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Coxsackievirus B3 (CVB3), is commonly implicated in myocarditis, which can lead to dilated cardiomyopathy, in addition to causing acute pancreatitis and meningitis. Yet, no vaccines are currently available to prevent this infection. Here, we describe the derivation of a live attenuated vaccine virus, termed mutant (Mt) 10, encoding a single amino acid substitution H790A within the viral protein 1, that prevents CVB3 infection in mice and protects from both myocarditis and pancreatitis in challenge studies. We noted that animals vaccinated with Mt 10 developed virus-neutralizing antibodies, predominantly containing IgG2a and IgG2b, and to a lesser extent IgG3 and IgG1. Furthermore, by using major histocompatibility complex class II dextramers and tetramers, we demonstrated that Mt 10 induces antigen-specific T cell responses that preferentially produce interferon-γ. Finally, neither vaccine recipients nor those challenged with the wild-type virus revealed evidence of autoimmunity or cardiac injury as determined by T cell response to cardiac myosin and measurement of circulating cardiac troponin I levels, respectively. Together, our data suggest that Mt 10 is a vaccine candidate that prevents CVB3 infection through the induction of neutralizing antibodies and antigen-specific T cell responses, the two critical components needed for complete protection against virus infections in vaccine studies.

The development of novel therapeutics that exploit alterations in the activation state of key cellular signaling pathways due to mutations in upstream regulators has generated the field of personalized medicine. These first-generation efforts have focused on actionable mutations identified by deep sequencing of large numbers of tumor samples. We propose that a second-generation opportunity exists by exploiting key downstream “nodes of control” that contribute to oncogenesis and are inappropriately activated due to loss of upstream regulation and microenvironmental influences. The RNA-binding protein HuR represents such a node. Because HuR functionality in cancer cells is dependent on HuR dimerization and its nuclear/cytoplasmic shuttling, we developed a new class of molecules targeting HuR protein dimerization. A structure–activity relationship algorithm enabled development of inhibitors of HuR multimer formation that were soluble, had micromolar activity, and penetrated the blood–brain barrier. These inhibitors were evaluated for activity validation and specificity in a robust cell-based assay of HuR dimerization. SRI-42127, a molecule that met these criteria, inhibited HuR multimer formation across primary patient-derived glioblastoma xenolines (PDGx), leading to arrest of proliferation, induction of apoptosis, and inhibition of colony formation. SRI-42127 had favorable attributes with central nervous system penetration and inhibited tumor growth in mouse models. RNA and protein analysis of SRI-42127–treated PDGx xenolines across glioblastoma molecular subtypes confirmed attenuation of targets upregulated by HuR. These results highlight how focusing on key attributes of HuR that contribute to cancer progression, namely cytoplasmic localization and multimerization, has led to the development of a novel, highly effective inhibitor. Significance: These findings utilize a cell-based mechanism of action assay with a structure–activity relationship compound development pathway to discover inhibitors that target HuR dimerization, a mechanism required for cancer promotion.

Autoreactive T cells may contribute to post-viral myocarditis induced with Coxsackievirus B3 (CVB3), but the underlying mechanisms of their generation are unclear. Here, we have comprehensively analyzed the generation of antigen-specific, autoreactive T cells in the mouse model of CVB3 infection for antigens implicated in patients with myocarditis/dilated cardiomyopathy. First, comparative analysis of CVB3 proteome with five autoantigens led us to identify three mimicry epitopes, one each from adenine nucleotide translocator 1 (ANT), sarcoplasmic/endoplasmic reticulum Ca(2+) ATPase 2a (SERCA2a) and cardiac troponin I. None of these induced cross-reactive T cell responses. Next, we generated major histocompatibility complex (MHC) class II dextramers to enumerate the frequencies of antigen-specific T cells to determine whether T cells with multiple antigen specificities are generated by CVB3 infection. These analyses revealed appearance of CD4 T cells positive for SERCA2a 971-990, and cardiac myosin heavy chain-α (Myhc) 334-352 dextramers, both in the periphery and also in the hearts of CVB3-infected animals. While ANT 21-40 dextramer(+) T cells were inconsistently detected, the β1-adrenergic receptor 181-200/211-230 or branched chain α-ketoacid dehydrogenase kinase 111-130 dextramer(+) cells were absent. Interestingly, SERCA2a 971-990, Myhc 334-352 and ANT 21-40 dextramer(+) cells were also detected in the liver indicating that they may have a pathogenic role. Finally, we demonstrate that the SERCA2a 971-990-reactive T cells generated in CVB3 infection could transfer disease to naïve mice. The data suggest that CVB3 infection can lead to the generation of autoreactive T cells for multiple antigens indicating a possibility that the autoreactive T cells localized in the liver can potentially circulate and contribute to the development of viral myocarditis.

Sarcoplasmic/endoplasmic reticulum Ca2+ adenosine triphosphatase (SERCA)2a, a critical regulator of calcium homeostasis, is known to be decreased in heart failure. Patients with myocarditis or dilated cardiomyopathy develop autoantibodies to SERCA2a suggesting that they may have pathogenetic significance. In this report, we describe epitope mapping analysis of SERCA2a in A/J mice that leads us to make five observations: 1) SERCA2a contains multiple T cell epitopes that induce varying degrees of myocarditis. One epitope, SERCA2a 971–990, induces widespread atrial inflammation without affecting noncardiac tissues; the cardiac abnormalities could be noninvasively captured by echocardiography, electrocardiography, and magnetic resonance microscopy imaging. 2) SERCA2a 971–990-induced disease was associated with the induction of CD4 T cell responses and the epitope preferentially binds MHC class II/IAk rather than IEk. By creating IAk/and IEk/SERCA2a 971–990 dextramers, the T cell responses were determined by flow cytometry to be Ag specific. 3) SERCA2a 971–990-sensitized T cells produce both Th1 and Th17 cytokines. 4) Animals immunized with SERCA2a 971–990 showed Ag-specific Abs with enhanced production of IgG2a and IgG2b isotypes, suggesting that SERCA2a 971–990 can potentially act as a common epitope for both T cells and B cells. 5) Finally, SERCA2a 971–990-sensitized T cells were able to transfer disease to naive recipients. Together, these data indicate that SERCA2a is a critical autoantigen in the mediation of atrial inflammation in mice and that our model may be helpful to study the inflammatory events that underlie the development of conditions such as atrial fibrillation in humans.

Introduction Organ-specific autoimmune diseases are believed to result from immune responses generated against self-antigens specific to each organ. However, when such responses target antigens expressed promiscuously in multiple tissues, then the immune-mediated damage may be wide spread. Methods In this report, we describe a mitochondrial protein, branched chain α-ketoacid dehydrogenase kinase (BCKDk) that can act as a target autoantigen in the development of autoimmune inflammatory reactions in both heart and liver. Results We demonstrate that BCKDk protein contains at least nine immunodominant epitopes, three of which, BCKDk 71–90, BCKDk 111–130 and BCKDk 141–160, were found to induce varying degrees of myocarditis in immunized mice. One of these, BCKDk 111–130, could also induce hepatitis without affecting lungs, kidneys, skeletal muscles, and brain. In immunogenicity testing, all three peptides induced antigen-specific T cell responses, as verified by proliferation assay and/or major histocompatibility complex class II/IAk dextramer staining. Finally, the disease-inducing abilities of BCKDk peptides were correlated with the production of interferon-γ, and the activated T cells could transfer disease to naive recipients. Conclusions The disease induced by BCKDk peptides could serve as a useful model to study the autoimmune events of inflammatory heart and liver diseases.

Heart failure, a leading cause of death in humans, can emanate from myocarditis. Although most individuals with myocarditis recover spontaneously, some develop chronic dilated cardiomyopathy. Myocarditis may result from both infectious and noninfectious causes, including autoimmune responses to cardiac antigens. In support of this notion, intracellular cardiac antigens, like cardiac myosin heavy chain-α, cardiac troponin-I, and adenine nucleotide translocator 1 (ANT1), have been identified as autoantigens in cardiac autoimmunity. Herein, we demonstrate that ANT1 can induce autoimmune myocarditis in A/J mice by generating autoreactive T cells. We show that ANT1 encompasses multiple immunodominant epitopes (namely, ANT1 21-40, ANT1 31-50, ANT1 171-190, and ANT1 181-200). Although all four peptides induce comparable T-cell responses, only ANT1 21-40 was found to be a major myocarditogenic epitope in immunized animals. The myocarditis-inducing ability of ANT1 21-40 was associated with the generation of T cells producing predominantly IL-17A, and the antigen-sensitized T cells could transfer the disease to naïve recipients. These data indicate that cardiac mitochondrial proteins can be target autoantigens in myocarditis, supporting the notion that the antigens released as a result of primary damage may contribute to the persistence of chronic inflammation through autoimmunity.

Several pharmacophore models have been proposed for 5-HT2A serotonin receptor antagonists. These typically consist of two aromatic/hydrophobic moieties separated by a given distance from each other, and from a basic amine. Although specified distances might vary, the models are relatively similar in their general construction. Because our preliminary data indicated that two aromatic (hydrophobic) moieties might not be required for such action, we deconstructed the serotonin-dopamine antipsychotic agent risperidone (1) into four smaller structural fragments that were thoroughly examined in 5-HT2A receptor binding and functional (i.e., two-electrode voltage clamp (TEVC) and intracellular calcium release) assays. It was apparent that truncated risperidone analogues behaved as antagonists. In particular, 6-fluoro-3-(1-methylpiperidin-4-yl)benzisoxazole (4) displayed high affinity for 5-HT2A receptors (Ki of ca. 12 nM) relative to risperidone (Ki of ca. 5 nM) and behaved as a potent 5-HT2A serotonin receptor antagonist. These results suggest that multiple aromatic (hydrophobic) moieties are not essential for high-affinity 5-HT2A receptor binding and antagonist activity and that current pharmacophore models for such agents are very much in need of revision.

The dopamine D2 receptor (D2R) is known to elicit effects through activating two major signaling pathways mediated by either G proteins (Gi/o) or β-arrestins. However, the specific role of each pathway in physiological or therapeutic activities is not known with certainty. One approach to the dissection of these pathways is through the use of drugs that can selectively modulate one pathway vs. the other through a mechanism known as functional selectivity or biased signaling. Our laboratory has previously described a G protein signaling-biased agonist, MLS1547, for the D2R using a variety of in vitro functional assays. To further evaluate the biased signaling activity of this compound, we investigated its ability to promote D2R internalization, a process known to be mediated by β-arrestin. Using multiple cellular systems and techniques, we found that MLS1547 promotes little D2R internalization, which is consistent with its inability to recruit β-arrestin. Importantly, we validated these results in primary striatal neurons where the D2R is most highly expressed suggesting that MLS1547 will exhibit biased signaling activity in vivo. In an effort to optimize and further explore structure-activity relationships (SAR) for this scaffold, we conducted an iterative chemistry campaign to synthesize and characterize novel analogs of MLS1547. The resulting analysis confirmed previously described SAR requirements for G protein-biased agonist activity and, importantly, elucidated new structural features that are critical for agonist efficacy and signaling bias of the MLS1547 scaffold. One of the most important determinants for G protein-biased signaling is the interaction of a hydrophobic moiety of the compound with a defined pocket formed by residues within transmembrane five and extracellular loop two of the D2R. These results shed new light on the mechanism of biased signaling of the D2R and may lead to improved functionally-selective molecules.

Myocarditis/dilated cardiomyopathy (DCM) patients can develop autoantibodies to various cardiac antigens and one major antigen is β1-adrenergic receptor (β1AR). Previous reports indicate that animals immunized with a β1AR fragment encompassing, 197–222 amino acids for a prolonged period can develop DCM by producing autoantibodies, but existence of T cell epitopes, if any, were unknown. Using A/J mice that are highly susceptible to lymphocytic myocarditis, we have identified β1AR 171–190, β1AR 181–200, and β1AR 211–230 as the major T cell epitopes that bind major histocompatibility complex class II/IAk or IEk alleles, and by creating IAk and IEk dextramers, we demonstrate that the CD4 T cell responses to be antigen-specific. Of note, all the three epitopes were found also to stimulate CD8 T cells suggesting that they can act as common epitopes for both CD4 and CD8 T cells. While, all epitopes induced only mild myocarditis, the disease-incidence was enhanced in animals immunized with all the three peptides together as a cocktail. Although, antigen-sensitized T cells produced mainly interleukin-17A, their transfer into naive animals yielded no disease. But, steering for T helper 1 response led the T cells reacting to one epitope, β1AR 181–200 to induce severe myocarditis in naive mice. Finally, we demonstrate that all three β1AR epitopes to be unique for T cells as none of them induced antibody responses. Conversely, animals immunized with a non-T cell activator, β1AR 201–220, an equivalent of β1AR 197–222, had antibodies comprising of all IgG isotypes and IgM except, IgA and IgE. Thus, identification of T cell and B cell epitopes of β1AR may be helpful to determine β1AR-reactive autoimmune responses in various experimental settings in A/J mice.

Background We recently reported the identification of Bacillus sp. NRRL B-14911 that induces heart autoimmunity by generating cardiac-reactive T cells through molecular mimicry. This marine bacterium was originally isolated from the Gulf of Mexico, but no associations with human diseases were reported. Therefore, to characterize its biological and medical significance, we sought to determine and analyze the complete genome sequence of Bacillus sp. NRRL B-14911. Results Based on the phylogenetic analysis of 16S ribosomal RNA (rRNA) genes, sequence analysis of the 16S-23S rDNA intergenic transcribed spacers, phenotypic microarray, and matrix-assisted laser desorption ionization time-of-flight mass spectrometry, we propose that this organism belongs to the species Bacillus infantis, previously shown to be associated with sepsis in a newborn child. Analysis of the complete genome of Bacillus sp. NRRL B-14911 revealed several virulence factors including adhesins, invasins, colonization factors, siderophores and transporters. Likewise, the bacterial genome encodes a wide range of methyl transferases, transporters, enzymatic and biochemical pathways, and insertion sequence elements that are distinct from other closely related bacilli. Conclusions The complete genome sequence of Bacillus sp. NRRL B-14911 provided in this study may facilitate genetic manipulations to assess gene functions associated with bacterial survival and virulence. Additionally, this bacterium may serve as a useful tool to establish a disease model that permits systematic analysis of autoimmune events in various susceptible rodent strains. Electronic supplementary material The online version of this article (doi:10.1186/s12864-016-2900-2) contains supplementary material, which is available to authorized users.

Cathinone and many of its analogs produce behavioral effects by promoting transporter-mediated release of the monoamine neurotransmitters dopamine, norepinephrine and/or serotonin. Stereoselectivity is one determinant of neurochemical and behavioral effects of cathinone analogs. This study compared effectiveness of the S(-) and R(+) enantiomers of cathinone and 4-methylcathinone to produce in vitro monoamine release and in vivo abuse-related behavioral effects in rats. For neurochemical studies, drug effects were evaluated on monoamine release through dopamine, norepinephrine, and serotonin transporters (DAT, NET and SERT, respectively) in rat brain synaptosomes. For behavioral studies, drug effects were evaluated on responding for electrical brain stimulation in an intracranial self-stimulation (ICSS) procedure. The cathinone enantiomers differed in potency [S(-)>R(+)], but both enantiomers were >50-fold selective at promoting monoamine release through DAT vs. SERT, and both enantiomers produced ICSS facilitation. The 4-methylcathinone enantiomers also differed in potency [S(-)>R(+)]; however, in neurochemical studies, the decrease in potency from S(-) to R(+)4-methylcathinone was less for DAT than for SERT, and as a result, DAT vs. SERT selectivity was greater for R(+) than for S(-)4-methylcathinone (4.1- vs. 1.2-fold). Moreover, in behavioral studies, S(-)4-methylcathinone produced only ICSS depression, whereas R(+)4-methylcathinone produced ICSS facilitation. This study provides further evidence for stereoselectivity in neurochemical and behavioral actions of cathinone analogs. More importantly, stereoselective 4-methylcathinone effects on ICSS illustrate the potential for diametrically opposite effects of enantiomers in a preclinical behavioral assay of abuse potential.

Background Cardiac myosin heavy chain-α (Myhc), an intracellular protein expressed in the cardiomyocytes, has been identified as a major autoantigen in cardiac autoimmunity. In our studies with Myhc 334–352 -induced experimental autoimmune myocarditis in A/J mice (H-2 a ), we discovered that Myhc 334–352 , supposedly a CD4 T cell epitope, also induced antigen-specific CD8 T cells that transfer disease to naive animals. Methods and results In our efforts to identify the CD8 T cell determinants, we localized Myhc 338–348 within the full length-Myhc 334–352 , leading to four key findings. (1) By acting as a dual epitope, Myhc 338–348 induces both CD4 and CD8 T cell responses. (2) In a major histocompatibility complex (MHC) class I-stabilization assay, Myhc 338–348 was found to bind H-2D d —but not H-2K k or H-2L d —alleles. (3) The CD8 T cell response induced by Myhc 338–348 was antigen-specific, as evaluated by MHC class I/H-2D d dextramer staining. The antigen-sensitized T cells predominantly produced interferon-γ, the critical cytokine of effector cytotoxic T lymphocytes. (4) Myhc 338–348 was found to induce myocarditis in immunized animals as determined by histology and magnetic resonance microscopy imaging. Conclusions Our data provide new insights as to how different immune cells can recognize the same antigen and inflict damage through different mechanisms.