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.

Cognitive theories in visual attention and perception, categorization, and memory often critically rely on concepts of similarity among objects, and empirically require measures of “sameness” among their stimuli. For instance, a researcher may require similarity estimates among multiple exemplars of a target category in visual search, or targets and lures in recognition memory. Quantifying similarity, however, is challenging when everyday items are the desired stimulus set, particularly when researchers require several different pictures from the same category. In this article, we document a new multidimensional scaling database with similarity ratings for 240 categories, each containing color photographs of 16–17 exemplar objects. We collected similarity ratings using the spatial arrangement method. Reports include: the multidimensional scaling solutions for each category, up to five dimensions, stress and fit measures, coordinate locations for each stimulus, and two new classifications. For each picture, we categorized the item's prototypicality, indexed by its proximity to other items in the space. We also classified pairs of images along a continuum of similarity, by assessing the overall arrangement of each MDS space. These similarity ratings will be useful to any researcher that wishes to control the similarity of experimental stimuli according to an objective quantification of “sameness.”

Background: To be effective, orally administered live Salmonella vaccines must first survive their encounter with the low pH environment of the stomach. To enhance survival, an antacid is often given to neutralize the acidic environment of the stomach just prior to or concomitant with administration of the vaccine. One drawback of this approach, from the perspective of the clinical trial volunteer, is that the taste of a bicarbonate-based acid neutralization system can be unpleasant. Thus, we explored an alternative method that would be at least as effective as bicarbonate and with a potentially more acceptable taste. Because ingestion of protein can rapidly buffer stomach pH, we examined the possibility that the protein-rich Ensure® Nutrition shakes would be effective alternatives to bicarbonate.

Results: We tested one Salmonella enterica serovar Typhimurium and three Salmonella Typhi vaccine strains and found that all strains survived equally well when incubated in either Ensure® or bicarbonate. In a low gastric pH mouse model, Ensure® worked as well or better than bicarbonate to enhance survival through the intestinal tract, although neither agent enhanced the survival of the S. Typhi test strain possessing a rpoS mutation.

Conclusions: Our data show that a protein-rich drink such as Ensure® Nutrition shakes can serve as an alternative to bicarbonate for reducing gastric pH prior to administration of a live Salmonella vaccine.

While expert groups often make recommendations on a range of non-controversial as well as controversial issues, little is known about how the level of expert consensus-the level of expert agreement-influences perceptions of the recommendations. This research illustrates that for non-controversial issues expert groups that exhibit high levels of agreement are more persuasive than expert groups that exhibit low levels of agreement. This effect is mediated by the perceived entitativity-the perceived cohesiveness or unification of the group-of the expert group. But for controversial issues, this effect is moderated by the perceivers' implicit assumptions about the group composition. When perceivers are provided no information about a group supporting the Affordable Care Act-a highly controversial piece of U.S. legislation that is divided by political party throughout the country-higher levels of agreement are less persuasive than lower levels of agreement because participants assume there were more democrats and fewer republicans in the group. But when explicitly told that the group was half republicans and half democrats, higher levels of agreement are more persuasive.

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.

Leucine-responsive regulatory protein (Lrp) is known to be an indirect activator of type 1 fimbriae synthesis in Salmonella enterica serovar Typhimurium via direct regulation of FimZ, a direct positive regulator for type 1 fimbriae production. Using RT-PCR, we have shown previously that fimA transcription is dramatically impaired in both lrp-deletion (Δlrp) and constitutive-lrp expression (lrp^C) mutant strains. In this work, we used chromosomal P_fimA-lacZ fusions and yeast agglutination assays to confirm and extend our previous results. Direct binding of Lrp to P_fimA was shown by an electrophoretic mobility shift assay (EMSA) and DNA footprinting assay. Site-directed mutagenesis revealed that the Lrp-binding motifs in P_fimA play a role in both activation and repression of type 1 fimbriae production. Overproduction of Lrp also abrogates fimZ expression. EMSA data showed that Lrp and FimZ proteins independently bind to P_fimA without competitive exclusion. In addition, both Lrp and FimZ binding to P_fimA caused a hyper retardation (supershift) of the DNA-protein complex compared to the shift when each protein was present alone. Nutrition-dependent cellular Lrp levels closely correlated with the amount of type 1 fimbriae production. These observations suggest that Lrp plays important roles in type 1 fimbriation by acting as both a positive and negative regulator and its effect depends, at least in part, on the cellular concentration of Lrp in response to the nutritional environment.

The low pH of the stomach serves as a barrier to ingested microbes and must be overcome or bypassed when delivering live bacteria for vaccine or probiotic applications. Typically, the impact of stomach acidity on bacterial survival is evaluated in vitro, as there are no small animal models to evaluate these effects in vivo. To better understand the effect of this low pH barrier to live attenuated Salmonella vaccines, which are often very sensitive to low pH, we investigated the value of the histamine mouse model for this application. A low pH gastric compartment was transiently induced in mice by the injection of histamine. This resulted in a gastric compartment of approximately pH 1.5 that was capable of distinguishing between acid-sensitive and acid-resistant microbes. Survival of enteric microbes during gastric transit in this model directly correlated with their in vitro acid resistance. Because many Salmonella enterica serotype Typhi vaccine strains are sensitive to acid, we have been investigating systems to enhance the acid resistance of these bacteria. Using the histamine mouse model, we demonstrate that the in vivo survival of S. Typhi vaccine strains increased approximately 10-fold when they carried a sugar-inducible arginine decarboxylase system. We conclude that this model will be a useful for evaluating live bacterial preparations prior to clinical trials.

Background: Counselor behaviors that mediate the efficacy of motivational interviewing (MI) are not well understood, especially when applied to health behavior promotion. We hypothesized that client change talk mediates the relationship between counselor variables and subsequent client behavior change.

Methods: Purposeful sampling identified individuals from a prospective randomized worksite trial using an MI intervention to promote firefighters’ healthy diet and regular exercise that increased dietary intake of fruits and vegetables (n = 21) or did not increase intake of fruits and vegetables (n = 22). MI interactions were coded using the Motivational Interviewing Skill Code (MISC 2.1) to categorize counselor and firefighter verbal utterances. Both Bayesian and frequentist mediation analyses were used to investigate whether client change talk mediated the relationship between counselor skills and behavior change.

Results: Counselors’ global spirit, empathy, and direction and MI-consistent behavioral counts (e.g., reflections, open questions, affirmations, emphasize control) significantly correlated with firefighters’ total client change talk utterances (rs = 0.42, 0.40, 0.30, and 0.61, respectively), which correlated significantly with their fruit and vegetable intake increase (r = 0.33). Both Bayesian and frequentist mediation analyses demonstrated that findings were consistent with hypotheses, such that total client change talk mediated the relationship between counselor’s skills—MI-consistent behaviors [Bayesian mediated effect: αβ = .06 (.03), 95% CI = .02, .12] and MI spirit [Bayesian mediated effect: αβ = .06 (.03), 95% CI = .01, .13]—and increased fruit and vegetable consumption.

Conclusion: Motivational interviewing is a resource- and time-intensive intervention, and is currently being applied in many arenas. Previous research has identified the importance of counselor behaviors and client change talk in the treatment of substance use disorders. Our results indicate that similar mechanisms may underlie the effects of MI for dietary change. These results inform MI training and application by identifying those processes critical for MI success in health promotion domains.

Background: The success of new sequencing technologies and informatic methods for identifying genes has made establishing gene product function a critical rate limiting step in progressing the molecular sciences. We present a method to functionally mine genomes for useful activities in vivo, using an unusual property of a member of the poxvirus family to demonstrate this screening approach.

Results: The genome of Parapoxvirus ovis (Orf virus) was sequenced, annotated, and then used to PCR-amplify its open-reading-frames. Employing a cloning-independent protocol, a viral expression-library was rapidly built and arrayed into sub-library pools. These were directly delivered into mice as expressible cassettes and assayed for an immune-modulating activity associated with parapoxvirus infection. The product of the B2L gene, a homolog of vaccinia F13L, was identified as the factor eliciting immune cell accumulation at sites of skin inoculation. Administration of purified B2 protein also elicited immune cell accumulation activity, and additionally was found to serve as an adjuvant for antigen-specific responses. Co-delivery of the B2L gene with an influenza gene-vaccine significantly improved protection in mice. Furthermore, delivery of the B2L expression construct, without antigen, non-specifically reduced tumor growth in murine models of cancer.

Conclusion: A streamlined, functional approach to genome-wide screening of a biological activity in vivo is presented. Its application to screening in mice for an immune activity elicited by the pathogen genome of Parapoxvirus ovis yielded a novel immunomodulator. In this inverted discovery method, it was possible to identify the adjuvant responsible for a function of interest prior to a mechanistic study of the adjuvant. The non-specific immune activity of this modulator, B2, is similar to that associated with administration of inactivated particles to a host or to a live viral infection. Administration of B2 may provide the opportunity to significantly impact host immunity while being itself only weakly recognized. The functional genomics method used to pinpoint B2 within an ORFeome may be more broadly applicable to screening for other biological activities in an animal.

Salmonella enterica serovar Typhimurium, a gram-negative facultative rod-shaped bacterium causing salmonellosis and foodborne disease, is one of the most common isolated Salmonella serovars in both developed and developing nations. Several S. Typhimurium genomes have been completed and many more genome-sequencing projects are underway. Comparative genome analysis of the multiple strains leads to a better understanding of the evolution of S. Typhimurium and its pathogenesis. S. Typhimurium strain UK-1 (belongs to phage type 1) is highly virulent when orally administered to mice and chickens and efficiently colonizes lymphoid tissues of these species. These characteristics make this strain a good choice for use in vaccine development. In fact, UK-1 has been used as the parent strain for a number of nonrecombinant and recombinant vaccine strains, including several commercial vaccines for poultry. In this study, we conducted a thorough comparative genome analysis of the UK-1 strain with other S. Typhimurium strains and examined the phenotypic impact of several genomic differences. Whole genomic comparison highlights an extremely close relationship between the UK-1 strain and other S. Typhimurium strains; however, many interesting genetic and genomic variations specific to UK-1 were explored. In particular, the deletion of a UK-1-specific gene that is highly similar to the gene encoding the T3SS effector protein NleC exhibited a significant decrease in oral virulence in BALB/c mice. The complete genetic complements in UK-1, especially those elements that contribute to virulence or aid in determining the diversity within bacterial species, provide key information in evaluating the functional characterization of important genetic determinants and for development of vaccines.