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Reducing the background fluorescence in mice receiving fluorophore/inhibitor DNA duplexes

Mon, 12/15/2014 - 1:55pm

In principle, a DNA duplex consisting of an antisense fluorophore-conjugated major strand hybridized to a shorter complementary inhibitor-conjugated minor strand should provide fluorescence only in the tumor after intravenous administration if designed to remain intact except in the presence in tumor of its mRNA target. While we have obtained impressive tumor images in mice using this approach, there remains some background fluorescence. In this study, tissue homogenates of selected mouse organs were incubated with a test duplex and the kinetics of duplex dissociation in normal tissues were measured. In this manner we were able to identify the liver as the likely major source responsible for the duplex dissociation providing this fluorescence background. Thereafter liver homogenates were used to screen a series of duplex candidates with variable-length minor strands, and dissociation was measured by gel electrophoresis. The selected fluorophore/inhibitor duplex with improved stability displayed an insignificant (P > 0.05) background fluorescence after administration to SKH-1 normal mice and apparently without affecting target mRNA binding in vitro in cell culture or in vivo in tumor bearing mice.

Human islet cell MORF/cMORF pretargeting in a xenogeneic murine transplant model

Mon, 12/15/2014 - 1:55pm

Noninvasive measurement of human islet cell mass in pancreas or following islet transplantation by nuclear imaging has yet to be achieved. It has been shown using mouse tumor models that pretargeting imaging strategies are sensitive and can greatly increase target to nontarget signal ratios. The objective now is to demonstrate the specific pretargeting of human islet cells in mice. Our pretargeting strategy uses an anti-human islet cell antibody HPi1, conjugated to a phosphorodiamidate morpholino oligomer (MORF) that binds specifically to a (99m)Tc labeled complementary MORF (cMORF). Sensitivity and specificity of the pretargeting were first validated in culture using a human beta cell line (betalox5) and a negative control human cell line (HEK293). Pretargeting was then used to target and visualize these two cell lines and human islets transplanted subcutaneously in NOD-scid IL2rgamma(null) mice. In culture, (99m)Tc accumulation on the betalox5 cells pretargeted by MORF-HPi1 was 100-fold higher than on untreated betalox5 cells or following treatment with native HPi1 and much higher than on the MORF-HPi1 pretargeted control HEK293 cells. Small animal imaging readily localized the transplanted betalox5 cells and human islets, but not the HEK293 cells. Ex vivo counting demonstrated 3-fold higher (99m)Tc accumulation in the transplanted betalox5 cells and human islets than in the control HEK293 cells. The target accumulation was also shown to increase linearly with increased numbers of the implanted betalox5 cells. These results demonstrate specific binding of radioactivity and successful imaging of human betalox5 cells and human islets transplanted in mice. Thus MORF/cMORF pretargeting may be useful to measure noninvasively human islet cell mass within the pancreas or following islet transplantation.

A brief evaluation of tumor imaging in mice with 99mTc-glucarate including a comparison with 18F-FDG

Mon, 12/15/2014 - 1:55pm

OBJECTIVE: Recently 99mTc-glucarate, a radiolabeled glucose analogue, has been considered as a SPECT alternative to 18F-FDG and PET for non-invasive detection of certain tumors. Thus far there have been few studies on (99m) Tcglucarate for tumor imaging and fewer, if any, studies comparing (99m)Tc-glucarate with 18F-FDG. As a preliminary indication of the properties of (99m)Tc-glucarate as a possible substitute for 18F-FDG in animal studies, we have imaged mice bearing xenografts of four tumor types with (99m)Tc-glucarate and have compared in two mice with one of these tumor types the 99mTc and 18F biodistributions.

METHODS: Two mice bearing SUM190 breast cancer xenografts received 1 mCi of (99m)Tc-glucarate and were imaged on a NanoSPECT/CT small animal camera. One day later, the same animals received 1 mCi of 18F-FDG and were imaged on a MosaicHP PET small animal camera. In addition, 0.5-1 mCi of (99m)Tc-glucarate only was administered to mice bearing xenografts induced by BxPC3 pancreatic cancer cells, HEK-293 renal cell carcinomas cells or HCT-116 colorectal tumor cells. NanoSPECT/CT acquisitions were performed in these mice to evaluate tumor accumulations.

RESULTS: In the SUM190 xenografted mice, the average tumor accumulation was 1.4 % (ID%/cm3) for (99m)Tc-glucarate and 2.1 % (ID%/cm3) for 18F-FDG. While slightly higher than (99m)Tc-glucarate, the tumor accumulation of 18F-FDG was accompanied by higher bone marrow and muscle accumulations at levels that could interfere with the tumor image depending upon location. The whole body clearance of (99m)Tc-glucarate was faster than that of 18F-FDG. Tumor accumulation of (99m)Tc-glucarate varied among tumor types but the tumors were readily visible in all images.

CONCLUSION: In a direct comparison in the same two SUM190 tumored animals, SPECT images obtained with (99m)Tcglucarate compared favorably with PET images obtained with 18F-FDG. Tumor images with 99mTc-glucarate were also positive in three additional tumor mouse models. While further comparison studies are necessary, we conclude that (99m)Tcglucarate may be a more convenient and less expensive alternative to 18F-FDG for tumored mouse studies.

Improving the quantitation accuracy in noninvasive small animal single photon emission computed tomography imaging

Mon, 12/15/2014 - 1:55pm

INTRODUCTION: Noninvasive imaging of small animals to measure biodistributions and pharmacokinetics of radiolabeled agents is increasingly seen as an effective alternative to external counting of tissues obtained by sacrifice and dissection. However, we have observed important disagreements in measuring the accumulation of (111)In-labeled antibodies in organs such as liver and kidneys when comparing imaging to ex vivo counting in the same animals. This study was conducted to establish whether this discrepancy could be minimized by selecting the region of interest (ROI) in images at the appropriate color threshold and by correcting for the estimated radioactivity within the blood pool of these organs during imaging.

METHODS: Vials with known concentrations of (111)In as phantoms were imaged on a Bioscan NanoSPECT/CT. Thereafter, an (111)In-DTPA-IgG antibody as the test agent was administered intravenously to normal rats, and whole body acquisitions were obtained at 2, 24 or 48 h. Immediately following imaging, the animals were sacrificed, the tissues were removed for ex vivo counting and the radioactivity accumulations were then compared.

RESULTS: The phantom measurements showed that accuracy depended upon setting the correct ROI and that, in turn, depended upon setting the appropriate threshold of the color scale. Under the most unfavorable conditions, this error did not exceed 60%. Compared to the results of ex vivo counting, quantitation by imaging provided high values in liver and kidneys at all three time points by as much as 140%. However, by using the blood radioactivity at the time of sacrifice and the known blood volume in these organs, the disagreement was reduced in all cases to below 25%.

CONCLUSION: In this study, the discrepancy in quantitating organ radioactivity accumulations between noninvasive imaging and necropsy was primarily due to blood pool radioactivity contributing to the in vivo images. The discrepancy may be minimized by subtracting an estimate of this contribution.

Comparing two TAG-72 binding peptides previously identified by phage display as potential imaging agents

Mon, 12/15/2014 - 1:55pm

AIM: To evaluate the targeting property in vitro and in vivo of two tumor-associated glycoprotein 72 (TAG-72) binding peptides, previously identified in this laboratory by phage selection using different elution conditions.

MATERIALS AND METHODS: The peptides GGVSCMQTSPVCENNL (A2-6) and NPGTCKDKWEICLLNGG (A3-10) were radiolabeled with technetium-99m ((99m)Tc) using N-hydroxysuccinimidyl-S-acetyl-mercaptoacetyltriglycine (NHS-MAG(3)) as a chelator or were biotinylated. The specificity of the two peptides for the TAG-72 positive LS-174T cancer cells was demonstrated in vitro both by flow cytometry analysis using the biotinylated peptides and by competitive binding using the (99m)Tc-labeled peptides. The in-vivo biodistributions of the peptides were evaluated in TAG-72 positive LS-174T tumor-bearing mice by small-animal single photon emission computed tomography/computed tomography imaging.

RESULTS: As evidence of specific binding, both peptides showed a significant increase in percentage binding with increasing peptide concentration by flow cytometry analysis to LS-174T cells, but not to TAG-72 negative HT-29 cells. The (99m)Tc-labeled A2-6 peptide bound LS-174T cells with an inhibition constant at 50% of 46.5 nmol/l compared with 420 nmol/l for the A3-10 peptide. In mice, accumulation of both peptides was highest in kidneys and gallbladder. Tumors were clearly visible by single photon emission computed tomography imaging for both (99m)Tc-labeled peptides through 60 min, although the tumor accumulation was higher for the A3-10 peptide.

CONCLUSION: The A3-10 peptide, with lower, yet reasonable binding affinity compared with the A2-6 peptide, showed sufficiently favorable specific binding and tumor accumulation to be considered further as a potential imaging agent for TAG-72 positive cancers.

Comparing the intracellular fate of components within a noncovalent streptavidin nanoparticle with covalent conjugation

Mon, 12/15/2014 - 1:55pm

INTRODUCTION: Auger radiotherapy requires adequate tumor delivery and high nuclear accumulation and retention. We hypothesize that the noncovalent nature of a streptavidin/biotin three-component nanoparticle possessing these qualities may be required for dissociation of the radiolabeled oligomer and its accumulation into the cell nucleus.

METHODS: As a test of our hypothesis, the intracellular fate of an antisense oligomer when incubated as the nanoparticle and when incubated while covalently conjugated to the antibody was compared. The three-component noncovalent nanoparticle consisted of streptavidin linking three biotinylated components: a Cy3-labeled anti-RIalpha antisense phosphorodiamidate morpholino (MORF) oligomer, a tat transfecting peptide and the anti-Her2 herceptin antibody. The covalent constructs included an anti-RIalpha antisense DNA conjugated to a radiolabeled herceptin and a fluorescent DNA conjugated to native herceptin. Fluorescence microscopy in SK-BR-3 (Her2+) cells was used to evaluate the fate of the fluorescent Cy5.5-DNA and Cy3-MORF, while the subcellular accumulation of the (111)In-labeled herceptin and herceptin-DNA in both SK-BR-3 and MDA-MB-231 (Her2) cells was determined by isolating and counting the nuclear fractions.

RESULTS: Previously, we demonstrated that when incubated as the three-component nanoparticle consisting of herceptin and streptavidin and (99m)Tc-labeled antisense MORF, only the MORF accumulated in the nucleus of Her2+ cells. In this investigation, clear evidence was observed of nuclear accumulation of the antisense oligomer within the noncovalent nanoparticle as before, but when incubated as the covalent construct, by both fluorescence microscopy and nuclear counting, no evidence of nuclear accumulation was observed.

CONCLUSION: The weaker noncovalent biotin-streptavidin bond may be essential for adequate delivery of the radiolabeled antisense oligomer to the nucleus of tumor cells.

90Y labeled phosphorodiamidate morpholino oligomer for pretargeting radiotherapy

Mon, 12/15/2014 - 1:55pm

While (188)Re has been used successfully in mice for tumor radiotherapy by MORF/cMORF pretargeting, previous radiolabeling of the amine-derivatized cMORF with (90)Y, a longer physical half-life nuclide, was not very successful. After developing a method involving a prepurification heating step during conjugation that increases labeling efficiency and label stability, the biodistribution of (90)Y-DOTA-Bn-SCN-cMORF ((90)Y-DOTA-cMORF) was measured in normal mice and in MORF-CC49 pretargeted mice that bear LS174T tumors. Absorbed radiation doses were then estimated and compared to those estimated for (188)Re. The pharmacokinetics of the (90)Y-DOTA-cMORF in normal mice and in the pretargeted nude mice was similar to that observed previously with (99m)Tc- and (188)Re-MAG(3)-cMORFs. While the (90)Y-DOTA-cMORF cleared rapidly from normal tissues, tumor clearance was very slow and tumor radioactivity accumulation was constant for at least 7 days such that the tumor/blood (T/B) ratio increased linearly from 6 to 25 over this period. Therefore, by extrapolation, normal tissue toxicities following administration of therapeutic doses of (90)Y may be comparable to that observed for (188)Re in which the T/B increased from 5 to 20. In conclusion, radiolabeling of DOTA-cMORF with (90)Y was improved by introducing a prepurification heating step during conjugation. The (90)Y-DOTA-cMORF provided a similar T/B ratio and biodistribution to that of (188)Re-MAG(3)-cMORF and was retained well in the tumor pretargeted with MORF-CC49. Because of the longer physical half-life, the T/NT absorbed radiation dose ratios were improved in most organs and especially in blood.

Pretargeting vs. direct targeting of human betalox5 islet cells subcutaneously implanted in mice using an anti-human islet cell antibody

Mon, 12/15/2014 - 1:55pm

INTRODUCTION: We previously demonstrated MORF/cMORF pretargeting of human islets and betalox 5 cells (a human beta cell line) transplanted subcutaneously in mice with the anti-human islet antibody, HPi1. We now compare pretargeting with direct targeting in the beta cell transplant model to evaluate the degree to which target/non-target (T/NT) ratios may be improved by pretargeting.

METHODS: Specific binding of an anti-human islet antibody HPi1 to the beta cells transplanted subcutaneously in mice was examined against a negative control antibody. We then compared pretargeting by MORF-HPi1 plus 111In-labeled cMORF to direct targeting by 111In-labeled HPi1.

RESULTS: HPi1 binding to betalox5 human cells in the transplant was shown by immunofluorescence. Normal organ 111In backgrounds by pretargeting were always lower, although target accumulations were similar. More importantly, the transplant to pancreas and liver ratios was, respectively, 26 and 10 by pretargeting as compared to 9 and 0.6 by direct targeting.

CONCLUSIONS: Pretargeting greatly improves the T/NT ratios, and based on the estimated endocrine to exocrine ratio within a pancreas, pretargeting may be approaching the sensitivity required for successful imaging of human islets within this organ.

(99m)Tc-MORF oligomers specific for bacterial ribosomal RNA as potential specific infection imaging agents

Mon, 12/15/2014 - 1:55pm

PURPOSE: Radiolabeled oligomers complementary to the 16S rRNA in bacteria were investigated as bacterial infection imaging agents.

METHODS AND RESULTS: Identical sequences with backbones phosphorodiamidate morpholino (MORF), peptide nucleic acid (PNA), and phosphorothioate DNA (PS-DNA) were (99m)Tc-labeled and evaluated for binding to bacterial RNA. MORF binding to RNA from Escherichia coli strains SM101 and K12 was 4- and 150-fold higher compared to PNA and PS-DNA, respectively. Subsequently MORF oligomer in fluorescence in situ hybridization showed a stronger signal with study MORF compared to control in fixed preparations of two E. coli strains and Klebsiella pneumoniae. Flow cytometry analysis showed study MORF accumulation to be 8- and 80-fold higher compared to the control in live K. pneumoniae and Staphylococcus aureus, respectively. Further, fluorescence microscopy showed increased accumulation of study MORF over control in live E. coli and K. pneumonia. Binding of (99m)Tc-study MORF to RNA from E. coli SM101 and K12 was 30.4 and 117.8pmol, respectively, per 10(10) cells. Mice with K. pneumoniae live or heat-killed (sterile inflammation) in one thigh at 90min for both (99m)Tc-study MORF and control showed higher accumulation in target thighs than in blood and all other organs expect for kidneys and small intestine. Accumulation of (99m)Tc-study MORF was significantly higher (p = 0.009) than that of the control in the thigh with sterile inflammation.

CONCLUSION: A (99m)Tc-MORF oligomer complimentary to the bacterial 16S rRNA demonstrated binding to bacterial RNA in vitro with specific accumulation into live bacteria. Radiolabeled MORF oligomers antisense to the bacterial rRNA may be useful to image bacterial infection.

Differentiation between temporary and real non-clearability of biotinylated IgG antibody by avidin in mice

Mon, 12/15/2014 - 1:55pm

Although an increasing number of antibody conjugates are being used in the clinic, there remain many unmet needs in antibody targeting. Normal tissue background is one of the key issues that limits the therapeutic efficacy and the detection sensitivity. Background reduction coupled with dose increase may provide the required target accumulation of the label or toxin at an acceptable normal tissue background. However, the knowledge about the in vivo interaction between antibody and a clearing agent is currently inadequate for designing a rational clearance regimen or system. The current investigation focuses on the clearability of antibody for background reduction, an important topic to antibody targeting in general. The investigation employs pretargeting as a research tool and avidin as a model clearing agent. By comparing the effects of natural clearance at a longer post-injection time and avidin clearance, we demonstrated that avidin clearance is much more effective. By directly attaching avidin to a biotinylated antibody prior to injection, we found that the biotinylated antibody in blood, once bound to the clearing agent, can be removed from the circulation immediately and completely, while the real non-clearable antibody without biotin stays. The study of multiple avidin injections confirmed that the presence of clearable biotinylated antibodies after an avidin injection is due to their temporary inaccessibility and subsequent return from tissue compartments. The collective clearance efficiency of 91% by three avidin injections indicates a continuous IV infusion would be recommended to remove all of the biotinylated IgG molecules. In conclusion, the use of antibody pretargeting as a tool in this study has improved understanding of the incomplete clearance by avidin and can aid in overcoming this obstacle.

Modality-specific occult intrarenal pseudoaneurysm in a renal allograft and the legacy of catheter angiography

Mon, 12/15/2014 - 1:55pm

A 69-year-old man with history of end-stage-renal disease (ESRD) underwent successful kidney transplantation from a cadaveric donor in November 2011. However, posttransplant recovery was complicated by delayed graft function and recurrent gross hematuria. Serial Doppler ultrasound (US) of the renal allograft demonstrated a pseudoaneurysm with interval increase in size. However, it could not be visualized with other modalities, including an initial angiogram (postoperative day 49) and a second angiogram (postoperative day 68), followed by surgical exploration (postoperative day 71), which demonstrated complete intra-aneurysmal thrombosis on intraoperative Doppler US. Unfortunately, the patient's hematuria continued and a repeat Doppler US 48 hours later demonstrated a persistent pseudoaneurysm. Therefore, on postoperative day 75, we performed targeted percutaneous intra-aneurysmal thrombin injection under dual image guidance, which showed complete intra-aneurysmal thrombosis on intraprocedural Doppler US. Hematuria recurred the next day. A third angiogram (postoperative day 77) finally illuminated the hidden pseudoaneurysm occult on the first and second angiographic studies (sensitivity [index case] 33%) and surgery. This allowed for successful coil embolization of a subsegmental feeding branch with an excellent outcome. We support a more aggressive management with serial angiography and embolization of the intrarenal symptomatic pseudoaneurysm rather than surgery in renal allograft recipients, with the benefits outweighing the risks.

Increased Risk for Vitamin D Deficiency in Obese Children with Both Celiac Disease and Type 1 Diabetes

Fri, 12/12/2014 - 9:35am

Background. It is unknown whether the coexistence of type 1 diabetes (T1D) and celiac disease (CD) increases the risk for vitamin D deficiency.

Aims. To determine the vitamin D status and the risk for vitamin D deficiency in prepubertal children with both T1D and CD compared to controls, TID, and CD.

Subjects and Methods. Characteristics of 62 prepubertal children of age 2–13 y with either CD + T1D (

Cytoplasmic HIV-1 RNA is mainly transported by diffusion in the presence or absence of Gag protein

Thu, 12/11/2014 - 11:38am

Full-length HIV-1 RNA plays a central role in viral replication by serving as the mRNA for essential viral proteins and as the genome packaged into infectious virions. Proper RNA trafficking is required for the functions of RNA and its encoded proteins; however, the mechanism by which HIV-1 RNA is transported within the cytoplasm remains undefined. Full-length HIV-1 RNA transport is further complicated when group-specific antigen (Gag) protein is expressed, because a significant portion of HIV-1 RNA may be transported as Gag-RNA complexes, whose properties could differ greatly from Gag-free RNA. In this report, we visualized HIV-1 RNA and monitored its movement in the cytoplasm by using single-molecule tracking. We observed that most of the HIV-1 RNA molecules move in a nondirectional, random-walk manner, which does not require an intact cytoskeletal structure, and that the mean-squared distance traveled by the RNA increases linearly with time, indicative of diffusive movement. We also observed that a single HIV-1 RNA molecule can move at various speeds when traveling through the cytoplasm, indicating that its movement is strongly affected by the immediate environment. To examine the effect of Gag protein on HIV-1 RNA transport, we analyzed the cytoplasmic HIV-1 RNA movement in the presence of sufficient Gag for virion assembly and found that HIV-1 RNA is still transported by diffusion with mobility similar to the mobility of RNAs unable to express functional Gag. These studies define a major mechanism of HIV-1 gene expression and resolve the long-standing question of how the RNA genome is transported to the assembly site.

Making Women's Health Connections: Between Researchers and to Resources

Wed, 12/10/2014 - 9:03am

The Lamar Soutter Library at the University of Massachusetts Medical School has recently completed the second year of a National Library of Medicine grant funded project; the Women’s Health Resources Dissemination Outreach Project. The goals include assisting women’s health researchers by providing them with access to information and making them more aware of opportunities and available resources. This, ultimately, enables both an improvement in women’s health and the advancement of women in academic medicine. Moreover, by supporting women’s health research and women researchers through the objectives of this project, women researchers build connections, knowledge, and skills. This facilitates meaningful contributions and fosters greater promotion and leadership opportunities for these women. This poster describes the goals, activities, and progress of the project through the completion of the second year. Specific programs and initiatives are highlighted. This includes the multi-year endeavor to build, recruit, and showcase women’s health researchers in a specific collection of eScholarship@UMMS, the library’s institutional repository. Promotion and outreach, of both the project and the resources, was major component of the second year. Additionally, programming was developed that helped researchers better communicate their work to the media and public. Other programs, lessons learned, successes, and future goals are noted.