Dominique Egrise

Myocardial Homing of Nonmobilized Peripheral‐Blood CD34+ Cells After Intracoronary Injection

Granulocyte– colony‐stimulating factor administered for autologous hematopoietic stem cell isolation from blood may favor restenosis in patients implanted after acute myocardial infarction (AMI). We therefore tested the isolation of peripheral‐blood CD34+ cells without mobilization in six patients with AMI. After large‐volume cytapheresis and positive CD34+ cell selection, 3.6 to 27.6 million CD34+ cells were obtained. We performed intra‐coronary implantation of these cells and recorded no restenosis or arrhythmia. We used positron emission tomography (PET) to assess myocardial‐labeled CD34+ cell homing, which accounted for 5.5% of injected cells 1 hour after implantation. In conclusion, large amounts of CD34+ cells, in the range reported in previous studies, can be obtained from nonmobilized peripheral blood. PET with [18F]‐fluorodeoxyglucose cell labeling is an efficient imaging method for homing assessment.

Bone blood flow and In vitro proliferation of bone marrow and trabecular bone osteoblast-like cells in ovariectomized rats

SummaryOvariectomy in the rat induces a rapid osteopenia associated with an elevated bone turnover. One hundred and twenty-day-old rats were ovariectomized (OVX) or sham-operated (n=6–8 per group and per time period studied). 45Ca accretion rate and bone blood flow (microspheres trapping technique) in the femurs were determined at 28, 42, 84, and 119 days after ovariectomy. Both parameters were markedly increased by 84 days and subsided thereafter. At the 42nd day, when bone turnover was maximal, bone marrow and trabecular bone cultures were obtained from shamoperated and ovariectomized animals (n=10/group). Proliferation rate of bone marrow cells and trabecular osteoblast-like cells estimated by fibroblast colony-forming units (FCFU) efficiency and cell counting was markedly increased in primary and secondary cultures in ovariectomy. These data fitted well with the enhanced number of osteoblasts observed in situ in the long bone metaphyses of estrogen-depleted animals. As estrogens were shown in the literature to inhibit proliferation of the red cell line and of other hemopoietic lines, it is possible that estrogens, through a general mechanism, inhibit hemopoietic and stromal lines and also the proliferation of bone marrow-derived trabecular bone cells.

Vitamin D deficiency-induced hypertension is associated with vascular oxidative stress and altered heart gene expression.

Vitamin D deficiency (VDD) is associated with an increased cardiovascular risk. We investigated the effect of VDD on the cardiovascular system of growing male rats fed with a vitamin D-deficient diet. Using isolated rat aorta, we assessed both superoxide anion and endothelial-dependent relaxations. Microarray technology was used to identify changes induced by VDD in cardiac gene expression. Compared with control, VDD increased systolic blood pressure (P < 0.05) and superoxide anion production in the aortic wall (P < 0.05) and tended to increase serum levels of angiotensin II and atrial natriuretic peptide (P < 0.15). However, VDD slightly improved maximal relaxation to acetylcholine from 75 % ± 3% to 83% ± 2% (P < 0.05). Incubation of aortic rings either with nitro-l-arginine methyl ester (l-NAME) or catalase did not eliminate the enhancement of endothelial-mediated relaxation observed in vitamin D-deficient rats. Only incubation with indometacin or calcium-activated potassium channels blockers suppressed this difference. Compared with control, the expression of 51 genes showed different expression, including several genes involved in the regulation of oxidative stress and myocardial hypertrophy. In conclusion, VDD in early life increases arterial blood pressure, promotes vascular oxidative stress, and induces changes in cardiac gene expression. However, the endothelial-mediated regulation of vasomotor tone is maintained throughout the enhancement of an NO-independent compensatory pathway.

Bidirectional MHC molecule exchange between migratory and resident dendritic cells

Dendritic cells (DCs) loaded extracorporeally with antigen can be used as an adjuvant in vivo. In this work, we analyzed the migration of transferred DC and monitored the phenotype of new migrants in the draining lymph nodes. It is surprising that we found that a majority of resident DCs expressed donor MHC molecules and that a proportion of injected DCs acquired host MHC molecules. These observations suggest that a bidirectional MHC molecule exchange occurs between migratory and resident DCs, a mechanism that may amplify antigen presentation in vivo.

Therapeutic efficacy of antitumor dendritic cell vaccinations correlates with persistent Th1 responses, high intratumor CD8+ T cell recruitment and low relative regulatory T cell infiltration

Despite the increasing number of immunotherapeutic strategies for the treatment of cancer, most approaches have failed to correlate the induction of an anti-tumor immune response with therapeutic efficacy. We therefore took advantage of a successful vaccination strategy—combining dendritic cells and irradiated GM-CSF secreting tumor cells—to compare the immune response induced against 9L gliosarcoma tumors in cured rats versus those with progressively growing tumors. At the systemic level, the tumor specific cytotoxic responses were quite heterogeneous in uncured vaccinated rats, and were surprisingly often high in animals with rapidly-growing tumors. IFN-γ secretion by activated splenic T cells was more discriminative as the CD4+ T cell-mediated production was weak in uncured rats whereas high in cured ones. At the tumor level, regressing tumors were strongly infiltrated by CD8+ T cells, which demonstrated lytic capacities as high as their splenic counterparts. In contrast, progressing tumors were weakly infiltrated by T cells showing impaired cytotoxic activities. Proportionately to the T cell infiltrate, the expression of Foxp3 was increased in progressive tumors suggesting inhibition by regulatory T cells. In conclusion, the main difference between cured and uncured vaccinated animals does not depend directly upon the induction of systemic cytotoxic responses. Rather the persistence of higher CD4+ Th1 responses, a high intratumoral recruitment of functional CD8+ T cells, and a low proportion of regulatory T cells correlate with tumor rejection.

PET/CT with 18F-FDG– and 18F-FBEM–Labeled Leukocytes for Metabolic Activity and Leukocyte Recruitment Monitoring in a Mouse Model of Pulmonary Fibrosis

Idiopathic pulmonary fibrosis is characterized by a progressive and irreversible respiratory failure. Validated noninvasive methods able to assess disease activity are essential for prognostic purposes as well as for the evaluation of emerging antifibrotic treatments. Methods: C57BL/6 mice were used in a murine model of pulmonary fibrosis induced by an intratracheal instillation of bleomycin (control mice were instilled with a saline solution). At different times after instillation, PET/CT with 18F-FDG– or 18F-4-fluorobenzamido-N-ethylamino-maleimide (18F-FBEM)–labeled leukocytes was performed to assess metabolic activity and leukocyte recruitment, respectively. Results: In bleomycin-treated mice, a higher metabolic activity was measured on 18F-FDG PET/CT scans from day 7 to day 24 after instillation, with a peak of activity measured at day 14. Of note, lung mean standardized uptake values correlated with bleomycin doses, histologic score of fibrosis, lung hydroxyproline content, and weight loss. Moreover, during the inflammatory phase of the model (day 7), but not the fibrotic phase (day 23), bleomycin-treated mice presented with an enhanced leukocyte recruitment as assessed by 18F-FBEM–labeled leukocyte PET/CT. Autoradiographic analysis of lung sections and CD45 immunostaining confirm the higher and early recruitment of leukocytes in bleomycin-treated mice, compared with control mice. Conclusion: 18F-FDG– and 18F-FBEM–labeled leukocyte PET/CT enable monitoring of metabolic activity and leukocyte recruitment in a mouse model of pulmonary fibrosis. Implications for preclinical evaluation of antifibrotic therapy are expected.

Fully automated radiosynthesis of N1-[18F]fluoroethyl-tryptophan and study of its biological activity as a new potential substrate for indoleamine 2,3-dioxygenase PET imaging

INTRODUCTION Indoleamine 2,3-dioxygenase (IDO) catalyzes the initial step in the catabolism of l-tryptophan along the kynurenine pathway and exerts immunosuppressive properties in inflammatory and tumor tissues by blocking locally T-lymphocyte proliferation. Recently, 1-(2-[(19)F]fluoroethyl)-dl-tryptophan (1-[(19)F]FE-dl-Trp) was reported as a good and specific substrate of this enzyme. Herein, the radiosynthesis of its radioactive isotopomer (1-[(18)F]FE-dl-Trp, dl-[(18)F]5) is presented along with in vitro enzymatic and cellular uptake studies. METHODS The one-pot n.c.a. radiosynthesis of this novel potential PET imaging tracer, including HPLC purification and formulation, has been fully automated on a FASTlab™ synthesizer. Chiral separation of both isomers and their formulation were implemented on a second cassette. In vitro enzymatic and cellular uptake studies were then conducted with the d-, l- and dl-radiotracers. RESULTS The radiolabeling of the tosylate precursor was performed in DMF (in 5min; RCY: 57% (d.c.), n=3). After hydrolysis, HPLC purification and formulation, dl-[(18)F]5 was obtained with a global radiochemical yield of 18±3% (not decay corrected, n=7, in 80min) and a specific activity of 600±180GBq/μmol (n=5). The subsequent separation of l- and d-enantiomers was performed by chiral HPLC and both were obtained after formulation with an RCY (d.c.) of 6.1% and 5.8%, respectively. In vitro enzymatic assays reveal that l-[(18)F]5 is a better substrate than d-[(18)F]5 for human IDO. In vitro cellular assays show an IDO-specific uptake of the racemate varying from 30% to 50% of that of l-[(18)F]5, and a negligible uptake of d-[(18)F]5. CONCLUSION In vitro studies show that l-[(18)F]5 is a good and specific substrate of hIDO, while presenting a very low efflux. These results confirm that l-[(18)F]5 could be a very useful PET radiotracer for IDO expressing cells in cancer imaging.

3D super-resolved in vitro multiphoton microscopy by saturation of excitation

We demonstrate a significant resolution enhancement beyond the conventional limit in multiphoton microscopy (MPM) using saturated excitation of fluorescence. Our technique achieves super-resolved imaging by temporally modulating the excitation laser-intensity and demodulating the higher harmonics from the saturated fluorescence signal. The improvement of the lateral and axial resolutions is measured on a sample of fluorescent microspheres. While the third harmonic already provides an enhanced resolution, we show that a further improvement can be obtained with an appropriate linear combination of the demodulated harmonics. Finally, we present in vitro imaging of fluorescent microspheres incorporated in HeLa cells to show that this technique performs well in biological samples.

[18F]-FBEM, a tracer targeting cell-surface protein thiols for cell trafficking imaging.

We used [(18)F]-4-fluorobenzamido-N-ethylamino-maleimide ([(18)F]-FBEM) to radiolabel cells ex vivo for in vivo positron emission tomography (PET) in order to assess cell trafficking in mice. In contrast to commonly used imaging agents, [(18)F]-FBEM forms a covalent bond with thiol groups present on the cells surface. The stability of the probe in aqueous medium was tested at different pH values and cross-experiment showed that thiol-labeling efficiency was retained (at least) up to pH 9. The labeling procedure did not affect significantly the cell viability. To illustrate the procedure, PET images of living mice injected intravenously with labeled T lymphocytes were obtained. They showed the expected cell homing in the spleen that was absent in mice injected with free label.

Protracted Systemic Changes in Bone Biology after Segmented Unloading in the Rat

To investigate whether the decreased bone formation observed in most experimental situations of disuse was caused by an increased inhibition by the bone microenvironment of osteoblast (OB) proliferation, we studied the inhibiting power on ROS 17/2.8 proliferation of the bone marrow extracellular fluid (IPEF) in loaded and unloaded bones of rats submitted to two situations of partial disuse: tail suspension (TS) for 3 days to 2 weeks and around the knee tenectomy (KT) for 2–10 weeks. Histomorphometric parameters and osteoblast precursors dynamics were studied in parallel. Bone volume was lost in the unloaded bones, but not in loaded bones, in both experimental situations. Bone formation was low at early times (7 - 14 days) in TS rats. However, in KT at later times (4 - 10 weeks), the osteoblastic index of the unloaded tibia was increased. IPEF was not increased in the unloaded bones 3–7 days after TS. It was decreased later in the course of unloading (after 2 weeks of TS and 2–10 weeks after KT). This decrease was observed in the loaded bones as well. Unexpectedly, we also found that the number of FCFUs was decreased in both loaded and unloaded limbs in TS and KT, and that the yield of cells obtained in primary culture from tibial metaphysis was decreased in both tibiae from KT animals. These data show that an increased IPEF does not play a role in the early inhibition of bone formation responsible for the loss of bone after unloading in the TS model. Its later decrease could be permissive for the increased osteoblastic index observed in the KT model. They also show that, contrary to the usual assumptions, bone biology is changed all over the skeleton after partial unloading, even if the changes result in bone loss in the unloaded bones only. Thus, as yet, unidentified systemic factors probably superimpose on the local factors that control bone volume.