ASU Scholarship Showcase

Background: Cancer diagnosis in both dogs and humans is complicated by the lack of a non-invasive diagnostic test. To meet this clinical need, we apply the recently developed immunosignature assay to spontaneous canine lymphoma as clinical proof-of-concept. Here we evaluate the immunosignature as a diagnostic for spontaneous canine lymphoma at both at initial diagnosis and evaluating the disease free interval following treatment.

Methods: Sera from dogs with confirmed lymphoma (B cell n = 38, T cell n = 11) and clinically normal dogs (n = 39) were analyzed. Serum antibody responses were characterized by analyzing the binding pattern, or immunosignature, of serum antibodies on a non-natural sequence peptide microarray. Peptides were selected and tested for the ability to distinguish healthy dogs from those with lymphoma and to distinguish lymphoma subtypes based on immunophenotype. The immunosignature of dogs with lymphoma were evaluated for individual signatures. Changes in the immunosignatures were evaluated following treatment and eventual relapse.

Results: Despite being a clonal disease, both an individual immunosignature and a generalized lymphoma immunosignature were observed in each dog. The general lymphoma immunosignature identified in the initial set of dogs (n = 32) was able to predict disease status in an independent set of dogs (n = 42, 97% accuracy). A separate immunosignature was able to distinguish the lymphoma based on immunophenotype (n = 25, 88% accuracy). The individual immunosignature was capable of confirming remission three months following diagnosis. Immunosignature at diagnosis was able to predict which dogs with B cell lymphoma would relapse in less than 120 days (n = 33, 97% accuracy).

Conclusion: We conclude that the immunosignature can serve as a multilevel diagnostic for canine, and potentially human, lymphoma.

Quantitative three-dimensional (3D) computed tomography (CT) imaging of living single cells enables orientation-independent morphometric analysis of the intricacies of cellular physiology. Since its invention, x-ray CT has become indispensable in the clinic for diagnostic and prognostic purposes due to its quantitative absorption-based imaging in true 3D that allows objects of interest to be viewed and measured from any orientation. However, x-ray CT has not been useful at the level of single cells because there is insufficient contrast to form an image. Recently, optical CT has been developed successfully for fixed cells, but this technology called Cell-CT is incompatible with live-cell imaging due to the use of stains, such as hematoxylin, that are not compatible with cell viability. We present a novel development of optical CT for quantitative, multispectral functional 4D (three spatial + one spectral dimension) imaging of living single cells. The method applied to immune system cells offers truly isotropic 3D spatial resolution and enables time-resolved imaging studies of cells suspended in aqueous medium. Using live-cell optical CT, we found a heterogeneous response to mitochondrial fission inhibition in mouse macrophages and differential basal remodeling of small (0.1 to 1 fl) and large (1 to 20 fl) nuclear and mitochondrial structures on a 20- to 30-s time scale in human myelogenous leukemia cells. Because of its robust 3D measurement capabilities, live-cell optical CT represents a powerful new tool in the biomedical research field.

There are an increasing variety of applications in which peptides are both synthesized and used attached to solid surfaces. This has created a need for high throughput sequence analysis directly on surfaces. However, common sequencing approaches that can be adapted to surface bound peptides lack the throughput often needed in library-based applications. Here we describe a simple approach for sequence analysis directly on solid surfaces that is both high speed and high throughput, utilizing equipment available in most protein analysis facilities. In this approach, surface bound peptides, selectively labeled at their N-termini with a positive charge-bearing group, are subjected to controlled degradation in ammonia gas, resulting in a set of fragments differing by a single amino acid that remain spatially confined on the surface they were bound to. These fragments can then be analyzed by MALDI mass spectrometry, and the peptide sequences read directly from the resulting spectra.

Recent infectious outbreaks highlight the need for platform technologies that can be quickly deployed to develop therapeutics needed to contain the outbreak. We present a simple concept for rapid development of new antimicrobials. The goal was to produce in as little as one week thousands of doses of an intervention for a new pathogen. We tested the feasibility of a system based on antimicrobial synbodies. The system involves creating an array of 100 peptides that have been selected for broad capability to bind and/or kill viruses and bacteria. The peptides are pre-screened for low cell toxicity prior to large scale synthesis. Any pathogen is then assayed on the chip to find peptides that bind or kill it. Peptides are combined in pairs as synbodies and further screened for activity and toxicity. The lead synbody can be quickly produced in large scale, with completion of the entire process in one week.

Antigen-antibody complexes are central players in an effective immune response. However, finding those interactions relevant to a particular disease state can be arduous. Nonetheless many paths to discovery have been explored since deciphering these interactions can greatly facilitate the development of new diagnostics, therapeutics, and vaccines. In silico B cell epitope mapping approaches have been widely pursued, though success has not been consistent. Antibody mixtures in immune sera have been used as handles for biologically relevant antigens, but these and other experimental approaches have proven resource intensive and time consuming. In addition, these methods are often tailored to individual diseases or a specific proteome, rather than providing a universal platform. Most of these methods are not able to identify the specific antibody’s epitopes from unknown antigens, such as un-annotated neo antigens in cancer. Alternatively, a peptide library comprised of sequences unrestricted by naturally-found protein space provides for a universal search for mimotopes of an antibody’s epitope. Here we present the utility of such a non-natural random sequence library of 10,000 peptides physically addressed on a microarray for mimotope discovery without sequence information of the specific antigen. The peptide arrays were probed with serum from an antigen-immunized rabbit, or alternatively probed with serum pre-absorbed with the same immunizing antigen. With this positive and negative screening scheme, we identified the library-peptides as the mimotopes of the antigen. The unique library peptides were successfully used to isolate antigen-specific antibodies from complete immune serum. Sequence analysis of these peptides revealed the epitopes in the immunized antigen. We present this method as an inexpensive, efficient method for identifying mimotopes of any antibody’s targets. These mimotopes should be useful in defining both components of the antigen-antibody complex.

The probiotic effects of Lactobacillus reuteri have been speculated to partly depend on its capacity to produce the antimicrobial substance reuterin during the reduction of glycerol in the gut. In this study, the potential of this process to protect human intestinal epithelial cells against infection with Salmonella enterica serovar Typhimurium was investigated. We used a three-dimensional (3-D) organotypic model of human colonic epithelium that was previously validated and applied to study interactions between S. Typhimurium and the intestinal epithelium that lead to enteric salmonellosis. Using this model system, we show that L. reuteri protects the intestinal cells against the early stages of Salmonella infection and that this effect is significantly increased when L. reuteri is stimulated to produce reuterin from glycerol. More specifically, the reuterin-containing ferment of L. reuteri caused a reduction in Salmonella adherence and invasion (1 log unit), and intracellular survival (2 log units). In contrast, the L. reuteri ferment without reuterin stimulated growth of the intracellular Salmonella population with 1 log unit. The short-term exposure to reuterin or the reuterin-containing ferment had no observed negative impact on intestinal epithelial cell health. However, long-term exposure (24 h) induced a complete loss of cell-cell contact within the epithelial aggregates and compromised cell viability. Collectively, these results shed light on a potential role for reuterin in inhibiting Salmonella-induced intestinal infections and may support the combined application of glycerol and L. reuteri. While future in vitro and in vivo studies of reuterin on intestinal health should fine-tune our understanding of the mechanistic effects, in particular in the presence of a complex gut microbiota, this the first report of a reuterin effect on the enteric infection process in any mammalian cell type.

Immunosignaturing shows promise as a general approach to diagnosis. It has been shown to detect immunological signs of infection early during the course of disease and to distinguish Alzheimer’s disease from healthy controls. Here we test whether immunosignatures correspond to clinical classifications of disease using samples from people with brain tumors. Blood samples from patients undergoing craniotomies for therapeutically naïve brain tumors with diagnoses of astrocytoma (23 samples), Glioblastoma multiforme (22 samples), mixed oligodendroglioma/astrocytoma (16 samples), oligodendroglioma (18 samples), and 34 otherwise healthy controls were tested by immunosignature. Because samples were taken prior to adjuvant therapy, they are unlikely to be perturbed by non-cancer related affects. The immunosignaturing platform distinguished not only brain cancer from controls, but also pathologically important features about the tumor including type, grade, and the presence or absence of O⁶-methyl-guanine-DNA methyltransferase methylation promoter (MGMT), an important biomarker that predicts response to temozolomide in Glioblastoma multiformae patients.

Hydrophobic platinum(II)-5,10,15,20-tetrakis-(2,3,4,5,6-pentafluorophenyl)-porphyrin (PtTFPP) was physically incorporated into micelles formed from poly(ε-caprolactone)-block-poly(ethylene glycol) to enable the application of PtTFPP in aqueous solution. Micelles were characterized using dynamic light scattering (DLS) and atomic force microscopy (AFM) to show an average diameter of about 140 nm. PtTFPP showed higher quantum efficiency in micellar solution than in tetrahydrofuran (THF) and dichloromethane (CH₂Cl₂). PtTFPP in micelles also exhibited higher photostability than that of PtTFPP suspended in water. PtTFPP in micelles exhibited good oxygen sensitivity and response time. This study provided an efficient approach to enable the application of hydrophobic oxygen sensors in a biological environment.

Extra-intestinal pathogenic E. coli (ExPEC), including avian pathogenic E. coli (APEC), pose a considerable threat to both human and animal health, with illness causing substantial economic loss. APEC strain χ7122 (O78∶K80∶H9), containing three large plasmids [pChi7122-1 (IncFIB/FIIA-FIC), pChi7122-2 (IncFII), and pChi7122-3 (IncI₂)]; and a small plasmid pChi7122-4 (ColE2-like), has been used for many years as a model strain to study the molecular mechanisms of ExPEC pathogenicity and zoonotic potential. We previously sequenced and characterized the plasmid pChi7122-1 and determined its importance in systemic APEC infection; however the roles of the other pChi7122 plasmids were still ambiguous. Herein we present the sequence of the remaining pChi7122 plasmids, confirming that pChi7122-2 and pChi7122-3 encode an ABC iron transport system (eitABCD) and a putative type IV fimbriae respectively, whereas pChi7122-4 is a cryptic plasmid. New features were also identified, including a gene cluster on pChi7122-2 that is not present in other E. coli strains but is found in Salmonella serovars and is predicted to encode the sugars catabolic pathways. In vitro evaluation of the APEC χ7122 derivative strains with the three large plasmids, either individually or in combinations, provided new insights into the role of plasmids in biofilm formation, bile and acid tolerance, and the interaction of E. coli strains with 3-D cultures of intestinal epithelial cells. In this study, we show that the nature and combinations of plasmids, as well as the background of the host strains, have an effect on these phenomena. Our data reveal new insights into the role of extra-chromosomal sequences in fitness and diversity of ExPEC in their phenotypes.

Strategies are needed to improve repopulation of decellularized lung scaffolds with stromal and functional epithelial cells. We demonstrate that decellularized mouse lungs recellularized in a dynamic low fluid shear suspension bioreactor, termed the rotating wall vessel (RWV), contained more cells with decreased apoptosis, increased proliferation and enhanced levels of total RNA compared to static recellularization conditions. These results were observed with two relevant mouse cell types: bone marrow-derived mesenchymal stromal (stem) cells (MSCs) and alveolar type II cells (C10). In addition, MSCs cultured in decellularized lungs under static but not bioreactor conditions formed multilayered aggregates. Gene expression and immunohistochemical analyses suggested differentiation of MSCs into collagen I-producing fibroblast-like cells in the bioreactor, indicating enhanced potential for remodeling of the decellularized scaffold matrix. In conclusion, dynamic suspension culture is promising for enhancing repopulation of decellularized lungs, and could contribute to remodeling the extracellular matrix of the scaffolds with subsequent effects on differentiation and functionality of inoculated cells.