Organ-specific Uptake Research Articles

Organ-Specific Uptake of Extracellular Vesicles Secreted by Urological Cancer Cells.

Simple SummaryExtracellular vesicles (EVs) play an important role in the communication of cancer cells with their local microenvironment and distant organ systems, in order to promote a supportive tumor microenvironment, as well as to prepare premetastatic niches. In this study, we aimed to analyze if the EVs secreted by urological cancer cells are taken up by specific organ systems, depending on their origin. After the intravenous injection of fluorescence-labeled EVs from benign and malignant prostate, kidney, and bladder cells in immunodeficient mice, their organs were harvested and analyzed for the presence of fluorescent EVs. We could show that (i) EVs are taken up not entirely organ-specifically but in different amounts, depending on their origin; (ii) EVs from malignant cells are taken up more efficiently than EVs from benign cells; and (iii) EVs are taken up very fast. These observations hint to an organotropism in EV uptake, which needs to be further investigated.Extracellular vesicles (EVs) secreted by cancer cells have been shown to take a pivotal part in the process of local and systemic tumor progression by promoting the formation of a supportive local tumor microenvironment and preparing premetastatic niches in distant organ systems. In this study, we analyzed the organ-specific uptake of EVs secreted by urological cancer cells using an innovative in-vivo approach. EVs from benign and malignant prostate, kidney, and bladder cells were isolated using ultracentrifugation, fluorescence-labeled and injected intravenously in immunodeficient mice. After 12 or 24 h, the animals were sacrificed, their organs were harvested and analyzed for the presence of EVs by high-resolution fluorescence microscopy. Across all entities, EVs were taken up fast (12 h > 24 h), and EVs from malignant cells were taken up more efficiently than EVs from benign cells. Though not entirely organ-specific, EVs were incorporated in different amounts, depending on the entity (prostate: lung > liver > brain; kidney: brain > lung > liver; bladder: lung > liver > brain). EV uptake in other organs than lung, liver, brain, and spleen was not observed. Our results suggest a role of EVs in the formation of premetastatic niches and an organotropism in EV uptake, which have to be examined in more detail in further studies.

P0537 - Organ-specific uptake of extracellular vesicles secreted by urological cancer cells

P0537 - Organ-specific uptake of extracellular vesicles secreted by urological cancer cells

Creatine transport and pathological changes in creatine transporter deficient mice.

The severe impact on brain function and lack of effective therapy for patients with creatine (Cr) transporter deficiency motivated the generation of three ubiquitous Slc6a8 deficient mice (-/y). While each mouse knock-out line has similar behavioral effects at 2 to 3 months of age, other features critical to the efficient use of these mice in drug discovery are unclear or lacking: the concentration of Cr in brain and heart differ widely between mouse lines, there are limited data on histopathologic changes, and no data on Cr uptake. Here, we determined survival, measured endogenous Cr and uptake of its deuterium-labeled analogue Cr-d3 using a liquid chromatography coupled with tandem mass spectrometry assay, and performed comprehensive histopathologic examination on the Slc6a8-/y mouse developed by Skelton et al. Our results show that Slc6a8-/y mice have widely varying organ-specific uptake of Cr-d3, significantly diminished growth with the exception of brain, progressive vacuolar myopathy, and markedly shortened lifespan.

Abstract LB-210: Chemotherapy promotes metastasis through extracellular vesicle secretion

Abstract Neoadjuvant chemotherapy treatment is the gold standard for children and adults with intermediate- to high-risk tumors. However, a subset of patients develop distant metastases following treatment, and evidence suggests that chemotherapy itself may promote metastasis in some patients. Indeed, utilizing xenograft mouse model of human triple-negative breast cancer, we confirm that treatment with the chemotherapeutic agent Doxorubicin decreases primary tumor growth while promoting pulmonary metastasis. The purpose of this research is to understand how chemotherapy treatment regulates tumor-derived exosomes, extracellular vesicles that are secreted by all cells and that function in cellular communication through the horizontal transfer of biologically-active molecules. Specifically, our goal is to determine how exosomes derived from chemotherapy-treated tumor cells, or ‘chemoexosomes,' regulate tumor metastasis in order to develop more effective chemotherapeutic options for cancer patients. Utilizing models of both breast cancer and pediatric neuroblastoma, we demonstrate that sublethal doses of DNA-damaging chemotherapeutic agents significantly enhance exosome secretion compared to vehicle control. Xenografted mice treated with exosomes derived from chemotherapy-treated cells demonstrate a significant acceleration in metastasis with no increase in primary breast tumor growth compared to mice treated with exosomes derived from control-treated cells or saline. Although chemoexosomes do not enhance tumor cell migration or invasion in vitro, they demonstrate tumor type-specific preferential organotropic localization to the most common sites of tumor metastasis in vivo. Mice treated with chemoexosomes prior to tail-vein injection of either neuroblastoma or breast tumor cells demonstrate a significant increase in metastatic tumor burden compared to mice pre-treated with control exosomes or saline. Through proteomic analysis, we identified differentially-expressed exosomal proteins upregulated by chemotherapy treatment. Utilizing CRISPR-Cas9-mediated gene knockout, we identified a component of the complement pathway upregulated in chemoexosomes that is critically involved in exosome-mediated metastasis. Taken together, this research proposes a novel mechanism of chemotherapy-induced metastasis by which organ-specific uptake of exosomes derived from chemotherapy-treated cells primes the pre-metastatic niche in order to create a favorable environment for metastatic tumor growth. Citation Format: Carson A. Wills, Jeffrey Sundstrom, Hong-Gang Wang. Chemotherapy promotes metastasis through extracellular vesicle secretion [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-210.

Tetrazine-Derived Near-Infrared Dye as a Facile Reagent for Developing Targeted Photoacoustic Imaging Agents.

A new photoacoustic (PA) dye was developed as a simple-to-use reagent for creating targeted PA imaging agents. The lead molecule was prepared via an efficient two-step synthesis from an inexpensive commercially available starting material. With the dye's innate albumin-binding properties, the resulting tetrazine-derived dye is capable of localizing to tumor and exhibits a biological half-life of a few hours, allowing for an optimized distribution profile. The presence of tetrazine in turn makes it possible to link the albumin-binding optoacoustic signaling agent to a wide range of targeting molecules. To demonstrate the utility and ease of use of the platform, a novel PA probe for imaging calcium accretion was generated using a single-step bioorthogonal coupling reaction where high-resolution PA images of the knee joint in mice were obtained as early as 1 h post injection. Whole-body distribution was subsequently determined by labeling the probe with 99mTc and performing tissue counting following necropsy. These studies, along with tumor imaging and in vitro albumin binding studies, revealed that the core PA contrast agent can be imaged in vivo and can be easily linked to targeting molecules for organ-specific uptake.

Establishment of a [18F]-FDG-PET/MRI Imaging Protocol for Gastric Cancer PDX as a Preclinical Research Tool.

PurposeThe utility of 18-fluordesoxyglucose positron emission tomography ([18F]-FDG-PET) combined with computer tomography or magnetic resonance imaging (MRI) in gastric cancer remains controversial and a rationale for patient selection is desired. This study aims to establish a preclinical patient-derived xenograft (PDX) based [18F]-FDG-PET/MRI protocol for gastric cancer and compare different PDX models regarding tumor growth and FDG uptake.Materials and MethodsFemale BALB/c nu/nu mice were implanted orthotopically and subcutaneously with gastric cancer PDX. [18F]-FDG-PET/MRI scanning protocol evaluation included different tumor sizes, FDG doses, scanning intervals, and organ-specific uptake. FDG avidity of similar PDX cases were compared between ortho- and heterotopic tumor implantation methods. Microscopic and immunohistochemical investigations were performed to confirm tumor growth and correlate the glycolysis markers glucose transporter 1 (GLUT1) and hexokinase 2 (HK2) with FDG uptake.ResultsOrgan-specific uptake analysis showed specific FDG avidity of the tumor tissue. Standard scanning protocol was determined to include 150 μCi FDG injection dose and scanning after one hour. Comparison of heterotopic and orthotopic implanted mice revealed a long growth interval for orthotopic models with a high uptake in similar PDX tissues. The H-score of GLUT1 and HK2 expression in tumor cells correlated with the measured maximal standardized uptake value values (GLUT1: Pearson r=0.743, P=0.009; HK2: Pearson r=0.605, P=0.049).ConclusionsThis preclinical gastric cancer PDX based [18F]-FDG-PET/MRI protocol reveals tumor specific FDG uptake and shows correlation to glucose metabolic proteins. Our findings provide a PET/MRI PDX model that can be applicable for translational gastric cancer research.

Biodistribution of orally administered 99mTc-heparin to assess eosinophilic esophagitis associated inflammation

Here we explore the biodistribution of 99mTc-heparin, an orally administered radiopharmaceutical candidate expected to identify localized inflammation in eosinophilic esophagitis (EoE). Heparin binds to eosinophil granule proteins in inflamed regions of gastrointestinal (GI) organs, and 99mTc signals their location. This article evaluates the biodistribution of this conjugate first within the esophagus by modeling the reactive transport of 99mTc-heparin based on the kinetics of 99mTc-heparin binding to non-human primate esophagi and second in the remainder of the body by measuring organ specific uptake using single-photon emission computed tomography/computed tomography imaging. Results show that binding and dissociation rate constants remain similar, and that optimal imaging occurs after drinking of the agent is complete. We also find that 99mTc-heparin remains within the GI tract simply transporting through the gut and out. These findings provide a new avenue to clinically explore the utility of 99mTc-heparin to detect eosinophil associated inflammation in the GI tract.

P23 - Organ-specific uptake of cancer-associated extracellular vesicles

P23 - Organ-specific uptake of cancer-associated extracellular vesicles

The adaptor protein PID1 regulates receptor-dependent endocytosis of postprandial triglyceride-rich lipoproteins

ObjectiveInsulin resistance is associated with impaired receptor dependent hepatic uptake of triglyceride-rich lipoproteins (TRL), promoting hypertriglyceridemia and atherosclerosis. Next to low-density lipoprotein (LDL) receptor (LDLR) and syndecan-1, the LDLR-related protein 1 (LRP1) stimulated by insulin action contributes to the rapid clearance of TRL in the postprandial state. Here, we investigated the hypothesis that the adaptor protein phosphotyrosine interacting domain-containing protein 1 (PID1) regulates LRP1 function, thereby controlling hepatic endocytosis of postprandial lipoproteins. MethodsLocalization and interaction of PID1 and LRP1 in cultured hepatocytes was studied by confocal microscopy of fluorescent tagged proteins, by indirect immunohistochemistry of endogenous proteins, by GST-based pull down and by immunoprecipitation experiments. The in vivo relevance of PID1 was assessed using whole body as well as liver-specific Pid1-deficient mice on a wild type or Ldlr-deficient (Ldlr−/−) background. Intravital microscopy was used to study LRP1 translocation in the liver. Lipoprotein metabolism was investigated by lipoprotein profiling, gene and protein expression as well as organ-specific uptake of radiolabelled TRL. ResultsPID1 co-localized in perinuclear endosomes and was found associated with LRP1 under fasting conditions. We identified the distal NPxY motif of the intracellular C-terminal domain (ICD) of LRP1 as the site critical for the interaction with PID1. Insulin-mediated NPxY-phosphorylation caused the dissociation of PID1 from the ICD, causing LRP1 translocation to the plasma membrane. PID1 deletion resulted in higher LRP1 abundance at the cell surface, higher LDLR protein levels and, paradoxically, reduced total LRP1. The latter can be explained by higher receptor shedding, which we observed in cultured Pid1-deficient hepatocytes. Consistently, PID1 deficiency alone led to increased LDLR-dependent endocytosis of postprandial lipoproteins and lower plasma triglycerides. In contrast, hepatic PID1 deletion on an Ldlr−/− background reduced lipoprotein uptake into liver and caused plasma TRL accumulation. ConclusionsBy acting as an insulin-dependent retention adaptor, PID1 serves as a regulator of LRP1 function controlling the disposal of postprandial lipoproteins. PID1 inhibition provides a novel approach to lower plasma levels of pro-atherogenic TRL remnants by stimulating endocytic function of both LRP1 and LDLR in the liver.

Uptake and effects of 2, 4, 6 - trinitrotoluene (TNT) in juvenile Atlantic salmon (Salmo salar)

Organ specific uptake and depuration, and biological effects in Atlantic salmon (Salmo salar) exposed to 2, 4, 6-trinitrotoluene (TNT) were studied. Two experiments were conducted, the first using radiolabeled TNT (14C-TNT, 0.16mg/L) to study uptake (48h) and depuration (48h), while the second experiment focused on physiological effects in fish exposed to increasing concentrations of unlabeled TNT (1μg–1mg/L) for 48h. The uptake of 14C-TNT in the gills and most of the organs increased rapidly during the first 6h of exposure (12h in the brain) followed by a rapid decrease even though the fish were still exposed to TNT in the water. The radioactivity in the gall bladder reached a maximum after 55h, 7h after the transfer to the clean water. A high concentration of 14C-TNT in the gall bladder indicates that TNT is excreted through the gall bladder. Mortality (2 out of 14) was observed at a concentration of 1mg/L, and the surviving fish had hemorrhages in the dorsal muscle tissue near the spine. Analysis of the physiological parameters in blood from the high exposure group revealed severe effects, with an increase in the levels of glucose, urea and HCO3, and a decrease in hematocrit and the levels of Cl and hemoglobin. No effects on blood physiology were observed in fish exposed to the lower concentrations of TNT (1–100μg/L). TNT and the metabolites 2-amino-4,6-dinitrotoluene (2-ADNT) and 4-amino-2,6-dinitrotoluene (4-ADNT) were found in the muscle tissue, whereas only 2-ADNT and 4-ADNT were found in the bile. The rapid excretion and estimated bioconcentration factors (range of 2–18 after 48h in gills, blood, liver, kidney, muscle and brain) indicated a low potential for bioaccumulation of TNT.

Total iodine quantification in fluids and tissues from iodine- or iodide-supplemented rats by ion chromatography following microwave-assisted digestion.

Iodine is a crucial component of thyroid hormones, and several reports have shown that iodine per se is implicated in the physiopathology of other organs. Innovative ion chromatography detection following a four-step temperature ramp microwave digestion in 25-50 mM nitric acid was developed to measure total iodine in biological fluids and tissue samples from female Sprague-Dawley rats supplemented with 0.05% molecular iodine (I2) or 0.05% potassium iodide (I(-)) in drinking water. The reported method allows the measurement of total iodine with a limit of quantification of 13.7 μg L(-1), recoveries of 96.3-100.3%, and intra- and inter-assay variations, of 3.5% and 7.4% respectively. Analysis of biological fluids showed that after 48 hours, iodine-supplemented animals exhibited significantly higher levels of total iodine in both serum and urine compared with those supplemented with iodide. The half-life of iodine in serum and urine measured over the first 48 h showed similar patterns for both the I2 (7.89 and 7.76 hours) and I(-) (8.27 and 8.90 hours) supplements. Differential uptake patterns were observed in tissues after 6 days of supplements, with I(-) preferentially retained by thyroid, lactating mammary gland, and milk, and a slightly but significantly higher capture of I2 in pituitary, ovary, and virgin mammary gland. We developed a rapid, selective, and accurate digestion method to process fluid and tissue samples that permits reproducible measurements of total iodine by ion chromatography; iodine or iodide supplement show a similar serum and urine half-life, but organ-specific uptake depends on the chemical form of the iodine supplement.

Selective X-ray contrast enhancement of the spleen of living mice mediated by gold nanorods.

Gold nanomaterials (AuNPs) represent a promising new class of contrast agents for X-ray computed tomographic (CT) imaging in both research and clinical settings. These materials exhibit superior X-ray absorption properties compared with other iodinated agents, and thus require lower injection doses. Gold is nonimmunogenic and therefore contributes to safety profile in living specimens. Unfortunately, most reports on the use of AuNPs as X-ray CT enhancers only demonstrate marginal enhancement of the intended anatomical structure. In this study, we demonstrate the dramatic properties of gold nanorods (GNR) to serve as robust X-ray CT contrast-enhancing agent for selective imaging of the spleen. These organ-specific uptake properties were delineated by performing longitudinal CT imaging of living mice that were dosed with GNR at 2 day intervals. Rapid uptake in spleen was noted within 12 h of first systemic administration with a change in contrast enhancement of 90 Hounsfield units (ΔHU = 90) and with two subsequent injections a total contrast enhancement of over 200 HU was observed. The resulting images provide excellent contrast that will enable the detailed anatomical visualization and study of a range of pre-clinical models of spleen disease including infection and cancer.

Preclinical evaluation of BAY 1075553, a novel 18F-labelled inhibitor of prostate-specific membrane antigen for PET imaging of prostate cancer

Prostate-specific membrane antigen (PSMA) is a transmembrane protein overexpressed in prostate cancer and is therefore being explored as a biomarker for diagnosing and staging of the disease. Here we report preclinical data on BAY 1075553 (a 9:1 mixture of (2S,4S)- and (2R,4S)-2-[(18)F]fluoro-4-phosphonomethyl-pentanedioic acid), a novel (18)F-labelled small molecule inhibitor of PSMA enzymatic activity, which can be efficiently synthesized from a direct radiolabelling precursor. The (18)F-radiolabelled stereoisomers of 2-[(18)F]fluoro-4-(phosphonomethyl)-pentanedioic acid were synthesized from their respective isomerically pure precursors dimethyl 2-{[bis(benzyloxy)phosphoryl]methyl}-4-(tosyloxy)pentanedioate. In vivo positron emission tomography (PET) imaging and biodistribution studies were conducted in mice bearing LNCaP, 22Rv1 and PC-3 tumours. Pharmacokinetic parameters and dosimetry estimates were calculated based on biodistribution studies in rodents. For non-clinical safety assessment (safety pharmacology, toxicology) to support a single-dose human microdose study, off-target effects in vitro, effects on vital organ functions (cardiovascular in dogs, nervous system in rats), mutagenicity screens and an extended single-dose study in rats were conducted with the non-radioactive racemic analogue of BAY 1075553. BAY 1075553 showed high tumour accumulation specific to PSMA-positive tumour-bearing mice and was superior to other stereoisomers tested. Fast clearance of BAY 1075553 resulted overall in low background signals in other organs except for high uptake into kidney and bladder which was mainly caused by renal elimination of BAY 1075553. A modest uptake into bone was observed which decreased over time indicating organ-specific uptake as opposed to defluorination of BAY 1075553 in vivo. Biodistribution studies found highest organ doses for kidneys and the urinary bladder wall resulting in a projected effective dose (ED) in humans of 0.0219 mSv/MBq. Non-clinical safety studies did not show off-target activity, effects on vital organs function or dose-dependent adverse effects. BAY 1075553 was identified as a promising PET tracer for PSMA-positive prostate tumours in preclinical studies. BAY 1075553 can be produced using a robust, direct radiosynthesis procedure. Pharmacokinetic, toxicology and safety pharmacology studies support the application of BAY 1075553 in a first-in-man microdose study with single i.v. administration.

Dynamic contrast-enhanced optical imaging of in vivo organ function

Conventional approaches to optical small animal molecular imaging suffer from poor resolution, limited sensitivity, and unreliable quantitation, often reducing their utility in practice. We previously demonstrated that the in vivo dynamics of an injected contrast agent could be exploited to provide high-contrast anatomical registration, owing to the temporal differences in each organ's response to the circulating fluorophore. This study extends this approach to explore whether dynamic contrast-enhanced optical imaging (DyCE) can allow noninvasive, in vivo assessment of organ function by quantifying the differing cellular uptake or wash-out dynamics of an agent in healthy and damaged organs. Specifically, we used DyCE to visualize and measure the organ-specific uptake dynamics of indocyanine green before and after induction of transient liver damage. DyCE imaging was performed longitudinally over nine days, and blood samples collected at each imaging session were analyzed for alanine aminotransferase (ALT), a liver enzyme assessed clinically as a measure of liver damage. We show that changes in DyCE-derived dynamics of liver and kidney dye uptake caused by liver damage correlate linearly with ALT concentrations, with an r2 value of 0.91. Our results demonstrate that DyCE can provide quantitative, in vivo, longitudinal measures of organ function with inexpensive and simple data acquisition.

Modification of OATP2B1-Mediated Transport by Steroid Hormones

The family of the organic anion transporting polypeptides forms an increasing group of uptake transport proteins with a wide substrate spectrum. Although the expression of some members of this group, such as organic anion transporting polypeptide (OATP)-A or C, is limited to special tissues (such as liver or brain), the organic anion transporting polypeptide 2B1 (OATPB/SLCO2B1) is expressed in many organs, including liver, placenta, mammary gland, brain, and intestine. However, little is known about its function in those tissues because only a limited number of compounds, such as dehydroepiandrosterone-sulfate (DHEAS) and estrone-3-sulfate (E3S), have been characterized as OATP2B1 substrates. To further elucidate the role of OATP2B1 on steroid transport, we examined the influence of steroid hormones on OATP2B1-mediated E3S and DHEAS uptake using OATP2B1-overexpressing Madin-Darby canine kidney II cells. We identified unconjugated androgens (e.g., testosterone) as potent inhibitors for OATP2B1. In contrast, gestagenes such as progesterone enhanced E3S uptake in a concentration-dependent manner to up to 300% of the control, accompanied by a significant decrease in the OATP2B1 K(m) value for E3S (control, K(m) = 14 microM; in the presence of 31.6 muM progesterone, K(m) = 3.6 microM). Moreover, we demonstrated that testosterone and progesterone are not substrates of OATP2B1, indicating an allosteric mechanism for the observed effects. Furthermore, we showed that progesterone enhances the OATP2B1-dependent pregnenolone sulfate transport. Taken together, the results indicate functional modification of OATP2B1-mediated E3S and DHEAS as well as pregnenolone sulfate transport through steroid hormones such as progesterone. These effects can have physiological consequences for the organ-specific uptake of steroids.

Localization of 131I-labelled monoclonal antibody ERIC1 in a subcutaneous xenograft model of neuroblastoma in SCID mice

To evaluate a novel strategy of immunolocalization of human neuroblastoma by targeting the neural cell adhesion molecule (NCAM), which is over-expressed on neuroblastoma. NCAM expression on the cell surface of established neuroblastoma cells was shown by flow cytometry. A SCID mouse model using IMR5-75 neuroblastoma cells to induce subcutaneous tumour growth was established. 131I was used to label monoclonal NCAM specific ERIC1 antibodies generating the 131I-ERIC1 antibody, which showed a high affinity to NCAM also after labelling (KD=9 x 10(-8) mol . l(-1)). Measurement of organ-specific radioactivity showed low organ-specific uptake (5.33%ID/g (percent of injected dose per gram of tissue) after 72 h), which continuously decreased over the 96 h investigation period, demonstrating clearance of radioactivity. In contrast, tumours accumulated radioactivity continuously up to a peak of 42.07%ID/g at the 96 h time point (31.07%ID/g at 72 h). This specific uptake could be blocked by application of unlabelled ERIC1 antibodies. Measurement of blood specific radioactivity revealed a characteristic clearance over the first 72 h. With 37 Gy, tumour-specific radioactivity reached therapeutic doses after 96 h. These results indicate that 131I-labelled ERIC1 has the ability to probe NCAM-expressing tumour cells in vivo with high efficiency and is a promising reagent for the diagnosis and treatment of NCAM-positive human tumours, especially for neuroblastoma.

HDLs in apoA-I transgenic Abca1 knockout mice are remodeled normally in plasma but are hypercatabolized by the kidney

Patients homozygous for Tangier disease have a near absence of plasma HDL as a result of mutations in ABCA1 and hypercatabolize normal HDL particles. To determine the relationship between ABCA1 expression and HDL catabolism, we investigated intravascular remodeling, plasma clearance, and organ-specific uptake of HDL in mice expressing the human apolipoprotein A-I (apoA-I) transgene in the Abca1 knockout background. Small HDL particles (7.5 nm), radiolabeled with (125)I-tyramine cellobiose, were injected into recipient mice to quantify plasma turnover and the organ uptake of tracer. Small HDL tracer was remodeled to 8.2 nm diameter particles within 5 min in human apolipoprotein A-I transgenic (hA-I(Tg)) mice (control) and knockout mice. Decay of tracer from plasma was 1.6-fold more rapid in knockout mice (P < 0.05) and kidney uptake was twice that of controls, with no difference in liver uptake. We also observed 2-fold greater hepatic expression of ABCA1 protein in hA-I(Tg) mice compared with nontransgenic mice, suggesting that overexpression of human apoA-I stabilized hepatic ABCA1 protein in vivo. We conclude that ABCA1 is not required for in vivo remodeling of small HDLs to larger HDL subfractions and that the hypercatabolism of normal HDL particles in knockout mice is attributable to a selective catabolism of HDL apoA-I by the kidney.

Titer determination of Ad5 in blood: a cautionary note.

Recombinant adenoviruses are presently the most efficient in vivo gene transfer system available. Targeting single organs or large tumors by adenoviral vectors requires an intravascular route of application. During the first pass of viral particles through the vascular bed of the target tissue, virus uptake is not quantitative and indefinite amounts of particles leak into circulation. To determine the amount of leaking particles and to calculate organ-specific uptake (in-/outflow ratio), it is necessary to titrate virus particles directly in blood. In preclinical and clinical trials titration is currently mostly done with blood plasma instead of full blood. However, this technique provides valid results only as long as there is no affinity between adenovirus particles and erythrocytes. In this study we demonstrate that Ad5 particles, as mostly employed for gene therapy, have a strong affinity to human erythrocytes. At 60 min after coincubation of human erythrocytes and Ad5 particles, more than 98% of the particles are attached to the surface of erythrocytes. Therefore, ignoring the amount of red cell bound particles by performing titration in plasma leads to severe miscalculation of organ-specific transfer rates or virus circulation half-life. The biological impact of an increased affinity between virus particles and erythrocytes will be discussed.