Tissue-engineered vascular grafts transform into mature blood vessels via an inflammation-mediated process of vascular remodeling.

Proc Natl Acad Sci U S A. 2010 Mar 5;

Authors: Roh JD, Sawh-Martinez R, Brennan MP, Jay SM, Devine L, Rao DA, Yi T, Mirensky TL, Nalbandian A, Udelsman B, Hibino N, Shinoka T, Saltzman WM, Snyder E, Kyriakides TR, Pober JS, Breuer CK

Biodegradable scaffolds seeded with bone marrow mononuclear cells (BMCs) are the earliest tissue-engineered vascular grafts (TEVGs) to be used clinically. These TEVGs transform into living blood vessels in vivo, with an endothelial cell (EC) lining invested by smooth muscle cells (SMCs); however, the process by which this occurs is unclear. To test if the seeded BMCs differentiate into the mature vascular cells of the neovessel, we implanted an immunodeficient mouse recipient with human BMC (hBMC)-seeded scaffolds. As in humans, TEVGs implanted in a mouse host as venous interposition grafts gradually transformed into living blood vessels over a 6-month time course. Seeded hBMCs, however, were no longer detectable within a few days of implantation. Instead, scaffolds were initially repopulated by mouse monocytes and subsequently repopulated by mouse SMCs and ECs. Seeded BMCs secreted significant amounts of monocyte chemoattractant protein-1 and increased early monocyte recruitment. These findings suggest TEVGs transform into functional neovessels via an inflammatory process of vascular remodeling.

PMID: 20207947 [PubMed - as supplied by publisher]

Genetic deficiency of Syk protects mice from autoantibody-induced arthritis.

Arthritis Rheum. 2010 Mar 3;

Authors: Jakus Z, Simon E, Balázs B, Mócsai A

OBJECTIVE:: The Syk tyrosine kinase plays an important role in diverse functions in hematopoietic lineage cells. Though prior in vitro studies and pharmacological approaches suggested Syk to be a possible player in the development of autoimmune arthritis, no in vivo genetic studies addressing that issue have yet been reported. The aim of the present work was to test whether the genetic deficiency of Syk affects autoantibody-induced experimental arthritis in the K/BxN serum transfer model. METHODS:: Syk(-/-) bone marrow chimeras carrying a Syk-deficient hematopoietic system were generated by transplanting Syk(-/-) fetal liver cells into lethally irradiated wild type recipients. After complete repopulation of the hematopoietic compartment, autoantibody-mediated arthritis was induced by injection of arthritogenic K/BxN serum. Arthritis development was followed by macroscopic and microscopic observation of the ankle joints, micro-CT analysis of bone morphology, as well as a joint functional assay. RESULTS:: Genetic deficiency of Syk in the hematopoietic compartment completely blocked the development of all macroscopic and microscopic signs of arthritis. The Syk(-/-) mutation also prevented the appearance of periarticular bone erosions. Finally, Syk(-/-) bone marrow chimeras were completely protected from arthritis-induced loss of articular function. CONCLUSION:: Our experiments indicate that Syk is critically involved in the development of all clinically relevant aspects of the autoantibody-mediated K/BxN serum transfer arthritis in experimental mice. These results provide the first in vivo genetic evidence for the role of Syk in the development of autoimmune arthritis.

PMID: 20201079 [PubMed - as supplied by publisher]

Apoptotic analysis of memory T cells and dendritic cells during the early stages of viral infection or exposure to TLR agonists.

J Virol. 2010 Mar 3;

Authors: Bahl K, Hüebner A, Davis RJ, Welsh RM

Profound type 1 interferon (IFN1)-dependent attrition of memory CD8 and CD4 T cells occurs early during many infections. It is dramatic 2-4 days following lymphocytic choriomeningitis virus (LCMV) infection of mice and can be elicited by the IFN-inducing Toll receptor agonist poly(I:C). We show that this attrition occurs in many organs, indicating that it is due to T cell loss rather than redistribution. This loss correlated with elevated intracellular staining of T cells ex vivo for activated caspases, but only with low levels of ex vivo staining with Annexin V, probably due to the rapid clearance of apoptotic cells in vivo. Instead, a high frequency of Annexin V-reactive CD8alpha(+) dendritic cells (DC), known to be highly phagocytic, accumulated in the spleen as the memory T cell populations disappeared. After short in vitro incubation, memory phenotype T cells isolated from LCMV-infected mice (day 3) or mice treated with poly(I:C) (12 hours) displayed substantial DNA fragmentation, as detected by TUNEL assay, when compared to T cells isolated from uninfected mice, indicating a role for apoptosis in the memory T cell attrition. This apoptosis of memory CD8 T cells early during LCMV infection was reduced in mice lacking the pro-apoptotic molecule Bim. Evidence is presented showing that high levels of T cell attrition, as found in young mice, correlates with reduced immunodomination by cross-reactive memory cells.

PMID: 20200235 [PubMed - as supplied by publisher]

Bortezomib inhibits hepatitis B virus replication in transgenic mice.

Antimicrob Agents Chemother. 2010 Feb;54(2):749-56

Authors: Bandi P, Garcia ML, Booth CJ, Chisari FV, Robek MD

Pharmacological modulation of cellular proteins as a means to block virus replication has been proposed as an alternative antiviral strategy that may be less susceptible than others to the development of viral drug resistance. Recent evidence indicates that the ubiquitin-proteasome pathway interacts with different aspects of the hepatitis B virus (HBV) life cycle in cell culture models of virus replication. We therefore examined the effect of proteasome inhibition on HBV replication in vivo using HBV transgenic mice. The proteasome inhibitor bortezomib (Velcade) inhibits proteasome activity in vivo and is used therapeutically for the clinical treatment of multiple myeloma. We found that a single intravenous dose of 1 mg of bortezomib/kg of body weight reduced virus replication for as long as 6 days. The inhibition of HBV by bortezomib was dose dependent and occurred at a step in replication subsequent to viral RNA and protein expression. The reduction in HBV replication did not result from nonspecific hepatocellular toxicity and was not mediated indirectly through the induction of an intrahepatic interferon response. Thus, pharmacological manipulation of the ubiquitin-proteasome pathway may represent an alternative therapeutic approach for the treatment of chronic HBV infection.

PMID: 19949053 [PubMed - indexed for MEDLINE]

Rapid Three-Dimensional Phenotyping of Cardiovascular Development in Mouse Embryos by Micro-CT with Iodine Staining.

Circ Cardiovasc Imaging. 2010 Feb 27;

Authors: Degenhardt K, Wright AC, Horng D, Padmanabhan A, Epstein JA

BACKGROUND: -Micro-computed tomography (micro-CT) has been used extensively in research to generate high-resolution three-dimensional images of calcified tissues in small animals non-destructively. It has been especially useful for the characterization of skeletal mutations, but limited in its utility for the analysis of soft tissue such as the cardiovascular system. Visualization of the cardiovascular system has been largely restricted to structures that can be filled with radiopaque intravascular contrast agents in adult animals. Recent ex vivo studies using osmium tetroxide, iodinated contrast agents, inorganic iodine and phosphotungstic acid have demonstrated the ability to stain soft tissues differentially, allowing for high inter-tissue contrast in micro-CT images. Here, we demonstrate the application of this technology for visualization of cardiovascular structures in developing mouse embryos using Lugol's solution (aqueous potassium iodide plus iodine). METHODS AND RESULTS: -We show the optimization of this method to obtain ex vivo micro-CT images of embryonic and neonatal mice with excellent soft-tissue contrast. We demonstrate the utility of this method to visualize key structures during cardiovascular development at various stages of embryogenesis. Our method benefits from the ease of sample preparation, low toxicity, and low cost. Furthermore, we show how multiple cardiac defects can be demonstrated by micro-CT in a single specimen with a known genetic lesion. Indeed, a previously undescribed cardiac venous abnormality is revealed in a PlexinD1 mutant mouse. CONCLUSIONS: -Micro-CT of iodine stained tissue is a valuable technique for the characterization of cardiovascular development and defects in mouse models of congenital heart disease.

PMID: 20190279 [PubMed - as supplied by publisher]

Comparison of the osteoinductivity of bioimplants containing recombinant human bone morphogenetic proteins 2 (Infuse) and 7 (OP-1).

Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2010 Feb 24;

Authors: Barr T, McNamara AJ, Sándor GK, Clokie CM, Peel SA

OBJECTIVES: Recent research has focused on application of growth factors such as bone morphogenetic proteins (BMPs) as alternatives to autogenous bone grafting. Two bone graft substitute bioimplants containing recombinant human BMPs (rhBMPs), Infuse (rhBMP-2) and OP-1 (rhBMP-7), are approved for human application but have never been compared side by side. The aim of this study was to provide a direct comparison of the osteoinductive activity of the 2 commercially available and approved rhBMP-containing bioimplants in their clinically available forms. STUDY DESIGN: The activity of rhBMP-2 and -7 in solution were compared in vitro using the C2C12 cell-based assay. The activity of Infuse and OP-1 bioimplants containing 52.5 mug of rhBMP-2 or rhBMP-7, respectively, were compared in vivo using a mouse muscle pouch assay and analyzed by microscopic CT (microCT) and histology. RESULTS: The in vitro results showed that rhBMP-2 stimulated greater alkaline phosphatase production than rhBMP-7 over various time points and concentrations. The in vivo results showed that OP-1 induced greater bone volume than Infuse. Both implants induced bone of equivalent quality based on microCT and histologic evaluation. CONCLUSION: In their clinically available forms, the rhBMP-7-containing OP-1 induced greater bone volume than the rhBMP-2-containing Infuse in the mouse muscle pouch model.

PMID: 20188607 [PubMed - as supplied by publisher]

Multi-slice computed tomography with N1177 identifies ruptured atherosclerotic plaques in rabbits.

Basic Res Cardiol. 2010 Jan;105(1):51-9

Authors: Van Herck JL, De Meyer GR, Martinet W, Salgado RA, Shivalkar B, De Mondt R, Van De Ven H, Ludwig A, Van Der Veken P, Van Vaeck L, Bult H, Herman AG, Vrints CJ

Rupture-prone and ruptured plaques are characterized by the presence of large numbers of macrophages. N1177 is a contrast agent consisting of iodinated nanoparticles that are selectively phagocytosed by macrophages. The aim of this study was to investigate the effect of N1177 on the CT attenuation of rupture-prone and ruptured plaques in rabbits. In addition, we examined in vitro whether uptake of N1177 occurred without cytotoxic or pro-inflammatory effects on macrophages. In vitro, the viability of J774 macrophages was not affected by treatment with N1177. Moreover, N1177 had no effect on the phagocytic capacity or cytokine production of macrophages. For the in vivo experiments, 6 New Zealand White rabbits were fed a cholesterol-supplemented diet for 12-15 months, resulting in the development of large atherosclerotic plaques that resembled rupture-prone plaques in humans. In three rabbits, mechanical plaque rupture was induced by retrograde pullback of an embolic protection device. N1177 had no effect on the median density of rupture-prone plaques [35 HU (range 3-85) before injection vs. 32 HU (range 1-93) 2 h after injection of N1177; P > 0.05]. However, after induction of mechanical plaque rupture, the median density of the atherosclerotic plaques increased from 40 HU (range 6-86) before injection to 74 HU (range 14-111) 2 h after injection of N1177 (P < 0.001). Using time-of-flight static secondary ion mass spectrometry, the presence of N1177 nanoparticles was demonstrated in macrophage-rich areas of ruptured plaques, but not of non-ruptured plaques. In conclusion, our results show that N1177 is a contrast agent that can identify ruptured atherosclerotic plaques.

PMID: 19693628 [PubMed - indexed for MEDLINE]

A standardised study to compare prostate cancer targeting efficacy of five radiolabelled bombesin analogues.

Eur J Nucl Med Mol Imaging. 2010 Feb 25;

Authors: Schroeder RP, Müller C, Reneman S, Melis ML, Breeman WA, de Blois E, Bangma CH, Krenning EP, van Weerden WM, de Jong M

PURPOSE: Prostate-specific antigen (PSA)-based screening for prostate cancer (PC) has dramatically increased early diagnosis. Current imaging techniques are not optimal to stage early PC adequately. A promising alternative to PC imaging is peptide-based scintigraphy using radiolabelled bombesin (BN) analogues that bind to gastrin-releasing peptide receptors (GRPR) being overexpressed in PC. When labelled to appropriate radionuclides BN targeting of GRPRs may also provide applications for peptide radionuclide receptor therapy (PRRT). Assessment studies under identical experimental conditions allowing a reliable comparison of the potential of such analogues are lacking. This study was performed to evaluate and directly compare five promising radiolabelled BN analogues for their targeting efficacy for PC under standardised conditions. METHODS: The BN agonists [(111)In]DOTA-PESIN, [(111)In]AMBA, [(111)In]MP2346 and [(111)In]MP2653 and one antagonist [(99m)Tc]Demobesin-1 were evaluated in GRPR-overexpressing human PC-3 tumour-bearing mice to determine peptide stability in vivo, biodistribution and GRPR targeting potential by animal SPECT/CT imaging and ex vivo autoradiography. RESULTS: HPLC analysis of blood showed intact Demobesin-1 at 5 and 15 min after injection (64.1 +/- 1.6% and 41.0 +/- 01%, respectively) being much less for the other compounds. AMBA, the second most stable analogue, showed 36.1 +/- 2.7% and 9.8 +/- 1.1% intact peptide after 5 and 15 min. PC-3 tumour uptake at 1 h was comparable for Demobesin-1, AMBA, PESIN and MP2346 (3.0 +/- 0.4, 2.7 +/- 0.5, 2.3 +/- 0.5 and 2.1 +/- 0.9%ID/g, respectively), but very low for MP2653 (0.9 +/- 0.2%ID/g). In addition, MP2346 showed undesirably high uptake in the kidneys (7.9 +/- 1.9%ID/g) being significantly less for the other analogues. AMBA, MP2346 and PESIN revealed favourable increases in tumour to blood ratios over time while changes in tumour to kidney and pancreas ratios for Demobesin-1 from 1 to 24 h after injection were significantly better than for the other analogues. All analogues visualised PC-3 tumours by SPECT/CT and autoradiography. CONCLUSION: In the present study the BN antagonist Demobesin-1 was the best performing analogue showing superior in vivo stability, highest tumour uptake and retention while pancreatic and renal clearance were rapid. PESIN and AMBA were the best GRP agonists with sufficient in vivo stabilities as well as high tumour uptake and retention. Based on these results all three analogues deserve further evaluation for clinical use in PC patients.

PMID: 20182713 [PubMed - as supplied by publisher]

Activation of p38 MAPK pathway in the skull abnormalities of Apert syndrome Fgfr2+/P253R mice.

BMC Dev Biol. 2010 Feb 22;10(1):22

Authors: Wang Y, Sun M, Uhlhorn VL, Zhou X, Peter I, Martinez-Abadias N, Hill CA, Percival CJ, Richtsmeier JT, Huso DL, Jabs EW

ABSTRACT: BACKGROUND: Apert syndrome is characterized by craniosynostosis and limb abnormalities and is primarily caused by FGFR2 +/P253R and +/S252W mutations. The former mutation is present in approximately one third whereas the latter mutation is present in two-thirds of the patients with this condition. We previously reported an inbred transgenic mouse model with the Fgfr2 +/S252W mutation on the C57BL/6J background for Apert syndrome. Here we present a mouse model for the Fgfr2+/P253R mutation. RESULTS: We generated inbred Fgfr2 +/P253R mice on the same C56BL/6J genetic background and analyzed their skeletal abnormalities. 3D micro-CT head scans of the Fgfr2+/P253R mice revealed that the skull length was shortened with the length of the anterior cranial base significantly shorter than that of the Fgfr2+/S252W mice at P0. The Fgfr2+/P253R mice presented with synostosis in coronal suture and proximate fronts with disorganized cellularity in sagittal and lambdoid sutures. Abnormal osteogenesis and proliferation were observed at the developing coronal suture and long bones of the Fgfr2+/P253R mice as in the Fgfr2+/S252W mice. Activation of mitogen-activated protein kinases (MAPK) was observed in the Fgfr2+/P253R neurocranium with an increase in phosphorylated p38 as well as ERK1/2, whereas phosphorylated AKT and PKCalpha were not obviously changed as compared to those of wild-type controls. There were localized phenotypic and molecular variations among individual embryos with different mutations and among those with the same mutation. CONCLUSIONS: Our in vivo studies demonstrated that the Fgfr2 +/P253R mutation resulted in mice with cranial features that resemble those of the Fgfr2+/S252W mice and human Apert syndrome. Activated p38 in addition to the ERK1/2 signaling pathways may mediate the mutant neurocranial phenotype. Though Apert syndrome is traditionally thought to be a consistent phenotype, our results suggest localized and regional variations in the phenotypes that characterize Apert syndrome.

PMID: 20175913 [PubMed - as supplied by publisher]

In vivo bioluminescence tomography with a blocking-off finite-difference SP3 method and MRI/CT coregistration.

Med Phys. 2010 Jan;37(1):329-38

Authors: Klose AD, Beattie BJ, Dehghani H, Vider L, Le C, Ponomarev V, Blasberg R

PURPOSE: Bioluminescence imaging is a research tool for studying gene expression levels in small animal models of human disease. Bioluminescence light, however, is strongly scattered in biological tissue and no direct image of the light-emitting reporter probe's location can be obtained. Therefore, the authors have developed a linear image reconstruction method for bioluminescence tomography (BLT) that recovers the three-dimensional spatial bioluminescent source distribution in small animals. METHODS: The proposed reconstruction method uses third-order simplified spherical harmonics (SP3) solutions to the equation of radiative transfer for modeling the bioluminescence light propagation in optically nonuniform tissue. The SP3 equations and boundary conditions are solved with a finite-difference (FD) technique on a regular grid. The curved geometry of the animal surface was taken into account with a blocking-off region method for regular grids. Coregistered computed tomography (CT) and magnetic resonance (MR) images provide information regarding the geometry of the skin surface and internal organs. The inverse source problem is defined as an algebraic system of linear equations for the unknown source distribution and is iteratively solved given multiview and multispectral boundary measurements. The average tissue absorption parameters, which are used for the image reconstruction process, were calculated with an evolution strategy (ES) from in vivo measurements using an implanted pointlike source of known location and spectrum. Moreover, anatomical information regarding the location of the internal organs and other tissue structures within the animal's body are provided by coregistered MR images. RESULTS: First, the authors recovered the wavelength-dependent absorption coefficients (average error of 14%) with the ES under ideal conditions by using a numerical mouse model. Next, they reconstructed the average absorption coefficient of a small animal by using an artificial implanted light source and the validated ES. Last, they conducted two in vivo animal experiments and recovered the spatial location of the implanted light source and the spatial distribution of a bioluminescent reporter system located in the kidneys. The source reconstruction results were coregistered to CT and MR images. They further found that accurate bioluminescence image reconstructions could be obtained when segmenting a voidlike cyst with low-scattering and absorption parameters, whereas inaccurate image reconstructions were obtained when assuming a uniform optical parameter distribution instead. The image reconstructions were completed within 23 min on a 3 GHz Intel processor. CONCLUSIONS: The authors demonstrated on in vivo examples that the combination of anatomical coregistration, accurate optical tissue properties, multispectral acquisition, and a blocking-off FD-SP3 solution of the radiative transfer model significantly improves the accuracy of the BLT reconstructions.

PMID: 20175496 [PubMed - in process]

In Vivo Requirement for Atg5 in Antigen Presentation by Dendritic Cells.

Immunity. 2010 Feb 17;

Authors: Lee HK, Mattei LM, Steinberg BE, Alberts P, Lee YH, Chervonsky A, Mizushima N, Grinstein S, Iwasaki A

Autophagy is known to be important in presentation of cytosolic antigens on MHC class II (MHC II). However, the role of autophagic process in antigen presentation in vivo is unclear. Mice with dendritic cell (DC)-conditional deletion in Atg5, a key autophagy gene, showed impaired CD4(+) T cell priming after herpes simplex virus infection and succumbed to rapid disease. The most pronounced defect of Atg5(-/-) DCs was the processing and presentation of phagocytosed antigens containing Toll-like receptor stimuli for MHC class II. In contrast, cross-presentation of peptides on MHC I was intact in the absence of Atg5. Although induction of metabolic autophagy did not enhance MHC II presentation, autophagic machinery was required for optimal phagosome-to-lysosome fusion and subsequent processing of antigen for MHC II loading. Thus, our study revealed that DCs utilize autophagic machinery to optimally process and present extracellular microbial antigens for MHC II presentation.

PMID: 20171125 [PubMed - as supplied by publisher]

Some attenuated variants of vesicular stomatitis virus show enhanced oncolytic activity against human glioblastoma cells relative to normal brain cells.

J Virol. 2010 Feb;84(3):1563-73

Authors: Wollmann G, Rogulin V, Simon I, Rose JK, van den Pol AN

Vesicular stomatitis virus (VSV) has been shown in laboratory studies to be effective against a variety of tumors, including malignant brain tumors. However, attenuation of VSV may be necessary to balance the potential toxicity toward normal cells, particularly when targeting brain tumors. Here we compared 10 recombinant VSV variants resulting from different attenuation strategies. Attenuations included gene shifting (VSV-p1-GFP/RFP), M protein mutation (VSV-M51), G protein cytoplasmic tail truncations (VSV-CT1/CT9), G protein deletions (VSV-dG-GFP/RFP), and combinations thereof (VSV-CT9-M51). Using in vitro viability and replication assays, the VSV variants were grouped into three categories, based on their antitumor activity and non-tumor-cell attenuation. In the first group, wild-type-based VSV-G/GFP, tumor-adapted VSV-rp30, and VSV-CT9 showed a strong antitumor profile but also retained some toxicity toward noncancer control cells. The second group, VSV-CT1, VSV-dG-GFP, and VSV-dG-RFP, had significantly diminished toxicity toward normal cells but showed little oncolytic action. The third group displayed a desired combination of diminished general toxicity and effective antitumor action; this group included VSV-M51, VSV-CT9-M51, VSV-p1-GFP, and VSV-p1-RFP. A member of the last group, VSV-p1-GFP, was then compared in vivo against wild-type-based VSV-G/GFP. Intranasal inoculation of young, postnatal day 16 mice with VSV-p1-GFP showed no adverse neurological effects, whereas VSV-G/GFP was associated with high lethality (80%). Using an intracranial tumor xenograft model, we further demonstrated that attenuated VSV-p1-GFP targets and kills human U87 glioblastoma cells after systemic application. We concluded that some, but not all, attenuated VSV mutants display a favorable oncolytic profile and merit further investigation.

PMID: 19906910 [PubMed - indexed for MEDLINE]

Enhancing in vivo vascularized bone formation by cobalt chloride-treated bone marrow stromal cells in a tissue engineered periosteum model.

Biomaterials. 2010 Feb 11;

Authors: Fan W, Crawford R, Xiao Y

The periosteum plays an indispensable role in both bone formation and bone defect healing. In this study we constructed an artificial in vitro periosteum by incorporating osteogenic differentiated bone marrow stromal cells (BMSCs) and cobalt chloride (CoCl(2))-treated BMSCs. The engineered periostea were implanted both subcutaneously and into skull bone defects in SCID mice to investigate ectopic and orthotopic osteogenesis and vascularization. After two weeks in subcutaneous and four weeks in bone defect areas, the implanted constructs were assessed for ectopic and orthotopic osteogenesis and vascularization by micro-CT, histomorphometrical and immunohistochemical methods. The results showed that CoCl(2) pre-treated BMSCs induced higher degree of vascularization and enhanced osteogenesis within the implants in both ectopic and orthotopic areas. This study provided a novel approach using BMSCs sourced from the same patient for both osteogenic and pro-angiogenic purposes in constructing tissue engineered periosteum to enhance vascularized osteogenesis.

PMID: 20153522 [PubMed - as supplied by publisher]

MicroSPECT/CT Imaging and Pharmacokinetics of 188Re-(DXR)-liposome in Human Colorectal Adenocarcinoma-bearing Mice.

Anticancer Res. 2010 Jan;30(1):65-72

Authors: Chen MH, Chang CH, Chang YJ, Chen LC, Yu CY, Wu YH, Lee WC, Yeh CH, Lin FH, Lee TW, Yang CS, Ting G

Nanoliposome can be designed as a drug delivery carrier to improve the pharmacological and therapeutic properties of drug administration. (188)Re-labeled nanoliposomes are useful for diagnostic imaging as well as for targeted radionuclide therapy. In this study, the in vivo nuclear imaging, pharmacokinetics and biodistribution of administered nanoliposomes were investigated as drug and radionuclide carriers for targeting solid tumor via intravenous (i.v.) administration. The radiotherapeutics ((188)Re-liposome) and radiochemotherapeutics ((188)Re-DXR-liposome) were i.v. administered to nude mice bearing human HT-29 colorectal adenocarcinoma xenografts. (188)Re-liposome and (188)Re-DXR-liposomes show similar biodistribution profile; both have higher tumor uptake, higher blood retention time, and lower excretion rate than (188)Re-N,N-bis(2-mercaptoethyl)-N',N'-diethylenediamine (BMEDA). In contrast to tumor uptake, the area under the curve (AUC) value of tumor for (188)Re-liposome and (188)Re-DXR-liposome was 16.5- and 11.5-fold higher than that of free (188)Re-BMEDA, respectively. Additionally, (188)Re-liposome and (188)Re-DXR-liposome had a higher tumor-to-muscle ratio at 24 h (14.4+/-2 .7 and 17.14+/-4.1, respectively) than (188)Re-BMEDA (1.6+/-0.1). The tumor targeting and distribution of (188)Re-(DXR)-liposome (representing (188)Re-DXR-liposome and (188)Re-liposome) can also be acquired by signal photon-emission computed tomography/computed tomography images as well as whole body autoradiograph. These results suggest that (188)Re-(DXR)-liposomes are potentially promising agents for passive targeting treatment of malignant disease.

PMID: 20150618 [PubMed - in process]

Targeting Prostate Cancer Cells In Vivo Using a Rapidly Internalizing Novel Human Single-Chain Antibody Fragment.

J Nucl Med. 2010 Feb 11;

Authors: He J, Wang Y, Feng J, Zhu X, Lan X, Iyer AK, Zhang N, Seo Y, Vanbrocklin HF, Liu B

Human antibodies targeting prostate cancer cell surface epitopes may be useful for imaging and therapy. The objective of this study was to evaluate the tumor targeting of an internalizing human antibody fragment, a small-size platform, to provide high contrast in a mouse model of human prostate carcinoma. METHODS: A prostate tumor-targeting single-chain antibody fragment (scFv), UA20, along with a nonbinding control scFv, N3M2, were labeled with (99m)Tc and evaluated for binding and rapid internalization into human prostate tumor cells in vitro and tumor homing in vivo using xenograft models. For the in vitro studies, the labeled UA20 scFv was incubated at 37 degrees C for 1 h with metastatic prostate cancer cells (DU145) to assess the total cellular uptake versus intracellular uptake. For the animal studies, labeled UA20 and N3M2 scFvs were administered to athymic mice implanted subcutaneously with DU145 cells. Mice were imaged with small-animal SPECT/CT with concomitant biodistribution at 1 and 3 h after injection. RESULTS: The UA20 scFv was labeled in 55%-65% yield and remained stable in phosphate buffer within 24 h. The labeled UA20 scFv was taken up specifically by prostate tumor cells. Internalization was rapid, because incubation at 37 degrees C for less than 1 h resulted in 93% internalization of total cell-associated scFvs. In animal studies, SPECT/CT showed significant tumor uptake as early as 1 h after injection. At 3 h after injection, tumor uptake was 4.4 percentage injected dose per gram (%ID/g), significantly greater than all organs or tissues studied (liver, 2.7 %ID/g; other organs or tissues, <1 %ID/g), except the kidneys (81.4 %ID/g), giving tumor-to-blood and tumor-to-muscle ratios of 12:1 and 70:1, respectively. In contrast, the control antibody exhibited a tumor uptake of only 0.26 %ID/g, similar to that of muscle and fat. Tumor-specific targeting was evidenced by reduced tumor uptake of nearly 70% on administration of a 10-fold excess of unlabeled UA20 scFv. Kidney uptake was nonspecific, consistent with the route of excretion by scFvs. CONCLUSION: The UA20 scFv showed rapid and specific internalization in prostate tumor cells in vitro and accumulation in prostate tumor xenografts in vivo, demonstrating the potential for future development for prostate cancer imaging and targeted therapy.

PMID: 20150269 [PubMed - as supplied by publisher]

A Novel Facile Method of Labeling Octreotide with 18F-Fluorine.

J Nucl Med. 2010 Feb 11;

Authors: Laverman P, McBride WJ, Sharkey RM, Eek A, Joosten L, Oyen WJ, Goldenberg DM, Boerman OC

Several methods have been developed to label peptides with (18)F. However, in general these are laborious and require a multistep synthesis. We present a facile method based on the chelation of (18)F-aluminum fluoride (Al(18)F) by 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA). The method is characterized by the labeling of NOTA-octreotide (NOTA-D-Phe-cyclo[Cys-Phe-D-Trp-Lys-Thr-Cys]-Throl (MH(+) 1305) [IMP466]) with (18)F. METHODS: Octreotide was conjugated with the NOTA chelate and labeled with (18)F in a 2-step, 1-pot method. The labeling procedure was optimized with regard to the labeling buffer, peptide, and aluminum concentration. Radiochemical yield, specific activity, in vitro stability, and receptor affinity were determined. Biodistribution of (18)F-IMP466 was studied in AR42J tumor-bearing mice and compared with that of (68)Ga-labeled IMP466. In addition, small-animal PET/CT images were acquired. RESULTS: IMP466 was labeled with Al(18)F in a single step with 50% yield. The labeled product was purified by high-performance liquid chromatography to remove unbound Al(18)F and unlabeled peptide. The radiolabeling, including purification, was performed in 45 min. The specific activity was 45,000 GBq/mmol, and the peptide was stable in serum for 4 h at 37 degrees C. Labeling was performed at pH 4.1 in sodium citrate, sodium acetate, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, and 2-(N-morpholino)ethanesulfonic acid buffer and was optimal in sodium acetate buffer. The apparent 50% inhibitory concentration of the (19)F-labeled IMP466 determined on AR42J cells was 3.6 nM. Biodistribution studies at 2 h after injection showed a high tumor uptake of (18)F-IMP466 (28.3 +/- 5.2 percentage injected dose per gram [%ID/g]; tumor-to-blood ratio, 300 +/- 90), which could be blocked by an excess of unlabeled peptide (8.6 +/- 0.7 %ID/g), indicating that the accumulation in the tumor was receptor-mediated. Biodistribution of (68)Ga-IMP466 was similar to that of (18)F-IMP466. (18)F-IMP466 was stable in vivo, because bone uptake was only 0.4 +/- 0.2 %ID/g, whereas free Al(18)F accumulated rapidly in the bone (36.9 +/- 5.0 %ID/g at 2 h after injection). Small-animal PET/CT scans showed excellent tumor delineation and high preferential accumulation in the tumor. CONCLUSION: NOTA-octreotide could be labeled rapidly and efficiently with (18)F using a 2-step, 1-pot method. The compound was stable in vivo and showed rapid accretion in somatostatin receptor subtype 2-expressing AR42J tumors in nude mice. This method can be used to label other NOTA-conjugated compounds with (18)F.

PMID: 20150268 [PubMed - as supplied by publisher]

Astrocyte- and endothelial-targeted CCL2 conditional knockout mice: critical tools for studying the pathogenesis of neuroinflammation.

J Mol Neurosci. 2009 Sep;39(1-2):269-83

Authors: Ge S, Murugesan N, Pachter JS

While the expression of the C-C chemokine ligand 2 (CCL2) in the central nervous system (CNS) is associated with numerous neuroinflammatory conditions, the critical cellular sources of this chemokine, which is responsible for disease processes-as well as associated pathogenic mechanisms, remain unresolved. As the potential for anti-CCL2 therapeutics in treating neuroinflammatory disease is likely to be contingent upon effective drug delivery to the source(s) and/or target(s) of CCL2 action in the CNS, tools to highlight the course of CCL2 action during neuroinflammation are imperative. In response to this need, we used the Cre/loxP and FLP-FRT recombination system to develop the first two, cell-conditional CCL2 knockout mice-separately targeting CCL2 gene elimination to astrocytes and endothelial cells, both of which have been considered to play crucial though undefined roles in neuroinflammatory disease. Specifically, mice containing a floxed CCL2 allele were intercrossed with GFAP-Cre or Tie2-Cre transgenic mice to generate mice with CCL2-deficient astrocytes (astrocyte KO) or endothelial cells (endothelial KO), respectively. Polymerase chain reaction, reverse transcription polymerase chain reaction/quantitative reverse transcriptase polymerase chain reaction, and enzyme-linked immunosorbent assay of CCL2 gene, RNA, and protein, respectively, from cultured astrocytes and brain microvascular endothelial cells (BMEC) established the efficiency and specificity of the CCL2 gene deletions and a CCL2 null phenotype in these CNS cells. Effective cell-conditional knockout of CCL2 was also confirmed in an in vivo setting, wherein astrocytes and BMEC were retrieved by immune-guided laser capture microdissection from their in situ positions in the brains of mice experiencing acute, lipopolysaccharide-mediated endotoxemia to induce CCL2 gene expression. In vivo analysis further revealed apparent cross-talk between BMEC and astrocytes regarding the regulation of astrocyte CCL2 expression. Use of astrocyte KO and endothelial KO mice should prove critical in elaborating the pathogenic mechanisms of and optimizing the treatments for neuroinflammatory disease.

PMID: 19340610 [PubMed - indexed for MEDLINE]

DAPK1 Interaction with NMDA Receptor NR2B Subunits Mediates Brain Damage in Stroke.

Cell. 2010 Jan 22;140(2):222-234

Authors: Tu W, Xu X, Peng L, Zhong X, Zhang W, Soundarapandian MM, Balel C, Wang M, Jia N, Zhang W, Lew F, Chan SL, Chen Y, Lu Y

N-methyl-D-aspartate (NMDA) receptors constitute a major subtype of glutamate receptors at extrasynaptic sites that link multiple intracellular catabolic processes responsible for irreversible neuronal death. Here, we report that cerebral ischemia recruits death-associated protein kinase 1 (DAPK1) into the NMDA receptor NR2B protein complex in the cortex of adult mice. DAPK1 directly binds with the NMDA receptor NR2B C-terminal tail consisting of amino acid 1292-1304 (NR2B(CT)). A constitutively active DAPK1 phosphorylates NR2B subunit at Ser-1303 and in turn enhances the NR1/NR2B receptor channel conductance. Genetic deletion of DAPK1 or administration of NR2B(CT) that uncouples an activated DAPK1 from an NMDA receptor NR2B subunit in vivo in mice blocks injurious Ca(2+) influx through NMDA receptor channels at extrasynaptic sites and protects neurons against cerebral ischemic insults. Thus, DAPK1 physically and functionally interacts with the NMDA receptor NR2B subunit at extrasynaptic sites and this interaction acts as a central mediator for stroke damage.

PMID: 20141836 [PubMed - as supplied by publisher]

Assessment of the Na/I symporter as a reporter gene to visualize oncolytic adenovirus propagation in peritoneal tumours.

Eur J Nucl Med Mol Imaging. 2010 Feb 6;

Authors: Merron A, Baril P, Martin-Duque P, de la Vieja A, Tran L, Briat A, Harrington KJ, McNeish IA, Vassaux G

PURPOSE: In vivo imaging of the spread of oncolytic viruses using the Na/I symporter (NIS) has been proposed. Here, we assessed whether the presence of NIS in the viral genome affects the therapeutic efficacy of the oncolytic adenovirus dl922-947 following intraperitoneal administration, in a mouse model of peritoneal ovarian carcinoma. METHODS: We generated AdAM7, a dl922-947 oncolytic adenovirus encoding the NIS coding sequence. Iodide uptake, NIS expression, infectivity and cell-killing activity of AdAM7, as well as that of relevant controls, were determined in vitro. In vivo, the propagation of this virus in the peritoneal cavity of tumour-bearing mice was determined using SPECT/CT imaging and its therapeutic efficacy was evaluated. RESULTS: In vitro infection of ovarian carcinoma IGROV-1 cells with ADAM7 led to functional expression of NIS. However, the insertion of NIS into the viral genome resulted in a loss of efficacy of the virus in terms of replication and cytotoxicity. In vivo, on SPECT/CT imaging AdAM7 was only detectable in the peritoneal cavity of animals bearing peritoneal ovarian tumours for up to 5 days after intraperitoneal administration. Therapeutic experiments in vivo demonstrated that AdAM7 is as potent as its NIS-negative counterpart. CONCLUSION: This study demonstrated that despite the detrimental effect observed in vitro, insertion of the reporter gene NIS in an oncolytic adenovirus did not affect its therapeutic efficacy in vivo. We conclude that NIS is a highly relevant reporter gene to monitor the fate of oncolytic adenovectors in live subjects.

PMID: 20140612 [PubMed - as supplied by publisher]

Analytical radiography for planar radiographic images implemented with a multi-modal system.

Comput Methods Programs Biomed. 2010 Feb 2;

Authors: Vizard DL, Wood DO, Papineni RV, Feke GD, Orton SP, McLaughlin WE

A radiographic system is optimized for the contrast inherent to small animals and is developed for a multi-modal imaging system devised for in-vivo studies. The range of X-ray energies utilized (generally considered "soft X-rays") enables enhanced spatial resolution and superior contrast for detailed study of the mouse anatomy and smaller specimens. Despite the difficulties presented by the complicated energy spectrum of soft X-rays, relevant system calibrations for bone measures are described in detail and applied to the mouse. Further, long-bone symmetry modeling using a cylindrical projection is applied to the planar density image, providing convenient bone density estimates that are consistent with other methodologies.

PMID: 20133007 [PubMed - as supplied by publisher]