Initial experiences with in vivo right coronary artery human MR vessel wall imaging at 3 tesla.

Due to their relatively small size and central location within the thorax, improvement in signal-to-noise (SNR) is of paramount importance for in vivo coronary vessel wall imaging. Thus, with higher field strengths, coronary vessel wall imaging is likely to benefit from the expected "near linear" proportional gain in SNR. In this study, we demonstrate the feasibility of in vivo human high field (3 T) coronary vessel wall imaging using a free-breathing black blood fast gradient echo technique with respiratory navigator gating and real-time motion correction. With the broader availability of more SNR efficient fast spin echo and spiral techniques, further improvements can be expected.

Staphylococcal biofilm formation on the surface of three different calcium phosphate bone grafts: a qualitative and quantitative in vivo analysis.

Differences in physico-chemical characteristics of bone grafts to fill bone defects have been demonstrated to influence in vitro bacterial biofilm formation. Aim of the study was to investigate in vivo staphylococcal biofilm formation on different calcium phosphate bone substitutes. A foreign-body guinea-pig infection model was used. Teflon cages prefilled with β-tricalcium phosphate, calcium-deficient hydroxyapatite, or dicalcium phosphate (DCP) scaffold were implanted subcutaneously. Scaffolds were infected with 2 × 10(3) colony-forming unit of Staphylococcus aureus (two strains) or S. epidermidis and explanted after 3, 24 or 72 h of biofilm formation. Quantitative and qualitative biofilm analysis was performed by sonication followed by viable counts, and microcalorimetry, respectively. Independently of the material, S. aureus formed increasing amounts of biofilm on the surface of all scaffolds over time as determined by both methods. For S. epidermidis, the biofilm amount decreased over time, and no biofilm was detected by microcalorimetry on the DCP scaffolds after 72 h of infection. However, when using a higher S. epidermidis inoculum, increasing amounts of biofilm were formed on all scaffolds as determined by microcalorimetry. No significant variation in staphylococcal in vivo biofilm formation was observed between the different materials tested. This study highlights the importance of in vivo studies, in addition to in vitro studies, when investigating biofilm formation of bone grafts.

Lentiviral vectors: a powerful tool to target astrocytes in vivo.

The morphological and functional diversity of astrocytes, and their essential contribution in physiological and pathological conditions, are starting to emerge. However, experimental systems to investigate neuron-glia interactions and develop innovative approaches for the treatment of central nervous system (CNS) disorders are still very limited. Fluorescent reporter genes have been used to visualize populations of astrocytes and produce an atlas of gene expression in the brain. Knock-down or knock-out of astrocytic proteins using transgenesis have also been developed, but these techniques remain complex and time-consuming. Viral vectors have been developed to overexpress or silence genes of interest as they can be used for both in vitro and in vivo studies in adult mammalian species. In most cases, high transduction efficiency and long-term transgene expression are observed in neurons but there is limited expression in astrocytes. Several strategies have been developed to shift the tropism of lentiviral vectors (LV) and allow local and controlled gene expression in glial cells. In this review, we describe how modifications of the interaction between the LV envelope glycoprotein and the surface receptor molecules on target cells, or the integration of cell-specific promoters and miRNA post-transcriptional regulatory elements have been used to selectively express transgenes in astrocytes.

Hierarchal utilization of different T-cell receptor Vbeta gene segments in the CD8(+)-T-cell response to an immunodominant Moloney leukemia virus-encoded epitope in vivo.

The CD8(+)-T-cell response to Moloney murine leukemia virus (M-MuLV)-associated antigens in C57BL/6 mice is directed against an immunodominant gag-encoded epitope (CCLCLTVFL) presented in the context of H-2D(b) and is restricted primarily to cytotoxic T lymphocytes (CTL) expressing the Valpha3.2 and Vbeta5.2 gene segments. We decided to examine the M-MuLV response in congenic C57BL/6 Vbeta(a) mice which are unable to express the dominant Valpha3.2(+) Vbeta5.2(+) T-cell receptor (TCR) due to a large deletion at the TCR locus that includes the Vbeta5.2 gene segment. Interestingly, M-MuLV-immune C57BL/6 Vbeta(a) mice were still able to reject M-MuLV-infected tumor cells and direct ex vivo analysis of peripheral blood lymphocytes from these immune mice revealed a dramatic increase in CD8(+) cells utilizing the same Valpha3.2 gene segment in association with two different Vbeta segments (Vbeta3 and Vbeta17). Surprisingly, all these CTL recognized the same immunodominant M-MuLV gag epitope. Analysis of the TCR repertoire of individual M-MuLV-immune (C57BL/6 x C57BL/6 Vbeta(a))F(1) mice revealed a clear hierarchy in Vbeta utilization, with a preferential usage of the Vbeta17 gene segment, whereas Vbeta3 and especially Vbeta5.2 were used to much lesser extents. Sequencing of TCRalpha- and -beta-chain junctional regions of CTL clones specific for the M-MuLV gag epitope revealed a diverse repertoire of TCRbeta chains in Vbeta(a) mice and a highly restricted TCRbeta-chain repertoire in Vbeta(b) mice, whereas TCRalpha-chain sequences were highly conserved in both cases. Collectively, our data indicate that the H-2D(b)-restricted M-MuLV gag epitope can be recognized in a hierarchal fashion by different Vbeta domains and that the degree of beta-chain diversity varies according to Vbeta utilization.

Localized in vivo hyperpolarization transfer sequences.

In vivo localized and fully adiabatic homonuclear and heteronuclear polarization transfer experiments were designed and performed in the rat brain at 9.4 T after infusion of hyperpolarized sodium [1,2-(13)C(2)] and sodium [1-(13)C] acetate. The method presented herein leads to highly enhanced in vivo detection of short-T(1) (13)C as well as attached protons. This indirect detection scheme allows for probing additional molecular sites in hyperpolarized substrates and their metabolites and can thus lead to improved spectral resolution such as in the case of (13)C-acetate metabolism.

Calcium-sensing receptors modulate renin release in vivo and in vitro in the rat.

OBJECTIVES: Calcium-sensing receptors (CaSRs) have been localized in the juxtaglomerular apparatus where they may contribute to the regulation of renin release. In the present study, we investigated the in-vitro and in-vivo effects of the calcimimetic R-568 on renin release. METHODS: In vitro, the effect of calcimimetics on renin release was assessed by incubating freshly isolated rat juxtaglomerular cells with or without R-568 (1 and 10 mumol/l) in serum-free medium in the presence or absence of forskolin or CaCl2. In vivo, we measured the impact of R-568 (20 ng/min intravenously) on the acute changes in plasma renin activity (PRA) induced by either a 90 min infusion of the angiotensin-converting enzyme inhibitor captopril, or the beta-receptor agonist isoproterenol, or of a vehicle in or after a furosemide challenge in conscious Wistar rats. RESULTS: In vitro, R-568 dose-dependently blunted renin release, but also reduced the increase in renin due to forskolin (P < 0.01). Both isoproterenol and enalapril increased in vivo PRA to 3.1 +/- 0.3 and 3.7 +/- 0.5 ng Ang I/ml per h, respectively (P < 0.01), compared with vehicle (1.5 +/- 0.2 ng Ang I/ml per h). R-568 significantly reduced PRA to 2.1 +/- 0.1 ng/ml per h in isoproterenol-treated rats and to 1.6 +/- 0.2 ng/ml per h in enalapril-treated rats (P < 0.05). In low-salt treated animals, acute infusion of furosemide increased PRA from 8.7 +/- 3.2 to 18.6 +/- 2.3, whereas R-568 partially blunted this rise to 11.2 +/- 1.5 (P = 0.02). In vivo, R-568 significantly lowered serum calcium and PTH1-84, but the drug-induced changes in PRA were independent of the changes in calcium and parathyroid hormone. CONCLUSION: After the recent discovery of CaSRs in juxtaglomerular cells of mice, our results confirm the presence of such receptors in rats and demonstrate that these receptors modulate renin release both in vitro and in vivo. This suggests that CaSRs play a role as a regulatory pathway of renin release.

Influence of chronic hyperglycemia on cerebral microvascular remodeling: an in vivo study using perfusion computed tomography in acute ischemic stroke patients.

BACKGROUND AND PURPOSE: To investigate the effect of chronic hyperglycemia on cerebral microvascular remodeling using perfusion computed tomography. METHODS: We retrospectively identified 26 patients from our registry of 2453 patients who underwent a perfusion computed tomographic study and had their hemoglobin A1c (HbA1c) measured. These 26 patients were divided into 2 groups: those with HbA1c>6.5% (n=15) and those with HbA1c≤6.5% (n=11). Perfusion computed tomographic studies were processed using a delay-corrected, deconvolution-based software. Perfusion computed tomographic values were compared between the 2 patient groups, including mean transit time, which relates to the cerebral capillary architecture and length. RESULTS: Mean transit time values in the nonischemic cerebral hemisphere were significantly longer in the patients with HbA1c>6.5% (P=0.033), especially in the white matter (P=0.005). Significant correlation (R=0.469; P=0.016) between mean transit time and HbA1c level was observed. CONCLUSIONS: Our results from a small sample suggest that chronic hyperglycemia may be associated with cerebral microvascular remodeling in humans. Additional prospective studies with larger sample size are required to confirm this observation.

In vivo mechanisms leading to transplantation tolerance induced by regulatory T cells

Purpose: The mechanisms by which CD4+CD25+Foxp3+ T cells (Tregs) regulate effector T cells in a transplantation setting and their in vivo homeostasis still remain to be clarified. Using a mouse adoptive transfer and skin transplantation model, we analyzed the in vivo expansion, effector function and trafficking of effector T cells and donor-specific Tregs, in response to an allograft. Methods and materials: Antigen-specific Tregs were generated and expanded in vitro by culturing freshly isolated Tregs from BALB/c mice (H2d) with syngeneic dendritic cells pulsed with an allopeptide (here the Kb peptide derived from the MHC class I molecule of allogeneic H2b mice). Fluorescent-labelled CD4+CD25- naive T cells and donor-antigen-specific Tregs were transferred alone or coinjected into syngeneic BALB/c-Nude recipients transplanted with allogeneic C57BL/6xBALB/c donor skin. Results: As opposed to their in vitro hyporesponsiveness, Tregs divided in vivo, migrated and accumulated in the allograft draining lymph nodes (drLN) and within the graft. The co-transfer of Tregs did not modify the early proliferation and homing of CD4+CD25- T cells to secondary lymphoid organs. But, in the presence of Tregs, effector T cells produced significantly less IFN- and IL-2 effector cytokines, while higher amounts of IL-10 were detected in the spleen and drLN of these mice. Furthermore, time-course studies showed that Tregs were recruited into the allograft at a very early stage posttransplantation and prevented infiltration by effector T cells. Conclusion: Overall, our results suggest that suppression of graft rejection involves the early recruitment of donor-specific Tregs at the sites of antigenic challenge and that Tregs mainly regulate the effector arm of T cell alloresponses.

Feasibility of in vivo 15N MRS detection of hyperpolarized 15N labeled choline in rats.

The increase of total choline in tumors has become an important biomarker in cancer diagnosis. Choline and choline metabolites can be measured in vivo and in vitro using multinuclear MRS. Recent in vivo(13)C MRS studies using labeled substrates enhanced via dynamic nuclear polarization demonstrated the tremendous potential of hyperpolarization for real-time metabolic studies. The present study demonstrates the feasibility of detecting hyperpolarized (15)N labeled choline in vivo in a rat head at 9.4 T. We furthermore report the in vitro (172 +/- 16 s) and in vivo (126 +/- 15 s) longitudinal relaxation times. We conclude that with appropriate infusion protocols it is feasible to detect hyperpolarized (15)N labeled choline in live animals.

Oral administration of a low dose of midazolam (75 microg) as an in vivo probe for CYP3A activity.

OBJECTIVE: We investigated whether the oral administration of a low dose (75 micro g) of midazolam, a CYP3A probe, can be used to measure the in vivo CYP3A activity. METHODS: Plasma concentrations of midazolam, 1'OH-midazolam and 4'OH-midazolam were measured after the oral administration of 7.5 mg and 75 micro g midazolam in 13 healthy subjects without medication, in four subjects pretreated for 2 days with ketoconazole (200 mg b.i.d.), a CYP3A inhibitor, and in four subjects pretreated for 4 days with rifampicin (450 mg q.d.), a CYP3A inducer. RESULTS: After oral administration of 75 micro g midazolam, the 30-min total (unconjugated + conjugated) 1'OH-midazolam/midazolam ratios measured in the groups without co-medication, with ketoconazole and with rifampicin were (mean+/-SD): 6.23+/-2.61, 0.79+/-0.39 and 56.1+/-12.4, respectively. No side effects were reported by the subjects taking this low dose of midazolam. Good correlations were observed between the 30-min total 1'OH-midazolam/midazolam ratio and midazolam clearance in the group without co-medication (r(2)=0.64, P<0.001) and in the three groups taken together (r(2)=0.91, P<0.0001). Good correlations were also observed between midazolam plasma levels and midazolam clearance, measured between 1.5 h and 4 h. CONCLUSION: A low oral dose of midazolam can be used to phenotype CYP3A, either by the determination of total 1'OH-midazolam/midazolam ratios at 30 min or by the determination of midazolam plasma levels between 1.5 h and 4 h after its administration.

Thalamic connectivity in patients with essential tremor treated with MR imaging-guided focused ultrasound: in vivo fiber tracking by using diffusion-tensor MR imaging.

PURPOSE: To use diffusion-tensor (DT) magnetic resonance (MR) imaging in patients with essential tremor who were treated with transcranial MR imaging-guided focused ultrasound lesion inducement to identify the structural connectivity of the ventralis intermedius nucleus of the thalamus and determine how DT imaging changes correlated with tremor changes after lesion inducement. MATERIALS AND METHODS: With institutional review board approval, and with prospective informed consent, 15 patients with medication-refractory essential tremor were enrolled in a HIPAA-compliant pilot study and were treated with transcranial MR imaging-guided focused ultrasound surgery targeting the ventralis intermedius nucleus of the thalamus contralateral to their dominant hand. Fourteen patients were ultimately included. DT MR imaging studies at 3.0 T were performed preoperatively and 24 hours, 1 week, 1 month, and 3 months after the procedure. Fractional anisotropy (FA) maps were calculated from the DT imaging data sets for all time points in all patients. Voxels where FA consistently decreased over time were identified, and FA change in these voxels was correlated with clinical changes in tremor over the same period by using Pearson correlation. RESULTS: Ipsilateral brain structures that showed prespecified negative correlation values of FA over time of -0.5 or less included the pre- and postcentral subcortical white matter in the hand knob area; the region of the corticospinal tract in the centrum semiovale, in the posterior limb of the internal capsule, and in the cerebral peduncle; the thalamus; the region of the red nucleus; the location of the central tegmental tract; and the region of the inferior olive. The contralateral middle cerebellar peduncle and bilateral portions of the superior vermis also showed persistent decrease in FA over time. There was strong correlation between decrease in FA and clinical improvement in hand tremor 3 months after lesion inducement (P < .001). CONCLUSION: DT MR imaging after MR imaging-guided focused ultrasound thalamotomy depicts changes in specific brain structures. The magnitude of the DT imaging changes after thalamic lesion inducement correlates with the degree of clinical improvement in essential tremor.

Increase of [(18)F]FLT Tumor Uptake In Vivo Mediated by FdUrd: Toward Improving Cell Proliferation Positron Emission Tomography.

PURPOSE: 3'-deoxy-3'-[(18)F]fluorothymidine ([(18)F]FLT), a cell proliferation positron emission tomography (PET) tracer, has been shown in numerous tumors to be more specific than 2-deoxy-2-[(18)F]fluoro-D: -glucose ([(18)F]FDG) but less sensitive. We studied the capacity of a nontoxic concentration of 5-fluoro-2'-deoxyuridine (FdUrd), a thymidine synthesis inhibitor, to increase uptake of [(18)F]FLT in tumor xenografts. METHODS: The duration of the FdUrd effect in vivo on tumor cell cycling and thymidine analogue uptake was studied by varying FdUrd pretreatment timing and holding constant the timing of subsequent flow cytometry and 5-[(125)I]iodo-2'-deoxyuridine biodistribution measurements. In [(18)F]FLT studies, FdUrd pretreatment was generally performed 1 h before radiotracer injection. [(18)F]FLT biodistributions were measured 1 to 3 h after radiotracer injection of mice grafted with five different human tumors and pretreated or not with FdUrd and compared with [(18)F]FDG tumor uptake. Using microPET, the dynamic distribution of [(18)F]FLT was followed for 1.5 h in FdUrd pretreated mice. High-field T2-weighted magnetic resonance imaging (MRI) and histology were used comparatively in assessing tumor viability and proliferation. RESULTS: FdUrd induced an immediate increase in tumor uptake of 5-[(125)I]iodo-2'-deoxyuridine, that vanished after 6 h, as also confirmed by flow cytometry. Biodistribution measurements showed that FdUrd pretreatment increased [(18)F]FLT uptake in all tumors by factors of 3.2 to 7.8 compared with controls, while [(18)F]FDG tumor uptake was about fourfold and sixfold lower in breast cancers and lymphoma. Dynamic PET in FdUrd pretreated mice showed that [(18)F]FLT uptake in all tumors increased steadily up to 1.5 h. MRI showed a well-vascularized homogenous lymphoma with high [(18)F]FLT uptake, while in breast cancer, a central necrosis shown by MRI was inactive in PET, consistent with the histomorphological analysis. CONCLUSION: We showed a reliable and significant uptake increase of [(18)F]FLT in different tumor xenografts after low-dose FdUrd pretreatment. These results show promise for a clinical application of FdUrd aimed at increasing the sensitivity of [(18)F]FLT PET.

Encapsulation of packaging cell line results in successful retroviral-mediated transfer of a suicide gene in vivo in an experimental model of glioblastoma.

AIMS: Retroviral-mediated gene therapy has been proposed as a primary or adjuvant treatment for advanced cancer, because retroviruses selectively infect dividing cells. Efficacy of retroviral-mediated gene transfer, however, is limited in vivo. Although packaging cell lines can produce viral vectors continuously, such allo- or xenogeneic cells are normally rejected when used in vivo. Encapsulation using microporous membranes can protect the packaging cells from rejection. In this study, we used an encapsulated murine packaging cell line to test the effects of in situ delivery of a retrovirus bearing the herpes simplex virus thymidine kinase suicide gene in a rat model of orthotopic glioblastoma. MATERIALS AND METHODS: To test gene transfer in vitro, encapsulated murine psi2-VIK packaging cells were co-cultured with baby hamster kidney (BHK) cells, and the percentage of transfected BHK cells was determined. For in vivo experiments, orthotopic C6 glioblastomas were established in Wistar rats. Capsules containing psi2-VIK cells were stereotaxically implanted into these tumours and the animals were treated with ganciclovir (GCV). Tumours were harvested 14 days after initiation of GCV therapy for morphometric analysis. RESULTS: Encapsulation of psi2-VIK cells increased transfection rates of BHK target cells significantly in vitro compared to psi2-VIK conditioned medium (3 x 10(6) vs 2.3 x 10(4) cells; P<0.001). In vivo treatment with encapsulated packaging cells resulted in 3% to 5% of C6 tumour cells transduced and 45% of tumour volume replaced by necrosis after GCV (P<0.01 compared to controls). CONCLUSION: In this experimental model of glioblastoma, encapsulation of a xenogeneic packaging cell line increased half-life and transduction efficacy of retrovirus-mediated gene transfer and caused significant tumour necrosis.

Drug-eluting beads loaded with antiangiogenic agents for chemoembolization: in vitro sunitinib loading and release and in vivo pharmacokinetics in an animal model.

PURPOSE: The combination of embolic beads with a multitargeted tyrosine kinase inhibitor that inhibits tumor vessel growth is suggested as an alternative and improvement to the current standard doxorubicin-eluting beads for use in transarterial chemoembolization. This study demonstrates the in vitro loading and release kinetics of sunitinib using commercially available embolization microspheres and evaluates the in vitro biologic efficacy on cell cultures and the resulting in vivo pharmacokinetics profiles in an animal model. MATERIALS AND METHODS: DC Bead microspheres, 70-150 µm and 100-300 µm (Biocompatibles Ltd., Farnham, United Kingdom), were loaded by immersion in sunitinib solution. Drug release was measured in saline in a USP-approved flow-through apparatus and quantified by spectrophotometry. Activity after release was confirmed in cell culture. For pharmacokinetics and in vivo toxicity evaluation, New Zealand white rabbits received sunitinib either by intraarterial injection of 100-300 µm sized beads or per os. Plasma and liver tissue drug concentrations were assessed by liquid chromatography-tandem mass spectroscopy. RESULTS: Sunitinib loading on beads was close to complete and homogeneous. A total release of 80% in saline was measured, with similar fast-release profiles for both sphere sizes. After embolization, drug plasma levels remained below the therapeutic threshold (< 50 ng/mL), but high concentrations at 6 hours (14.9 µg/g) and 24 hours (3.4 µg/g) were found in the liver tissue. CONCLUSIONS: DC Bead microspheres of two sizes were efficiently loaded with sunitinib and displayed a fast and almost complete release in saline. High liver drug concentrations and low systemic levels indicated the potential of sunitinib-eluting beads for use in embolization.

Ultra-High Field (7 Tesla) MRI for Gamma Knife Surgery Targeting of the Ventro-Intermediate Nucleus: A Pilot in Vivo Study

Aim: Gamma Knife surgery (GKS) is a non-invasive neurosurgical stereotactic procedure, increasingly used as an alternative to open functional procedures. This includes the targeting of the ventro-intermediate (Vim) nucleus of the thalamus for tremor. We currently perform an indirect targeting, using the "quadrilatere of Guyot," as the Vim nucleus is not visible on current 3 Tesla (T) MRI acquisitions. The primary objective of the current study was to enhance anatomic imaging for Vim GKS using high-field (7 T) MRI, with the aim of refining the visualization and precision of anatomical targeting. Method: Five young healthy subjects (mean age 23 years) were scanned both on 3 and 7 T MRI in Lausanne University Hospital (CHUV) and Center for Biomedical Imaging (CIBM). Classical T1-weighted MPRAGE, T2 CISS sequences (replacing former ventriculography) and diffusion tensor imaging were acquired at 3T. We obtained high-resolution susceptibility weighted images (SWI) at 7T for the visualization of thalamic subparts. SWI was further integrated for the first time into Leksell Gamma Plan® (LGP) software and co-registered with the 3T images. A simulation of targeting of the Vim was done using the "quadrilatere of Guyot" methodology on the 3T images. Furthermore, a correlation with the position of the found target on SWI was performed. The atlas of Morel et al. was used to confirm the findings on a detailed computer analysis outside LGP. Also, 3T and 7T MRI of one patient undergoing GKS Vim thalamotomy, were obtained before and 2 years after the procedure, and studied similarly. Results: The use of SWI provided a superior resolution and improved image contrast within the central gray matter. This allowed visualization and direct delineation of groups of thalamic nuclei in vivo, including the Vim. The position of the target, as assessed with the "quadrilatere of Guyot" method on 3 T, perfectly matched with the supposed one of the Vim on the SWI. Furthermore, a 3-dimensional model of the Vim target area was created on the basis of 3T and 7T images. Conclusion: This is the first report of the integration of SWI high-field MRI into the LGP in healthy subjects and in one patient treated GKS Vim thalamotomy. This approach aims at the improvement of targeting validation and further direct targeting of the Vim in tremor. The anatomical correlation between the direct visualization on 7T and the current targeting methods on 3T seems to show a very good anatomical matching.