSION: A novel method employing the designed SR, which generates opposing magnetic fields to partially shield a sample, has been proposed to mitigate the risk of induced-RF heating at the VoI through numerical simulations and corresponding experiments under various conditions at 3.0 T.

Growth arrest of PPP2R5C and PPP2R5D double knockout mice indicates a genetic interaction and conserved function for these PP2A B subunits.

Protein phosphatase 2A (PP2A) is a heterotrimeric phosphatase that controls a wide range of cellular functions. The catalytic activity and intracellular location of PP2A are modulated by its association with regulatory B subunits, including B56 proteins, which are encoded by five separate genes in humans and mice. The specific effects of each B56 protein on PP2A activity and function are largely unknown. As part of an effort to identify specific PP2A-B56 functions, we created knockout strains of B56ß, B56δ, and B56ε using CRISPR/Cas9n. We found that none of the individual B56 genes are essential for mouse survival. However, mice that have both B56δ and B56γ inactivated (B56δγ-), arrest fetal development around Day E12. The hearts of B56δγ- mice have a single outflow vessel rather than having both an aorta and a pulmonary artery. Thus, there appears to be strong genetic interaction between B56δ and B56γ, and together they are necessary for heart development. Of note, both these proteins have been shown to localize to the nucleus and have the most related peptide sequences of the B56 family members. Our results suggest there are B56 subfamilies, which work in conjunction to regulate specific PP2A functions.

Sensitivity and uniformity improvement of phased array MR images using inductive coupling and RF detuning circuits.

OBJECTIVE: To improve sensitivity and uniformity of MR images obtained using a phased array RF coil, an inductively coupled secondary resonator with RF detuning circuits at 300 MHz was designed. MATERIALS AND METHODS: A secondary resonator having detuning circuits to turn off the resonator during the transmit mode was constructed. The secondary resonator was located at the opposite side of the four-channel phased array to improve sensitivity and uniformity of the acquired MR images. Numerical simulations along with phantom and in vivo experiments were conducted to evaluate the designed secondary resonator. RESULTS: The numerical simulation results of |B1+| in a transmit mode showed that magnetic field uniformity would be decreased with a secondary resonator having no detuning circuits because of unwanted interferences between the transmit birdcage coil and the secondary resonator. The standard deviation (SD) of |B1+| was decreased 57% with a secondary resonator containing detuning circuits. The sensitivity and uniformity of |B1-| in the receive mode using a four-channel phased array were improved with the secondary resonator. Phantom experiments using a uniform saline phantom had 20% improvement of the mean signal intensity and 50% decrease in the SD with the secondary resonator. Mice with excess adipose tissue were imaged to demonstrate the utility of the secondary resonator. CONCLUSION: The designed secondary resonator having detuning circuits improved sensitivity and uniformity of mouse MR images acquired using the four-channel phased array.

Improvement of 19F MR image uniformity in a mouse model of cellular therapy using inductive coupling.

OBJECTIVE: Improve 19F magnetic resonance imaging uniformity of perfluorocarbon (PFC)-labeled cells by using a secondary inductive resonator tuned to 287 MHz to enhance the induced radio frequency (RF) magnetic field (B1) at 7.05 T. MATERIALS AND METHODS: Following Faraday's induction law, the sign of induced B1 made by the secondary resonator can be changed depending on the tuning of the resonator. A secondary resonator located on the opposite side of the phantom of the 19F surface coil can be shown to enhance or subtract the induced B1 field, depending upon its tuning. RESULTS: The numerical simulation results of rotating transmit B1 magnitude (|B 1 + |) and corresponding experimental 19F images were compared without and with the secondary resonator. With the secondary resonator tuned to 287 MHz, improvements of |B 1 + | and 19F image uniformity were demonstrated. The use of the secondary resonator improved our ability to visualize transplanted cell location non-invasively over a period of 6 weeks. CONCLUSION: The secondary resonator tuned to enhance the induced B1 results in improved image uniformity in a pre-clinical application, enabling cell tracking of PFC-labeled cells with the secondary resonator.

Evaluation of the differentiation status of neural stem cells based on cell morphology and the expression of Notch and Sox2.

Neural stem cells (NSCs) isolated from a variety of sources are being developed as cellular therapies aimed at treating neurodegenerative diseases. During NSC culture and expansion it is important the cells do not differentiate prematurely because this may have an unfavorable effect on product quality and yield. In our study, we evaluated the use of Notch and Sox2 as markers for undifferentiated human and mouse NSCs. The expression of Notch2 and Sox2 during extensive-passage, low-oxygen culture and differentiation conditions were analyzed to confirm that the presence of these signature proteins directly correlates with the ability of NSCs to form new neurospheres and differentiate into multiple cell types. Using expression of Notch1, Notch2 and Sox2 as a reference, we then used flow cytometry to identify a specific morphological profile for undifferentiated murine and human NSCs. Our studies show that Notch and Sox2 expression, along with flow cytometry analysis, can be used to monitor the differentiation status of NSCs grown in culture for use in cellular therapies.

PP2A-B56γ is required for an efficient spindle assembly checkpoint.

The Spindle Assembly Checkpoint (SAC) is part of a complex feedback system designed to ensure that cells do not proceed through mitosis unless all chromosomal kinetochores have attached to spindle microtubules. The formation of the kinetochore complex and the implementation of the SAC are regulated by multiple kinases and phosphatases. BubR1 is a phosphoprotein that is part of the Cdc20 containing mitotic checkpoint complex that inhibits the APC/C so that Cyclin B1 and Securin are not degraded, thus preventing cells going into anaphase. In this study, we found that PP2A in association with its B56γ regulatory subunit, are needed for the stability of BubR1 during nocodazole induced cell cycle arrest. In primary cells that lack B56γ, BubR1 is prematurely degraded and the cells proceed through mitosis. The reduced SAC efficiency results in cells with abnormal chromosomal segregation, a hallmark of transformed cells. Previous studies on PP2A's role in the SAC and kinetochore formation were done using siRNAs to all 5 of the B56 family members. In our study we show that inactivation of only the PP2A-B56γ subunit can affect the efficiency of the SAC. We also provide data that show the intracellular locations of the B56 subunits varies between family members, which is consistent with the hypothesis that they are not completely functionally redundant.

Improvement of Electromagnetic Field Distributions Using High Dielectric Constant (HDC) Materials for CTL-Spine MRI: Numerical Simulations and Experiments.

This study investigates the use of pads with high dielectric constant (HDC) materials to alter electromagnetic field distributions in patients during magnetic resonance imaging (MRI). The study was performed with numerical simulations and phantom measurements. An initial proof-of-concept and validation was performed using a phantom at 64 MHz, showing increases of up to 10% in electromagnetic field when using distilled water as the high dielectric material. Additionally, numerical simulations with computational models of human anatomy were performed at 128 MHz. Results of these simulations using barium titanate (BaTiO3) beads showed a 61% increase of [Formula: see text] with a quadrature driven RF coil and a 64% increase with a dual-transmit array. The presence of the HDC material also allowed for a decrease of SAR up to twofold (e.g., peak 10 g-averaged SAR from 54 to 22 W/kg with a quadrature driven RF coil and from 27 to 22 W/kg with a dual-transmit array using CaTiO3 powder at 128 MHz). The results of this study show that the use of HDC pads at 128 MHz for MRI spine applications could result in improved magnetic fields within the region of interest, while decreasing SAR outside the region.

RF Safety Evaluation of a Breast Tissue Expander Device for MRI: Numerical Simulation and Experiment.

This study describes the MRI-related radio frequency (RF) safety evaluation of breast tissue expander devices to establish safety criteria. Numerical simulations and experimental measurements were performed at 64 MHz with a gel phantom containing a breast expander. Additionally, computational modeling was performed (64 and 128 MHz) with an adult female model, containing a virtually implanted breast tissue expander device for four imaging landmark positions. The presence of the breast tissue expander device led to significant alterations in specific absorption rate (SAR) and|B1+|distributions. The main source of SAR alterations with the use of the breast expander device was the saline-filled pouch of the expander. Conversely, the variation of RF magnetic field (B1+) was mainly caused by the metallic port. The measured values of electric field magnitude did not increase significantly due to the introduction of the expander device. The maximum 1g- or 10g-averaged SAR values in tissues near the implant were lower than those expected in other regions of the patient body with normalization of both|B1+|equal to 2 µT at the coil isocenter and whole body averaged SAR equal to 4W/kg.

The protein phosphatase 2A B56γ regulatory subunit is required for heart development.

BACKGROUND: Protein Phosphatase 2A (PP2A) function is controlled by regulatory subunits that modulate the activity of the catalytic subunit and direct the PP2A complex to specific intracellular locations. To study PP2A's role in signal transduction pathways that control growth and differentiation in vivo, a transgenic mouse lacking the B56γ regulatory subunit of PP2A was made. RESULTS: Lack of PP2A activity specific to the PP2A-B56γ holoenzyme, resulted in the formation of an incomplete ventricular septum and a decrease in the number of ventricular cardiomyocytes. During cardiac development, B56γ is expressed in the nucleus of α-actinin-positive cardiomyocytes that contain Z-bands. The pattern of B56γ expression correlated with the cardiomyocyte apoptosis we observed in B56γ-deficient mice during mid to late gestation. In addition to the cardiac phenotypes, mice lacking B56γ have a decrease in locomotive coordination and gripping strength, indicating that B56γ has a role in controlling PP2A activity required for efficient neuromuscular function. CONCLUSIONS: PP2A-B56γ activity is required for efficient cardiomyocyte maturation and survival. The PP2A B56γ regulatory subunit controls PP2A substrate specificity in vivo in a manner that cannot be fully compensated for by other B56 subunits.

A disintegrin and metalloproteinase 10 regulates antibody production and maintenance of lymphoid architecture.

A disintegrin and metalloproteinase 10 (ADAM10) is a zinc-dependent proteinase related to matrix metalloproteinases. ADAM10 has emerged as a key regulator of cellular processes by cleaving and shedding extracellular domains of multiple transmembrane receptors and ligands. We have developed B cell-specific ADAM10-deficient mice (ADAM10(B-/-)). In this study, we show that ADAM10 levels are significantly enhanced on germinal center B cells. Moreover, ADAM10(B-/-) mice had severely diminished primary and secondary responses after T-dependent immunization. ADAM10(B-/-) displayed impaired germinal center formation, had fewer follicular Th cells, decreased follicular dendritic cell networks, and altered chemokine expression in draining lymph nodes (LNs). Interestingly, when spleen and LN structures from immunized mice were analyzed for B and T cell localization, tissues structure was aberrant in ADAM10(B-/-) mice. Importantly, when ADAM10-deficient B cells were stimulated in vitro, they produced comparable Ab as wild type B cells. This result demonstrates that the defects in humoral responses in vivo result from inadequate B cell activation, likely because of the decrease in follicular Th cells and the changes in structure. Thus, ADAM10 is essential for the maintenance of lymphoid structure after Ag challenge.

The contribution of Notch1 to nephron segmentation in the developing kidney is revealed in a sensitized Notch2 background and can be augmented by reducing Mint dosage.

We previously determined that Notch2, and not Notch1, was required for forming proximal nephron segments. The dominance of Notch2 may be conserved in humans, since Notch2 mutations occur in Alagille syndrome (ALGS) 2 patients, which includes renal complications. To test whether mutations in Notch1 could increase the severity of renal complications in ALGS, we inactivated conditional Notch1 and Notch2 alleles in mice using a Six2-GFP::Cre. This BAC transgene is expressed mosaically in renal epithelial progenitors but uniformly in cells exiting the progenitor pool to undergo mesenchymal-to-epithelial transition. Although delaying Notch2 inactivation had a marginal effect on nephron numbers, it created a sensitized background in which the inactivation of Notch1 severely compromised nephron formation, function, and survival. These and additional observations indicate that Notch1 in concert with Notch2 contributes to the morphogenesis of renal vesicles into S-shaped bodies in a RBP-J-dependent manner. A significant implication is that elevating Notch1 activity could improve renal functions in ALGS2 patients. As proof of principle, we determined that conditional inactivation of Mint, an inhibitor of Notch-RBP-J interaction, resulted in a moderate rescue of Notch2 null kidneys, implying that temporal blockage of Notch signaling inhibitors downstream of receptor activation may have therapeutic benefits for ALGS patients.

Generation of mice that conditionally express the activation domain of Notch2.

The Notch pathway is an intercellular signaling mechanism frequently used for controlling cell fate during organogenesis. There are four structurally related Notch receptors in mice and humans, and Notch1 and Notch2 are essential genes. In this report we describe the construction of a transgenic mouse strain that expresses the Notch2 intracellular domain in response to cell lineage specific expression of Cre recombinase. This approach bypasses the requirement for ligand- receptor interaction and allows the direct determination of the consequences of Notch2 activation in vivo. Exogenous expression of the Notch2 intracellular domain resulted in the developmental arrest of secondary heart field derived cardiomyocytes during the transition from immature alpha-Smooth Muscle Actin expressing cells to mature alpha-Actinin positive cardiomyocytes. In contrast, a cell nonautonomous mesenchymal expansion was observed in semilunar valves. This new conditionally expressed allele of Notch2 can be used in studies by investigators interested in the effects of Notch2 activation in vivo.

Notch signaling regulates bile duct morphogenesis in mice.

BACKGROUND: Alagille syndrome is a developmental disorder caused predominantly by mutations in the Jagged1 (JAG1) gene, which encodes a ligand for Notch family receptors. A characteristic feature of Alagille syndrome is intrahepatic bile duct paucity. We described previously that mice doubly heterozygous for Jag1 and Notch2 mutations are an excellent model for Alagille syndrome. However, our previous study did not establish whether bile duct paucity in Jag1/Notch2 double heterozygous mice resulted from impaired differentiation of bile duct precursor cells, or from defects in bile duct morphogenesis. METHODOLOGY/PRINCIPAL FINDINGS: Here we characterize embryonic biliary tract formation in our previously described Jag1/Notch2 double heterozygous Alagille syndrome model, and describe another mouse model of bile duct paucity resulting from liver-specific deletion of the Notch2 gene. CONCLUSIONS/SIGNIFICANCE: Our data support a model in which bile duct paucity in Notch pathway loss of function mutant mice results from defects in bile duct morphogenesis rather than cell fate specification.

Notch2 is required for the proliferation of cardiac neural crest-derived smooth muscle cells.

Mutations in Notch receptors and their ligands have been identified as the cause of human congenital heart diseases, indicating the importance of the Notch signaling pathway during heart development. In our study, we use Cre-Lox technology to inactivate Notch2 in several cardiac cell lineages to determine the functional requirements for Notch2 during mammalian heart development. Inactivation of Notch2 in cardiac neural crest cells resulted in abnormally narrow aortas and pulmonary arteries due to a decrease in smooth muscle tissue. The reduction in smooth muscle tissue was not due to cell migration defects but instead was found to be caused by less proliferation in smooth muscle cells during mid to late gestation. Our findings demonstrate that Notch2 is required cell autonomously for proper formation of the heart outflow tract and provides insights into the role of Notch2 in vascular smooth muscle development and the cardiovascular defects associated with Alagille syndrome.

Direct regulation of Gata3 expression determines the T helper differentiation potential of Notch.

CD4(+) T helper cells differentiate into T helper 1 (Th1) or Th2 effector lineages, which orchestrate immunity to different types of microbes. Both Th1 and Th2 differentiation can be induced by Notch, but what dictates which of these programs is activated in response to Notch is not known. By using T cell-specific gene ablation of the Notch effector RBP-J or the Notch1 and 2 receptors, we showed here that Notch was required on CD4(+) T cells for physiological Th2 responses to parasite antigens. GATA-3 was necessary for Notch-induced Th2 differentiation, and we identified an upstream Gata3 promoter as a direct target for Notch signaling. Moreover, absence of GATA-3 turned Notch from a Th2 inducer into a powerful inducer of Th1 differentiation. Therefore, Gata3 is a critical element determining inductive Th2 differentiation and limiting Th1 differentiation by Notch.

Generation of new Notch2 mutant alleles.

The Notch signaling pathway is an evolutionarily conserved intercellular signaling mechanism, and mutations in its components disrupt embryonic development in many organisms and cause inherited diseases in humans. We previously described construction and analysis of a hypomorphic allele of the Notch2 gene. Homozygosity for this allele leads to embryonic and perinatal lethality due to cardiovascular and kidney defects. We report here novel Notch2 mutant alleles generated by gene targeting in embryonic stem cells, including a conditional null allele in which exon 3 of the Notch2 gene is flanked by loxP sequences. These new Notch2 mutant alleles expand the set of tools available for studying the myriad roles of the Notch pathway during mammalian development and will enable analysis of Notch2 function at additional stages of embryogenesis and in adult mice.

Functional conservation of Notch1 and Notch2 intracellular domains.

The Notch receptor is a key component of a highly conserved signaling pathway that regulates cell fate determination during development. In Drosophila, where Notch signaling was first identified and studied, there is only one Notch receptor. In contrast, mammals have four Notch receptor genes, Notch1-4. Notch1 and Notch2 are both required for embryo viability, are widely expressed in mammals, and are structurally conserved. It is presently unknown if these two receptors are functionally redundant or if they have unique capabilities related to differences in their amino acid sequences. In contrast to the rest of the molecule, the amino acid sequences of a large region of the Notch intracellular domain are not highly conserved and thus may be able to interact with distinct transcription factors and mediate the expression of different sets of genes. To determine if the function of this region is conserved, the last 426 amino acids of the Notch2 receptor have been replaced with the corresponding region of Notch1 in mice by using gene targeting. We have determined that even though the amino acid sequences of this region are only 37% identical (137/426), the C-terminal region of the Notch1 intracellular domain can functionally replace that of Notch2 in vivo.

Notch signaling in kidney development.

PURPOSE OF REVIEW: Notch signaling is a highly conserved mechanism used by multicellular animals to specify cell fate decisions during the formation of complex structures such as the kidney. A number of studies have recently identified requirements for Notch signaling during kidney organogenesis and tissue repair. This review will summarize these studies and compare Notch signaling in the mammalian kidney with Notch signaling in other organ systems. RECENT FINDINGS: A targeted mutation in the mouse Notch2 receptor resulted in kidneys that are devoid of glomerular endothelial and mesangial cells. The mutant epithelial cells of the developing glomerulus have reduced amounts of vascular endothelial growth factor expression, which may be responsible for the lack of vascularization observed in these glomeruli. Notch2 is expressed in the epithelial cells of the developing glomerulus, and a potential ligand, Jagged1 is expressed in the endothelial cells of the glomerulus. Mice simultaneously heterozygous for mutations in both Notch2 and Jagged1 phenocopy the kidney defects seen in mice homozygous for the Notch2 mutation. These doubly heterozygous mice also display liver and heart developmental abnormalities reminiscent of Alagille's syndrome. SUMMARY: Notch signaling is required for kidney development, and the expression of Notch genes is increased in response to kidney damage. Further studies of Notch signaling will be important in order to understand kidney development and tissue repair.

A mouse model of Alagille syndrome: Notch2 as a genetic modifier of Jag1 haploinsufficiency.

Alagille syndrome is a human autosomal dominant developmental disorder characterized by liver, heart, eye, skeletal, craniofacial and kidney abnormalities. Alagille syndrome is caused by mutations in the Jagged 1 (JAG1) gene, which encodes a ligand for Notch family receptors. The majority of JAG1 mutations seen in Alagille syndrome patients are null alleles, suggesting JAG1 haploinsufficiency as a primary cause of this disorder. Mice homozygous for a Jag1 null mutation die during embryogenesis and Jag1/+ heterozygous mice exhibit eye defects but do not exhibit other phenotypes characteristic of Alagille syndrome patients ( Xue, Y., Gao, X., Lindsell, C. E., Norton, C. R., Chang, B., Hicks, C., Gendron-Maguire, M., Rand, E. B., Weinmaster, G. and Gridley, T. (1999) HUM: Mol. Genet. 8, 723-730). Here we report that mice doubly heterozygous for the Jag1 null allele and a Notch2 hypomorphic allele exhibit developmental abnormalities characteristic of Alagille syndrome. Double heterozygous mice exhibit jaundice, growth retardation, impaired differentiation of intrahepatic bile ducts and defects in heart, eye and kidney development. The defects in bile duct epithelial cell differentiation and morphogenesis in the double heterozygous mice are similar to defects in epithelial morphogenesis of Notch pathway mutants in Drosophila, suggesting that a role for the Notch signaling pathway in regulating epithelial morphogenesis has been conserved between insects and mammals. This work also demonstrates that the Notch2 and Jag1 mutations interact to create a more representative mouse model of Alagille syndrome and provides a possible explanation of the variable phenotypic expression observed in Alagille syndrome patients.