Mri Methods For Imaging The Feto-Placental Vasculature And Blood

Fetal magnetic resonance imaging (MRI) in recent times has become a well-established adjunct to ultrasound (US) in routine clinical prenatal care and diagnostics. The majority of fetal MRI is restricted to T2-weighted scans, where the diagnosis is based on the appearance of normal and abnormal tissue. Although there have been many advancements in MRI and a plethora of sequences, that probe different anatomical and different physiological process, the adaptation of these in fetal imaging has been rather slow. Many of these can extract quantitative parameters that can throw light on the underlying tissue’s normal/patho-physiology. But the use of such quantitative MRI methods has been extremely limited in fetal imaging due to its unique and dynamic physiological milieu that pose several technical challenges including low signal to noise and/or resolution, artifacts associated with abdominal imaging and most importantly fetal motion. These limitations are expected to be overcome by (a) optimizing and (b) developing novel MR imaging sequences, both of which constitute the primary aim of my work. This work develops a framework that allows for vascular imaging in the fetus and placenta. This includes both qualitative vascular imaging and blood flow quantification. Towards this, three broad directions were explored (a) Moving to higher field imaging, while optimizing parameters for low energy deposition and (b) application of non-gated phase contrast MRI and (c) optimization of conventional time-of-flight angiography for fetal applications

Tristetraprolin Mediates Radiation-Induced TNF-α Production in Lung Macrophages

<div><p>The efficacy of radiation therapy for lung cancer is limited by radiation-induced lung toxicity (RILT). Although tumor necrosis factor-alpha (TNF-α) signaling plays a critical role in RILT, the molecular regulators of radiation-induced TNF-α production remain unknown. We investigated the role of a major TNF-α regulator, Tristetraprolin (TTP), in radiation-induced TNF-α production by macrophages. For <i>in vitro</i> studies we irradiated (4 Gy) either a mouse lung macrophage cell line, MH-S or macrophages isolated from TTP knockout mice, and studied the effects of radiation on TTP and TNF-α levels. To study the <i>in vivo</i> relevance, mouse lungs were irradiated with a single dose (15 Gy) and assessed at varying times for TTP alterations. Irradiation of MH-S cells caused TTP to undergo an inhibitory phosphorylation at Ser-178 and proteasome-mediated degradation, which resulted in increased TNF-α mRNA stabilization and secretion. Similarly, MH-S cells treated with TTP siRNA or macrophages isolated from <i>ttp</i> (−/−) mice had higher basal levels of TNF-α, which was increased minimally after irradiation. Conversely, cells overexpressing TTP mutants defective in undergoing phosphorylation released significantly lower levels of TNF-α. Inhibition of p38, a known kinase for TTP, by either siRNA or a small molecule inhibitor abrogated radiation-induced TNF-α release by MH-S cells. Lung irradiation induced TTP^Ser178 phosphorylation and protein degradation and a simultaneous increase in TNF-α production in C57BL/6 mice starting 24 h post-radiation. In conclusion, irradiation of lung macrophages causes TTP inactivation via p38-mediated phosphorylation and proteasome-mediated degradation, leading to TNF-α production. These findings suggest that agents capable of blocking TTP phosphorylation or stabilizing TTP after irradiation could decrease RILT.</p> </div

Schematic model explaining the role of post-translational TTP modifications (phosphorylation and degradation) in radiation-induced TTP inactivation as an upstream regulator involved in increased TNF-α secretion by mouse lung macrophages.

<p>Schematic model explaining the role of post-translational TTP modifications (phosphorylation and degradation) in radiation-induced TTP inactivation as an upstream regulator involved in increased TNF-α secretion by mouse lung macrophages.</p

Radiation-induced TTP degradation and TNF-α secretion is inhibited significantly by the proteasome inhibitor MG132.

<p>(A) MH-S cells were metabolically labeled with ³⁵S-methionine, were either sham-irradiated or irradiated with 4 Gy, and chased with cold methionine for indicated time periods. After the chase period cell lysates were subjected to immunoprecipitation using TTP antibody, immunocomplexes were resolved by SDS-PAGE and autoradiography. (B) TTP protein’s half life in sham-irradiated (-RT) and irradiated (4 Gy) groups were determined by densitometric scanning of the autoradiographs followed by quantitation using Image J1.32j software (NIH, Bethesda, MD). Relative protein levels were determined in comparison to sample isolated immediately after the pulse labeling (0 h chase). (C) MH-S cells were either sham-irradiated or radiated with 4 Gy, and 44 h after radiation 2 µM of MG132 was added. Cell lysates were collected 4 h after MG132 addition and immunoblotted for TTP and TNF-α. GAPDH was used as loading control.</p

Radiation causes significant down-regulation of TTP in irradiated mouse lung.

<p>(A) C57BL/6 mice were either sham-irradiated or the whole lung was irradiated with a single dose of 15 Gy. Tissue lysates were prepared from three individual animals at each indicated time points post-radiation and immunoblotted using indicated antibodies. (B) Mouse lung cryo-sections from sham-radiated or 15 Gy single fraction radiated mice were isolated at indicated time points and immune-staining were performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057290#s2" target="_blank">materials and methods</a>.</p

p38 kinase controls radiation-induced TTP phosphorylation and TNF-α secretion by MH-S cells.

<p>(A) CHO cells overexpressing TTP were treated with 4 Gy in the presence of either DMSO (vehicle control) or p38 inhibitor (SB203580), or PI3K inhibitor (Wortmannin), or GSK3ß inhibitors (SB415286, SB216763). Cell lysates were prepared 10 min post-radiation and immunoblotted using indicated antibodies. (B) MH-S cells were pretreated with either p38 or GSK3ß inhibitors as above and cell lysates were prepared 10 min post-radiation and immunoblotted with the indicated antibodies. (C) MH-S cells were irradiated with 4 Gy in the presence or absence of a p38 inhibitor (SB203580), and radiation-induced TNF-α secretion was quantified using ELISA. (D) MH-S cells were treated with either control (C) or TTP (T) siRNA. 24 h post-transfection, cells were either left un-irradiated or radiated with 4 Gy. Culture supernatants were collected 48 h post-radiation, and TNF-α levels were quantified. In the inset, the effectiveness of p38 siRNA is shown in cell lysates isolated from C or T siRNA treated cells.</p

Radiation increased the TNF-α transcript and its release by MH-S cells 48 h post–radiation.

<p>(A) MH-S cells were left untreated (0 Gy) or irradiated (4 Gy) and culture supernatants were collected at the indicated time points. Released TNF-α levels were quantified using ELISA kits according to the manufacturer’s instruction. (B) Cells were treated as above, and RNA was isolated and quantified at the indicated time points as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057290#s2" target="_blank">materials and methods</a>. (C) MH-S cells were either sham irradiated or irradiated with 4 Gy. 48 h post-irradiation TNF-α mRNA level, stability and synthesis was determined using BrU pulse-chase labeling experiment as described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057290#pone.0057290-Fu1" target="_blank">[28]</a>.</p