scholarly journals 4081 Quantifying pH buffering capacity and kinetics of tumor and healthy tissue to understand and exploit differences in biology

2020 ◽  
Vol 4 (s1) ◽  
pp. 15-15
Author(s):  
A. Colleen Crouch ◽  
Emily A. Thompson ◽  
Mark D. Pagel ◽  
Erik N.K. Cressman
Keyword(s):  
Tumor Tissue ◽  
Ex Vivo ◽  
Buffering Capacity ◽  
The Body ◽  
Healthy Tissue ◽  
Buffer System ◽  
Bicarbonate Buffer ◽  
Ph Buffering ◽  
Acidocest Mri

OBJECTIVES/GOALS: The purpose of this work is to investigate natural buffering capacity of liver tissue and tumors, to understand and exploit differences for therapy. Using this work, we will determine the concentrations of reagents (acids or bases) used in ablation treatment to optimize treatment by increasing tumor toxicity and minimizing healthy tissue toxicity. METHODS/STUDY POPULATION: For this preliminary study, two methods will be used: benchtop pH experiments ex vivo and non-invasive imaging using acidoCEST MRI in vivo. For ex vivo, two types of tissues will be tested: non-cancerous liver and tumor tissue from HepG2 inoculated mice (n = 10). After mice are euthanized, pH will be measured in tissue homogenates at baseline and then the homogenates will be placed in either acidic (acetic acid) or basic (sodium hydroxide) solutions with varied concentrations (0.5–10M) and time recorded until pH returns to baseline. For in vivo imaging, Mia PaCA-2 flank model mice (n = 10) will be imaged with acidoCEST MRI to quantify pH at baseline. Mice will then be injected intratumorally with (up to 100 μL of) acid or base at increasing concentrations and imaged to quantify pH changes in the tumor. RESULTS/ANTICIPATED RESULTS: For this study, buffering capacity is defined as the concentration threshold for which tissue can buffer pH back to within normal range. Non-cancerous tissue is likely to buffer a wider range of concentrations compared to tumor tissue. From the benchtop experiment, comparison of time-to-buffer will be made for each concentration of acid/base for the two tissue types. AcidoCEST MRI will provide in vivo buffering capacity and potentially demonstrate tumor heterogeneity of buffering capacity. For both experiments, a pH vs. concentration curve for the two tissue types will allow for comparison of ex vivo to in vivo experiments, which will differentiate contributions of local tissue buffering capacity from the full body’s natural bicarbonate buffer system that depends on respiration and blood flow. DISCUSSION/SIGNIFICANCE OF IMPACT: The pH of the body must be maintained within a narrow range. With cancer, impairment in regulation of tumor metabolism causes acidosis, lowering extracellular pH in tumors. It remains unclear if pH plays a role in local recurrence or tumor toxicity. This work will determine if acidoCEST MRI can measure deliberate alteration of pH and how this change affects biology.

The Role of Polyphenols in the Modulation of Plasma Non-Enzymatic Antioxidant Capacity (NEAC)

2012 ◽  
Vol 82 (3) ◽  
pp. 228-232 ◽  
Author(s):  
Mauro Serafini ◽  
Giuseppa Morabito
Keyword(s):  
Antioxidant Capacity ◽  
Dietary Intervention ◽  
Ex Vivo ◽  
The Body ◽  
Dietary Polyphenols ◽  
Enzymatic Antioxidant ◽  
Endogenous Antioxidant

Dietary polyphenols have been shown to scavenge free radicals, modulating cellular redox transcription factors in different in vitro and ex vivo models. Dietary intervention studies have shown that consumption of plant foods modulates plasma Non-Enzymatic Antioxidant Capacity (NEAC), a biomarker of the endogenous antioxidant network, in human subjects. However, the identification of the molecules responsible for this effect are yet to be obtained and evidences of an antioxidant in vivo action of polyphenols are conflicting. There is a clear discrepancy between polyphenols (PP) concentration in body fluids and the extent of increase of plasma NEAC. The low degree of absorption and the extensive metabolism of PP within the body have raised questions about their contribution to the endogenous antioxidant network. This work will discuss the role of polyphenols from galenic preparation, food extracts, and selected dietary sources as modulators of plasma NEAC in humans.

scholarly journals Highly Potent GalNAc-Conjugated Tiny LNA Anti-miRNA-122 Antisense Oligonucleotides

Pharmaceutics ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 817
Author(s):  
Tsuyoshi Yamamoto ◽  
Yahiro Mukai ◽  
Fumito Wada ◽  
Chisato Terada ◽  
Yukina Kayaba ◽  
...  
Keyword(s):  
Antisense Oligonucleotides ◽  
Ex Vivo ◽  
The Body ◽  
Locked Nucleic Acid ◽  
Imaging Studies ◽  
Phosphodiester Bond ◽  
Chemically Modified ◽  
Ex Vivo Imaging ◽  
Mirna Targeting

The development of clinically relevant anti-microRNA antisense oligonucleotides (anti-miRNA ASOs) remains a major challenge. One promising configuration of anti-miRNA ASOs called “tiny LNA (tiny Locked Nucleic Acid)” is an unusually small (~8-mer), highly chemically modified anti-miRNA ASO with high activity and specificity. Within this platform, we achieved a great enhancement of the in vivo activity of miRNA-122-targeting tiny LNA by developing a series of N-acetylgalactosamine (GalNAc)-conjugated tiny LNAs. Specifically, the median effective dose (ED50) of the most potent construct, tL-5G3, was estimated to be ~12 nmol/kg, which is ~300–500 times more potent than the original unconjugated tiny LNA. Through in vivo/ex vivo imaging studies, we have confirmed that the major advantage of GalNAc over tiny LNAs can be ascribed to the improvement of their originally poor pharmacokinetics. We also showed that the GalNAc ligand should be introduced into its 5′ terminus rather than its 3′ end via a biolabile phosphodiester bond. This result suggests that tiny LNA can unexpectedly be recognized by endogenous nucleases and is required to be digested to liberate the parent tiny LNA at an appropriate time in the body. We believe that our strategy will pave the way for the clinical application of miRNA-targeting small ASO therapy.

scholarly journals Regional distribution of SGLT activity in rat brain in vivo

2013 ◽  
Vol 304 (3) ◽  
pp. C240-C247 ◽  
Author(s):  
Amy S. Yu ◽  
Bruce A. Hirayama ◽  
Gerald Timbol ◽  
Jie Liu ◽  
Ana Diez-Sampedro ◽  
...  
Keyword(s):  
Rat Brain ◽  
Ex Vivo ◽  
Osmotic Shock ◽  
Regional Distribution ◽  
The Body ◽  
Specific Substrate ◽  
Ex Vivo Autoradiography ◽  
Molecular Imaging Probe ◽  
The Brain

Na+-glucose cotransporter (SGLT) mRNAs have been detected in many organs of the body, but, apart from kidney and intestine, transporter expression, localization, and functional activity, as well as physiological significance, remain elusive. Using a SGLT-specific molecular imaging probe, α-methyl-4-deoxy-4-[18F]fluoro-d-glucopyranoside (Me-4-FDG) with ex vivo autoradiography and immunohistochemistry, we mapped in vivo the regional distribution of functional SGLTs in rat brain. Since Me-4-FDG is not a substrate for GLUT1 at the blood-brain barrier (BBB), in vivo delivery of the probe into the brain was achieved after opening of the BBB by an established procedure, osmotic shock. Ex vivo autoradiography showed that Me-4-FDG accumulated in regions of the cerebellum, hippocampus, frontal cortex, caudate nucleus, putamen, amygdala, parietal cortex, and paraventricular nucleus of the hypothalamus. Little or no Me-4-FDG accumulated in the brain stem. The regional accumulation of Me-4-FDG overlapped the distribution of SGLT1 protein detected by immunohistochemistry. In summary, after the BBB is opened, the specific substrate for SGLTs, Me-4-FDG, enters the brain and accumulates in selected regions shown to express SGLT1 protein. This localization and the sensitivity of these neurons to anoxia prompt the speculation that SGLTs may play an essential role in glucose utilization under stress such as ischemia. The expression of SGLTs in the brain raises questions about the potential effects of SGLT inhibitors under development for the treatment of diabetes.

scholarly journals Highly Sensitive Identification of Lymphatic and Hematogenous Metastasis Routes of Novel Radiolabeled Exosomes Using Non-invasive PET Imaging

2020 ◽  
Author(s):  
Kyung Oh Jung ◽  
Young-Hwa Kim ◽  
Seock-Jin Chung ◽  
Keon Wook Kang ◽  
Siyeon Rhee ◽  
...  
Keyword(s):  
Lymph Nodes ◽  
Optical Imaging ◽  
Pet Imaging ◽  
Ex Vivo ◽  
The Body ◽  
Hematogenous Metastasis ◽  
Axillary Lymph Nodes ◽  
Axillary Lymph ◽  
Gamma Counter

Clinically, there has been significant interest in the use of exosomes for diagnostic applications as promising biomarkers and therapeutic applications as therapeutic vehicles. However, knowledge of in vivo physiological biodistribution of exosomes was difficult to assess until now. Physiological distribution of exosomes in the body must be elucidated for clinical application. In this study, we aimed to develop reliable and novel methods to monitor biodistribution of exosomes using in vivo PET and optical imaging.MethodsExosomes were isolated from cultured medium of 4T1, mouse breast cancer cells. Exosomes were labeled with Cy7 and 64Cu (or 68Ga). In mice, radio/fluorescent dye-labeled exosomes were injected through the lymphatic routes (footpad injection) and hematogenous metastatic routes (tail vein injection). Fluorescence and PET images were obtained and quantified. Radio-activity of ex vivo organs was measured by gamma counter.ResultsPET signals from exosomes in the lymphatic metastatic route were observed in the draining lymph nodes, which are not distinguishable with optical imaging. Immunohistochemistry revealed greater uptake of exosomes in brachial and axillary lymph nodes than inguinal lymph node. After administration through the hematogenous metastasis pathway, accumulation of exosomes was clearly observed in PET images in the lungs, liver, and spleen, showing results similar to ex vivo gamma counter data.ConclusionExosomes from tumor cells were successfully labeled with 64Cu (or 68Ga) and visualized by PET imaging. These results suggest that this cell type-independent, quick, and easy exosome labeling method using PET isotopes could provide valuable information for further application of exosomes in the clinic.

scholarly journals [68Ga]ABY-028: an albumin-binding domain (ABD) protein-based imaging tracer for positron emission tomography (PET) studies of altered vascular permeability and predictions of albumin-drug conjugate transport

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Emma Jussing ◽  
Li Lu ◽  
Jonas Grafström ◽  
Tetyana Tegnebratt ◽  
Fabian Arnberg ◽  
...  
Keyword(s):  
Positron Emission Tomography ◽  
Vascular Permeability ◽  
Ex Vivo ◽  
Circulatory System ◽  
Binding Domain ◽  
The Body ◽  
Emission Tomography ◽  
Albumin Binding ◽  
Positron Emission

Abstract Background Albumin is commonly used as a carrier platform for drugs to extend their circulatory half-lives and influence their uptake into tissues that have altered permeability to the plasma protein. The albumin-binding domain (ABD) protein, which binds in vivo to serum albumin with high affinity, has proven to be a versatile scaffold for engineering biopharmaceuticals with a range of binding capabilities. In this study, the ABD protein equipped with a mal-DOTA chelator (denoted ABY-028) was radiolabeled with gallium-68 (68Ga). This novel radiotracer was then used together with positron emission tomography (PET) imaging to examine variations in the uptake of the ABD-albumin conjugate with variations in endothelial permeability. Results ABY-028, produced by peptide synthesis in excellent purity and stored at − 20 °C, was stable for 24 months (end of study). [68Ga]ABY-028 could be obtained with labeling yields of > 80% and approximately 95% radiochemical purity. [68Ga]ABY-028 distributed in vivo with the plasma pool, with highest radioactivity in the heart ventricles and major vessels of the body, a gradual transport over time from the circulatory system into tissues and elimination via the kidneys. Early [68Ga]ABY-028 uptake differed in xenografts with different vascular properties: mean standard uptake values (SUVmean) were initially 5 times larger in FaDu than in A431 xenografts, but the difference decreased to 3 after 1 h. Cutaneously administered, vasoactive nitroglycerin increased radioactivity in the A431 xenografts. Heterogeneity in the levels and rates of increases of radioactivity uptake was observed in sub-regions of individual MMTV-PyMT mammary tumors and in FaDu xenografts. Higher uptake early after tracer administration could be observed in lower metabolic regions. Fluctuations in the increased permeability for the tracer across the blood-brain-barrier (BBB) direct after experimentally induced stroke were monitored by PET and the increased uptake was confirmed by ex vivo phosphorimaging. Conclusions [68Ga]ABY-028 is a promising new tracer for visualization of changes in albumin uptake due to disease- and pharmacologically altered vascular permeability and their potential effects on the passive uptake of targeting therapeutics based on the ABD protein technology.

scholarly journals Knockdown of microRNA-135b in Mammary Carcinoma by Targeted Nanodiamonds: Potentials and Pitfalls of In Vivo Applications

Nanomaterials ◽  
2019 ◽  
Vol 9 (6) ◽  
pp. 866 ◽  
Author(s):  
Romana Křivohlavá ◽  
Eva Neuhӧferová ◽  
Katrine Q. Jakobsen ◽  
Veronika Benson
Keyword(s):  
Antisense Rna ◽  
Tumor Tissue ◽  
Immune Cell ◽  
Ex Vivo ◽  
Tumor Bearing ◽  
Intravenous Application ◽  
Effective Inhibition ◽  
Tumor Accumulation ◽  
Safe Approach

Nanodiamonds (ND) serve as RNA carriers with potential for in vivo application. ND coatings and their administration strategy significantly change their fate, toxicity, and effectivity within a multicellular system. Our goal was to develop multiple ND coating for effective RNA delivery in vivo. Our final complex (NDA135b) consisted of ND, polymer, antisense RNA, and transferrin. We aimed (i) to assess if a tumor-specific coating promotes NDA135b tumor accumulation and effective inhibition of oncogenic microRNA-135b and (ii) to outline off-targets and immune cell interactions. First, we tested NDA135b toxicity and effectivity in tumorospheres co-cultured with immune cells ex vivo. We found NDA135b to target tumor cells, but it binds also to granulocytes. Then, we followed with NDA135b intravenous and intratumoral applications in tumor-bearing animals in vivo. Application of NDA135b in vivo led to the effective knockdown of microRNA-135b in tumor tissue regardless administration. Only intravenous application resulted in NDA135b circulation in peripheral blood and urine and the decreased granularity of splenocytes. Our data show that localized intratumoral application of NDA135b represents a suitable and safe approach for in vivo application of nanodiamond-based constructs. Systemic intravenous application led to an interaction of NDA135b with bio-interface, and needs further examination regarding its safety.

scholarly journals Hyperoxia in portal vein causes enhanced vasoconstriction in arterial vascular bed

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Dilmurodjon Eshmuminov ◽  
Dustin Becker ◽  
Max L. Hefti ◽  
Matteo Mueller ◽  
Catherine Hagedorn ◽  
...  
Keyword(s):  
Portal Vein ◽  
Ex Vivo ◽  
Liver Perfusion ◽  
Venous Blood ◽  
Poor Quality ◽  
The Body ◽  
Mrna Levels ◽  
Blood Oxygen Saturation ◽  
Arterial Blood

AbstractLong-term perfusion of liver grafts outside of the body may enable repair of poor-quality livers that are currently declined for transplantation, mitigating the global shortage of donor livers. In current ex vivo liver perfusion protocols, hyperoxic blood (arterial blood) is commonly delivered in the portal vein (PV). We perfused porcine livers for one week and investigated the effect of and mechanisms behind hyperoxia in the PV on hepatic arterial resistance. Applying PV hyperoxia in porcine livers (n = 5, arterial PV group), we observed an increased need for vasodilator Nitroprussiat (285 ± 162 ml/week) to maintain the reference hepatic artery flow of 0.25 l/min during ex vivo perfusion. With physiologic oxygenation (venous blood) in the PV the need for vasodilator could be reduced to 41 ± 34 ml/week (p = 0.011; n = 5, venous PV group). This phenomenon has not been reported previously, owing to the fact that such experiments are not feasible practically in vivo. We investigated the mechanism of the variation in HA resistance in response to blood oxygen saturation with a focus on the release of vasoactive substances, such as Endothelin 1 (ET-1) and nitric oxide (NO), at the protein and mRNA levels. However, no difference was found between groups for ET-1 and NO release. We propose direct oxygen sensing of endothelial cells and/or increased NO break down rate with hyperoxia as possible explanations for enhanced HA resistance.

Recovery and Biodistribution of Ex-Vivo Expanded Human Erythroblasts Injected Into NOD/SCID/IL2Rγnull Mice

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 338-338 ◽  
Author(s):  
Barbara Ghinassi ◽  
Leda Ferro ◽  
Stefan Kachala ◽  
Isabelle Riviere ◽  
Michel Sadelain ◽  
...  
Keyword(s):  
Cord Blood ◽  
Blood Cells ◽  
Conflicts Of Interest ◽  
Therapeutic Potential ◽  
Ex Vivo ◽  
The Body ◽  
Transduction Efficiency ◽  
Control Group ◽  
Retroviral Transduction

Abstract Abstract 338 Ex vivo expanded erythroblasts (EBs) are red blood cell precursors with proliferative capacity that have the potential to serve as alternative transfusion product. In the present study, we investigated the biodistribution and persistence of human EBs expanded ex vivo from cord blood following intravenous administration to NOD/SCID/IL2Rγnull mice. In the first experiment, 107 EBs generated ex vivo from cord blood under Human Erythroid Massive Amplification (HEMA) culture conditions (Migliaccio G et al. Blood Cells Mol Dis. 2002;28:169) were labeled with CFSE and transfused via the tail vein into NOD/SCID/IL2Rγnull mice which had been bled (200 μL) 24 hrs earlier to increase erythropoietin (EPO) levels. The presence of human EBs in bone marrow (BM), spleen and blood of the transfused recipient mice was analyzed by flow cytometry for CSFE and human CD235a. At day 4, 1.5 – 5% of cells in BM and spleen of the animals were CD235apos but no human cells were detectable in blood. To clarify failure of human EBs to generate red blood cells in mice, a second cohort of mice was given 25×106 expanded EBs and sacrificed 4 days thereafter. Their tissues, including BM, liver and spleen, were examined by immunohistochemistry for expression of human markers. Human CD235apos cells were found in the spleen, representing up to 18% of total cells spleen cells of transfused recipients, but the cells were trapped inside larger CD235aneg cells, probably of murine origin. These results indicate that lodging of human EBs in the spleen, where they are probably destroyed by the macrophages, may represent a barrier to using mouse models as a surrogate assay for investigating transfused human EBs. To test this hypothesis, we analyzed the fate and biodistribution of human EBs into normal vs. splenectomized NOD/SCID/IL2Rγnull mice. Cell biodistribution was analyzed using bioluminescence imaging (BLI), following retroviral-mediated transfer of eGFP and the external Gaussia luciferase genes (Santos et al Nat Med 15: 338, 2009) into expanding cord blood-derived EBs. Cord blood-derived CD34pos cells were either expanded in HEMA culture (as control) or cultured for 3 days with TPO, SCF and FLT3L before retroviral transduction. After 3 days, the cells were cultured under HEMA conditions to induce EBs expansion. Mature EBs were detectable after 11 days of culture in the untransduced, control group and the cells expanded 67-fold. By contrast, transduced cord blood cells matured by day 5–7 and amplified only 24-fold (see Figure). Transduction efficiency, as reflected by GFP expression, was on the order of 32–50% in expanded EBs. All the recipient mice were bled 10 hrs before injection (200 μL). Half of them were splenectomized 24 hrs earlier. Mice were given 15×106 each expanded EBs together with 20 units of EPO and the cell biodistribution analyzed by imaging 24 hrs later (see Figure). In intact mice, BLI signal was virtually undetectable (other than in the tail). In contrast, in the splenectomized mice, significant signal levels were also observed in the body of the animals (see Figure). Four days after injection, mice were sacrificed and the presence of human CD235apos cells in the marrow, liver, spleen (intact animals only) and blood analyzed. CD235apos cells were detectable in the marrow (20%) and liver (6%) of the splenectomized mice while in intact animals they were mainly detected in spleen (15%). Detection of human red cells in blood, however, remained low in all the cases. Overall, we have established a model for the tracking and quantification of human EBs transfused into NOD/SCID/IL2Rγnull mice. This model will be very valuable to investigate the in vivo function and persistence of human EBs expanded under different conditions and thereby define the therapeutic potential of ex vivo generated human EBs derived from different stem cell sources. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Confocal Laser Endomicroscopy Assessment of Pituitary Tumor Microstructure: A Feasibility Study

2020 ◽  
Vol 9 (10) ◽  
pp. 3146 ◽  
Author(s):  
Evgenii Belykh ◽  
Brandon Ngo ◽  
Dara S. Farhadi ◽  
Xiaochun Zhao ◽  
Michael A. Mooney ◽  
...  
Keyword(s):  
Pituitary Adenoma ◽  
Pituitary Tumor ◽  
Tumor Tissue ◽  
Ex Vivo ◽  
In Vivo Studies ◽  
Confocal Laser Endomicroscopy ◽  
Confocal Laser ◽  
Frozen Sections ◽  
Tuberculum Sellae

This is the first study to assess confocal laser endomicroscopy (CLE) use within the transsphenoidal approach and show the feasibility of obtaining digital diagnostic biopsies of pituitary tumor tissue after intravenous fluorescein injection. We confirmed that the CLE probe reaches the tuberculum sellae through the transnasal transsphenoidal corridor in cadaveric heads. Next, we confirmed that CLE provides images with identifiable histological features of pituitary adenoma. Biopsies from nine patients who underwent pituitary adenoma surgery were imaged ex vivo at various times after fluorescein injection and were assessed by a blinded board-certified neuropathologist. With frozen sections used as the standard, pituitary adenoma was diagnosed as “definitively” for 13 and as “favoring” in 3 of 16 specimens. CLE digital biopsies were diagnostic for pituitary adenoma in 10 of 16 specimens. The reasons for nondiagnostic CLE images were biopsy acquisition <1 min or >10 min after fluorescein injection (n = 5) and blood artifacts (n = 1). In conclusion, fluorescein provided sufficient contrast for CLE at a dose of 2 mg/kg, optimally 1–10 min after injection. These results provide a basis for further in vivo studies using CLE in transsphenoidal surgery.

scholarly journals Frictional internal work of damped limbs oscillation in human locomotion

2020 ◽  
Vol 287 (1931) ◽  
pp. 20201410 ◽  
Author(s):  
Alberto E. Minetti ◽  
Alex P. Moorhead ◽  
Gaspare Pavei
Keyword(s):  
Energy Balance ◽  
Time Course ◽  
Ex Vivo ◽  
Biological Tissues ◽  
The Body ◽  
Lower Limbs ◽  
Metabolic Energy ◽  
External Work ◽  
Internal Work

Joint friction has never previously been considered in the computation of mechanical and metabolic energy balance of human and animal (loco)motion, which heretofore included just muscle work to move the body centre of mass (external work) and body segments with respect to it. This happened mainly because, having been previously measured ex vivo , friction was considered to be almost negligible. Present evidences of in vivo damping of limb oscillations, motion captured and processed by a suited mathematical model, show that: (a) the time course is exponential, suggesting a viscous friction operated by the all biological tissues involved; (b) during the swing phase, upper limbs report a friction close to one-sixth of the lower limbs; (c) when lower limbs are loaded, in an upside-down body posture allowing to investigate the hip joint subjected to compressive forces as during the stance phase, friction is much higher and load dependent; and (d) the friction of the four limbs during locomotion leads to an additional internal work that is a remarkable fraction of the mechanical external work. These unprecedented results redefine the partitioning of the energy balance of locomotion, the internal work components, muscle and transmission efficiency, and potentially readjust the mechanical paradigm of the different gaits.

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