Mouse Albumin Research Articles

Species-Specific Unbound Fraction Differences in Highly Bound PFAS: A Comparative Study across Human, Rat, and Mouse Plasma and Albumin.

Per- and polyfluoroalkyl substances (PFAS) are a diverse group of fluorinated compounds which have yet to undergo comprehensive investigation regarding potential adverse health effects and bioaccumulative properties. With long half-lives and accumulative properties, PFAS have been linked to several toxic effects in both non-clinical species such as rat and mouse as well as human. Although biological impacts and specific protein binding of PFAS have been examined, there is no study focusing on the species-specific fraction unbound (fu) in plasma and related toxicokinetics. Herein, a presaturation equilibrium dialysis method was used to measure and validate the binding of 14 individual PFAS with carbon chains containing 4 to 12 perfluorinated carbon atoms and several functional head-groups to albumin and plasma of mouse (C57BL/6 and CD-1), rat, and human. Equivalence testing between each species-matrix combination showed positive correlation between rat and human when comparing fu in plasma and binding to albumin. Similar trends in binding were also observed for mouse plasma and albumin. Relatively high Spearman correlations for all combinations indicate high concordance of PFAS binding regardless of matrix. Physiochemical properties of PFAS such as molecular weight, chain length, and lipophilicity were found to have important roles in plasma protein binding of PFAS.

Albumin promoter-driven FlpO expression induces efficient genetic recombination in mouse liver.

Tissue-specific gene manipulations are widely used in genetically engineered mouse models. A single recombinase system, such as the one using Alb-Cre, has been commonly used for liver-specific genetic manipulations. However, most diseases are complex, involving multiple genetic changes and various cell types. A dual recombinase system is required for conditionally modifying different genes sequentially in the same cell or inducing genetic changes in different cell types within the same organism. A FlpO cDNA was inserted between the last exon and 3'-UTR of the mouse albumin gene in a bacterial artificial chromosome (BAC-Alb-FlpO). The founders were crossed with various reporter mice to examine the efficiency of recombination. Liver cancer tumorigenesis was investigated by crossing the FlpO mice with FSF-KrasG12D mice and p53frt mice (KPF mice). BAC-Alb-FlpO mice exhibited highly efficient recombination capability in both hepatocytes and intrahepatic cholangiocytes. No recombination was observed in the duodenum and pancreatic cells. BAC-Alb-FlpO-mediated liver-specific expression of mutant KrasG12D and conditional deletion of p53 gene caused the development of liver cancer. Remarkably, liver cancer in these KPF mice manifested a distinctive mixed hepatocellular carcinoma and cholangiocarcinoma phenotype. A highly efficient and liver-specific BAC-Alb-FlpO mouse model was developed. In combination with other Cre lines, different genes can be manipulated sequentially in the same cell, or distinct genetic changes can be induced in different cell types of the same organism.NEW & NOTEWORTHY A liver-specific Alb-FlpO mouse line was generated. By coupling it with other existing CreERT or Cre lines, the dual recombinase approach can enable sequential gene modifications within the same cell or across various cell types in an organism for liver research through temporal and spatial gene manipulations.

Plasma Exchange Reduces Aβ Levels in Plasma and Decreases Amyloid Plaques in the Brain in a Mouse Model of Alzheimer’s Disease

Alzheimer’s disease (AD) is the most common type of dementia, characterized by the abnormal accumulation of protein aggregates in the brain, known as neurofibrillary tangles and amyloid-β (Aβ) plaques. It is believed that an imbalance between cerebral and peripheral pools of Aβ may play a relevant role in the deposition of Aβ aggregates. Therefore, in this study, we aimed to evaluate the effect of the removal of Aβ from blood plasma on the accumulation of amyloid plaques in the brain. We performed monthly plasma exchange with a 5% mouse albumin solution in the APP/PS1 mouse model from 3 to 7 months old. At the endpoint, total Aβ levels were measured in the plasma, and soluble and insoluble brain fractions were analyzed using ELISA. Brains were also analyzed histologically for amyloid plaque burden, plaque size distributions, and gliosis. Our results showed a reduction in the levels of Aβ in the plasma and insoluble brain fractions. Interestingly, histological analysis showed a reduction in thioflavin-S (ThS) and amyloid immunoreactivity in the cortex and hippocampus, accompanied by a change in the size distribution of amyloid plaques, and a reduction in Iba1-positive cells. Our results provide preclinical evidence supporting the relevance of targeting Aβ in the periphery and reinforcing the potential use of plasma exchange as an alternative non-pharmacological strategy for slowing down AD pathogenesis.

Propensities of Fatty Acid-Modified ASOs: Self-Assembly vs Albumin Binding

We conducted a biophysical study to investigate the self-assembling and albumin-binding propensities of a series of fatty acid-modified locked nucleic acid (LNA) antisense oligonucleotide (ASO) gapmers specific to the MALAT1 gene. To this end, a series of biophysical techniques were applied using label-free ASOs that were covalently modified with saturated fatty acids (FAs) of varying length, branching, and 5'/3' attachment. Using analytical ultracentrifugation (AUC), we demonstrate that ASOs conjugated with fatty acids longer than C16 exhibit an increasing tendency to form self-assembled vesicular structures. The C16 to C24 conjugates interacted via the fatty acid chains with mouse and human serum albumin (MSA/HSA) to form stable adducts with near-linear correlation between FA-ASO hydrophobicity and binding strength to mouse albumin. This was not observed for the longer fatty acid chain ASO conjugates (>C24) under the experimental conditions applied. The longer FA-ASO however adopted self-assembled structures with increasing intrinsic stabilities proportional to the fatty acid chain length. For instance, FA chain lengths smaller than C24 readily formed self-assembled structures containing 2 (C16), 6 (C22, bis-C12), and 12 (C24) monomers, as measured by analytical ultracentrifugation (AUC). Incubation with albumin disrupted these supramolecular architectures to form FA-ASO/albumin complexes mostly with 2:1 stoichiometry and binding affinities in the low micromolar range, as determined by isothermal titration calorimetry (ITC) and analytical ultracentrifugation (AUC). Binding of FA-ASOs underwent a biphasic pattern for medium-length FA chain lengths (>C16) with an initial endothermic phase of particulate disruption, followed by an exothermic binding event to the albumin. Conversely, ASO modified with di-palmitic acid (C32) formed a strong, hexameric complex. This structure was not disrupted when incubated with albumin under conditions above the critical nanoparticle concentration (CNC; <0.4 μM). It is noteworthy that the interaction of parent, fatty acid-free malat1 ASO to albumin was below detectability by ITC (KD ≫150 μM). This work demonstrates that the nature of mono- vs multimeric structures of hydrophobically modified ASOs is governed by the hydrophobic effect. Consequently, supramolecular assembly to form particulate structures is a direct consequence of the fatty acid chain length. This provides opportunities to exploit the concept of hydrophobic modification to influence pharmacokinetics (PK) and biodistribution for ASOs in two ways: (1) binding of the FA-ASO to albumin as a carrier vehicle and (2) self-assembly resulting in albumin-inert, supramolecular architectures. Both concepts create opportunities to influence biodistribution, receptor interaction, uptake mechanism, and pharmacokinetics/pharmacodynamics (PK/PD) properties in vivo, potentially enabling access to extrahepatic tissues in sufficient concentration to treat disease.

Abstract 920: Melanoma differentiation associated gene-9/syndecan binding protein (mda-9/sdcbp): A positive regulator of hepatocellular carcinoma

Abstract Background: Hepatocellular carcinoma (HCC) is a major clinical challenge because of increasing rates of incidence and mortality, and high resistance to standard radio- and chemotherapy. Identification of novel molecules regulating HCC facilitates development of targeted therapies having a lasting impact on HCC patient survival. The oncogene MDA-9/SDCBP promotes tumorigenesis and metastasis in many cancers. In The Cancer Genome Atlas (TCGA), 8% of HCC patients show amplification of MDA-9 gene. However, the role of MDA-9 in regulating HCC has not been well-studied. Objective: To unravel the function of MDA-9 in HCC using a transgenic mouse model with hepatocyte-specific overexpression of MDA-9 (Alb/MDA-9). Study design: Alb/MDA-9 mice were generated in B6/CBA background by using the mouse albumin promoter/enhancer element to drive human MDA-9 expression. N-nitrosodiethylamine (DEN)/phenobarbital (PB) was used for induction of HCC. Tumor immune profile was analyzed by Opal multiplex staining and flow cytometry. RNA-sequencing (RNA-seq) was performed to analyze MDA-9-mediated gene regulation. Results: At 18 months of age, none of the WT mice developed liver tumor, while one male and one female Alb/MDA-9 mice (n = 11) developed one isolated tumor each, suggesting that MDA-9 may not be a strong driver oncogene, rather it might promote HCC progression once tumor is initiated. Indeed, at 32 weeks after induction of HCC using DEN/PB, tumor burden in the livers of Alb/MDA-9 mice was significantly higher compared to WT. These tumors showed loss of hepatic architecture and hepatocyte ballooning, features of HCC. Increased staining for the proliferation marker PCNA, HCC markers AFP and cytokeratin, and angiogenesis marker CD31 in tumor sections, and significantly increased levels of serum liver enzymes, AST and ALT, were observed in Alb/MDA-9 mice vs WT. Macrophage-mediated inflammation plays a central role in HCC. Compared to WT, Alb/MDA-9 tumors showed marked infiltration of CD11b+ myeloid cells, FoxP3+ T regulatory cells and F4/80+ macrophages. RNA-seq in naïve WT and Alb/MDA-9 hepatocytes identified activation of signaling pathways associated with invasion, angiogenesis and inflammation. Conclusions: MDA-9 overexpression in hepatocytes promotes HCC by stimulating inflammation, angiogenesis and immune modulation. Studies are ongoing to obtain in-depth understanding of the molecular mechanisms by which MDA-9 exerts these pleiotropic effects. This study was supported by NIH/NCI Grant 1R01CA244993-01 (DS and PBF). Citation Format: Debashri Manna, Saranya Chidambaranathan Reghupaty, Rachel Gredler Mendoza, Maria Del Camarena, Mark A. Subler, Jennifer Koblinski, Rebecca Martin, Mikhail G. Dozmorov, Swadesh K. Das, Jolene J. Windle, Paul B. Fisher, Devanand Sarkar. Melanoma differentiation associated gene-9/syndecan binding protein (mda-9/sdcbp): A positive regulator of hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 920.

Susceptibility to kidney fibrosis in mice is associated with early growth response-2 protein and tissue inhibitor of metalloproteinase-1 expression

Patients with chronic kidney disease and experimental animal models of kidney fibrosis manifest diverse progression rates. Genetic susceptibility may contribute to this diversity, but the causes remain largely unknown. We have previously described kidney fibrosis with a mild or severe phenotype in mice expressing transforming growth factor-beta1 (TGF-β1) under the control of a mouse albumin promoter (Alb/TGF-β1), on a mixed genetic background with CBAxC57Bl6 mice. Here, we aimed to examine how genetic background may influence kidney fibrosis in TGF-β1 transgenic mice, and in the unilateral ureteral obstruction (UUO) and subtotal nephrectomy (SNX) mouse models. Congenic C57Bl6(B6)-TGFβ and CBAxB6-TGFβ (F1) transgenic mice were generated and survival, proteinuria, kidney histology, transcriptome and protein expressions were analyzed. We investigated the kidneys of B6 and CBA mice subjected to UUO and SNX, and the effects of tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) neutralization on the fibrotic process. CBAxB6-TGFβ mice developed severe kidney fibrosis and premature death, while B6-TGF-β mice had mild fibrosis and prolonged survival. Kidney early growth response factor-2 (EGR2) and TIMP-1 expression were induced only in CBAxB6-TGFβ mice. Similar strain-dependent early changes in EGR2 and TIMP-1 of mice subjected to UUO or SNX were observed. TIMP-1 neutralization invivo hindered fibrosis both in transgenic mice and the SNX model. EGR2 over-expression in cultured HEK293 cells induced TIMP-1 while EGR2 silencing hindered TGF-β induced TIMP-1 production in HK-2 cells and ureteral obstructed kidneys. Finally, EGR2 and TIMP1 was increased in human kidneys manifesting focal segmental glomerulosclerosis suggesting a correlation between animal studies and patient clinical settings. Thus, our observations demonstrate a strong relationship between genetic background and the progression of kidney fibrosis, which might involve early altered EGR2 and TIMP-1 response, but the relationship to patient genetics remains to be explored.

Simultaneous measurement of mouse and human albumin in chimeric mice with humanized livers.

Background: The degree of human hepatocyte replacement inchimeric mice with humanized liver has previously been shown to correlate with human plasma albumin measurements. However, there are no reportsthatdirectly comparetheremaining endogenous mouse albumin with the newly expressed human albumin following engraftment. To better understand the disposition of serum albumin in PXB-mice, we developed aliquid chromatographytandem mass spectrometry (LC-MS/MS) method to simultaneously quantitate both human and mouse albumin from plasma. Results: A robust correlation was observed between the serum human albumin levels measured by LC-MS/MS and the estimated replacement index of PXB-mice. Conclusion: All data wereshown to be specific and suitable to accurately quantify both human and mouse albumin from plasma of chimeric mice with humanized livers.

Multimodality molecular imaging of the alveolar-capillary barrier in lung disease using albumin based optical and PET tracers

Inflammatory changes caused by viruses, bacteria, exposure to toxins, commonly used drugs and even surgical intervention have the potential of causing abnormal epithelial permeability, which is manifest as infiltrative processes on computed tomography (CT), including the widespread infiltrates seen in COVID-19 pneumonia and acute respiratory distress syndrome (ARDS). We utilized a previously published mouse model of ARDS, intranasal delivery of LPS, to induce the alveolar-capillary barrier permeability seen in lung disease. We intravenously injected mice with Cy7 or 68-Gallium (68Ga) labeled mouse albumin and imaged using optical imaging (OI)/CT and PET. We observed significantly increased lung levels of Cy7-albumin on 3D OI/CT, which matched the abnormal appearance on microCT. This uptake correlated with fluorescence seen on sectioned lungs. To examine the translational potential of these findings, we radiolabeled albumin with 68Ga. We found that in mice with LPS-induced lung injury, 68Ga-albumin PET correlated with our optical imaging findings and demonstrated abnormal activity in the lung fields, indicative of abnormal epithelial permeability. These findings indicate 68Ga-albumin can be utilized as a sensitive translational radiotracer for quantifying the abnormal epithelial permeability that is seen in various lung pathologies, including COVID-19 induced pneumonia and ARDS. The ability to use Cy7-albumin 3D OI/CT imaging as a preclinical translational surrogate for 68Ga-albumin offers an accessible high throughput means to rapidly screen potential therapeutics against lung diseases that clinically manifest with endothelial permeability.

Engineered Anti-GPC3 Immunotoxin, HN3-ABD-T20, Produces Regression in Mouse Liver Cancer Xenografts Through Prolonged Serum Retention.

Treatment of hepatocellular carcinomas using our glypican-3 (GPC3)-targeting human nanobody (HN3) immunotoxins causes potent tumor regression by blocking protein synthesis and down-regulating the Wnt signaling pathway. However, immunogenicity and a short serum half-life may limit the ability of immunotoxins to transition to the clinic. To address these concerns, we engineered HN3-based immunotoxins to contain various deimmunized Pseudomonas exotoxin (PE) domains. This included HN3-T20, which was modified to remove T-cell epitopes and contains a PE domain II truncation. We compared them to our previously reported B-cell deimmunized immunotoxin (HN3-mPE24) and our original HN3-immunotoxin with a wild-type PE domain (HN3-PE38). All of our immunotoxins displayed high affinity to human GPC3, with HN3-T20 having a KD value of 7.4nM. HN3-T20 retained 73% enzymatic activity when compared with the wild-type immunotoxin in an adenosine diphosphate-ribosylation assay. Interestingly, a real-time cell growth inhibition assay demonstrated that a single dose of HN3-T20 at 62.5ng/mL (1.6nM) was capable of inhibiting nearly all cell proliferation during the 10-day experiment. To enhance HN3-T20's serum retention, we tested the effect of adding a streptococcal albumin-binding domain (ABD) and a llama single-domain antibody fragment specific for mouse and human serum albumin. For the detection of immunotoxin in mouse serum, we developed a highly sensitive enzyme-linked immunosorbent assay and found that HN3-ABD-T20 had a 45-fold higher serum half-life than HN3-T20 (326 minutes vs. 7.3minutes); consequently, addition of an ABD resulted in HN3-ABD-T20-mediated tumor regression at 1mg/kg. These data indicate that ABD-containing deimmunized HN3-T20 immunotoxins are high-potency therapeutics ready to be evaluated in clinical trials for the treatment of liver cancer.

3D optical/CT as a preclinical companion imaging platform for glioblastoma drug development

Multimodality 3D Optical Imaging (OI)/CT has the potential to play a major role in drug development for glioblastomas (GBM), as it is an accessible preclinical method. To demonstrate the potential of 3D OI/CT to visualize orthotopic GBM implantation, we labeled GBM cells with Cy7 and imaged their location using 3D OI/CT. To confirm the accuracy of the spatial localization and demonstrate the ability to image locoregionally delivered therapies, we labeled mouse albumin with Cy7 (Cy7ALB) and delivered it via locoregional infusion 1 mm or 3 mm into the brain and demonstrated correlation of signal between the 3D OI/CT and post necropsy brain slices. In addition, we demonstrated the potential of systemically delivered Cy7ALB contrast to detect blood–brain barrier (BBB) permeability caused by orthotopic GBMs using 3D OI/CT. We also tested the potential of 3D OI/CT to assess focal BBB permeability induced by high intensity focused ultrasound (HIFU), a methodology being used in clinical trials to noninvasively permeabilize the BBB for systemic therapeutic delivery to GBM. We demonstrated the ability of systemic Cy7ALB contrast together with 3D OI/CT to accurately assess real-time HIFU-induced BBB permeability, which correlated to post necropsy imaging of brains. Furthermore, we demonstrated that 3D OI/CT can also image the therapeutic distribution of a Cy7-labeled anti-PD-1 antibody, a prototype translational antibody therapy. We successfully imaged real-time antibody distribution after HIFU-induced BBB permeability, which correlated with post necropsy Cy7 signal and translational PET imaging after injection of [89Zr] anti-PD-1 antibody. Thus, we demonstrated the broad potential of using 3D OI/CT as an accessible preclinical tool to develop anti-GBM therapies.

Conjugate of Doxorubicin to Albumin-Binding Peptide Outperforms Aldoxorubicin.

Short circulation time and off-target toxicity are the main challenges faced by small-molecule chemotherapeutics. To overcome these shortcomings, an albumin-binding peptide conjugate of chemotherapeutics is developed that binds specifically to endogenous albumin and harnesses its favorable pharmacokinetics and pharmacodynamics for drug delivery to tumors. A protein-G-derived albumin-binding domain (ABD) is conjugated with doxorubicin (Dox) via a pH-sensitive linker. One to two Dox molecules are conjugated to ABD without loss of aqueous solubility. The albumin-binding ABD-Dox conjugate exhibits nanomolar affinity for human and mouse albumin, and upon administration in mice, shows a plasma half-life of 29.4 h, which is close to that of mouse albumin. Additionally, 2 h after administration, ABD-Dox exhibits an approximately 4-fold higher concentration in the tumor than free Dox. Free Dox clears quickly from the tumor, while ABD-Dox maintains a steady concentration in the tumor for at least 72 h, so that its relative accumulation at 72 h is ≈120-fold greater than that of free Dox. The improved pharmacokinetics and biodistribution of ABD-Dox result in enhanced therapeutic efficacy in syngeneic C26 colon carcinoma and MIA PaCa-2 pancreatic tumor xenografts, compared with free Dox and aldoxorubicin, an albumin-reactive Dox prodrug currently in clinical development.

Human and mouse albumin bind their respective neonatal Fc receptors differently

Albumin has a serum half-life of three weeks in humans and is utilized to extend the serum persistence of drugs that are genetically fused or conjugated directly to albumin or albumin-binding molecules. Responsible for the long half-life is FcRn that protects albumin from intracellular degradation. An in-depth understanding of how FcRn binds albumin across species is of importance for design and evaluation of albumin-based therapeutics. Albumin consists of three homologous domains where domain I and domain III of human albumin are crucial for binding to human FcRn. Here, we show that swapping of two loops in domain I or the whole domain with the corresponding sequence in mouse albumin results in reduced binding to human FcRn. In contrast, humanizing domain I of mouse albumin improves binding. We reveal that domain I of mouse albumin plays a minor role in the interaction with the mouse and human receptors, as domain III on its own binds with similar affinity as full-length mouse albumin. Further, we show that P573 in domain III of mouse albumin is required for strong receptor binding. Our study highlights distinct differences in structural requirements for the interactions between mouse and human albumin with their respective receptor, which should be taken into consideration in design of albumin-based drugs and evaluation in mouse models.

Different Nanoformulations Alter the Tissue Distribution of Paclitaxel, Which Aligns with Reported Distinct Efficacy and Safety Profiles.

Previous studies have shown that different paclitaxel formulations produce distinct anticancer efficacy and safety profiles in animals and humans. This study aimed to investigate the distinct pharmacokinetics and tissue distribution of various nanoformulations of paclitaxel, which may translate into potential differences in safety and efficacy. Four nanoparticle formulations ( nab-paclitaxel, mouse albumin nab-paclitaxel [m -nab-paclitaxel], micellar paclitaxel, and polymeric nanoparticle paclitaxel) as well as solvent-based paclitaxel were intravenously administered to mice. Seventeen blood and tissue samples were collected at different time points. The total paclitaxel concentration in each tissue specimen was measured with liquid chromatography-tandem mass spectrometry. Compared with solvent-based paclitaxel, all four nanoformulations demonstrated decreased paclitaxel exposure in plasma. All nanoformulations were associated with paclitaxel blood-cell accumulation in mice; however, m- nab-paclitaxel was associated with the lowest accumulation. Five minutes after dosing, the total paclitaxel in the tissues and blood was approximately 44% to 57% of the administered dose of all paclitaxel formulations. Paclitaxel was primarily distributed to liver, muscle, intestine, kidney, skin, and bone. Compared with solvent-based paclitaxel, the different nanocarriers altered the distribution of paclitaxel in all tissues with distinct paclitaxel concentration-time profiles. nab-paclitaxel was associated with increased delivery efficiency of paclitaxel in the pancreas compared with the other formulations, consistent with the demonstrated efficacy of nab-paclitaxel in pancreatic cancer. All the nanoformulations led to high penetration in the lungs and fat pad, which potentially points to efficacy in lung and breast cancers. Micellar paclitaxel and polymeric nanoparticle paclitaxel were associated with high paclitaxel accumulation in the heart; thus, the risk of cardiovascular toxicity with these formulations may warrant further investigation. The solvent-based formulation was associated with the poorest paclitaxel penetration in all tissues and the lowest tissue-to-plasma ratio. The different nanocarriers of paclitaxel were associated with distinct pharmacokinetics and tissue distribution, which largely align with the observed efficacy and toxicity profiles in clinical trials.

Graft remodelling after transplantation of chimeric mouse/rat livers into rats

Background: The efficacy of transplanting chimeric organs lacks of any evidence, even in preclinical studies involving small animals. We challenged this idea by transplanting mouse-rat chimeric livers into baby rats. Method: Chimeric livers were created by transplanting Lewis rat hepatocytes into FRG® mice (C57Bl/6 Fah-/-/Rag2-/-/Il2rg-/-). These grafts were transplanted into newly weaned Lewis rats (45±3g) without or with immunosuppression (IS) (Tacrolimus 0.6mg/kg/day, for 56 or 112 days). Rejection was assessed by graft biopsies and immune cells activation. Rat and mouse albumin was measured weekly. Wild type C57Bl/6 mice were used as control donors. Results: All non-immunosuppressed recipients experienced acute rejection and died from 8 to 11 days after transplantation. Under tacrolimus all chimeric liver recipients survived having normal development and weight gain. Banff score was 2-3, rejection being mostly driven by endothelium and cholangiocytes. Rat albumin production was within physiologic ranges. Chimeric livers grew on average 670% (from 1.3±0.2g to 8.7±1.4g), in line with syngeneic rat controls. By contrast, xenogeneic controls showed impaired growth and died before or shortly after the immunosuppression was stopped, in the 56-day IS group (p=0.0014). In the 112-day IS group they all died while still on Tacrolimus (p=0.0013). Banff score was never inferior to 6. Four months after implantation into rats, chimeric livers were partially repopulated by portal endothelial cells and some rat hepatocytes had undergone complete transdifferentiation into cholangiocytes. Conclusion: We show robust survival of orthotopically transplanted chimeric livers. The transplanted organs not only sustained normal animal growth, but also underwent profound recipient-oriented remodelling.

Protein mass spectrometry extends temporal blood meal detection over polymerase chain reaction in mouse-fed Chagas disease vectors.

BACKGROUND Chagas disease is highly prevalent in Latin America, and vector control is the most effective control strategy to date. We have previously shown that liquid chromatography tandem mass spectrometry (LC-MS/MS) is a valuable tool for identifying triatomine vector blood meals.OBJECTIVES The purpose of this study was to determine blood meal detection ability as a function of method [polymerase chain reaction (PCR) vs. LC-MS/MS], time since feeding, and the effect of molting in mouse-fed triatomine insect vectors targeting hemoglobin and albumin proteins with LC-MS/MS and short interspersed nuclear elements (SINE)-based PCR.METHODS We experimentally fed Triatoma protracta on mice and used LC-MS/MS to detect hemoglobin and albumin peptides over time post-feeding and post-molting (≤ 12 weeks). We compared LC-MS/MS results with those of a standard PCR method based on SINEs.FINDINGS Hemoglobin-based LC-MS/MS detected blood meals most robustly at all time points post-feeding. Post-molting, no blood meals were detected with PCR, whereas LC-MS/MS detected mouse hemoglobin and albumin up to 12 weeks.MAIN CONCLUSIONS In our study, the hemoglobin signature in the insect abdomen lasted longer than that of albumin and DNA. LC-MS/MS using hemoglobin shows promise for identifying triatomine blood meals over long temporal scales and even post-molting. Clarifying the frequency of blood-feeding on different hosts can foster our understanding of vector behavior and may help devise sounder disease-control strategies, including Ecohealth (community based ecosystem management) approaches.

Leucine rich α-2 glycoprotein is a potential urinary biomarker for renal tubular injury

Recent evidence suggests that renal tubular injury plays a key role in deterioration of renal function in both chronic kidney disease (CKD) and acute kidney injury (AKI). Since commonly used biochemical indicators such as GFR, serum creatinine, blood urea nitrogen and creatinine clearance are inappropriate for detecting alteration in renal tubules, biomarkers reflecting renal tubular injury have been extensively explored.Our research group identified leucine rich α-2 glycoprotein (LRG) as a novel serum biomarker for various inflammatory diseases such as rheumatoid arthritis and inflammatory bowel disease. In inflammatory diseases, LRG expression is up-regulated at the site of inflammation, in accordance with the induction of LRG in many cell types by various inflammatory cytokines. Recently, urinary LRG was reported as a possible biomarker for several renal diseases, but the mechanism of LRG excretion in urine is still unclear. In this study, by analyzing a mouse albumin (ALB) overload model that is commonly used to study proteinuria-induced renal tubular injury, we provided evidence that urinary LRG is produced in renal tubular epithelial cells by interleukin-1β (IL-1β) that is released during proteinuria-induced renal damage. In this model, urinary LRG became detectable after ALB overload. In kidney, mRNA expression of LRG together with that of NACHT LRR and PYD domains-containing protein 3 (NLRP3) and IL-1β was significantly up-regulated in ALB-overloaded mice, compared to PBS-treated mice. By pathological analysis of kidney, LRG was detected in the injured proximal tubules, distal tubules and collecting ducts in ALB-overloaded mice. Accordingly, in vitro stimulation of mouse renal cortical tubular epithelial cells with excessive ALB led to LRG mRNA up-regulation and its protein secretion, which was effectively blocked by IL-1 receptor antagonist. These results suggest that urinary LRG could be applied to a biomarker detecting renal tubular injury in various renal diseases.

Abstract B004: Enhanced efficacy of immune modulators with albumin binding domains

Abstract Numerous approaches to improve the biologic half-life and tumor targeting of small therapeutic proteins (e.g., interleukins), peptides, and fragment-based antibodies (scFv) have been described. The attractiveness of these smaller biologic formats includes improved tumor penetration and the ability to engineer a single molecule that can modulate several tumor targets. However, the limitation of these smaller biologic-based therapeutics (less than 150kd) is their short half-life and lack of tumor targeting. Here we describe a unique (24Kd) single-chain fragment antibody (ScFv) that binds to serum albumin across a range of pHs and that retains binding to, and recycling by, the neonatal Fc-receptor (FcRn), resulting in a significant extension of the biologic half-life of three different therapeutic proteins that were molecularly linked to this albumin binding domain (ABD) fragment antibody. A second advantage to having an ABD construct is enhanced tumor targeting. Numerous studies have shown that albumin-mediated delivery of therapeutic proteins and small molecules results in tumor accumulation that was higher than that produced by PEGylation, Thus, fusion with an ABD is a promising approach for improving the therapeutic/safety window of small recombinant therapeutic proteins (e.g., interleukins, receptor ligands) or enhancing the targeting of receptor-directed scFv therapeutic proteins. Using the XOMA phage library, we discovered an scFv ABD that has high binding affinity to mouse, human, and cynomolgus serum albumin. We demonstrate that fusion constructs of this scFv ABD with several different small therapeutic proteins (recombinant interleukin proteins and scFvs targeting relevant immune-oncology receptors) demonstrated improved serum half-life and greater efficacy compared to the naked proteins without the ABD present. We have further characterized our scFV-ABD and have demonstrated that (1) biologic activity is retained when the therapeutic protein is attached via the N- or C-terminus, suggesting utility for delivering more than one therapeutic protein; and (2) half-life in mouse serum in vivo was extended from minutes to hours/days for three different recombinant proteins with MWs of 10-80Kd. Additionally, we have demonstrated markedly superior reductions in tumor growth and improved overall survival with our ABD constructs vs free recombinant protein in an established B6F10 melanoma model. Moreover, superior efficacy was observed at lower doses and with a single dose of the ABD constructs vs free recombinant protein. The versatility of this ABD construct to attached combinations of various types of recombinant immune stimulators and/or scFv to immune checkpoint inhibitors can result in significantly enhanced clinical efficacy. Citation Format: John Cini. Enhanced efficacy of immune modulators with albumin binding domains [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B004.

Dapsone and Nitroso Dapsone Activation of Naı̈ve T-Cells from Healthy Donors.

Dapsone (DDS) causes hypersensitivity reactions in 0.5-3.6% of patients. Although clinical diagnosis is indicative of a hypersensitivity reaction, studies have not been performed to define whether dapsone or a metabolite activates specific T-cells. Thus, the aims of this study were to explore the immunogenicity DDS and nitroso DDS (DDS-NO) using peripheral blood mononuclear cells from healthy donors and splenocytes from mice and generate human T-cell clones to characterize mechanisms of T-cell activation. DDS-NO was synthesized from DDS-hydroxylamine and shown to bind to the thiol group of glutathione and human and mouse albumin through sulfonamide and N-hydroxyl sulphonamide adducts. Naïve T-cell priming to DDS and DDS-NO was successful in three human donors. DDS-specific CD4+ T-cell clones were stimulated to proliferate in response to drug via a MHC class II restricted direct binding interaction. Cross reactivity with DDS-NO, DDS-analogues, and sulfonamides was not observed. DDS-NO clones were CD4+ and CD8+, MHC class II and I restricted, respectively, and activated via a pathway dependent on covalent binding and antigen processing. DDS and DDS-NO-specific clones secreted a mixture of Th1 and Th2 cytokines, but not granzyme-B. Splenocytes from mice immunized with DDS-NO were stimulated to proliferate in vitro with the nitroso metabolite, but not DDS. In contrast, immunization with DDS did not activate T-cells. These data show that DDS- and DDS-NO-specific T-cell responses are readily detectable.

Abolishment of proximal tubule albumin endocytosis does not affect plasma albumin during nephrotic syndrome in mice

The megalin/cubilin receptor complex is required for proximal tubular endocytosis and degradation of filtered albumin. An additional high-capacity retrieval pathway of intact albumin for the recovery of large amounts of filtered albumin has been proposed, possibly involving cooperation between megalin/cubilin and the neonatal Fc receptor. To clarify the potential role of such a pathway, we examined the effects of megalin/cubilin gene inactivation on tubular albumin uptake and plasma albumin levels in nephrotic, podocin knockout mice. Immunofluorescence microscopy of megalin/cubilin/podocin knockout mouse kidneys demonstrated abolishment of proximal tubule albumin uptake, in contrast to the excessive albumin accumulation observed in podocin knockout mice compared to controls. Correspondingly, urinary albumin excretion was increased 1.4 fold in megalin/cubilin/podocin compared to podocin knockout mice (albumin/creatinine: 226 vs. 157 mg/mg). However, no difference in plasma albumin levels was observed between megalin/cubilin/podocin and podocin knockout mice, as both were reduced to approximately 40% of controls. There were no differences in liver albumin synthesis by mRNA levels and protein abundance. Thus, megalin/cubilin knockout efficiently blocks proximal tubular albumin uptake in nephrotic mice but plasma albumin levels did not differ as a result of megalin/cubilin-deficiency, suggesting no significance of the megalin/cubilin-pathway for albumin homeostasis by retrieval of intact albumin.

Construction of a general albumin promoter reporter system for real-time monitoring of the differentiation status of functional hepatocytes from stem cells in mouse, rat and human.

Genetic constructs with promoters fused to reporter genes for simultaneous monitoring of cellular events have been the focus of attention in recent years. Adenoviral vectors, which have distinctive characteristics, have been used to monitor the differentiation of stem cells in vitro. In the present study, a modified adenoviral vector was constructed, containing a mouse, rat, and human general albumin promoter sequence fused to a ZsGreen reporter gene, and evaluated its efficiency in different cell types. Two hepatocyte cell lines (Hepa1-6 and HepG2), rat primary hepatocytes, rat bone marrow mesenchymal stem cells (BM-MSCs) and rat BM-MSCs-derived hepatocyte-like cells were transduced with this vector, and the transfection efficiency and functional capabilities of the promoter were evaluated by fluorescent microscopy. The results demonstrated efficient expression of ZsGreen in Hepa1-6 cells, HepG2 cells, rat primary hepatocytes, and rat BM-MSCs-derived hepatocyte-like cells, but not in rat BM-MSCs. In conclusion, the current study demonstrates a simple, high-efficiency, general tool for real-time monitoring of the differentiation status of hepatocytes from stem cells in mice, rats, and humans. This tool may be useful for evaluating different protocols to generate functional hepatocytes from stem cells in multiple species.