Uptake Of Radiolabel Research Articles

The riboflavin (vitamin B2) transporter protein (SmaRT) of the human intravascular parasitic trematode Schistosoma mansoni

Schistosomes are intravascular parasitic worms infecting >200 million people globally. Here we examine how the worms acquire an essential nutrient - vitamin B2 (riboflavin). We demonstrate that all intravascular life stages (schistosomula, adult males and females) take up radiolabeled riboflavin. This process is impeded in the presence of excess unlabeled riboflavin and at 4 °C. We have identified a transporter homolog in worms designated SmaRT (Schistosoma mansoni riboflavin transporter) that localizes to the tegument and internal tissues of adults. CHO–S cells transfected with plasmid encoding SmaRT import significantly more radiolabeled riboflavin compared to controls. Uptake of radiolabel is impeded when SmaRT-expressing cells are incubated in an excess of unlabeled riboflavin but not by an excess of an irrelevant metabolite. Uptake is mediated in a sodium-independent manner and over a wide range of pH values (pH 5.5–9). This is the first identification of a bone fide riboflavin transporter in any platyhelminth.

The prevalence and significance of nonuniform thyroid radio-isotope uptake in patients with Graves' disease.

ObjectiveTo evaluate the prevalence and clinical significance of nonuniform technetium (99mTc) uptake among patients with Graves’ disease (GD).Design, Patients and MeasurementsPatients with GD, referred between July 2005 and March 2018, had Tc99‐ uptake scans and TSH‐receptor antibody (TRAb) measured before antithyroid drug (ATD) therapy. Risk of relapse after ATD cessation was monitored until June 2021 and compared between GD patients based on uptake patterns.ResultsOf the 276 GD patients (mean age, 49.8 years; 84% female), 25 (9.0%) had nonuniform Tc99 uptake. At diagnosis, individuals with nonuniform uptake were older (mean age of 61.8 vs. 48.5 years, p < .001), had lower mean thyroid hormone levels (free thyroxine: 36.3 vs. 45.4 pmol/L, p = .04 and free triiodothyronine: 10.0 vs. 17.8 pmol/L, p < .001) and median TRAb levels (4.2 vs. 6.6 U/L, p = .04) compared with those with a uniform uptake. Older age was a significant predictor for the presence of nonuniform uptake in GD patients; odds ratio (95% confidence intervals) of 1.07 (1.03 – 1.10). The risk of relapse was similar in both groups after a median (IQR) follow‐up of 41 (13–74) months after ATD cessation (56.0% vs. 46.3%, respectively); hazard ratio (95% confidence intervals) of 1.74 (0.96–3.15).ConclusionsNonuniform radio‐isotope uptake is seen in 1 in 11 patients with GD which could be misdiagnosed as toxic multinodular goitre if TRAb levels are not measured. Treatment of GD patients with nonuniform radio‐isotope uptake with ATD therapy as first‐line appears to be equally effective as compared with those with uniform uptake. TRAb testing should be the main diagnostic test for patients with suspected GD with radio‐labelled uptake scans being reserved for those who are TRAb negative.

Functional characterization of the adenine transporter EaAdeP from the fire blight pathogen Erwinia amylovora and its effect on disease establishment in apples and pears.

Erwinia amylovora is the causal agent of fire blight, an economically important disease of apples and pears. As part of the infection process, Er. amylovora propagates on different plant tissues each with distinct nutrient environments. Here, the biochemical properties of the Er. amylovora adenine permease (EaAdeP) are investigated. Heterologous expression of EaAdeP in nucleobase transporter-deficient Escherichia coli strains, coupled with radiolabel uptake studies, revealed that EaAdeP is a high affinity adenine transporter with a Km of 0.43± 0.09 μM. Both Es. coli and Er. amylovora carrying extra copies of EaAdeP are sensitive to growth on the toxic analog 8-azaadenine. EaAdeP is expressed during immature pear fruit infection. Immature pear and apple fruit virulence assays reveal that an E. amylovora ΔadeP::Camr mutant is still able to cause disease symptoms, however, with growth at a lower level, indicating that external adenine is utilized in disease establishment.

Targeted Deletion of Hepatocyte Abca1 Increases Plasma HDL (High-Density Lipoprotein) Reverse Cholesterol Transport via the LDL (Low-Density Lipoprotein) Receptor.

The role of hepatocyte Abca1 (ATP binding cassette transporter A1) in trafficking hepatic free cholesterol (FC) into plasma versus bile for reverse cholesterol transport (RCT) is poorly understood. We hypothesized that hepatocyte Abca1 recycles plasma HDL-C (high-density lipoprotein cholesterol) taken up by the liver back into plasma, maintaining the plasma HDL-C pool, and decreasing HDL-mediated RCT into feces. Approach and Results: Chow-fed hepatocyte-specific Abca1 knockout (HSKO) and control mice were injected with human HDL radiolabeled with 125I-tyramine cellobiose (125I-TC; protein) and 3H-cholesteryl oleate (3H-CO). 125I-TC and 3H-CO plasma decay, plasma HDL 3H-CO selective clearance (ie, 3H-125I fractional catabolic rate), liver radiolabel uptake, and fecal 3H-sterol were significantly greater in HSKO versus control mice, supporting increased plasma HDL RCT. Twenty-four hours after 3H-CO-HDL injection, HSKO mice had reduced total hepatic 3H-FC (ie, 3H-CO hydrolyzed to 3H-FC in liver) resecretion into plasma, demonstrating Abca1 recycled HDL-derived hepatic 3H-FC back into plasma. Despite similar liver LDLr (low-density lipoprotein receptor) expression between genotypes, HSKO mice treated with LDLr-targeting versus control antisense oligonucleotide had slower plasma 3H-CO-HDL decay, reduced selective 3H-CO clearance, and decreased fecal 3H-sterol excretion that was indistinguishable from control mice. Increased RCT in HSKO mice was selective for 3H-CO-HDL, since macrophage RCT was similar between genotypes. Hepatocyte Abca1 deletion unmasks a novel and selective FC trafficking pathway that requires LDLr expression, accelerating plasma HDL-selective CE uptake by the liver and promoting HDL RCT into feces, consequently reducing HDL-derived hepatic FC recycling into plasma.

Abstract PD4-12: Use of 64Cu-DOTA-trastuzumab positron emission tomography (PET) to predict response to ado-trastuzumab emtansine (TDM1)

Abstract Background: We have demonstrated that 64Cu-DOTA trastuzumab is an effective PET imaging agent for HER2 positive breast cancer and are now seeking to evaluate the methodology for prediction of response and benefit to ado-trastuzumab emtansine (TDM1) in women with metastatic disease. Methods: Patients with metastatic breast cancer were eligible if they had biopsy confirmed HER2 + disease, at least 1 site of metastasis 2.0 cm or larger outside the biopsy site, and were to receive TDM1 as therapy. Pretreatment staging included 18F-FDG/PET. Prior to injection of 64Cu-DOTA-trastuzumab, patients received 45 mg of cold trastuzumab to reduce liver uptake. PET-CT scans were obtained at 21-25 h and 47-48 h over fields of view chosen in reference to 18F-FDG scans. TDM1 was administered at a dose of 3.6 mg/kg every 3 weeks. Restaging 18F-FDG/PET was performed every 2 cycles, and response to therapy was determined by PERCIST criteria. The radiolabel uptake was measured in terms of maximum voxel standardized uptake value (SUVmax). Results: Ten women enrolled on study and are evaluable for response; three continue on TDM1 for 22-40 months and four patients remained on treatment for at least 1 year. The median age was 54.5 years (48-83 years); seven received prior trastuzumab-containing chemotherapy 3 wks to 55 months prior to study entry. HER2 was positive by IHC in 5 and by FISH in 5 (3 were 2+ by IHC; 1 was 1+ and 1 indeterminate). Complete or partial metabolic response was observed in 5 patients. Patients had their average SUVmax on 64Cu-DOTA trastuzumab PET (aSUVmax) assessed in addition to individual assessments on up to 8 lesions on both Day1 and Day2. The mean aSUVmax was (6.3, 8.8) for responding patients and (4.4, 5.2) for non-responder (day1, day2). The difference between responders and non-responders on Day1 aSUVmax was marginally significant (p=0.06), but significant on Day2 (p=0.04). The three highest aSUVmax on both day1 and day2 were three of the four patients with PFS&amp;gt;1 year. Data on the relationship of 64Cu-DOTA trastuzumab PET to IHC and FISH, and individual lesion SUVmax including evidence suggesting a potential threshold effect will be presented. Conclusions: In women with biopsy confirmed HER2 positive metastatic disease, 64Cu-DOTA-trastuzumab PET imaging is predictive for response to TDM1. Citation Format: Mortimer JE, Bading JR, Frankel P, Tumyan L, Tran TT, Rockne RC, Shively JE, Gidwaney N, Park J, Colcher DM. Use of 64Cu-DOTA-trastuzumab positron emission tomography (PET) to predict response to ado-trastuzumab emtansine (TDM1) [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr PD4-12.

Impact of Ga-68 DOTATATE imaging on clinical management of neuroendocrine tumor patients: Post-FDA-approval analysis of first 200 clinical patients.

238 Background: Gallium 68 dotatate (Ga-68 DOTA) provides physiologic imaging and assists in the localization of disease in somatostatin (SSA) receptor positive neuroendocrine tumor (NET) patients. Questions regarding the value of Ga-68 DOTA imaging in the FDA post-approval era for NET patients include: usefulness in disease monitoring, correlation of Ga-68 DOTA avidity to Ki-67 index and its impact on clinical decision making. We present our experience with 200 Ga-DOTATATE scans performed at University of Kentucky. Methods: A retrospective review of the first 200 patients who had undergone Ga-68 DOTA imaging at the Markey Cancer Center from Dec 2016 to Dec 2017 was conducted. Results: 59.5% were females and the median age was 62 (30-84 years). Primary tumor sites included: small bowel 37.5%, pancreas 18.5%, bronchial 14%, colon 3.5%, rectum 2%, appendix 1.5%, adrenal 0.5%, prostate 0.5%, others 3% and unknown primary 19%. Ga-68 DOTA scan influenced clinical decisions in 39% (n = 78) patients. Ga-68 DOTA imaging identified primary tumors in 17 of 38 patients who were classified as NET of unknown primary based on CT imaging. Subgroup analysis of mean standardized uptake value (SUV) for hepatic metastatic lesions revealed 37.3 for G1 (n = 20) as compared to 32.3 for G2 (n = 37) and 17.46 for G3 (n = 4). Mean hepatic SUV of the lesion with the greatest radiolabel uptake in 96 patients was similar irrespective of exposure to SSA LAR; 31.3 versus 27.8 for SSA versus the no SSA cohorts. Conclusions: Ga-68 DOTA imaging impacted clinical decision making in 39% of NET patients (n = 200) and identified the primary site in 17 of 38 patients with unknown primary. Systemic exposure to long acting SSA does not seem to impact quality of Ga-68 DOTA scan.

F18-choline/C11-choline PET/CT thyroid incidentalomas.

Thyroid incidentaloma is defined as a thyroid lesion incidentally and newly detected by imaging techniques performed for an unrelated purpose and especially for a non-thyroid disease. Aim of this review is to evaluate the prevalence and clinical significance of focal incidental radiolabelled choline uptake in the thyroid gland (CTI) revealed by PET or PET/CT. A comprehensive computer literature search of the PubMed/MEDLINE, Scopus, and Embase databases was conducted to find relevant published articles about the prevalence and clinical significance of CTIs detected by PET or PET/CT in patients studied for other oncologic purposes. Fifteen articles (14 case reports, one retrospective study on a larger population sample) were included in the systematic review. Considering the case reports, 7/14 CTIs were benign and 7/14 malignant. In the retrospective study on a larger population sample, 14/15 CTIs which underwent further investigations were benign. Despite very rare but probably underestimated, CTIs frequently signal in the presence of unexpected lesions in the thyroid that differ from the indicated reason for which the patient was initially scanned, and the risk of malignancy is not negligible.

Radiometal-Dependent Biological Profile of the Radiolabeled Gastrin-Releasing Peptide Receptor Antagonist SB3 in Cancer Theranostics: Metabolic and Biodistribution Patterns Defined by Neprilysin.

Recent advances in oncology involve the use of diagnostic/therapeutic radionuclide-carrier pairs that target cancer cells, offering exciting opportunities for personalized patient treatment. Theranostic gastrin-releasing peptide receptor (GRPR)-directed radiopeptides have been proposed for the management of GRPR-expressing prostate and breast cancers. We have recently introduced the PET tracer 68Ga-SB3 (SB3, DOTA- p-aminomethylaniline-diglycolic acid-DPhe-Gln-Trp-Ala-Val-Gly-His-Leu-NHEt), a receptor-radioantagonist that enables the visualization of GRPR-positive lesions in humans. Aiming to fully assess the theranostic potential of SB3, we herein report on the impact of switching 68Ga to 111In/177Lu-label on the biological properties of resulting radiopeptides. Notably, the bioavailability of 111In/177Lu-SB3 in mice drastically deteriorated compared with metabolically robust 68Ga-SB3, and as a result led to poorer 111In/177Lu-SB3 uptake in GRPR-positive PC-3 xenografts. The peptide cleavage sites were identified by chromatographic comparison of blood samples from mice intravenously receiving 111In/177Lu-SB3 with each of newly synthesized 111In/177Lu-SB3-fragments. Coinjection of the radioconjugates with the neprilysin (NEP)-inhibitor phosphoramidon led to full stabilization of 111In/177Lu-SB3 in peripheral mouse blood and resulted in markedly enhanced radiolabel uptake in the PC-3 tumors. In conclusion, in situ NEP-inhibition led to indistinguishable 68Ga/111In/177Lu-SB3 profiles in mice emphasizing the theranostic prospects of SB3 for clinical use.

Tumor Uptake of 64Cu-DOTA-Trastuzumab in Patients with Metastatic Breast Cancer.

The goal of this study was to characterize the relationship between tumor uptake of 64Cu-DOTA-trastuzumab as measured by PET/CT and standard, immunohistochemistry (IHC)-based, histopathologic classification of human epidermal growth factor receptor 2 (HER2) status in women with metastatic breast cancer (MBC). Methods: Women with biopsy-confirmed MBC and not given trastuzumab for 2 mo or more underwent complete staging, including 18F-FDG PET/CT. Patients were classified as HER2-positive (HER2+) or -negative (HER2-) based on fluorescence in situ hybridization (FISH)-supplemented immunohistochemistry of biopsied tumor tissue. Eighteen patients underwent 64Cu-DOTA-trastuzumab injection, preceded in 16 cases by trastuzumab infusion (45 mg). PET/CT was performed 21-25 (day 1) and 47-49 (day 2) h after 64Cu-DOTA-trastuzumab injection. Radiolabel uptake in prominent lesions was measured as SUVmax Average intrapatient SUVmax (<SUVmax>pt) was compared between HER2+ and HER2- patients. Results: Eleven women were HER2+ (8 immunohistochemistry 3+; 3 immunohistochemistry 2+/FISH amplified), whereas 7 were HER2- (3 immunohistochemistry 2+/FISH nonamplified; 4 immunohistochemistry 1+). Median <SUVmax>pt for day 1 and day 2 was 6.6 and 6.8 g/mL for HER 2+ and 3.7 and 4.3 g/mL for HER2- patients (P < 0.005 either day). The distributions of <SUVmax>pt overlapped between the 2 groups, and interpatient variability was greater for HER2+ than HER2- disease (P < 0.005 and 0.001, respectively, on days 1 and 2). Conclusion: By 1 d after injection, uptake of 64Cu-DOTA-trastuzumab in MBC is strongly associated with patient HER2 status and is indicative of binding to HER2. The variability within and among HER2+ patients, as well as the overlap between the HER2+ and HER2- groups, suggests a role for 64Cu-DOTA-trastuzumab PET/CT in optimizing treatments that include trastuzumab.

64Cu-DOTA-trastuzumab PET/CT to predict response to ado-trastuzumab emtansine (TDM1) in HER2-positive metastatic breast cancer.

e12525 Background: Having demonstrated that 64Cu-DOTA trastuzumab is an effective PET imaging agent for HER2 positive (HER2+) breast cancer, we now evaluate the methodology for prediction of response and benefit from TDM1 in women with metastatic disease. Methods: Patients eligible to receive TDM1 as therapy were chosen on the basis of biopsy confirmed HER2+ disease, and at least 1 site of metastasis ≥ 2.0 cm outside the biopsy site. Pretreatment staging included 18F-FDG PET/CT. Prior to injection of 64Cu-DOTA-trastuzumab, patients received 45 mg of cold trastuzumab to reduce liver uptake. PET-CT scans were obtained at 16-28 h (Day1) and 39-49 h (Day 2) over fields of view chosen in reference to the 18F-FDG scans. TDM1 (3.6 mg/kg) was administered every 3 weeks. Restaging 18F-FDG PET/CT was performed every 2 cycles, and response to therapy was determined by PERCIST (solid tumor) criteria. Radiolabel uptake was measured in terms of maximum voxel, standardized uptake value (SUVmax). Results: Ten women between the ages of 48-83 years old (median 55 years) qualified for study, and have been evaluated for response; 4 continue on TDM1 with treatment durations of 3-27 months.Three were trastuzumab-naïve, while 7 had received trastuzumab-containing chemotherapy 3 wks to 55 mo prior to study entry. HER2 was positive by ImmunoHistoChemistry, IHC (3+) in 5 patients, and by FISH testing in the other 5 (3 were 2+ by IHC; 1 was 1+, and 1 was indeterminate). Complete or partial metabolic response was observed in 5 patients. Median Day 2 SUVmax for 64Cu-DOTA trastuzumab was 9.3 g/ml in responding patients, and 4.6 g/ml in non-responders ( P= 0.03). Progression-free survival was longer for patients with higher SUVmax, with a hazard ratio of 0.73 (95% confidence interval 0.46-1.16) for each 1 unit increase in SUVmax. This was not statistically significant, although we can select a threshold SUVmaxfor which the effect is significant even for this small study. Further data is required to confirm such a threshold effect. Conclusions: 64Cu-DOTA-trastuzumab PET imaging predicts benefit from TDM1 in women with biopsy-confirmed HER2+ metastatic disease. Supported by NCI Clinical trial information: NCT02827877.

Rapid uptake, biotransformation, esterification and lack of depuration of testosterone and its metabolites by the common mussel, Mytilus spp.

The presence of the vertebrate steroids, testosterone (T) and 17β-estradiol in mollusks is often cited as evidence that they are involved in the control of their reproduction. In this paper, we show that a likely source of T in at least one species, the common mussel (Mytilus spp.), is from uptake from water. When mussels were exposed to waterborne tritiated T ([3H]-T) in a closed container, the radioactivity decreased rapidly and exponentially until, by 24h, approximately 35% remained in the water. The rate of uptake of radiolabel could not be saturated by concentrations as high as 16.5μgL−1 (mean measured) of non-radiolabeled T, showing that the animals have a very high capacity for uptake of T. At least 30% of the applied radioactivity could be extracted from the tissues of the animals with organic solvents and most of this (26% of the total applied radioactivity) was in the fatty acid ester fraction. Following alkaline hydrolysis, reverse phase HPLC and TLC, this fraction was shown to consist predominantly of 5α-dihydrotestosterone and 5α-androstane-3β,17β-diol, while T was a minor component. These steroids were definitively identified in the fatty acid ester fraction by mass spectrometry. Overall, less than 5% of the [3H]-T applied to the system remained untransformed at the end of exposure. After ten days of depuration there was no reduction in the total amount of radioactivity in the tissues, nor any changes in the ratio of the metabolites in the ester fraction. These findings show that any association between T presence and reproductive status or sex is confounded by their significant capacity for uptake, and that T undergoes extensive metabolism in mussels in vivo and therefore may not be representative of the androgenic burden of the animals. Consequently, measurements of T in mussel tissue offer little utility as an indicator of reproductive status or sex.

Brain uptake and metabolism of the endocannabinoid anandamide labeled in either the arachidonoyl or ethanolamine moiety

IntroductionAnandamide (N-arachidonoylethanolamine) is a retrograde neuromodulator that activates cannabinoid receptors. The concentration of anandamide in the brain is controlled by fatty acid amide hydrolase (FAAH), which has been the focus of recent drug discovery efforts. Previous studies in C57BL/6 mice using [3H-arachidonoyl]anandamide demonstrated deposition of tritium in thalamus and cortical areas that was blocked by treatment with an FAAH inhibitor and that was not seen in FAAH-knockout mice. This suggested that long chain fatty acid amides radiolabeled in the fatty acid moiety might be useful as ex vivo and in vivo radiotracers for FAAH, since labeled fatty acid released by hydrolysis would be rapidly incorporated into phospholipids with long metabolic turnover periods. MethodsRadiotracers were administered intravenously to conscious Swiss–Webster mice, and radioactivity concentrations in brain areas was quantified and radiolabeled metabolites determined by radiochromatography. Results[14C]Arachidonic acid, [14C-arachidonoyl]anandamide and [14C-ethanolamine]anandamide, and also [14C]myristic acid, [14C-myristoyl]myristoylethanolamine and [14C-ethanolamine]myristoyl-ethanolamine all had very similar distribution patterns, with whole brain radioactivity concentrations of 2–4% injected dose per gram. Pretreatment with the potent selective FAAH inhibitor URB597 did not significantly alter distribution patterns although radiochromatography demonstrated that the rate of incorporation of label from [14C]anandamide into phospholipids was decreased. Pretreatment with the muscarinic agonist arecoline which increases cerebral perfusion increased brain uptake of radiolabel from [14C]arachidonic acid and [14C-ethanolamine]anandamide, and (in dual isotope studies) from the unrelated tracer [125I]RTI-55. ConclusionsTogether with our previously published study with [18F-palmitoyl]16-fluoro-palmitoylethanolamine, the data show that the primary determinant of brain uptake for these tracers in Swiss–Webster mice is initial distribution according to blood flow. It is possible that recently reported strain differences in long chain fatty acid trafficking between C57BL/6 and Swiss–Webster mice are responsible for the differences between our results using [14C]anandamide and the earlier studies using [3H]anandamide.

Functional Imaging of Human Epidermal Growth Factor Receptor 2–Positive Metastatic Breast Cancer Using 64Cu-DOTA-Trastuzumab PET

Women with human epidermal growth factor receptor 2 (HER2)-positive breast cancer are candidates for treatment with the anti-HER2 antibody trastuzumab. Assessment of HER2 status in recurrent disease is usually made by core needle biopsy of a single lesion, which may not represent the larger tumor mass or other sites of disease. Our long-range goal is to develop PET of radiolabeled trastuzumab for systemically assessing tumor HER2 expression and identifying appropriate use of anti-HER2 therapies. The purpose of this study was to evaluate PET/CT of (64)Cu-DOTA-trastuzumab for detecting and measuring tumor uptake of trastuzumab in patients with HER2-positive metastatic breast cancer. Eight women with biopsy-confirmed HER2-positive metastatic breast cancer and no anti-HER2 therapy for 4 mo or longer underwent complete staging, including (18)F-FDG PET/CT. For 6 of the 8 patients, (64)Cu-DOTA-trastuzumab injection (364-512 MBq, 5 mg of trastuzumab) was preceded by trastuzumab infusion (45 mg). PET/CT (PET scan duration 1 h) was performed 21-25 (day 1) and 47-49 (day 2) h after (64)Cu-DOTA-trastuzumab injection. Scan fields of view were chosen on the basis of (18)F-FDG PET/CT. Tumor detection sensitivity and uptake analyses were limited to lesions identifiable on CT; lesions visualized relative to adjacent tissue on PET were considered PET-positive. Radiolabel uptake in prominent lesions was measured as maximum single-voxel standardized uptake value (SUVmax). Liver uptake of (64)Cu was reduced approximately 75% with the 45-mg trastuzumab predose, without significant effect on tumor uptake. The study included 89 CT-positive lesions. Detection sensitivity was 77%, 89%, and 93% for day 1, day 2, and (18)F-FDG, respectively. On average, tumor uptake was similar for (64)Cu-DOTA-trastuzumab and (18)F-FDG (SUVmax and range, 8.1 and 3.0-22.5 for day 1 [n = 48]; 8.9 and 0.9-28.9 for day 2 [n = 38]; 9.7 and 3.3-25.4 for (18)F-FDG [n = 56]), but same-lesion SUVmax was not correlated between the 2 radiotracers. No toxicities were observed, and estimated radiation dose from (64)Cu-DOTA-trastuzumab was similar to (18)F-FDG. (64)Cu-DOTA-trastuzumab visualizes HER2-positive metastatic breast cancer with high sensitivity and is effective in surveying disseminated disease. A 45-mg trastuzumab predose provides a (64)Cu-DOTA-trastuzumab biodistribution favorable for tumor imaging. (64)Cu-DOTA-trastuzumab PET/CT warrants further evaluation for assessing tumor HER2 expression and individualizing treatments that include trastuzumab.

175. EFFECTS OF SPECIES DIFFERENCES ON OOCYTE REGULATION OF GRANULOSA CELL PROLIFERATION

The role of oocytes in regulating ovulation quota between species is not fully understood. In sheep, the oocyte-derived growth factors, growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) have profound effects on ovarian follicular development and ovulation-quota. The aim of these studies was to compare the ability of oocytes from sheep (low ovulation-rate) and rat (poly-ovulator) to stimulate radiolabelled thymidine uptake by granulosa cells (GC) both within and between the two species. For these experiments, 32 oocytes denuded of cumulus-cells were co-incubated with 20 000 GC from either species. Rat or sheep oocytes stimulated thymidine uptake by GC from the same species (P &lt; 0.005). Sheep oocytes also stimulated thymidine uptake by rat GC (P &lt; 0.001) but not vice versa. To investigate this further, oocytes and GC were co-incubated with 3.2 µg/mL or 7.6 mg/mL monoclonal antibodies specific to GDF9 or BMP15 respectively and to a hydatids antigen (control). Both sheep and rat oocyte stimulation of thymidine uptake by GC was inhibited with the GDF9 antibody (P &lt; 0.05) but not control, irrespective of species of GC. Sheep oocyte stimulation of rat GC was also inhibited using an antibody to BMP15 (P &lt; 0.05). However, when using the BMP15 antibody to block the effects of rat oocytes on rat GC, the inhibition of thymidine uptake was modest (i.e. ~10%) albeit significant (P &lt; 0.05). The molecular forms of GDF9 and BMP15 in spent media were examined by Western blotting under reducing conditions. For both species, oocyte-secreted GDF9 was present in the mature form. For sheep oocytes, secreted BMP15 was present as promature and monomeric mature forms whereas from rats, trace amounts of mature form was sometimes, but not always, detected. Thus, in sheep both BMP15 and GDF9 are essential for regulating GC proliferation whereas in rats, although responsive to BMP15, GC proliferation is likely regulated primarily by GDF9.

Transport of the Photodynamic Therapy Agent 5-Aminolevulinic Acid by Distinct H+-Coupled Nutrient Carriers Coexpressed in the Small Intestine

5-Aminolevulinic acid (ALA) is a prodrug used in photodynamic therapy, fluorescent diagnosis, and fluorescent-guided resection because it leads to accumulation of the photosensitizer protoporphyrin IX (PpIX) in tumor tissues. ALA has good oral bioavailability, but high oral doses are required to obtain selective PpIX accumulation in colonic tumors because accumulation is also observed in normal gut mucosa. Structural similarities between ALA and GABA led us to test the hypothesis that the H(+)-coupled amino acid transporter PAT1 (SLC36A1) will contribute to luminal ALA uptake. Radiolabel uptake and electrophysiological measurements identified PAT1-mediated H(+)-coupled ALA symport after heterologous expression in Xenopus oocytes. The selectivity of the nontransported inhibitors 5-hydroxytryptophan and 4-aminomethylbenzoic acid for, respectively, PAT1 and the H(+)-coupled di/tripeptide transporter PepT1 (SLC15A1) were examined. 5-Hydroxytryptophan selectively inhibited PAT1-mediated amino acid uptake across the brush-border membrane of the human intestinal (Caco-2) epithelium whereas 4-aminomethylbenzoic acid selectively inhibited PepT1-mediated dipeptide uptake. The inhibitory effects of 5-hydroxytryptophan and 4-aminomethylbenzoic acid were additive, demonstrating that both PAT1 and PepT1 contribute to intestinal transport of ALA. This is the first demonstration of overlap in substrate specificity between these distinct transporters for amino acids and dipeptides. PAT1 and PepT1 expression was monitored by reverse transcriptase-polymerase chain reaction using paired samples of normal and cancer tissue from human colon. mRNA for both transporters was detected. PepT1 mRNA was increased 2.3-fold in cancer tissues. Thus, increased PepT1 expression in colonic cancer could contribute to the increased PpIX accumulation observed. Selective inhibition of PAT1 could enhance PpIX loading in tumor tissue relative to that in normal tissue.

Myocardial fatty acid imaging identifies a group of hemodialysis patients at high risk for cardiac death after coronary revascularization

We prospectively evaluated if impaired myocardial fatty acid metabolism is involved in cardiac death after revascularization by percutaneous coronary artery intervention in dialysis patients. A cohort of hemodialysis patients was assessed by dual single-photon emission computed tomography using the radioiodinated fatty acid analogue BMIPP and radiolabeled thallium chloride. Tomography was done within one month before the first coronary intervention and at the last follow-up angiography at which neither restenosis nor de novo lesions were detected. Radiolabel uptake on tomography images was graded in segments and calculated as summed BMIPP or thallium scores. Among the 90 hemodialysis patients in the study, 19 died of cardiac events. Multivariate Cox hazard analysis found a significant association of cardiac death with the BMIPP summed scores at the last follow-up angiography. Kaplan-Meier analysis showed the cardiac death-free survival rates at 3 years of follow-up were significantly higher in patients with lower BMIPP summed scores. These results suggest that myocardial fatty acid imaging may be a useful test to identify high risk groups of cardiac death in hemodialysis patients.

Imaging of Gene Expression in Live Pancreatic Islet Cell Lines Using Dual-Isotope SPECT

We are combining nuclear medicine with molecular biology to establish a sensitive, quantitative, and tomographic method with which to detect gene expression in pancreatic islet cells in vivo. Dual-isotope SPECT can be used to image multiple molecular events simultaneously, and coregistration of SPECT and CT images enables visualization of reporter gene expression in the correct anatomic context. We have engineered pancreatic islet cell lines for imaging with SPECT/CT after transplantation under the kidney capsule. INS-1 832/13 and alphaTC1-6 cells were stably transfected with a herpes simplex virus type 1-thymidine kinase-green fluorescent protein (HSV1-thymidine kinase-GFP) fusion construct (tkgfp). After clonal selection, radiolabel uptake was determined by incubation with 5-(131)I-iodo-1-(2-deoxy-2-fluoro-beta-d-arabinofuranosyl)uracil ((131)I-FIAU) (alphaTC1-6 cells) or (123)I-FIAU (INS-1 832/13 cells). For the first set of in vivo experiments, SPECT was conducted after alphaTC1-6/tkgfp cells had been labeled with either (131)I-FIAU or (111)In-tropolone and transplanted under the left kidney capsule of CD1 mice. Reconstructed SPECT images were coregistered to CT. In a second study using simultaneous acquisition dual-isotope SPECT, INS-1 832/13 clone 9 cells were labeled with (111)In-tropolone before transplantation. Mice were then systemically administered (123)I-FIAU and data for both (131)I and (111)In were acquired simultaneously. alphaTC1-6/tkgfp cells showed a 15-fold greater uptake of (131)I-FIAU, and INS-1/tkgfp cells showed a 12-fold greater uptake of (123)I-FIAU, compared with that of wild-type cells. After transplantation under the kidney capsule, both reporter gene expression and location of cells could be visualized in vivo with dual-isotope SPECT. Immunohistochemistry confirmed the presence of glucagon- and insulin-positive cells at the site of transplantation. Dual-isotope SPECT is a promising method to detect gene expression in and location of transplanted pancreatic cells in vivo.

Establishing a Definitive Stoichiometry for the Na +/Monocarboxylate Cotransporter SMCT1

Several different stoichiometries have been proposed for the Na +/monocarboxylate cotransporter SMCT1, including variable Na +/substrate stoichiometry. In this work, we have definitively established an invariant 2:1 cotransport stoichiometry for SMCT1. By using two independent means of assay, we first showed that SMCT1 exhibits a 2:1 stoichiometry for Na +/lactate cotransport. Radiolabel uptake experiments proved that, unlike lactate, propionic acid diffuses passively through oocyte membranes and, consequently, propionate is a poor candidate for stoichiometric determination by these methods. Although we previously determined SMCT1 stoichiometry by measuring reversal potentials, this technique produced erroneous values, because SMCT1 simultaneously mediates both an inwardly rectifying cotransport current and an outwardly rectifying anionic leak current; the leak current predominates in the range where reversal potentials are observed. We therefore employed a method that compared the effect of halving the external Na + concentration to the effect of halving the external substrate concentration on zero-current potentials. Both lactate and propionate were cotransported through SMCT1 using 2:1 stoichiometries. The leak current passing through the protein has a 1 osmolyte/charge stoichiometry. Identification of cotransporter stoichiometry is not always a trivial task and it can lead to a much better understanding of the transport activity mediated by the protein in question.

Evaluation of CD20, CD22, and HLA-DR Targeting for Radioimmunotherapy of B-Cell Lymphomas

Despite the promise of radioimmunotherapy using anti-CD20 antibodies (Ab) for the treatment of relapsed patients with indolent non-Hodgkin lymphoma (NHL), most patients treated with conventional doses of (131)I-tositumomab or (90)Y-ibritumomab eventually relapse. We did comparative assessments using conventional radioimmunotherapy targeting CD20, CD22, and HLA-DR on human Ramos, Raji, and FL-18 lymphoma xenografts in athymic mice to assess the potential for improving the efficacy of radioimmunotherapy by targeting other NHL cell surface antigens. Results of biodistribution studies showed significant differences in tumor localization consistent with variable antigenic expression on the different lymphoma cell lines. Interestingly, the radioimmunoconjugate that yielded the best tumor-to-normal organ ratios differed in each tumor model. We also explored administering all three (111)In-1,4,7,10-tetra-azacylododecane N,N',N'',N'''-tetraacetic acid antibodies in combination, but discovered, surprisingly, that this approach did not augment the localization of radioactivity to tumors compared with the administration of the best single radiolabeled Ab alone. These data suggest that conventional radioimmunotherapy using anti-CD20, anti-HLA-DR, or anti-CD22 Abs is effective when used singly and provides targeted uptake of radiolabel into the tumor that is dependent on the levels of antigen expression. Improvements in tumor-to-normal organ ratios of radioactivity cannot be achieved using directly labeled Abs in combination but may be afforded by novel pretargeting methods.

Medical Therapy of Children With Fibrous Dysplasia

FIBROUS DYSPLASIA (FD) is a sporadic developmental disorder characterized by expanding fibrous lesions throughout the skeleton. On X-rays, the dysplastic lesions frequently have a lytic or cystic appearance. At the tissue level, the lesions consist of abnormal fibrous tissue in the marrow space intertwined with poorly defined and irregular trabeculae reminiscent of woven bone. Bone scans usually show increased radiolabel uptake in the affected areas. Biochemical indices of bone turnover are usually high (particularly serum tissue nonspecific alkaline phosphatase activity) and correlate with the extent of the lesions. Hypophosphatemia is a common finding, probably caused by a circulating phosphaturic factor. The sites involved are typically the long bones and the skull. They are often unilateral. In some cases, a single lesion is present (monostotic form). Polyostotic (more than one site) forms are the most frequent, with, in rare instances, FD involving the whole skeleton (panostotic form). When bone lesions are associated with various endocrinopathies (mostly precocious puberty) and skin lesions (cafe-au-lait-spots), it is referred to as the McCuneAlbright syndrome (OMIM #174800).