Engineering attenuated virulence of a Theileria annulata-infected macrophage.

Live attenuated vaccines are used to combat tropical theileriosis in North Africa, the Middle East, India, and China. The attenuation process is empirical and occurs only after many months, sometimes years, of in vitro culture of virulent clinical isolates. During this extensive culturing, attenuated lines lose their vaccine potential. To circumvent this we engineered the rapid ablation of the host cell transcription factor c-Jun, and within only 3 weeks the line engineered for loss of c-Jun activation displayed in vitro correlates of attenuation such as loss of adhesion, reduced MMP9 gelatinase activity, and diminished capacity to traverse Matrigel. Specific ablation of a single infected host cell virulence trait (c-Jun) induced a complete failure of Theileria annulata-transformed macrophages to disseminate, whereas virulent macrophages disseminated to the kidneys, spleen, and lungs of Rag2/γC mice. Thus, in this heterologous mouse model loss of c-Jun expression led to ablation of dissemination of T. annulata-infected and transformed macrophages. The generation of Theileria-infected macrophages genetically engineered for ablation of a specific host cell virulence trait now makes possible experimental vaccination of calves to address how loss of macrophage dissemination impacts the disease pathology of tropical theileriosis.

Netrin-4 promotes mural cell adhesion and recruitment to endothelial cells.

Netrins are secreted molecules involved in axon guidance and angiogenesis. We previously showed that Netrin-4 acts as an anti-angiogenic factor by inhibiting endothelial cell (EC) functions. In this study, we investigated the effects of Netrin-4 on vascular smooth muscle cell (VSMC) activity in vitro and in vivo. We show that exogenous Netrin-4 stimulated VSMC adhesion and migration, and increased their coverage on EC tubes (grown on a Matrigel substrate). siRNA knock-down of endogenous Netrin-4 expression in VSMC decreased their recruitment to EC tubes. VSMC expressed Netrin-4 and three of the six Netrin-1 cognate receptors: DCC, Neogenin, and Unc5B. Silencing of these receptors reduced Netrin-4 adhesion to VSMC, strongly suggesting that these receptors were involved in the recruitment process. We previously showed that Netrin-4 overexpression in PC3 cancer cells delayed tumor growth in a model of subcutaneous xenograft by reducing tumor vessel density. Here, we show that Netrin-4 overexpression improved tumor blood vessel structure and increased VSMC coverage. Thus, Netrin-4 induced mural cell recruitment may play a role in the inhibition of tumor growth. Our data suggest that Netrin-4 is important for blood vessel normalization through the regulation of both endothelial and perivascular cells.

Maternofetal pharmacokinetics of a gadolinium chelate contrast agent in mice.

PURPOSE: To determine the maternofetal pharmacokinetics of gadoterate meglumine in mice during the first 48 hours following maternal intravenous injection of a high dose of 0.5 mmol of gadolinium per kilogram. MATERIALS AND METHODS: All the studies complied with French law and the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. Balb/C mice (n = 23) at 16 days of gestation were examined for 48 hours after maternal intravenous administration of 0.5 mmol gadolinium per kilogram of gadoterate meglumine. Gadolinium concentration in the placentas, fetuses, and amniotic fluid was determined by using mass spectrometry, and the total placental and fetal gadolinium content was calculated. Gadoterate meglumine half-life in the different compartments was estimated with one- and two-compartment models. Kruskal-Wallis and Wilcoxon signed-rank tests were used to compare the pharmacokinetic profiles. RESULTS: Gadoterate meglumine passed the placental barrier, entering the fetuses and amniotic fluid before being redistributed back to the mother. The placental gadolinium concentration showed two-compartmental decay, with a first half-life of distribution of 47 minutes and a second half-life of elimination of 107 hours. The half-lives in the fetuses and amniotic fluid were, respectively, 4 and 5 hours and followed a monocompartmental model after the initial peak. The maximal gadolinium fetal concentration (31.8 nmol/g) was observed 30 minutes after injection, which corresponded to a total fetal content of 0.077% of the injected dose. CONCLUSION: In mice, gadoterate meglumine, an extracellular nonspecific gadolinium chelate contrast medium, passed the placenta before being redistributed back to the mother, resulting in undetectable fetal concentrations after 48 hours.

New cerebellar phenotypes in YAC transgenic mouse in vivo library of human Down syndrome critical region-1.

Down syndrome (DS) is the most frequent genetic cause of mental retardation (MR) associated with neurological alterations. To allow a genetic dissection of DS phenotype, we studied eight transgenic mouse lines carrying YACs containing human DNA fragments covering DS critical region (DCR-1), as an in vivo library. Herein, we found an increased brain size in the 152F7-mice containing DYRK1A gene. We also identified a new cerebellar alteration in two independent lines carrying 230E8-YAC. These mice showed significant elongation of the cerebellar antero-posterior axis (p<0.001), determined by increased length of rostral folia of the vermis (lobule II-V, p<0.0001; lobule VI, p<0.001). In addition, we identified a major neurological defect in culmen and declivus lobules in the 230E8-mice. We analyzed P30, P12, and P9 stages and detected high significant increased lengths of anterior lobules (II-VI) of 230E8-mice at P30 and P12 (lobule II-V, p<0.0001; lobule VI, p<0.05), but not at P9, indicating that this new phenotype appears between P9 and P12. Interestingly, 230E8-mice also present increased cortical cell density and mild learning defects. 230E8-YAC contains seven genes, some of which could be potentially responsible for this phenotype. Between them, we proposed DOPEY2 as potential candidate gene for these cerebellar alterations considering its high expression in the brain and that its homologous genes in yeast, Caenorhabditis elegans and Drosophila are involved in morphogenesis, suggesting a conserved role of DOPEY2 as a patterning gene.

Placental perfusion and permeability: simultaneous assessment with dual-echo contrast-enhanced MR imaging in mice.

PURPOSE: To assess placental perfusion and permeability in mice with magnetic resonance (MR) imaging. MATERIALS AND METHODS: This study was conducted according to French law and National Institutes of Health recommendations for animal care. Twenty-two pregnant BALB/c mice were examined at 1.5 T with a single-section dual-echo fast spoiled gradient-echo sequence. Two injection protocols were used: monophasic injection (double the clinical dose of contrast agent) and biphasic injection (quadruple the clinical dose). Signal intensities (SIs) were measured in the maternal left ventricle, placenta, and fetus (n = 16). At these high gadolinium doses, a T2* effect correction was used. SIs were converted to gadolinium concentrations and were analyzed by using a three-compartment model. Quantitative microcirculation parameters were calculated. Results with the monophasic and biphasic protocols were compared, and final arterial concentrations determined with MR imaging were compared with those determined with atomic emission spectrophotometry by using the unpaired Student t test. RESULTS: Perfusion and permeability parameters for monophasic and biphasic injections were similar: Mean placental blood flow was 180 mL/min/100 g, mean permeability surface coefficient from maternal placental to fetal placental compartment was 10.3 x 10(-4) sec(-1) +/- 6.81 (standard deviation), mean permeability surface coefficient from fetal placental to maternal placental compartment was 4.65 x 10(-4) sec(-1) +/- 4.37, and mean fractional volume of the maternal vascular placental compartment was 36.5% +/- 0.9. Placental (146 vs 105 micromol/L, P < .004) and fetal (33.3 vs 19.1 micromol/L, P < .001) gadolinium concentrations were higher with the biphasic than with the monophasic protocol. Arterial gadolinium concentrations at MR imaging did not differ significantly from those at spectrophotometry for the monophasic (P = .254) or biphasic (P = .776) injection protocol. CONCLUSION: Placental perfusion and permeability can be measured in vivo by using high gadolinium doses and a dual-echo MR imaging sequence.

Placental perfusion MR imaging with contrast agents in a mouse model.

PURPOSE: To quantitatively analyze placental perfusion by using magnetic resonance (MR) imaging with contrast agents in a mouse model. MATERIALS AND METHODS: Study was conducted according to French law and in full compliance with National Institutes of Health recommendations for animal care. Thirty-six pregnant Balb/c mice at 16 days of gestation were injected intravenously with either a conventional or macromolecular gadolinium chelate, and 1.5-T single-section T1-weighted two-dimensional fast spoiled gradient-echo sequential MR imaging was then performed for 14 minutes. Images were analyzed qualitatively, and parametric map analysis was performed in the resultant 25 mice included in the study. Signal intensity was measured in maternal left ventricle (input function), placenta, and fetus on all images. After converting signal intensity into contrast agent tissue concentrations, a three-compartment model was developed with compartmental and numeric modeling software. Placental perfusion was calculated for conventional (n = 12) and macromolecular (n = 13) gadolinium chelates. Finally, placental and fetal gadolinium concentrations were assayed by means of atomic emission spectrophotometry (n = 15). Perfusion values and placental and fetal gadolinium concentrations for conventional and macromolecular chelates were compared by using an unpaired t test. RESULTS: Based on a constant transfer parameter, estimated placental perfusion did not differ between procedures with conventional and macromolecular gadolinium chelates (0.99 mL/min/g +/- 0.5 [standard deviation] and 1.28 mL/min/g +/- 0.6, respectively, P = .22). Likewise, mean placental gadolinium concentrations did not differ after injection of conventional and macromolecular chelates. In contrast, mean fetal gadolinium concentration was 9.83 micromol/L after conventional chelate injection and below detection limit after macromolecular chelate injection. CONCLUSION: Placental perfusion can be calculated by using dynamic contrast-enhanced MR imaging, as shown in this mouse model.

Cystathionine beta synthase deficiency affects mouse endochondral ossification.

Cystathionine beta synthase (CBS) is a crucial regulator of plasma concentrations of homocysteine. Severe hyperhomocysteinemia due to CBS deficiency confers diverse clinical manifestations, notably characteristic skeletal abnormalities. To investigate this aspect of hyperhomocysteinemia, we analyzed the skeleton of CBS-deficient mice, a murine model of severe hyperhomocysteinemia. Radiography, Alcian Blue/Alizarin Red S-stained whole skeletal preparations, and histological comparisons were used to determine the extent, pattern, and distribution of skeletal abnormalities in CBS-deficient mice. Disruption of the murine CBS gene leads to skeletal abnormalities, notably kyphoscoliosis, with temporal shortening of long bones due to impaired cartilage differentiation, albeit to differing degrees.

In vivo cellular imaging of magnetically labeled hybridomas in the spleen with a 1.5-T clinical MRI system.

The feasibility of in vivo cellular imaging using a 1.5 T clinical magnet was studied in the mouse. Hybridoma cells were labeled with anionic gamma-Fe2O3 superparamagnetic iron oxide nanoparticles. These were internalized by the endocytose pathway. Both electron spin resonance and magnetophoresis as a measure of the labeled cells migration velocity under a magnetic field were used to quantify particle uptake. A fast (< 2 hr) and substantial (up to 5 pg of iron per cell) internalization of nanoparticles by hybridomas was found, with good agreement between the two methods used. Hybridomas labeled with 2.5 pg iron per cell were injected intraperitoneally to male Swiss nude mice. A decrease in the spleen signal, suggesting a "homing" of labeled hybridomas to this organ, was found 24 hr later by MRI performed at 1.5 T. Furthermore, in labeled cells recovered from the spleen by ex vivo magnetic sorting, a mean of 0.5 pg iron per cell was found, i.e., a value five times lower than that of the injected hybridomas. This finding is consistent with in vivo proliferation of these cells. In addition, the amount of labeled hybridomas present in the spleen was found to correlate with MRI signal intensity.

Altered gene expression in liver from a murine model of hyperhomocysteinemia.

Cystathionine beta-synthase (CBS) deficiency causes severe hyperhomocysteinemia and other signs of homocystinuria syndrome, in particular a premature atherosclerosis with multiple thrombosis. However, the molecular mechanisms by which homocysteine could interfere with normal cell function are poorly understood in a whole organ like the liver, which is central to the catabolism of homocysteine. We used a combination of differential display and cDNA arrays to analyze differential gene expression in association with elevated hepatic homocysteine levels in CBS-deficient mice, a murine model of hyperhomocysteinemia. Expression of several genes was found to be reproducibly abnormal in the livers of heterozygous and homozygous CBS-deficient mice. We report altered expression of genes encoding ribosomal protein S3a and methylthioadenosine phosphorylase, suggesting such cellular growth and proliferation perturbations may occur in homozygous CBS-deficient mice liver. Many up- or down-regulated genes encoded cytochromes P450, evidence of perturbations of the redox potential in heterozygous and homozygous CBS-deficient mice liver. The expression of various genes involved in severe oxidative processes was also abnormal in homozygous CBS-deficient mice liver. Among them, the expression of heme oxygenase 1 gene was increased, concomitant with overexpression of heme oxygenase 1 at the protein level. Commensurate with the difference in hepatic mRNA paraoxonase 1 abundance, the mean hepatic activity of paraoxonase 1, an enzyme that protects low density lipoprotein from oxidation, was 3-fold lower in homozygous CBS-deficient mice. Heterozygous CBS-deficient mice, when fed a hyperhomocysteinemic diet, have also reduced PON1 activity, which demonstrates the effect of hyperhomocysteinemia in the paraoxonase 1 activity.

PCP4 is highly expressed in ectoderm and particularly in neuroectoderm derivatives during mouse embryogenesis.

PCP4 (PEP-19) belongs to a family of proteins involved in calcium transduction signals and binds calmodulin via an IQ motif, in a calcium independent manner. PCP4 gene maps to murine chromosome 16 and in human to chromosome 21. Murine PCP4 expression in the brain has been detected by Northern blot analysis to be mainly post-natal and in the adult to have a neuronal pattern. To investigate if it might have a role earlier in development, we analyzed its expression during mouse embryogenesis by in situ hybridization from E7.5 post-coitum (p.c.) to E17.5 p.c., and in P0 brain. Early, at E7.5, a high expression is restricted to the extra embryonic ectoderm. Embryonic expression starts at E9.5. At E10.5, PCP4 shows a strong signal in the post-mitotic cells of the diencephalon, the metencephalon and the myelencephalon and in the dorsal and cranial ganglia. The floor plate is also densely labelled. At E17.5, PCP4 is expressed in the central nervous system, in the myenteric plexus, and in other ectoderm derivatives, for instance the lens, the hairy cells of the cochlea, the enamel organ and the hair follicles. Thus, during embryogenesis PCP4 is mainly expressed in ectoderm and neuroectoderm comprising neural crest derived cells.