Hypoxia promotes tau hyperphosphorylation with associated neuropathology in vascular dysfunction.

BACKGROUND: Hypertension-induced microvascular brain injury is a major vascular contributor to cognitive impairment and dementia. We hypothesized that chronic hypoxia promotes the hyperphosphorylation of tau and cell death in an accelerated spontaneously hypertensive stroke prone rat model of vascular cognitive impairment. METHODS: Hypertensive male rats (n = 13) were fed a high salt, low protein Japanese permissive diet and were compared to Wistar Kyoto control rats (n = 5). RESULTS: Using electron paramagnetic resonance oximetry to measure in vivo tissue oxygen levels and magnetic resonance imaging to assess structural brain damage, we found compromised gray (dorsolateral cortex: p = .018) and white matter (corpus callosum: p = .016; external capsule: p = .049) structural integrity, reduced cerebral blood flow (dorsolateral cortex: p = .005; hippocampus: p < .001; corpus callosum: p = .001; external capsule: p < .001) and a significant drop in cortical oxygen levels (p < .05). Consistently, we found reduced oxygen carrying neuronal neuroglobin (p = .008), suggestive of chronic cerebral hypoperfusion in high salt-fed rats. We also observed a corresponding increase in free radicals (NADPH oxidase: p = .013), p-Tau (pThr231) in dorsolateral cortex (p = .011) and hippocampus (p = .003), active interleukin-1ß (p < .001) and neurodegeneration (dorsolateral cortex: p = .043, hippocampus: p = .044). Human patients with subcortical ischemic vascular disease, a type of vascular dementia (n = 38; mean age = 68; male/female ratio = 23/15) showed reduced hippocampal volumes and cortical shrinking (p < .05) consistent with the neuronal cell death observed in our hypertensive rat model as compared to healthy controls (n = 47; mean age = 63; male/female ratio = 18/29). CONCLUSIONS: Our data support an association between hypertension-induced vascular dysfunction and the sporadic occurrence of phosphorylated tau and cell death in the rat model, correlating with patient brain atrophy, which is relevant to vascular disease.

Quantitative [Fe]MRI determination of the dynamics of PSMA-targeted SPIONs discriminates among prostate tumor xenografts based on their PSMA expression.

BACKGROUND: There is a need for a quantitative MRI method for iron concentration magnetic resonance imaging suitable for measuring the delivery of targeted superparamagnetic iron oxide nanoparticles (SPIONs) to tumors. PURPOSE: To apply our newly developed [Fe]MRI method to the quantitative imaging in both space and time of the iron dynamics of anti-prostate specific membrane antigen (PSMA) conjugated SPIONs within human prostate tumor xenografts in nude mice. STUDY TYPE: Longitudinal. ANIMAL MODEL: 45 Harlan Sprague Dawley athymic nude mice bearing xenografts from PSMA-positive LNCaP, C4-2 and PSMA-negative DU145 tumors from human prostate tumor cell lines. FIELD STRENGTH/SEQUENCE: 1.0 Tesla/ T1 and T2 weighted spin echo. ASSESSMENT: Image intensity and contrast measurements. STATISTICAL TESTS: Student's t-test. RESULTS: The SPION diffusion coefficient within tumors was D = 44.8 ± 2.4 × 10-6 mm2 /s. The iron taken up by PSMA-positive LNCaP and C4-2 tumors was proportional to the tail-vein injected dose from 60 nmol to 1.6 µmol; injection of 1 µmol of iron in anti-PSMA conjugated SPIONs resulted in a tumor [Fe] of 76 µM. Even at the highest iron dose of 1.6 µmol, the PSMA-negative DU145 tumors took up no significant iron from the anti-PSMA conjugated SPIONs. A similar lack of nonspecific uptake was observed when the antibodies against PSMA were omitted from the injected SPION preparation. The fraction of the initial iron dose that was taken up by PSMA-positive tumors was 2.32 ± 0.75% (n = 10); uptake by the PSMA-negative DU145 tumors and for SPIONs without anti-PSMA antibodies was 0.16 ± 0.34% (n = 7) giving a ratio of [Fe] in PSMA + versus PSMA- tumors greater than 15:1 (P = 0.01). DATA CONCLUSION: Quantitative [Fe]MRI of anti-PSMA conjugated SPIONs discriminated between PSMA-positive LNCaP and C4-2 and PSMA-negative DU145 human prostate tumor xenografts in vivo. LEVEL OF EVIDENCE: 1 Technical Efficacy: Stage 2 J. Magn. Reson. Imaging 2017. J. MAGN. RESON. IMAGING 2018;48:469-481.

Microstructural and microglial changes after repetitive mild traumatic brain injury in mice.

Traumatic brain injury (TBI) is a major public health issue, with recently increased awareness of the potential long-term sequelae of repetitive injury. Although TBI is common, objective diagnostic tools with sound neurobiological predictors of outcome are lacking. Indeed, such tools could help to identify those at risk for more severe outcomes after repetitive injury and improve understanding of biological underpinnings to provide important mechanistic insights. We tested the hypothesis that acute and subacute pathological injury, including the microgliosis that results from repeated mild closed head injury (rmCHI), is reflected in susceptibility-weighted magnetic resonance imaging and diffusion-tensor imaging microstructural abnormalities. Using a combination of high-resolution magnetic resonance imaging, stereology, and quantitative PCR, we studied the pathophysiology of male mice that sustained seven consecutive mild traumatic brain injuries over 9 days in acute (24 hr) and subacute (1 week) time periods. rmCHI induced focal cortical microhemorrhages and impaired axial diffusivity at 1 week postinjury. These microstructural abnormalities were associated with a significant increase in microglia. Notably, microgliosis was accompanied by a change in inflammatory microenvironment defined by robust spatiotemporal alterations in tumor necrosis factor-α receptor mRNA. Together these data contribute novel insight into the fundamental biological processes associated with repeated mild brain injury concomitant with subacute imaging abnormalities in a clinically relevant animal model of repeated mild TBI. These findings suggest new diagnostic techniques that can be used as biomarkers to guide the use of future protective or reparative interventions. © 2016 Wiley Periodicals, Inc.

Superparamagnetic nanoparticle-enhanced MRI of Alzheimer's disease plaques and activated microglia in 3X transgenic mouse brains: Contrast optimization.

PURPOSE: To optimize magnetic resonance imaging (MRI) of antibody-conjugated superparamagnetic nanoparticles for detecting amyloid-ß plaques and activated microglia in a 3X transgenic mouse model of Alzheimer's disease. MATERIALS AND METHODS: Ten 3X Tg mice were fed either chow or chow containing 100 ppm resveratrol. Four brains, selected from animals injected with either anti-amyloid targeted superparamagnetic iron oxide nanoparticles, or anti-Iba-1-conjugated FePt-nanoparticles, were excised, fixed with formalin, and placed in Fomblin for ex vivo MRI (11.7T) using multislice-multiecho, multiple gradient echo, rapid acquisition with relaxation enhancement, and susceptibility-weighted imaging (SWI). Aß plaques and areas of neuroinflammation appeared as hypointense regions whose number, location, and Z-score were measured as a function of sequence type and echo time. RESULTS: The MR contrast was due to the shortening of the transverse relaxation time of the plaque-adjacent tissue water. A theoretical analysis of this effect showed that the echo time was the primary determinant of plaque contrast and was used to optimize Z-scores. The Z-scores of the detected lesions varied from 21 to 34 as the echo times varied from 4 to 25 msec, with SWI providing the highest Z-score and number of detected lesions. Computation of the entire plaque and activated microglial distributions in 3D showed that resveratrol treatment led to a reduction of ∼24-fold of Aß plaque density and ∼4-fold in microglial activation. CONCLUSION: Optimized MRI of antibody-conjugated superparamagnetic nanoparticles served to reveal the 3D distributions of both Aß plaques and activated microglia and to measure the effects of drug treatments in this 3X Tg model. LEVEL OF EVIDENCE: 1 Technical Efficacy: Stage 2 J. MAGN. RESON. IMAGING 2017;46:574-588.

Iron-based superparamagnetic nanoparticle contrast agents for MRI of infection and inflammation.

OBJECTIVE. In this article, we summarize the progress to date on the use of superparamagnetic iron oxide nanoparticles (SPIONs) as contrast agents for MRI of inflammatory processes. CONCLUSION. Phagocytosis by macrophages of injected SPIONs results in a prolonged shortening of both T2 and T2* leading to hypointensity of macrophage-infiltrated tissues in contrast-enhanced MR images. SPIONs as contrast agents are therefore useful for the in vivo MRI detection of macrophage infiltration, and there is substantial research and clinical interest in the use of SPION-based contrast agents for MRI of infection and inflammation. This technique has been used to identify active infection in patients with septic arthritis and osteomyelitis; importantly, the MRI signal intensity of the tissue has been found to return to its unenhanced value on successful treatment of the infection. In SPION contrast-enhanced MRI of vascular inflammation, animal studies have shown decreased macrophage uptake in atherosclerotic plaques after treatment with statin drugs. Human studies have shown that both coronary and carotid plaques that take up SPIONs are more prone to rupture and that abdominal aneurysms with increased SPION uptake are more likely to grow. Studies of patients with multiple sclerosis suggest that MRI using SPIONs may have increased sensitivity over gadolinium for plaque detection. Finally, SPIONs have enabled the tracking and imaging of transplanted stem cells in a recipient host.

Autophagy regulates lipolysis and cell survival through lipid droplet degradation in androgen-sensitive prostate cancer cells.

BACKGROUND: Androgen deprivation therapy, one of the standard treatments for prostate cancer (PCa) induces apoptosis, as well as autophagy in androgen-responsive PCa cells. As autophagy can promote either cell survival or death, it is important to understand its role in PCa treatment. The objective of this study was to elucidate the function of autophagy in lipid droplet (LD) homeostasis and survival in androgen-sensitive PCa cells. METHODS: To produce androgen deprivation, charcoal filtered serum or the androgen inhibitor casodex were used in LNCaP and LAPC4 cells. Autophagy was monitored by immunofluorescence/confocal microscopy and immunoblot analysis. Levels of intracellular LDs and triacyglycerols after the inhibition of autophagy by 3-methyladenine, bafilomycin A(1) , or si-ATG5 were quantified by three independent methods, Oil Red O staining, triacyglycerols lipase assay, and nuclear magnetic resonance. RESULTS: Androgen deprivation induced autophagy and the depletion of LDs in both of the androgen-sensitive PCa cell lines examined, whereas the blockage of autophagy by pharmacological or genetic means inhibited LD degradation and therefore lipolysis and cell growth. In addition, under androgen deprivation, increased colocalization of LDs and autophagic vesicles was observed in LNCaP cells, which can be further enhanced by blocking the autophagic flux. CONCLUSION: Autophagy mediates LD degradation and lipolysis in androgen-sensitive PCa cells during androgen deprivation which aids the survival of PCa cells during hormone therapy.

Prostate cancer targeting motifs: expression of αν ß3, neurotensin receptor 1, prostate specific membrane antigen, and prostate stem cell antigen in human prostate cancer cell lines and xenografts.

BACKGROUND: Membrane receptors are frequent targets of cancer therapeutic and imaging agents. However, promising in vitro results often do not translate to in vivo clinical applications. To better understand this obstacle, we measured the expression differences in receptor signatures among several human prostate cancer cell lines and xenografts as a function of tumorigenicity. METHODS: Messenger RNA and protein expression levels for integrin α(ν) ß(3), neurotensin receptor 1 (NTSR1), prostate specific membrane antigen (PSMA), and prostate stem cell antigen (PSCA) were measured in LNCaP, C4-2, and PC-3 human prostate cancer cell lines and in murine xenografts using quantitative reverse transcriptase polymerase chain reaction, flow cytometry, and immunohistochemistry. RESULTS: Stable expression patterns were observed for integrin α(ν) and PSMA in all cells and corresponding xenografts. Integrin ß(3) mRNA expression was greatly reduced in C4-2 xenografts and greatly elevated in PC-3 xenografts compared with the corresponding cultured cells. NTSR1 mRNA expression was greatly elevated in LNCaP and PC-3 xenografts. PSCA mRNA expression was elevated in C4-2 xenografts when compared with C4-2 cells cultured in vitro. Furthermore, at the protein level, PSCA was re-expressed in all xenografts compared with cells in culture. CONCLUSIONS: The regulation of mRNA and protein expression of the cell-surface target proteins α(ν) ß(3), NTSR1, PSMA, and PSCA, in prostate cancer cells with different tumorigenic potential, was influenced by factors of the microenvironment, differing between cell cultures and murine xenotransplants. Integrin α(ν) ß(3), NTRS1 and PSCA mRNA expression increased with tumorigenic potential, but mRNA expression levels for these proteins do not translate directly to equivalent expression levels of membrane bound protein.

Repurposing Ferumoxytol as a Breast Cancer-Associated Macrophage Tracer with Five-Dimensional Quantitative [Fe]MRI of SPION Dynamics.

Tumor-associated macrophages (TAMs) in breast cancer regulate inflammation, immunosuppression, angiogenesis, and metastasis. However, TAM imaging remains a clinical challenge. Ferumoxytol has long been an FDA-approved superparamagnetic iron oxide nanoparticle (SPION) preparation used as an intravenous (IV) treatment for iron-deficiency anemia. Given its high transverse relaxivity, ferumoxytol produces a negative image contrast upon cellular uptake in T2-weighted magnetic resonance imaging (MRI) studies. Here we evaluated ferumoxytol as a contrast agent to image/quantify TAMs in an aggressive mouse model of breast cancer: We developed [Fe]MRI to measure the 5-dimensional function c(x,y,z,t), where c is the concentration of nanoparticle iron and {x,y,z,t} is the 4-dimensional set of tumor space-time coordinates. Ferumoxytol SPIONs are readily phagocytosed (~104/cell) by the F4/80+CD11b+ TAMs within breast tumors. Quantitative [Fe]MRIs served to determine both the spatial and the temporal distribution of the SPION iron, and hence to measure [Fe] = c(x,y,z,t), a surrogate for TAM density. In single-dose pharmacokinetic studies, after an IV dose of 5 mg/Kg iron, [Fe]MRI measurements showed that c(x,y,z,t) within breast tumors peaked around [Fe] = 70 µM at 42 h post-administration, and decayed below the [Fe]MRI detection limit (~2 µM) by day 7. There was no SPION uptake in control organs (muscle and adipose tissue). Optical microscopy of tissue sections confirmed that F4/80+CD11b+ TAMs infiltrated the tumors and accumulated SPION iron. Our methodology and findings have translational applications for breast cancer patients.

Longitudinal monitoring of microglial/macrophage activation in ischemic rat brain using Iba-1-specific nanoparticle-enhanced magnetic resonance imaging.

Microglial/macrophage activation plays a dual role in response to brain injury after a stroke, promoting early neuroinflammation and benefit for neurovascular recovery. Therefore, the dynamics of stroke-induced cerebral microglial/macrophage activation are of substantial interest. This study used novel anti-Iba-1-targeted superparamagnetic iron-platinum (FePt) nanoparticles in conjunction with magnetic resonance imaging (MRI) to measure the spatiotemporal changes of the microglial/macrophage activation in living rat brain for four weeks post-stroke. Ischemic lesion areas were identified and measured using T2-weighted MR images. After injection of the FePt-nanoparticles, T2*-weighted MR images showed that the nanoparticles were seen solely in brain regions that coincided with areas of active microglia/macrophages detected by post-mortem immunohistochemistry. Good agreement in morphological and distributive dynamic changes was also observed between the Fe+-cells and the Iba-1+-microglia/macrophages. The spatiotemporal changes of nanoparticle detected by T2*-weighted images paralleled the changes of microglial/macrophage activation and phenotypes measured by post-mortem immunohistochemistry over the four weeks post-stroke. Maximum microglial/macrophage activation occurred seven days post-stroke for both measures, and the diminished activation found after two weeks continued to four weeks. Our results suggest that nanoparticle-enhanced MRI may constitute a novel approach for monitoring the dynamic development of neuroinflammation in living animals during the progression and treatment of stroke.

A comparison of ferumoxytol with gadolinium as contrast agents for the diagnostic magnetic resonance imaging of osteomyelitis.

BACKGROUND: Ferumoxytol, an FDA-approved superparamagnetic iron oxide nanoparticle (SPION) preparation used for the treatment of iron deficiency anemia, is also known to be taken up by macrophages in areas of infection or inflammation, where it produces negative contrast changes on T2-weighted MR images. PURPOSE: We sought to compare Ferumoxytol-induced MRI contrast changes with those observed using standard-of-care Gadolinium in patients presenting with symptoms suggestive of osteomyelitis. SUBJECTS: Out of eighteen enrolled patients, 15 had MR imaging with both ferumoxytol and gadolinium. Based on clinical and/or pathologic criteria, 7 patients were diagnosed with osteomyelitis, 5 patients had osteomyelitis ruled out, and in 3 patients a definitive diagnosis could not be made. FIELD STRENGTH: 1.5 Tesla. SEQUENCES: Used included STIR, T1-weighted and T2-weighted spin echo. ASSESSMENT: The mean contrast changes upon ferumoxytol and gadolinium administration were measured from lesion regions of interest and compared with control regions. STATISTICAL TESTS: Student's t-test, propagation of errors. Data are reported as means ± S.E. RESULTS: The mean contrast changes, ΔC, associated with a diagnosis of osteomyelitis were found to be ΔCFe = -2.7 ± 0.7 when Ferumoxytol and T2w imaging sequences were used and ΔCGd = +3.1 ± 1.1 (P < 0.001) when Gadolinium and a T1w imaging sequence was used. The MRI contrast changes for both agents correlated with systemic markers of inflammation, such as the erythrocyte sedimentation rate. In patients without osteomyelitis, no significant contrast changes were observed in T2-weighted, Ferumoxytol-contrasted MRI. The macrophages in osteomyelitic lesions were found to take up at least 16 times as much iron as benign bone marrow. DATA CONCLUSION: We conclude that in terms of its MRI diagnostic accuracy for osteomyelitis Ferumoxytol-contrasted MRI is a promising approach for diagnosing osteomyelitis that merits further study.

Qß Virus-like particle-based vaccine induces robust immunity and protects against tauopathy.

Tauopathies, including frontotemporal dementia (FTD) and Alzheimer's disease (AD) are progressive neurodegenerative diseases clinically characterized by cognitive decline and could be caused by the aggregation of hyperphosphorylated pathological tau (pTau) as neurofibrillary tangles (NFTs) inside neurons. There is currently no FDA-approved treatment that cures, slows or prevents tauopathies. Current immunotherapy strategies targeting pTau have generated encouraging data but may pose concerns about scalability, affordability, and efficacy. Here, we engineered a virus-like particle (VLP)-based vaccine in which tau peptide, phosphorylated at threonine 181, was linked at high valency to Qß bacteriophage VLPs (pT181-Qß). We demonstrate that vaccination with pT181-Qß is sufficient to induce a robust and long-lived anti-pT181 antibody response in the sera and the brains of both Non-Tg and rTg4510 mice. Only sera from pT181-Qß vaccinated mice are reactive to classical somatodendritic pTau in human FTD and AD post-mortem brain sections. Finally, we demonstrate that pT181-Qß vaccination reduces both soluble and insoluble species of hyperphosphorylated pTau in the hippocampus and cortex, avoids a Th1-mediated pro-inflammatory cell response, prevents hippocampal and corpus callosum atrophy and rescues cognitive dysfunction in a 4-month-old rTg4510 mouse model of FTD. These studies provide a valid scientific premise for the development of VLP-based immunotherapy to target pTau and potentially prevent Alzheimer's diseases and related tauopathies.

Neonatal erythropoietin mitigates impaired gait, social interaction and diffusion tensor imaging abnormalities in a rat model of prenatal brain injury.

Children who are born preterm are at risk for encephalopathy of prematurity, a leading cause of cerebral palsy, cognitive delay and behavioral disorders. Current interventions are limited and none have been shown to reverse cognitive and behavioral impairments, a primary determinant of poor quality of life for these children. Moreover, the mechanisms of perinatal brain injury that result in functional deficits and imaging abnormalities in the mature brain are poorly defined, limiting the potential to target interventions to those who may benefit most. To determine whether impairments are reversible after a prenatal insult, we investigated a spectrum of functional deficits and diffusion tensor imaging (DTI) abnormalities in young adult animals. We hypothesized that prenatal transient systemic hypoxia-ischemia (TSHI) would induce multiple functional deficits concomitant with reduced microstructural white and gray matter integrity, and tested whether these abnormalities could be ameliorated using postnatal erythropoietin (EPO), an emerging neurorestorative intervention. On embryonic day 18 uterine arteries were transiently occluded for 60min via laparotomy. Shams underwent anesthesia and laparotomy for 60min. Pups were born and TSHI pups were randomized to receive EPO or vehicle via intraperitoneal injection on postnatal days 1 to 5. Gait, social interaction, olfaction and open field testing was performed from postnatal day 25-35 before brains underwent ex vivo DTI to measure fractional anisotropy, axial diffusivity and radial diffusivity. Prenatal TSHI injury causes hyperactivity, impaired gait and poor social interaction in young adult rats that mimic the spectrum of deficits observed in children born preterm. Collectively, these data show for the first time in a model of encephalopathy of prematurity that postnatal EPO treatment mitigates impairments in social interaction, in addition to gait deficits. EPO also normalizes TSHI-induced microstructural abnormalities in fractional anisotropy and radial diffusivity in multiple regions, consistent with improved structural integrity and recovery of myelination. Taken together, these results show behavioral and memory deficits from perinatal brain injury are reversible. Furthermore, resolution of DTI abnormalities may predict responsiveness to emerging interventions, and serve as a biomarker of CNS injury and recovery.

Extended Erythropoietin Treatment Prevents Chronic Executive Functional and Microstructural Deficits Following Early Severe Traumatic Brain Injury in Rats.

Survivors of infant traumatic brain injury (TBI) are prone to chronic neurological deficits that impose lifelong individual and societal burdens. Translation of novel interventions to clinical trials is hampered in part by the lack of truly representative preclinical tests of cognition and corresponding biomarkers of functional outcomes. To address this gap, the ability of a high-dose, extended, post-injury regimen of erythropoietin (EPO, 3000U/kg/dose × 6d) to prevent chronic cognitive and imaging deficits was tested in a postnatal day 12 (P12) controlled-cortical impact (CCI) model in rats, using touchscreen operant chambers and regional analysis of diffusion tensor imaging (DTI). Results indicate that EPO prevents functional injury and MRI injury after infant TBI. Specifically, subacute DTI at P30 revealed widespread microstructural damage that is prevented by EPO. Assessment of visual discrimination on a touchscreen operant chamber platform demonstrated that all groups can perform visual discrimination. However, CCI rats treated with vehicle failed to pass reversal learning, and perseverated, in contrast to sham and CCI-EPO rats. Chronic DTI at P90 showed EPO treatment prevented contralateral white matter and ipsilateral lateral prefrontal cortex damage. This DTI improvement correlated with cognitive performance. Taken together, extended EPO treatment restores executive function and prevents microstructural brain abnormalities in adult rats with cognitive deficits in a translational preclinical model of infant TBI. Sophisticated testing with touchscreen operant chambers and regional DTI analyses may expedite translation and effective yield of interventions from preclinical studies to clinical trials. Collectively, these data support the use of EPO in clinical trials for human infants with TBI.

1H NMR Detection of superparamagnetic nanoparticles at 1T using a microcoil and novel tuning circuit.

Magnetic beads containing superparamagnetic iron oxide nanoparticles (SPIONs) have been shown to measurably change the nuclear magnetic resonance (NMR) relaxation properties of nearby protons in aqueous solution at distances up to approximately 50 microm. Therefore, the NMR sensitivity for the in vitro detection of single cells or biomolecules labeled with magnetic beads will be maximized with microcoils of this dimension. We have constructed a prototype 550 microm diameter solenoidal microcoil using focused gallium ion milling of a gold/chromium layer. The NMR coil was brought to resonance by means of a novel auxiliary tuning circuit, and used to detect water with a spectral resolution of 2.5 Hz in a 1.04 T (44.2MHz) permanent magnet. The single-scan SNR for water was 137, for a 200 micros pi/2 pulse produced with an RF power of 0.25 mW. The nutation performance of the microcoil was sufficiently good so that the effects of magnetic beads on the relaxation characteristics of the surrounding water could be accurately measured. A solution of magnetic beads (Dynabeads MyOne Streptavidin) in deionized water at a concentration of 1000 beads per nL lowered the T(1) from 1.0 to 0.64 s and the T2 * from 110 to 0.91 ms. Lower concentrations (100 and 10 beads/nL) also resulted in measurable reductions in T2 *, suggesting that low-field, microcoil NMR detection using permanent magnets can serve as a high-sensitivity, miniaturizable detection mechanism for very low concentrations of magnetic beads in biological fluids.

Nuclear magnetic resonance-based screening methods for drug discovery.

Nuclear magnetic resonance (NMR) techniques are widely used in the drug discovery process. The primary feature exploited in these investigations is the large difference in mass between drugs and receptors (usually proteins) and the effect that this has on the rotational or translational correlation times for drugs bound to their targets. Many NMR parameters, such as the diffusion coefficient, spin diffusion, nuclear Overhauser enhancement, and transverse and longitudinal relaxation times, are strong functions of either the overall tumbling or translation of molecules in solution. This has led to the development of a wide variety of NMR techniques applicable to the elucidation of protein and nucleic acid structure in solution, the screening of drug candidates for binding to a target of choice, and the study of the conformational changes that occur in a target on drug binding. High-throughput screening by NMR methods has recently received a boost from the introduction of sophisticated computational techniques for reducing the time needed for the acquistion of the primary NMR data for multidimensional studies.

Quantitative [Fe]MRI of PSMA-targeted SPIONs specifically discriminates among prostate tumor cell types based on their PSMA expression levels.

We report the development, experimental verification, and application of a general theory called [Fe]MRI (pronounced fem-ree) for the non-invasive, quantitative molecular magnetic resonance imaging (MRI) of added magnetic nanoparticles or other magnetic contrast agents in biological tissues and other sites. [Fe]MRI can easily be implemented on any MRI instrument, requiring only measurements of the background nuclear magnetic relaxation times (T1, T2) of the tissue of interest, injection of the magnetic particles, and the subsequent acquisition of a pair of T1-weighted and T2-weighted images. These images, converted into contrast images, are subtracted to yield a contrast difference image proportional to the absolute nanoparticle, iron concentration, ([Fe]) image. [Fe]MRI was validated with the samples of superparamagnetic iron oxide nanoparticles (SPIONs) both in agarose gels and bound to human prostate tumor cells. The [Fe]MRI measurement of the binding of anti-prostate specific membrane antigen (PSMA) conjugated SPIONs to PSMA-positive LNCaP and PSMA-negative DU145 cells in vitro allowed a facile discrimination among prostate tumor cell types based on their PSMA expression level. The low [Fe] detection limit of ~2 µM for SPIONs allows sensitive MRI of added iron at concentrations considerably below the US Food and Drug Administration's human iron dosage guidelines (<90 µM, 5 mg/kg).

Transient increase of fractional anisotropy in reversible vasogenic edema.

Brain vasogenic edema, involving disruption of the blood-brain barrier, is a common pathological condition in several neurological diseases, with a heterogeneous prognosis. It is sometimes reversible, as in posterior reversible encephalopathy syndrome, but often irreversible and our current clinical tools are insufficient to reveal its reversibility. Here, we show that increased fractional anisotropy in magnetic resonance imaging is associated with the reversibility of vasogenic edema. Spontaneously, hypertensive rats-stroke prone demonstrated posterior reversible encephalopathy syndrome-like acute encephalopathy in response to high-dose cyclosporine A treatment; the deteriorating neurological symptoms and worsening scores in behavioral tests, which were seen in acute phase, dissappered after recovery by cessation of cyclosporine A. In the acute phase of encephalopathy, the fractional anisotropy and apparent diffusion coefficient increased in areas with IgG leakage. This increase of fractional anisotropy occurred in the absence of demyelination: fluid leakage into the myelinated space increased the axial, but not the radial, diffusivity, resulting in the increased fractional anisotropy. This increased fractional anisotropy returned to pre-encephalopathy values in the recovery phase. Our results highlight the importance of the fractional anisotropy increase as a marker for the reversibility of brain edema, which can delineate the brain areas for which recovery is possible.

Imaging and serum biomarkers reflecting the functional efficacy of extended erythropoietin treatment in rats following infantile traumatic brain injury.

OBJECTIVE Traumatic brain injury (TBI) is a leading cause of death and severe morbidity for otherwise healthy full-term infants around the world. Currently, the primary treatment for infant TBI is supportive, as no targeted therapies exist to actively promote recovery. The developing infant brain, in particular, has a unique response to injury and the potential for repair, both of which vary with maturation. Targeted interventions and objective measures of therapeutic efficacy are needed in this special population. The authors hypothesized that MRI and serum biomarkers can be used to quantify outcomes following infantile TBI in a preclinical rat model and that the potential efficacy of the neuro-reparative agent erythropoietin (EPO) in promoting recovery can be tested using these biomarkers as surrogates for functional outcomes. METHODS With institutional approval, a controlled cortical impact (CCI) was delivered to postnatal Day (P)12 rats of both sexes (76 rats). On postinjury Day (PID)1, the 49 CCI rats designated for chronic studies were randomized to EPO (3000 U/kg/dose, CCI-EPO, 24 rats) or vehicle (CCI-veh, 25 rats) administered intraperitoneally on PID1-4, 6, and 8. Acute injury (PID3) was evaluated with an immunoassay of injured cortex and serum, and chronic injury (PID13-28) was evaluated with digitized gait analyses, MRI, and serum immunoassay. The CCI-veh and CCI-EPO rats were compared with shams (49 rats) primarily using 2-way ANOVA with Bonferroni post hoc correction. RESULTS Following CCI, there was 4.8% mortality and 55% of injured rats exhibited convulsions. Of the injured rats designated for chronic analyses, 8.1% developed leptomeningeal cyst-like lesions verified with MRI and were excluded from further study. On PID3, Western blot showed that EPO receptor expression was increased in the injured cortex (p = 0.008). These Western blots also showed elevated ipsilateral cortex calpain degradation products for αII-spectrin (αII-SDPs; p < 0.001), potassium chloride cotransporter 2 (KCC2-DPs; p = 0.037), and glial fibrillary acidic protein (GFAP-DPs; p = 0.002), as well as serum GFAP (serum GFAP-DPs; p = 0.001). In injured rats multiplex electrochemiluminescence analyses on PID3 revealed elevated serum tumor necrosis factor alpha (TNFα p = 0.01) and chemokine (CXC) ligand 1 (CXCL1). Chronically, that is, in PID13-16 CCI-veh rats, as compared with sham rats, gait deficits were demonstrated (p = 0.033) but then were reversed (p = 0.022) with EPO treatment. Diffusion tensor MRI of the ipsilateral and contralateral cortex and white matter in PID16-23 CCI-veh rats showed widespread injury and significant abnormalities of functional anisotropy (FA), mean diffusivity (MD), axial diffusivity (AD), and radial diffusivity (RD); MD, AD, and RD improved after EPO treatment. Chronically, P13-P28 CCI-veh rats also had elevated serum CXCL1 levels, which normalized in CCI-EPO rats. CONCLUSIONS Efficient translation of emerging neuro-reparative interventions dictates the use of age-appropriate preclinical models with human clinical trial-compatible biomarkers. In the present study, the authors showed that CCI produced chronic gait deficits in P12 rats that resolved with EPO treatment and that chronic imaging and serum biomarkers correlated with this improvement.

Design and structure of peptide and peptidomimetic antagonists of protein-protein interaction.

Peptides based on the amino acid sequences found at protein-protein interaction sites make excellent leads for antagonist development. A statistical picture of amino acids involved in protein-protein interactions indicates that proteins recognize and interact with one another through the restricted set of specialized interface amino acid residues, Pro, Ile, Tyr, Trp, Asp and Arg. These amino acids represent residues from each of the three classes of amino acids, hydrophobic, aromatic and charged, with one anionic and one cationic residue at neutral pH. The use of peptides as drug leads has been successfully used to search for antagonists of cell-surface receptors. Peptide, peptidomimetic, and non-peptide organic inhibitors of a class of cell surface receptors, the integrins, currently serve as therapeutic and diagnostic imaging agents. In this review, we discuss the structural features of protein-protein interactions as well as the design of peptides, peptidomimetics, and small organic molecules for the inhibition of protein-protein interactions. Information gained from studying inhibitors of integrin functions is now being applied to the design and testing of inhibitors of other protein-protein interactions. Most drug development progress in the past several decades has been made using the enzyme binding-pocket model of drug targets. Small molecules are designed to fit into the substrate-binding pockets of proteins based on a lock-and-key, induced-fit, or conformational ensemble model of the protein binding site. Traditionally, enzymes have been used as therapeutic drug targets because it was easier to develop rapid, sensitive screening assays, and to find low molecular weight inhibitors that blocked the active site. However, for proteins which interact with other proteins, rather than with small substrate molecules, the lack of binding pockets means that this approach will not generally succeed. There exist many diseases in which the inhibition of protein-protein interactions would provide therapeutic benefit, but there are no general methods available to address such problems. The focus of the first part of this review is to discuss the features of protein-protein interactions which may serve as general guidelines for the development and design of inhibitors for protein-protein interactions. In the second part we focus on the design of peptides (lead compounds) and their conversion into peptidomimetics or small organic molecules for the inhibition of protein-protein interactions. We draw examples from the important and emerging area of integrin-based cell adhesion and show how the principles of protein-protein interactions are followed in the discovery, optimization and usage of specific protein interface peptides as drug leads.