Characterization of companion animal pluripotent stem cells.

Pluripotent stem cells have the capacity to grow indefinitely in culture and differentiate into derivatives of the three germ layers. These properties underpin their potential to be used in regenerative medicine. Originally derived from early embryos, pluripotent stem cells can now be derived by reprogramming an adult cell back to a pluripotent state. Companion animals such as horses, dogs, and cats suffer from many injuries and diseases for which regenerative medicine may offer new treatments. As many of the injuries and diseases are similar to conditions in humans the use of companion animals for the experimental and clinical testing of stem cell and regenerative medicine products would provide relevant animal models for the translation of therapies to the human field. In order to fully utilize companion animal pluripotent stem cells robust, standardized methods of characterization must be developed to ensure that safe and effective treatments can be delivered. In this review we discuss the methods that are available for characterizing pluripotent stem cells and the techniques that have been applied in cells from companion animals. We describe characteristics which have been described consistently across reports as well as highlighting discrepant results. Significant steps have been made to define the in vitro culture requirements and drive lineage specific differentiation of pluripotent stem cells in companion animal species. However, additional basic research to compare pluripotent stem cell types and define characteristics of pluripotency in companion animal species is still required. © 2017 International Society for Advancement of Cytometry.

Evaluation of the safety and adjuvant effect of a detoxified listeriolysin O mutant on the humoral response to dengue virus antigens.

Listeriolysin O (LLO) has been proposed as a potential carrier or adjuvant molecule in the vaccination field. However, the cytotoxic and pro-apoptotic effects of LLO are the major limitations for this purpose. Here, we have performed a preclinical safety evaluation and characterized a new potential adjuvant application for a non-cytolytic LLO mutant (dtLLO) to enhance and modulate the immune response against the envelope (E) protein from dengue virus. In addition, we have studied the adjuvant effects of dtLLO on human immune cells and the role of membrane cholesterol for the binding and proinflammatory property of the toxoid. Our in-vivo results in the murine model confirmed that dtLLO is a safer molecule than wild-type LLO (wtLLO), with a significantly increased survival rate for mice challenged with dtLLO compared with mice challenged with wtLLO (P < 0·001). Histopathological analysis showed non-toxic effects in key target organs such as brain, heart, liver, spleen, kidney and lung after challenge with dtLLO. In vitro, dtLLO retained the capacity of binding to plasma membrane cholesterol on the surface of murine and human immune cells. Immunization of 6-8-week-old female BALB/c mice with a combination of dtLLO mixed with E protein elicited a robust specific humoral response with isotype diversification of immunoglobulin (Ig)G antibodies (IgG1 and IgG2a). Finally, we demonstrated that cholesterol and lipid raft integrity are required to induce a proinflammatory response by human cells. Taken together, these findings support a potential use of the dtLLO mutant as a safe and effective adjuvant molecule in vaccination.

A recombinant Listeria monocytogenes vaccine expressing a model tumour antigen protects mice against lethal tumour cell challenge and causes regression of established tumours.

Listeria monocytogenes is an intracellular organism that has the unusual ability to live in the cytoplasm of the cell. It is thus a good vector for targeting protein antigens to the cellular arm of the immune response. Here we use a model system, consisting of colon and renal carcinomas that express the influenza virus nucleoprotein and a recombinant L. monocytogenes that secrets this antigen, to test the potential of this organism as a cancer immunotherapeutic agent. We show that this recombinant organism can not only protect mice against lethal challenge with tumour cells that express the antigen, but can also cause regression of established macroscopic tumours in an antigen-specific T-cell-dependent manner.

Analysis of peptide binding patterns in different major histocompatibility complex/T cell receptor complexes using pigeon cytochrome c-specific T cell hybridomas. Evidence that a single peptide binds major histocompatibility complex in different conformations.

The interaction of TCR, antigen, and MHC complex has been analyzed using synthetic peptide antigens and a series of single amino acid-substituted analogues. Two similar antigens, mouse cytochrome c (mcyt c) and pigeon cytochrome c (pcyt c), elicit T cell responses in strains of mice bearing MHC class II Ek beta Ek alpha (B10.A), Eb beta Ek alpha [B10.A(5R)], and Es beta Ek alpha [B10.S(9R)]. The immunogenic regions of these antigens are located in the peptide sequence p88-104 for pcyt c and m88-103 for mcyt c. The limited T cell repertoire for these antigens is comprised of four groups of T cell phenotypes that have very few differences in their TCR gene make up. In this paper, we examine the diversity in their fine specificity for each of the antigens, m88-103 and p88-104, complexed with each of the I-Ek haplotypes. Epitopes, i.e., residues that interact with the TCR, and agretopes, i.e., residues in the MHC-binding site, were assigned for the two peptide antigens in the presence of APC bearing E beta kEk alpha, Eb beta Ek alpha, or Eb beta Ek alpha using T cell hybridomas of the phenotypes I, IIIa, and IV. From our results, we conclude that first, the substitution of any residue between 95 and 104 of the cytochrome c peptide changed the antigenic potency of the peptide for at least one of the hybridomas. Second, each T cell type has a different recognition pattern of epitopes and agretopes for a particular antigen-MHC complex, thus, ruling out a static model of T cell recognition, which assigns certain, invariant agretopic residues to the peptide by which it interacts with the MHC molecule independently of the TCR. Third, the same T cell hybridoma responded to the antigens differently when presented on various MHC molecules, implying that overall changes in the MHC groove, as displayed by the three haplotypes, may affect the efficiency in binding the peptide. Fourth, since most of the residues are used as epitopes by at least one of the T cell specificities, the peptide appears to be recognized in a different conformation by each T cell hybridoma phenotype; and, finally, the epitopic and agretopic residues do not segregate, for any one of the T cell specificities, in such a way that suggests they are recognized in a helical conformation. In summary, our results suggest that a single peptide may generate diversity in the T cell response by virtue of its conformational flexibility within the TCR-MHC-antigen complex.

The heme moiety of cytochrome c is an autoreactive Ir gene-restricted T cell epitope.

In these studies, we have shown that the heme moiety of cyt c is a dominant T cell epitope that induces a large proliferative response in lymph node T cells derived from SJL and B10.A mice when presented on either unfixed or fixed syngeneic APCs. Not only is this vigorous response observed for cyt c-primed T cell populations but also for populations obtained from naive SJL or B10.A mice. The reactivity to the heme moiety falls under strict MHC restriction, in that it is present only in murine strains bearing either the I-Ak or I-As molecule and can be blocked by antibodies specific for these class II molecules. Therefore, these findings require that the current models describing the nature of T cell epitopes be extended to include nonpeptide molecules. Furthermore, as the heme moiety is ubiquitous throughout the organism, although sequestered within proteins, the existence of heme-reactive T cell populations in unprimed animals provides another example of the existence of self-reactive T cell clones.

Delivery of a viral antigen to the class I processing and presentation pathway by Listeria monocytogenes.

Listeria monocytogenes is a facultative intracellular pathogen that grows in the cytoplasm of infected host cells. We examined the capacity of L. monocytogenes to introduce influenza nucleoprotein (NP) into the class I pathway of antigen presentation both in vitro and in vivo. Recombinant L. monocytogenes secreting a fusion of listeriolysin O and NP (LLO-NP) targeted infected cells for lysis by NP-specific class I-restricted cytotoxic T cells. Antigen presentation occurred in the context of three different class I haplotypes in vitro. A hemolysin-negative L. monocytogenes strain expressing LLO-NP was able to present in a class II-restricted manner. However, it failed to target infected cells for lysis by CD8+ T cells, indicating that hemolysin-dependent bacterial escape from the vacuole is necessary for class I presentation in vitro. Immunization of mice with a recombinant L. monocytogenes strain that stably expressed and secreted LLO-NP induced NP-specific CD8+ cytotoxic T lymphocytes. These studies have implications for the use of L. monocytogenes to deliver potentially any antigen to the class I pathway in vivo.

Identification of an autologous insulin B chain peptide as a target antigen for H-2Kb-restricted cytotoxic T lymphocytes.

We have examined the CD8+ peripheral T cell repertoire of C57BL/6 (H-2b) mice for cytotoxic T lymphocyte (CTL) reactivities to insulin, using in vitro immunization with a chymotryptic digest of reduced bovine insulin. The results presented in this study demonstrate that potentially autoreactive H-2Kb-restricted cytotoxic T cells specific for an autologous insulin B chain peptide are present in the preimmune splenic T cell repertoire. The immunogenic peptide comprises residues 7-15 from the insulin B chain and has features in common with naturally processed Kb-restricted peptides identified by others. The minimal peptide sequence recognized by these cytotoxic T cells is 10-15, which is highly conserved in mammalian species and constitutes a self-peptide in mice. The presence of class I major histocompatibility complex-restricted CTLs with potentially autoreactive specificities in preimmune animals raises the possibility of a role for such cells in autoimmune disease states. Possible mechanisms for the in vivo expansion of insulin peptide-specific CTLs are discussed.

Delayed expulsion of the nematode Trichinella spiralis in mice lacking the mucosal mast cell-specific granule chymase, mouse mast cell protease-1.

Expulsion of gastrointestinal nematodes is associated with pronounced mucosal mast cell (MMC) hyperplasia, differentiation, and activation, accompanied by the systemic release of MMC granule chymases (chymotrypsin-like serine proteases). The beta-chymase mouse mast cell protease-1 (mMCP-1) is expressed predominantly by intraepithelial MMCs, and levels in the bloodstream and intestinal lumen are maximal at the time of worm expulsion in parasitized mice. To address the in vivo functions of MMC-specific beta-chymases, we have generated transgenic mice that lack the mMCP-1 gene. They were backcrossed onto a congenic BALB/c background to investigate the response to nematode infection. The deletion of the mMCP-1 gene is associated with significantly delayed expulsion of Trichinella spiralis and increased deposition of muscle larvae in BALB/c mice despite the presence of normal and sometimes increased numbers of MMCs. Neither worm fecundity nor worm burdens were altered in Nippostrongylus-infected mMCP-1(-/)- BALB/c mice. These data demonstrate, for the first time, that the ablation of an MMC-derived effector molecule compromises the expulsion process.

The transcription factor scleraxis differentially regulates gene expression in tenocytes isolated at different developmental stages.

The transcription factor scleraxis (SCX) is expressed throughout tendon development and plays a key role in directing tendon wound healing. However, little is known regarding its role in fetal or young postnatal tendons, stages in development that are known for their enhanced regenerative capabilities. Here we used RNA-sequencing to compare the transcriptome of adult and fetal tenocytes following SCX knockdown. SCX knockdown had a larger effect on gene expression in fetal tenocytes, affecting 477 genes in comparison to the 183 genes affected in adult tenocytes, indicating that scleraxis-dependent processes may differ in these two developmental stages. Gene ontology, network and pathway analysis revealed an overrepresentation of extracellular matrix (ECM) remodelling processes within both comparisons. These included several matrix metalloproteinases, proteoglycans and collagens, some of which were also investigated in SCX knockdown tenocytes from young postnatal foals. Using chromatin immunoprecipitation, we also identified novel genes that SCX differentially interacts with in adult and fetal tenocytes. These results indicate a role for SCX in modulating ECM synthesis and breakdown and provide a useful dataset for further study into SCX gene regulation.

Genome-wide transcriptome analysis reveals equine embryonic stem cell-derived tenocytes resemble fetal, not adult tenocytes.

BACKGROUND: Tendon injuries occur frequently in human and equine athletes. Treatment options are limited, and the prognosis is often poor with functionally deficient scar tissue resulting. Fetal tendon injuries in contrast are capable of healing without forming scar tissue. Embryonic stem cells (ESCs) may provide a potential cellular therapeutic to improve adult tendon regeneration; however, whether they can mimic the properties of fetal tenocytes is unknown. To this end, understanding the unique expression profile of normal adult and fetal tenocytes is crucial to allow validation of ESC-derived tenocytes as a cellular therapeutic. METHODS: Equine adult, fetal and ESC-derived tenocytes were cultured in a three-dimensional environment, with histological, morphological and transcriptomic differences compared. Additionally, the effects on gene expression of culturing adult and fetal tenocytes in either conventional two-dimensional monolayer culture or three-dimensional culture were compared using RNA sequencing. RESULTS: No qualitative differences in three-dimensional tendon constructs generated from adult, fetal and ESCs were found using histological and morphological analysis. However, genome-wide transcriptomic analysis using RNA sequencing revealed that ESC-derived tenocytes' transcriptomic profile more closely resembled fetal tenocytes as opposed to adult tenocytes. Furthermore, this study adds to the growing evidence that monolayer cultured cells' gene expression profiles converge, with adult and fetal tenocytes having only 10 significantly different genes when cultured in this manner. In contrast, when adult and fetal tenocytes were cultured in 3D, large distinctions in gene expression between these two developmental stages were found, with 542 genes being differentially expressed. CONCLUSION: The information provided in this study makes a significant contribution to the investigation into the differences between adult reparative and fetal regenerative cells and supports the concept of using ESC-derived tenocytes as a cellular therapy. Comparing two- and three-dimensional culture also indicates three-dimensional culture as being a more physiologically relevant culture system for determining transcriptomic difference between the same cell types from different developmental stages.

Mapping epitopes on a protein antigen by the proteolysis of antigen-antibody complexes.

A monoclonal antibody bound to a protein antigen decreases the rate of proteolytic cleavage of the antigen, having the greatest effect on those regions involved in antibody contact. Thus, an epitope can be identified by the ability of the antibody to protect one region of the antigen more than others from proteolysis. By means of this approach, two distinct epitopes, both conformationally well-ordered, were characterized on horse cytochrome c.

An antibody binding site on cytochrome c defined by hydrogen exchange and two-dimensional NMR.

The interaction of a protein antigen, horse cytochrome c (cyt c), with a monoclonal antibody has been studied by hydrogen-deuterium (H-D) exchange labeling and two-dimensional nuclear magnetic resonance (2D NMR) methods. The H-exchange rate of residues in three discontiguous regions of the cyt c polypeptide backbone was slowed by factors up to 340-fold in the antibody-antigen complex compared with free cyt c. The protected residues, 36 to 38, 59, 60, 64 to 67, 100, and 101, and their hydrogen-bond acceptors, are brought together in the three-dimensional structure to form a contiguous, largely exposed protein surface with an area of about 750 square angstroms. The interaction site determined in this way is consistent with prior epitope mapping studies and includes several residues that were not previously identified. The hydrogen exchange labeling approach can be used to map binding sites on small proteins in antibody-antigen complexes and may be applicable to protein-protein and protein-ligand interactions in general.

Mage-b vaccine delivered by recombinant Listeria monocytogenes is highly effective against breast cancer metastases.

New therapies are needed that target breast cancer metastases. In previous studies, we have shown that vaccination with pcDNA3.1-Mage-b DNA vaccine is effective against breast cancer metastases. In the study presented here, we have further enhanced the efficacy of Mage-b vaccination through the improved delivery of the vaccine using recombinant Listeria monocytogenes (LM). Three overlapping fragments of Mage-b as well as the complete protein-encoding region of Mage-b have been expressed as a fusion protein with a truncated non-cytolytic form of listeriolysin O (LLO) in recombinant LM. These different Mage-b vaccine strains were preventively tested for their efficacy against breast cancer metastases in a syngeneic mouse tumour model 4T1. The LM-LLO-Mage-b/2nd, expressing position 311-660 of the cDNA of Mage-b, was the most effective vaccine strain against metastases in the 4T1 mouse breast tumour model. Vaccination with LM-LLO-Mage-b/2nd dramatically reduced the number of metastases by 96% compared with the saline group and by 88% compared with the vector control group (LM-LLO), and this correlated with strong Mage-b-specific CD8 T-cell responses in the spleen, after restimulation with Mage-b. However, no effect of LM-LLO-Mage-b/2nd was observed on 4T1 primary tumours, which may be the result of a complete absence of Mage-b-specific immune responses in the draining lymph nodes. Vaccination with LM-LLO-Mage-b/2nd could be an excellent follow-up after removal of the primary tumour, to eliminate metastases and residual tumour cells.

Recombinant Listeria monocytogenes cancer vaccines.

Listeria monocytogenes has recently been exploited as a vector for targeting antigens to the immune system. In the past year it has been shown to be particularly effective as a tumor antigen vector. Here we set its potency as a cancer vaccine in the context of the type of immunity induced by this facultative intracellular bacterium.

In vivo bactofection: listeria can function as a DNA-cancer vaccine.

The development of an effective therapeutic vaccine to induce cancer-specific immunity remains an unsolved yet pressing priority requiring novel vaccine strategies. Here we have generated a series of vaccines in which bacteria deliver a plasmid encoding a tumor antigen under the control of a mammalian promoter in an attempt to induce an antitumor immune response. Utilizing a plasmid release mechanism involving the suicide of the carrier bacteria, we were able to engineer Listeria monocytogenes to induce antitumor immunity to a physiologically relevant tumor antigen, the cervical cancer oncoprotein E7. In a mouse model of cervical cancer, we were able to slow tumor growth and induce an effector CD8(+) T-cell response against the immunodominant epitope for E7. The CD8(+) T cells generated could both home to and penetrate the tumor. This is the first demonstration of in vivo efficacy of bactofection vectors in treating solid tumors. However, although this delivery system was more effective than administering plasmid alone, it was not as effective as L. monocytogenes engineered to deliver the E7 protein in impacting on established tumor growth.

Regression of established tumors in mice mediated by the oral administration of a recombinant Listeria monocytogenes vaccine.

We have shown previously that Listeria monocytogenes, a gram-positive, facultative intracellular bacterium, is a potent vector for targeting tumor-specific antigens to the immune system. After parenteral administration, we observed protection against both renal and colorectal mouse tumors and regression of established renal tumors. In the present study, we have exploited the fact that the normal route of infection of this organism is through the gut. We show that an L. monocytogenes recombinant that expresses a model tumor antigen is an effective cancer immunotherapeutic agent when delivered orally in that it causes regression of established, macroscopic mouse renal and colorectal tumors expressing the same antigen.

Regression of established B16F10 melanoma with a recombinant Listeria monocytogenes vaccine.

We have previously shown that Listeria monocytogenes, a gram-positive, facultative intracellular bacterium, is a potent vector for targeting tumor-specific antigens to the immune system. In the present study, we extend these studies to the highly tumorigenic mouse melanoma B16F10, transduced with a model tumor antigen. We are able to induce the regression of primary tumors and established lung metastases by parenteral immunization with a L. monocytogenes recombinant that expresses the same antigen. Adjunctive therapy with granulocyte macrophage colony-stimulating factor or a vaccinia-based vaccine does not result in an improved cure rate over the L. monocytogenes vaccine alone. Tumor regression is accompanied by the expression of inflammatory cytokines in the tumor.

Structural basis for the binding of an anti-cytochrome c antibody to its antigen: crystal structures of FabE8-cytochrome c complex to 1.8 A resolution and FabE8 to 2.26 A resolution.

A complete understanding of antibody-antigen association and specificity requires the stereochemical description of both antigen and antibody before and upon complex formation. The structural mechanism involved in the binding of the IgG1 monoclonal antibody E8 to its highly charged protein antigen horse cytochrome c (cyt c) is revealed by crystallographic structures of the antigen-binding fragment (Fab) of E8 bound to cyt c (FabE8-cytc), determined to 1.8 A resolution, and of uncomplexed Fab E8 (FabE8), determined to 2.26 A resolution. E8 antibody binds to three major discontiguous segments (33 to 39; 56 to 66; 96 to 104), and two minor sites on cyt c opposite to the exposed haem edge. Crystallographic definition of the E8 epitope complements and extends biochemical mapping and two-dimensional nuclear magnetic resonance with hydrogen-deuterium exchange studies. These combined results demonstrate that antibody-induced stabilization of secondary structural elements within the antigen can propagate locally to adjacent residues outside the epitope. Pre-existing shape complementarity at the FabE8-cytc interface is enhanced by 48 bound water molecules, and by local movements of up to 4.2 A for E8 antibody and 8.9 A for cyt c. Glu62, Asn103 and the C-terminal Glu104 of cyt c adjust to fit the pre-formed VL "hill" and VH "valley" shape of the grooved E8 paratope. All six E8 complementarity determining regions (CDRs) contact the antigen, with CDR L1 forming 46% of the total atomic contacts, and CDRs L1 (29%) and H3 (20%) contributing the highest percentage of the total surface area of E8 buried by cyt c (550 A2). The E8 antibody covers 534 A2 of the cyt c surface. The formation of five ion pairs between E8 and flexible cyt c residues Lys60, Glu62 and Glu104 suggests the importance of mobile regions and electrostatic interactions in providing the exquisite specificity needed for recognition of this extremely conserved protein antigen. The highly homologous VL domains of E8 and anti-lysozyme antibody D1. 3 achieve their distinct antigen-binding specificities by expanding the impact of their limited sequence differences through the recruitment of different sets of conserved residues and distinctly different CDR L3 conformations.

Biochemical implications from the variable gene sequences of an anti-cytochrome c antibody and crystallographic characterization of its antigen-binding fragment in free and antigen-complexed forms.

To study the nature of antibody-antigen interactions, we have determined the variable gene sequences of the anti-cytochrome c immunoglobulin G1 (IgG1) monoclonal antibody E8, and obtained diffraction-quality crystals of the E8 antigen-binding fragment (Fab), both free and bound to its antigen, horse cytochrome c. The FabE8 crystals belong to space group P21 with unit cell dimensions of a = 45.0 A, b = 85.1 A, c = 63.3 A and beta = 105.5 degrees, have one FabE8 molecule per asymmetric unit and diffract to at least 2.1 A resolution. Crystals of the FabE8-cytochrome c complex belong to space group P212121 with unit cell dimensions of a = 84.3 A, b = 73.3 A and c = 94.9 A, accommodate one complex per asymmetric unit and diffract to 2.4 A resolution. In the nucleotide-derived amino acid sequences, the light-chain variable domain (VL) but not the heavy-chain variable domain (VH) of E8 is nearly identical to that of the anti-lysozyme antibody D1.3, differing by only five amino acid residues. Only one of these interacts with lysozyme in the D1.3-lysozyme crystal structure. Six negative and four positive charges in the VH complementarity determining regions of E8 complement four positive and three negative charges in the E8 epitope on cytochrome c. These data suggest that only a subset of the residues in an antibody-protein interface may be critical for binding and that the VH may play a dominant role in antigenic recognition.

Nuclear Magnetic Resonance Spectroscopy for the Study of B-Cell Epitopes

High-resolution nuclear magnetic resonance (NMR) spectroscopy is a structural technique that is finding increasing use in the study of antibody-antigen interactions. In this review we describe how the dynamic structural parameters obtained from NMR spectroscopy can further our understanding of B-cell epitopes and their function. Specific applications of NMR spectroscopy to examine the residues on peptides and proteins that contact the antibody combining site are also described. These include "footprinting" techniques using H-D exchange-COSY NMR spectroscopy, which are particularly useful for epitope mapping of protein antigens. For smaller systems, such as Fab-or Fv-peptide complexes, nuclear magnetization transfer difference NMR spectroscopy, transferred nuclear Overhauser effect spectroscopy, double-quantum-filtered NOE spectroscopy, and isotope editing techniques have been applied. The interpretation and limitations of the data obtained from these procedures and anticipated improvements in these applications in the future are discussed.