Effects of FGFR inhibitors TKI258, BGJ398 and AZD4547 on breast cancer cells in 2D, 3D and tissue explant cultures.

PURPOSE: Fibroblast growth factor receptors (FGFR) and pathways are important players in breast cancer (BC) development. They are commonly altered, and BCs exhibiting FGFR gene amplification are currently being studied for drug development. Here, we aimed to compare the effects of three FGFR inhibitors (FGFRis), i.e., non-selective TKI258 and selective BGJ398 and AZD4547, on different BC-derived cell lines (BCCs) and primary tissues. METHODS: The human BCCs MCF-7 and MDA-MB-231(SA) (wild-type FGFR) and MFM223 (amplified FGFR1 and FGFR2) were analyzed for FGFR expression using qRT-PCR, and the effects of FGFRis on FGFR signaling by Western blotting. The effects of FGFRis on proliferation, viability, migration and invasion of BCCs were assessed in 2D cultures using live-cell imaging, and in 3D cultures using phenotypic analysis of organoids. To study radio-sensitization, FGFRi treatment was combined with irradiation. Patient-derived BC samples were treated with FGFRis in explant cultures and immunostained for Ki67 and cleaved caspase 3. RESULTS: We found that all FGFRis tested decreased the growth and viability of BC cells in 2D and 3D cultures. BGJ398 and AZD4547 were found to be potent at low concentrations in FGFR-amplified MFM233 cells, whereas higher concentrations were required in non-amplified MCF7 and MDA-MB-231(SA) cells. TKI258 inhibited the migration and invasion, whereas BGJ398 and AZD4547 only inhibited the invasion of MDA-MB-231(SA) cells. FGFRi treatment of MCF7 and MFM223 cells enhanced the inhibitory effect of radiotherapy, but this effect was not observed in MDA-MB-231(SA) cells. FGFRi-treated primary BC explants with moderate FGFR levels showed a tendency towards decreased proliferation and increased apoptosis. CONCLUSIONS: Our results indicate that, besides targeting FGFR-amplified BCs with selective FGFRis, also BCs without FGFR amplification/activation may benefit from FGFRi-treatment. Combination with other treatment modalities, such as radiotherapy, may allow the use of FGFRis at relatively low concentrations and, thereby, contribute to better BC treatment outcomes.

Quantitative trait loci affecting milk production traits in Finnish Ayrshire dairy cattle.

A whole genome scan of Finnish Ayrshire was conducted to map quantitative trait loci (QTL) affecting milk production. The analysis included 12 half-sib families containing a total of 494 bulls in a granddaughter design. The families were genotyped with 150 markers to construct a 2764 cM (Haldane) male linkage map. In this study interval mapping with multiple-marker regression approach was extended to analyse multiple chromosomes simultaneously. The method uses identified QTL on other chromosomes as cofactors to increase mapping power. The existence of multiple QTL on the same linkage group was also analyzed by fitting a two-QTL model to the analysis. Empirical values for chromosome-wise significance thresholds were determined using a permutation test. Two genome-wise significant QTL were identified when chromosomes were analyzed individually, one affecting fat percentage on chromosome (BTA) 14 and another affecting fat yield on BTA12. The cofactor analysis revealed in total 31 genome-wise significant QTL. The result of two-QTL analysis suggests the existence of two QTL for fat percentage on BTA3. In general, most of the identified QTL confirm results from previous studies of Holstein-Friesian cattle. A new QTL for all yield components was identified on BTA12 in Finnish Ayrshire.

Water diffusion changes in Wallerian degeneration and their dependence on white matter architecture.

This study investigates water diffusion changes in Wallerian degeneration. We measured indices derived from the diffusion tensor (DT) and T2-weighted signal intensities in the descending motor pathways of patients with small chronic lacunar infarcts of the posterior limb of the internal capsule on one side. We compared these measurements in the healthy and lesioned sides at different levels in the brainstem caudal to the primary lesion. We found that secondary white matter degeneration is revealed by a large reduction in diffusion anisotropy only in regions where fibers are arranged in isolated bundles of parallel fibers, such as in the cerebral peduncle. In regions where the degenerated pathway crosses other tracts, such as in the rostral pons, paradoxically there is almost no change in diffusion anisotropy, but a significant change in the measured orientation of fibers. The trace of the diffusion tensor is moderately increased in all affected regions. This allows one to differentiate secondary and primary fiber loss where the increase in trace is considerably higher. We show that DT-MRI is more sensitive than T2-weighted MRI in detecting Wallerian degeneration. Significant diffusion abnormalities are observed over the entire trajectory of the affected pathway in each patient. This finding suggests that mapping degenerated pathways noninvasively with DT-MRI is feasible. However, the interpretation of water diffusion data is complex and requires a priori information about anatomy and architecture of the pathway under investigation. In particular, our study shows that in regions where fibers cross, existing DT-MRI-based fiber tractography algorithms may lead to erroneous conclusion about brain connectivity.

Evidence for neuroaxonal injury in patients with proteolipid protein gene mutations.

The authors used proton MRS to investigate neuropathologic correlates in nine patients with proteolipid protein (PLP) gene mutations who did not show cerebral atrophy on cranial MRI. When compared with 16 age-matched control participants, patients with PLP mutations had significant and widespread decreased brain N-acetyl aspartate, a neuronal marker. The authors conclude that PLP mutations cause neuroaxonal injury, which in turn contributes to the neurologic deficit observed in these patients.

Spectroscopic imaging of radiation-induced effects in the white matter of glioma patients.

External radiation therapy of brain tumors may cause adverse effects on normal brain tissue, resulting in severe neuropsychological and cognitive impairment. We investigated the late delayed radiation effects in the white matter (WM) using (1)H magnetic resonance spectroscopic imaging ((1)HMRSI). Nine glioma patients with local radiation-induced signal abnormalities in the T(2)-weighted MR images were studied with nine age- and sex-matched controls. The metabolite ratios in the radiation-induced hyper intensity area (RIHA) and in the normal appearing white matter (NAWM) of the patients were compared with respective WM areas of the controls. In RIHA, choline/creatine (Cho/Cr) was 17% decreased (1.22 +/- 0.13 vs 1.47 +/- 0.16, p = 0.0027, significant (s), unpaired Student's t test with Bonferroni correction) in the patients compared to the controls, while there was no difference in N-acetyl aspartate/Cr (NAA/Cr) (2.49 +/- 0.57 vs 2.98 +/- 0.32, p = 0.039) or NAA/Cho (2. 03 +/- 0.40 vs 2.04 +/- 0.17, p = 0.95). In NAWM, Cho/Cr was 24% decreased (1.21 +/- 0.15 vs 1.59 +/- 0.13, p < 0.0001, s) and NAA/Cho was 20% increased (2.49 +/- 0.49 vs 1.98 +/- 0.15, p = 0. 0082, s) in the patients compared to the controls, while there was no difference in NAA/Cr (2.99 +/- 0.46 vs 3.16 +/- 0.32, p = 0.38). NAA(RIHA)/NAA(NAWM) was 25% decreased (0.75 +/- 0.20 vs 1.00 +/- 0. 12, p = 0.0043, s) and Cr(RIHA)/Cr(NAWM) was 16% decreased (0.89 +/- 0.15 vs 1.06 +/- 0.10, p = 0.013, s) in the patients compared to the controls, while there was no difference in Cho(RIHA)/Cho(NAWM) (0.92 +/- 0.23 vs 0.98 +/- 0.10, p = 0.47). (1)HMRSI reveals widespread chemical changes in the WM after radiation therapy. In RIHA, there is loss of NAA, Cho, and Cr implying axonal and membrane damage and in NAWM, there is loss of Cho, reflecting membrane damage.

Visualizing and characterizing white matter fiber structure and architecture in the human pyramidal tract using diffusion tensor MRI.

We used diffusion tensor imaging to assess diffusion anisotropy in the pyramidal tract in ten young, and ten elderly subjects (five males and five females in each group). The purpose of this study was to define normative values for anisotropy at different anatomic levels of the brainstem as well as to assess differences due to age, gender, and laterality. In all subjects, anisotropy was highest in the cerebral peduncle, lowest in the caudal pons, and intermediate in the medulla. In the pons and medulla the regional variability was high, with significant differences in anisotropy even between contiguous slices. Multifactorial ANOVA (performed using the average value of anisotropy within each region of interest) revealed that elderly subjects had significantly lower values than young subjects in the cerebral peduncle, with no differences in the pons and medulla. No significant differences in anisotropy due to gender and side were found. The differences in anisotropy at different levels of the brainstem reflect differences in the local architecture of white matter fibers. Anisotropy is high in the cerebral peduncle because fibers have a highly ordered arrangement, while in the pons and medulla, anisotropy is lower because the local fiber architecture is less coherent due to the presence of other fibers and nuclei. The biologic meaning of the intergroup differences in anisotropy is discussed in light of the structure and architecture of the tissue under investigation. We also consider potential sources of artifacts, such as noise and motion, partial volume contamination, anatomic mismatching, and the use of inappropriate statistical tests. We conclude that the age-related decrease in anisotropy in the cerebral peduncle is not artifactual but rather reflects subtle structural changes of the aging white matter. Our study however shows that caution must be exercised in interpreting diffusion anisotropy data.

T1rho dispersion of rat tissues in vitro.

The purpose of this study was to demonstrate T1rho dispersion in different rat tissues (liver, brain, spleen, kidney, heart, and skeletal muscle), and to compare the 1/T1rho data to previous 1/T1 data and magnetization transfer of rat tissues at low (0.1 T) B0 field. The 1/T1rho dispersion showed a fairly similar pattern in all tissues. The highest 1/T1rho relaxation rates were seen in liver and muscle followed by heart, whereas the values for spleen, kidney, and brain were quite similar. Compared to 1/T2 relaxation rate, the greatest difference was seen in liver and muscle. The rank order 1/T1rho value at each locking field B1 was the same as the transfer rate of magnetization from the water to the macromolecular pool (Rwm) for liver, muscle, heart, and brain. The potential value T1rho imaging is to combine high T1 contrast of low field imaging with the high signal to noise ratio of high static field imaging. When the T1rho value for a given tissue is known, the contrast between different tissues can be optimized by adjusting the locking time TL. Further studies are encouraged to fully exploit this. Targets for more detailed research include brain infarct, brain and liver tumors.

Diffuse central neuronal involvement in Fabry disease: a proton MRS imaging study.

BACKGROUND: The in vivo determination of parenchymal involvement is important to evaluate disease burden. Proton MRS imaging (1H-MRSI) permits simultaneous measurement of N-acetylaspartate (NA), a putative neuron-specific molecule, choline-containing compounds, creatine-phosphocreatine, and lactate from four 15-mm slices divided into 0.84-mL single-volume elements. OBJECTIVE: To assess the cortical and subcortical neuropathology in Fabry disease (FD). METHODS: Regions of interest (ROIs) were selected from several cortical and subcortical locations in nine FD patients. Mean ROI metabolite ratios were compared with control values. RESULTS: FD patients showed a widespread pattern of cortical and subcortical NA reduction. Seven patients showed discrete MRI abnormalities consisting of white matter hyperintensities or basal ganglia infarcts. CONCLUSION: We found diffuse neuronal involvement in FD extending beyond the areas of MRI-visible cerebrovascular abnormalities. 1H-MRSI may become useful in therapeutic trials.

Low field T1rho imaging of myositis.

The purpose of this study was to evaluate 1/T1rho in relation to 1/T1 and 1/T2 in characterizing normal and diseased muscle. We measured the muscle relaxation rates 1/T1 and 1/T2 at 0.1 T and 1/T1rho at on-resonance locking fields B1 between 10 and 160 microT in myositis patients and normal volunteers. 1/T2 and 1/T1rho of muscle were lower in the patients than in the volunteers, whereas there was no difference in the 1/T1 values. The lower relaxation rates 1/T2 and 1/T1rho in the diseased muscle may be due to fat and connective tissue infiltrations and edema. 1/T1rho contrast between muscle and subcutaneous fat was higher than 1/T2 and 1/T1 contrast. This may be explained by the different B1 dispersion behavior of these two tissue types. 1/T1rho of fat is B1 field independent, whereas 1/T1rho of muscle decreases clearly with increasing B1 field. In conclusion, 1/T1rho provides a useful tool in manipulating contrast in magnetic resonance imaging of diseased muscle.

T1 rho MR imaging characteristics of human anterior tibial and gastrocnemius muscles.

RATIONALE AND OBJECTIVES: The authors evaluated the value of T1 rho in relation to T1 and T2 in the characterization of human muscles. MATERIALS AND METHODS: The authors studied the effect of muscle type (anterior tibial [AT] and gastrocnemius [GC]), sex, and age on 1/T1 and 1/T2 at 0.1 T and on 1/ T1 rho at locking-field B1s (spin-locking radio-frequency magnetic induction field) of 10-160 microT in 38 healthy volunteers. The contrast-to-noise ratio (CNR) between muscle and fat was evaluated with different T1-, T2-, and T1 rho-weighted magnetic resonance (MR) sequences. RESULTS: The 1/T1, 1/T2, and 1/T1 rho were slightly higher in AT than in GC muscles. The 1/T2 and 1/T1 rho of AT muscles showed a sex dependence, whereas no correlation with age was found. The CNR of the T1 rho-weighted images did not markedly differ from that of the T1- and T2-weighted images. CONCLUSION: T1 rho is as sensitive as T2 to the composition of muscle, whereas T1 is less sensitive. In MR imaging of normal muscle, T1 rho and T2 provide a relatively similar tissue contrast.

T1rho of protein solutions at very low fields: dependence on molecular weight, concentration, and structure.

The effect of molecular weight, concentration, and structure on 1/T1rho, the rotating frame relaxation rate, was investigated for several proteins using the on-resonance spin-lock technique, for locking fields B1 < 200 microT. The measured values of 1/T1rho were fitted to a simple theoretical model to obtain the dispersion curves 1/T1rho(omega1) and the relaxation rate at zero B1 field, 1/T1rho(0). 1/T1rho was highly sensitive to the molecular weight, concentration, and structure of the protein. The amount of intra- and intermolecular hydrogen and disulfide bonds especially contributed to 1/T1rho. In all samples, 1/T1rho(0) was equal to 1/T2 measured at the main magnetic field Bo = 0.1 T, but at higher locking fields the dispersion curves monotonically decreased. The results of this work indicate that a model considering the effective correlation time of molecular motions as the main determinant for T1rho relaxation in protein solutions is not valid at very low B1 fields. The underlying mechanism for the relaxation rate 1/T1rho at B1 fields below 200 microT is discussed.

A prospective radiologic and neurologic follow-up study of 61 HIV-1 -infected subjects: early beginning and slow progression of brain atrophy.

The course of the organic brain disease caused by human immunodeficency virus (HIV-1) was evaluated in a follow-up study. The primary material included 200 consecutive HIV-1 infected persons. Sixty-one subjects, in whom other brain-affecting factors were excluded, consented to the follow-up. They underwent 278 radiologic examinations: computed tomography, magnetic resonance imaging, or a combination of both (mean 4.6 examinations/subject). Clinical neurologic status and, in 40 subjects, cognitive performance were repeatedly evaluated. Sixteen subjects were followed up until death and 11 of them were autopsied. Median follow-up time was 27 mo (range 2.5-66 mo). The most common radiologic finding was atrophy, found in 19 subjects at study entry and developing in 10 subjects during the study. Twenty-four subjects (39%) showed the development and/or progression of atrophy. Atrophic changes progressed most rapidly in acquired immunodeficiency syndrome (AIDS), but mild developing/progressive atrophy was found even in 33% of asymptomatic or neurologically intact subjects. Cognitive and radiologic worsening were simultaneous in 6/7 subjects with declining neuropsychologic test performance. Signal intensity changes including HIV-1 leukoencephalopathy appeared in AIDS patients with clear cognitive decline.

Magnetization transfer of pure DNA and purified sperm nuclei.

Magnetization transfer (MT) imaging provides a novel opportunity to characterize interactions between tissue water and macromolecules. Although several in vitro investigations have shown that proteins and lipids are important determinants of MT, the contribution of DNA is still unknown. This study was designed to determine whether DNA and cell nuclear material exhibit MT. We measured the magnetization transfer effect of pure DNA strands and purified bovine sperm head nuclei. Although no transfer of magnetization could be detected in samples of pure DNA strands, the sperm head nuclei exhibited a strong MT effect that increased with increasing solid content of the samples. Since the purified bovine sperm head samples consist of large nuclei with only minor traces of perinuclear matrix, the measured MT effect arises from the chromatin of the nuclei. The DNA fills 90% of the nuclear volume and it is extremely tightly packed as chromatin fibers by nucleoproteins. We hypothesize that the numerous intra- and intermolecular disulfide bonds that stabilize the chromatin fibers restrict the movement of the surface water binding sites of both DNA and protamines and thus facilitate the transfer of magnetization. Therefore, the results indicate that the amount of nuclear material may positively contribute to MT in tissues.

Magnetization transfer in protein solutions at 0.1 T: dependence on concentration, molecular weight, and structure.

RATIONALE AND OBJECTIVES: We observed the magnetization transfer rates in a variety of protein solutions at 0.1-T magnetic field and compared our results with previous investigations at high magnetic fields (> 0.5 T). The effects of protein concentration, size, pH, denaturation, cross linking, and fiber formation were investigated. METHODS: We used the saturation transfer technique to determine the transfer of magnetization in gamma globulin, fibronectin, collagen, fibrinogen, and albumin solutions. RESULTS: The observed transfer rate increased with increasing concentration and size of the protein. Protein degradation decreased the transfer rate. Cross linking and fiber formation each increased the transfer rate, whereas buffer pH had no effect. CONCLUSION: Protein denaturation, aggregation, and fiber formation are important determinants of magnetization transfer in vitro. The size, concentration, and cross linking of the proteins contribute strongly to the transfer of magnetization at low fields, and the effect seems to be at least as important as at the higher fields.

Zidovudine reduces intrathecal immunoactivation in patients with early human immunodeficiency virus type 1 infection.

OBJECTIVE: To evaluate the effect of zidovudine on human immunodeficiency virus type 1 (HIV-1)-associated central nervous system infection in Centers for Disease Control and Prevention stage II or III disease. DESIGN: In an open-ended trial, patients received 500 mg of zidovudine twice a day for 12 months. Lumbar punctures, neurological, neuropsychological, and neuroradiological examinations were repeatedly performed during the trial period and were compared with pretrial values. In 11 patients post-trial neurological follow-up of 10 to 20 months was performed. PATIENTS: Initially, 14 volunteers with stage II or III disease and intrathecal synthesis of HIV-1-specific antibodies were enrolled. Additionally, patients had slight neuropsychological disturbance or brain atrophy unrelated to other agents than HIV-1. Two patients dropped out because of poor compliance. MAIN OUTCOME MEASURES: Intrathecal and systemic immune and virological responses, cognitive performance, and brain images were repeatedly monitored. RESULTS: After 6 weeks of zidovudine therapy, initial low-grade pleocytosis and elevated levels of beta 2-microglobulin, both in cerebrospinal fluid and in serum samples, declined. Intrathecal HIV-1 antibody synthesis could no longer be detected in half of the patients after 12 months of zidovudine therapy. Patients with defective cognition transiently improved cognitive speed and flexibility after 6 months of therapy. Slight atrophic brain changes, however, remained unchanged. CONCLUSIONS: Zidovudine reduces intrathecal immuno-activation and transiently improves cognitive functioning in HIV-1-infected subjects who show evidence of central nervous system involvement by HIV-1 but are otherwise asymptomatic.

Radiological study of the brain at various stages of human immunodeficiency virus infection: early development of brain atrophy.

One hundred and one persons infected with human immunodeficiency virus (HIV-1), in whom other central nervous system infections or diseases were excluded, underwent brain CT and/or MRI at various stages of HIV-1 infection: 29 were asymptomatic (ASX), 35 had lymphadenopathy syndrome (LAS), 17 had AIDS-related complex (ARC), and 20 had AIDS. A control group of 32 HIV-1-seronegative healthy persons underwent brain MRI. The most common finding was brain atrophy, found in 9% of controls, and 31% of ASX cases, 29% of LAS, 59% of ARC and 70% of AIDS. Even the difference between the ASX or LAS groups and controls was significant. The changes were bilateral and symmetrical, and they were more severe at later stages of infection. Infratentorial atrophy was seen in the early stages; supratentorial atrophy became more pronounced at ARC, and generalized atrophy was typical of AIDS. Non-specific small hyperintense foci were found on MRI in 13% of controls and 6-15% of the infected groups. Larger, diffuse, bilateral white matter infiltrates were detected in 4 demented patients with AIDS. Four patients with AIDS and 1 with LAS had focal hyperintense lesions in the internal capsules, lentiform nuclei or thalamus, often bilateral on MRI. One patient with AIDS, examined with CT only, had low density in the lentiform nucleus. Loss of brain parenchyma can occur at an early stage of HIV-1 infection, and the atrophic process becomes more intense at later stages (ARC and AIDS). Parenchymal infiltration, seen as hyperintense areas on MRI, is most often associated with severe clinical symptoms, in the later stages of the disease.

Mild brain atrophy in early HIV infection: the lack of association with cognitive deficits and HIV-specific intrathecal immune response.

Brain MRI and/or CT were performed on 72 HIV-infected patients at various stages of the disease, and on 34 controls. The neuroradiological findings were related to duration of the infection, neurological symptoms, and cognitive abnormalities as well as to immunological findings in the CSF and blood. All types of brain atrophy were more severe and more frequent in HIV-infected subjects than in controls. Patients with neurological symptoms, those with advanced HIV infection, and patients with a duration of HIV infection of more than 4 years showed the most severe and most frequent neuroradiological abnormalities, including central and cortical atrophy, brain stem atrophy, and cerebellar atrophy. Subjects with cognitive defects exhibited more severe central atrophy than cognitively intact patients. However, slight brain atrophy and/or parenchymal lesions were found in 57% of cognitively intact HIV-seropositive individuals. Patients with brain atrophy and those with radiologically normal brain, both showed increased intrathecal synthesis of total IgG, and intrathecal HIV-antibody synthesis. However, a declined general immune response and a lowered CSF leukocyte count were seen predominantly in patients with brain atrophy. The results suggest that subcortical, neurologically "silent" areas of brain white matter are an early target of HIV infection.