Responses In Transgenic Mice Research Articles

A safe and potentiated multi-type HPV L2-E7 nanoparticle vaccine with combined prophylactic and therapeutic activity

Persistent infection with high-risk human papillomavirus (HPV) is widely recognized as the primary cause of cervical and other malignant cancers. There are six licensed prophylactic vaccines available against HPV, but none of them shows any significant therapeutic effect on pre-existing infections or lesions. Thus, a prophylactic vaccine also endowed with therapeutic activity would afford protection regardless of the vaccine recipients HPV-infection status. Here, we describe the refinement and further potentiation of a dual-purpose HPV nanoparticle vaccine (hereafter referred to as cPANHPVAX) relying on eight different HPV L2 peptide epitopes and on the E7 oncoantigens from HPV16 and 18. cPANHPVAX not only induces anti-HPV16 E7 cytotoxic T-cell responses in C57BL/6 mice, but also anti-HPV18 E7 T-cell responses in transgenic mice with the A2.DR1 haplotype. These cytotoxic responses add up to a potent, broad-coverage humoral (HPV-neutralizing) response. cPANHPVAX safety was further improved by deletion of the pRb-binding domains of E7. Our dual-purpose vaccine holds great potential for clinical translation as an immune-treatment capable of targeting active infections as well as established HPV-related malignancies, thus benefiting both uninfected and infected individuals.

Beneficial islet inflammation in health depends on pericytic TLR/MyD88 signaling.

While inflammation is beneficial for insulin secretion during homeostasis, its transformation adversely affects β cells and contributes to diabetes. However, the regulation of islet inflammation for maintaining glucose homeostasis remains largely unknown. Here, we identified pericytes as pivotal regulators of islet immune and β cell function in health. Islets and pancreatic pericytes express various cytokines in healthy humans and mice. To interfere with the pericytic inflammatory response, we selectively inhibited the TLR/MyD88 pathway in these cells in transgenic mice. The loss of MyD88 impaired pericytic cytokine production. Furthermore, MyD88-deficient mice exhibited skewed islet inflammation with fewer cells, an impaired macrophage phenotype, and reduced IL-1β production. This aberrant pericyte-orchestrated islet inflammation was associated with β cell dedifferentiation and impaired glucose response. Additionally, we found that Cxcl1, a pericytic MyD88-dependent cytokine, promoted immune IL-1β production. Treatment with either Cxcl1 or IL-1β restored the mature β cell phenotype and glucose response in transgenic mice, suggesting a potential mechanism through which pericytes and immune cells regulate glucose homeostasis. Our study revealed pericyte-orchestrated islet inflammation as a crucial element in glucose regulation, implicating this process as a potential therapeutic target for diabetes.

Chronic chemogenetic activation of hippocampal progenitors enhances adult neurogenesis and modulates anxiety-like behavior and fear extinction learning

Adult hippocampal neurogenesis is a lifelong process that involves the integration of newborn neurons into the hippocampal network, and plays a role in cognitive function and the modulation of mood-related behavior. Here, we sought to address the impact of chemogenetic activation of adult hippocampal progenitors on distinct stages of progenitor development, including quiescent stem cell activation, progenitor turnover, differentiation and morphological maturation. We find that hM3Dq-DREADD-mediated activation of nestin-positive adult hippocampal progenitors recruits quiescent stem cells, enhances progenitor proliferation, increases doublecortin-positive newborn neuron number, accompanied by an acceleration of differentiation and morphological maturation, associated with increased dendritic complexity. Behavioral analysis indicated anxiolytic behavioral responses in transgenic mice subjected to chemogenetic activation of adult hippocampal progenitors at timepoints when newborn neurons are predicted to integrate into the mature hippocampal network. Furthermore, we noted an enhanced fear memory extinction on a contextual fear memory learning task in transgenic mice subjected to chemogenetic activation of adult hippocampal progenitors. Our findings indicate that hM3Dq-DREAD-mediated chemogenetic activation of adult hippocampal progenitors impacts distinct aspects of hippocampal neurogenesis, associated with the regulation of anxiety-like behavior and fear memory extinction.

Semisynthesis of homogeneous spike RBD glycoforms from SARS-CoV-2 for profiling the correlations between glycan composition and function

ABSTRACT Vaccines have been the primary remedy in the global fight against coronavirus disease 2019 (COVID-19). The receptor-binding domain (RBD) of the spike protein, a critical viral immunogen, is affected by the heterogeneity of its glycan structures and relatively low immunogenicity. Here, we describe a scalable synthetic platform that enables the precise synthesis of homogeneously glycosylated RBD, facilitating the elucidation of carbohydrate structure–function relationships. Five homogeneously glycosylated RBDs bearing biantennary glycans were prepared, three of which were conjugated to T-helper epitope (Tpep) from tetanus toxoid to improve their weak immune response. Relative to natural HEK293-derived RBD, synthetic RBDs with biantennary N-glycan elicited a higher level of neutralising antibodies against SARS-CoV-2 in mice. Furthermore, RBDs containing Tpep elicited significant immune responses in transgenic mice expressing human angiotensin-converting enzyme 2. Our collective data suggest that trimming the N-glycans and Tpep conjugation of RBD could potentially serve as an effective strategy for developing subunit vaccines providing efficient protection.

Enhancing T Cell and Antibody Response in Mucin-1 Transgenic Mice through Co-Delivery of Tumor-Associated Mucin-1 Antigen and TLR Agonists in C3-Liposomes.

Mucin-1 (MUC1) is a highly relevant antigen for cancer vaccination due to its overexpression and hypo-glycosylation in a high percentage of carcinomas. To enhance the immune response to MUC1, our group has developed C3-liposomes that encapsulate the MUC1 antigen along with immunostimulatory compounds for direct delivery to antigen-presenting cells (APCs). C3-liposomes bind complement C3, which interacts with C3-receptors on APCs, resulting in liposomal uptake and the delivery of tumor antigens to APCs in a manner that mimics pathogenic uptake. In this study, MUC1 and Toll-like receptor (TLR) agonists were encapsulated in C3-liposomes to provoke an immune response in transgenic mice tolerant to MUC1. The immune response to the C3-bound MUC1 liposomal vaccine was assessed by ELISA, ELISpot, and flow cytometry. Co-administering TLR 7/8 agonists with MUC1 encapsulated in C3-liposomes resulted in a significant antibody response compared to non-encapsulated MUC1. This antibody response was significantly higher in females than in males. The co-encapsulation of three TLR agonists with MUC1 in C3-liposomes significantly increased antibody responses and eliminated sex-based differences. Furthermore, this immunization strategy resulted in a significantly increased T cell-response compared to other treatment groups. In conclusion, the co-delivery of MUC1 and TLR agonists via C3-liposomes greatly enhances the immune response to MUC1, highlighting its potential for antigen-specific cancer immunotherapy.

Optogenetic cortical spreading depolarization induces headache-related behaviour and neuroinflammatory responses some prolonged in familial hemiplegic migraine type 1 mice

BackgroundCortical spreading depolarization (CSD), the neurophysiological correlate of the migraine aura, can activate trigeminal pain pathways, but the neurobiological mechanisms and behavioural consequences remain unclear. Here we investigated effects of optogenetically-induced CSDs on headache-related behaviour and neuroinflammatory responses in transgenic mice carrying a familial hemiplegic migraine type 1 (FHM1) mutation.MethodsCSD events (3 in total) were evoked in a minimally invasive manner by optogenetic stimulation through the intact skull in freely behaving wildtype (WT) and FHM1 mutant mice. Related behaviours were analysed using mouse grimace scale (MGS) scoring, head grooming, and nest building behaviour. Neuroinflammatory changes were investigated by assessing HMGB1 release with immunohistochemistry and by pre-treating mice with a selective Pannexin-1 channel inhibitor.ResultsIn both WT and FHM1 mutant mice, CSDs induced headache-related behaviour, as evidenced by increased MGS scores and the occurrence of oculotemporal strokes, at 30 min. Mice of both genotypes also showed decreased nest building behaviour after CSD. Whereas in WT mice MGS scores had normalized at 24 h after CSD, in FHM1 mutant mice scores were normalized only at 48 h. Of note, oculotemporal stroke behaviour already normalized 5 h after CSD, whereas nest building behaviour remained impaired at 72 h; no genotype differences were observed for either readout. Nuclear HMGB1 release in the cortex of FHM1 mutant mice, at 30 min after CSD, was increased bilaterally in both WT and FHM1 mutant mice, albeit that contralateral release was more pronounced in the mutant mice. Only in FHM1 mutant mice, contralateral release remained higher at 24 h after CSD, but at 48 h had returned to abnormal, elevated, baseline values, when compared to WT mice. Blocking Panx1 channels by TAT-Panx308 inhibited CSD-induced headache related behaviour and HMGB1 release.ConclusionsCSDs, induced in a minimally invasive manner by optogenetics, investigated in freely behaving mice, cause various migraine relevant behavioural and neuroinflammatory phenotypes that are more pronounced and longer-lasting in FHM1 mutant compared to WT mice. Prevention of CSD-related neuroinflammatory changes may have therapeutic potential in the treatment of migraine.

Aged mice are less susceptible to motion sickness and show decreased efferent vestibular activity compared to young adults.

The efferent vestibular system (EVS) is a feedback circuit thought to modulate vestibular afferent activity by inhibiting type II hair cells and exciting calyx-bearing afferents in the peripheral vestibular organs. In a previous study, we suggested EVS activity may contribute to the effects of motion sickness. To determine an association between motion sickness and EVS activity, we examined the effects of provocative motion (PM) on c-Fos expression in brainstem efferent vestibular nucleus (EVN) neurons that are the source of efferent innervation in the peripheral vestibular organs. c-Fos is an immediate early gene product expressed in stimulated neurons and is a well-established marker of neuronal activation. To study the effects of PM, young adult C57/BL6 wild-type (WT), aged WT, and young adult transgenic Chat-gCaMP6f mice were exposed to PM, and tail temperature (Ttail ) was monitored using infrared imaging. After PM, we used immunohistochemistry to label EVN neurons to determine any changes in c-Fos expression. All tissue was imaged using laser scanning confocal microscopy. Infrared recording of Ttail during PM indicated that young adult WT and transgenic mice displayed a typical motion sickness response (tail warming), but not in aged WT mice. Similarly, brainstem EVN neurons showed increased expression of c-Fos protein after PM in young adult WT and transgenic mice but not in aged cohorts. We present evidence that motion sickness symptoms and increased activation of EVN neurons occur in young adult WT and transgenic mice in response to PM. In contrast, aged WT mice showed no signs of motion sickness and no change in c-Fos expression when exposed to the same provocative stimulus.

An adenoviral-vectored vaccine confers seroprotection against capsular group B meningococcal disease.

Adenoviral-vectored vaccines are licensed for prevention of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and Ebola virus, but, for bacterial proteins, expression in a eukaryotic cell may affect the antigen's localization and conformation or lead to unwanted glycosylation. Here, we investigated the potential use of an adenoviral-vectored vaccine platform for capsular group B meningococcus (MenB). Vector-based candidate vaccines expressing MenB antigen factor H binding protein (fHbp) were generated, and immunogenicity was assessed in mouse models, including the functional antibody response by serum bactericidal assay (SBA) using human complement. All adenovirus-based vaccine candidates induced high antigen-specific antibody and T cell responses. A single dose induced functional serum bactericidal responses with titers superior or equal to those induced by two doses of protein-based comparators, as well as longer persistence and a similar breadth. The fHbp transgene was further optimized for human use by incorporating a mutation abrogating binding to the human complement inhibitor factor H.The resulting vaccine candidate induced high and persistent SBA responses in transgenic mice expressing human factor H.The optimized transgene was inserted into the clinically relevant ChAdOx1 backbone, and this vaccine has now progressed to clinical development. The results of this preclinical vaccine development study underline the potential of vaccines based on genetic material to induce functional antibody responses against bacterial outer membrane proteins.

Targeting the recurrent Rac1P29S neoepitope in melanoma with heterologous high-affinity T cell receptors.

Recurrent neoepitopes are cancer-specific antigens common among groups of patients and therefore ideal targets for adoptive T cell therapy. The neoepitope FSGEYIPTV carries the Rac1P29S amino acid change caused by a c.85C>T missense mutation, which is the third most common hotspot mutation in melanoma. Here, we isolated and characterized TCRs to target this HLA-A*02:01-binding neoepitope by adoptive T cell therapy. Peptide immunization elicited immune responses in transgenic mice expressing a diverse human TCR repertoire restricted to HLA-A*02:01, which enabled isolation of high-affinity TCRs. TCR-transduced T cells induced cytotoxicity against Rac1P29S expressing melanoma cells and we observed regression of Rac1P29S expressing tumors in vivo after adoptive T cell therapy (ATT). Here we found that a TCR raised against a heterologous mutation with higher peptide-MHC affinity (Rac2P29L) more efficiently targeted the common melanoma mutation Rac1P29S. Overall, our study provides evidence for the therapeutic potential of Rac1P29S-specific TCR-transduced T cells and reveal a novel strategy by generating more efficient TCRs by heterologous peptides.

Sex-Specific Differences in Resolution of Airway Inflammation in Fat-1 Transgenic Mice Following Repetitive Agricultural Dust Exposure.

In agriculture industries, workers are at increased risk for developing pulmonary diseases due to inhalation of agricultural dusts, particularly when working in enclosed confinement facilities. Agricultural dusts inhalation leads to unresolved airway inflammation that precedes the development and progression of lung disease. We have previously shown beneficial effects of the omega-3 polyunsaturated fatty acid (ω-3 PUFA) DHA in protecting against the negative inflammatory effects of repetitive dust exposure in the lung. Dietary manipulation of pulmonary disease risk is an attractive and timely approach given the contribution of an increased ω-6 to ω-3 PUFA ratio to low grade inflammation and chronic disease in the Western diet. To prevent any confounding factors that comes with dietary supplementation of ω-3 PUFA (different sources, purity, dose, and duration), we employed a Fat-1 transgenic mouse model that convert ω-6 PUFA to ω-3 PUFA, leading to a tissue ω-6 to ω-3 PUFA ratio of approximately 1:1. Building on our initial findings, we hypothesized that attaining elevated tissue levels of ω-3 PUFA would attenuate agricultural dust-induced lung inflammation and its resolution. To test this hypothesis, we compared wild-type (WT) and Fat-1 transgenic mice in their response to aqueous extracts of agricultural dust (DE). We also used a soluble epoxide hydrolase inhibitor (sEH) to potentiate the effects of ω-3 PUFA, since sEH inhibitors have been shown to stabilize the anti-inflammatory P450 metabolites derived from both ω-3 and ω-6 PUFA and promote generation of specialized pro-resolving lipid mediators from ω-3 PUFA. Over a three-week period, mice were exposed to a total of 15 intranasal instillations of DE obtained from swine confinement buildings in the Midwest. We observed genotype and sex-specific differences between the WT vs. Fat-1 transgenic mice in response to repetitive dust exposure, where three-way ANOVA revealed significant main effects of treatment, genotype, and sex. Also, Fat-1 transgenic mice displayed reduced lymphoid aggregates in the lung following DE exposure as compared to WT animals exposed to DE, suggesting improved resilience to the DE-induced inflammatory effects. Overall, our data implicate a protective role of ω-3 FA in the lung following repetitive dust exposure.

Personal Neoantigens From Patients With NSCLC Induce Efficient Antitumor Responses.

ObjectiveTo develop a neoantigen-targeted personalized cancer treatment for non-small cell lung cancer (NSCLC), neoantigens were obtained from collected human lung cancer samples, and the utility of neoantigen and neoantigen-reactive T cells (NRTs) was assessed.MethodsTumor specimens from three patients with NSCLC were obtained and analyzed by whole-exome sequencing, and neoantigens were predicted accordingly. Dendritic cells and T lymphocytes were isolated, NRTs were elicited and IFN-γ ELISPOT tests were conducted. HLA-A2.1/Kb transgenic mice were immunized with peptides from HLA-A*02:01+patient with high immunogenicity, and NRTs were subjected to IFN-γ, IL-2 and TNF-α ELISPOT as well as time-resolved fluorescence assay for cytotoxicity assays to verify the immunogenicity in vitro. The HLA-A*02:01+lung cancer cell line was transfected with minigene and inoculated into the flanks of C57BL/6nu/nu mice and the NRTs induced by the immunogenic polypeptides from autologous HLA-A2.1/Kb transgenic mice were adoptively transfused to verify their immunogenicity in vivo.ResultsMultiple putative mutation-associated neoantigens with strong affinity for HLA were selected from each patient. Immunogenic neoantigen were identified in all three NSCLC patients, the potency of ACAD8-T105I, BCAR1-G23V and PLCG1-M425L as effective neoantigen to active T cells in suppressing tumor growth was further proven both in vitro and in vivo using HLA-A2.1/Kb transgenic mice and tumor-bearing mouse models.ConclusionNeoantigens with strong immunogenicity can be screened from NSCLC patients through the whole-exome sequencing of patient specimens and machine-learning-based neoantigen predictions. NRTs shown efficient antitumor responses in transgenic mice and tumor-bearing mouse models. Our results indicate that the development of neoantigen-based personalized immunotherapies in NSCLC is possible.PrecisNeoantigens with strong immunogenicity were screened from NSCLC patients. This research provides evidence suggesting that neoantigen-based therapy might serve as feasible treatment for NSCLC.

C9orf72-associated SMCR8 protein binds in the ubiquitin pathway and with proteins linked with neurological disease

A pathogenic GGGCCC hexanucleotide expansion in the first intron/promoter region of the C9orf72 gene is the most common mutation associated with amyotrophic lateral sclerosis (ALS). The C9orf72 gene product forms a complex with SMCR8 (Smith-Magenis Syndrome Chromosome Region, Candidate 8) and WDR41 (WD Repeat domain 41) proteins. Recent studies have indicated roles for the complex in autophagy regulation, vesicle trafficking, and immune response in transgenic mice, however a direct connection with ALS etiology remains unclear. With the aim of increasing understanding of the multi-functional C9orf72-SMCR8-WDR41 complex, we determined by mass spectrometry analysis the proteins that directly associate with SMCR8. SMCR8 protein binds many components of the ubiquitin-proteasome system, and we demonstrate its poly-ubiquitination without obvious degradation. Evidence is also presented for localization of endogenous SMCR8 protein to cytoplasmic stress granules. However, in several cell lines we failed to reproduce previous observations that C9orf72 protein enters these granules. SMCR8 protein associates with many products of genes associated with various Mendelian neurological disorders in addition to ALS, implicating SMCR8-containing complexes in a range of neuropathologies. We reinforce previous observations that SMCR8 and C9orf72 protein levels are positively linked, and now show in vivo that SMCR8 protein levels are greatly reduced in brain tissues of C9orf72 gene expansion carrier individuals. While further study is required, these data suggest that SMCR8 protein level might prove a useful biomarker for the C9orf72 expansion in ALS.

Distinctive Roles and Mechanisms of Human Neutrophil Peptides in Experimental Sepsis and Acute Respiratory Distress Syndrome.

To examine the effects and mechanisms of human neutrophil peptides in systemic infection and noninfectious inflammatory lung injury. Prospective experimental study. University hospital-based research laboratory. In vitro human cells and in vivo mouse models. Wild-type (Friend virus B-type) and conditional leukocyte human neutrophil peptides transgenic mice were subjected to either sepsis induced by cecal ligation and puncture or acute lung injury by intratracheal instillation of hydrochloric acid followed by mechanical ventilation. Using human neutrophil peptides as bait, the basal cell adhesion molecule (CD239) and the purinergic P2Y purinoceptor 6 receptor were identified as the putative human neutrophil peptides receptor complex in human lung epithelial cells. In the cecal ligation and puncture sepsis model, Friend virus B-type mice exhibited higher systemic bacterial load, cytokine production, and lung injury than human neutrophil peptides transgenic mice. Conversely, an increased lung cytokine production was seen in Friend virus B-type mice, which was further enhanced in human neutrophil peptides transgenic mice in response to two-hit lung injury induced by hydrochloric acid and mechanical ventilation. The human neutrophil peptides-mediated inflammatory response was mediated through the basal cell adhesion molecule-P2Y purinoceptor 6 receptor signal pathway in human lung epithelial cells. Human neutrophil peptides are critical in host defense against infectious sepsis by their cationic antimicrobial properties but may exacerbate tissue injury when neutrophil-mediated inflammatory responses are excessive in noninfectious lung injury. Targeting the basal cell adhesion molecule/P2Y purinoceptor 6 signaling pathway may serve as a novel approach to attenuate the neutrophil-mediated inflammatory responses and injury while maintaining the antimicrobial function of human neutrophil peptides in critical illness.

Altered cerebral insulin response in transgenic mice expressing the epsilon-4 allele of the human apolipoprotein E gene

Apolipoprotein E epsilon-4 (APOEε4 or APOE4), an allelic variation of the APOE gene, not only increases the risk of developing the late-onset form of Alzheimer’s disease (AD), but also influences the outcome of treatment. Indeed, data from clinical studies show that the beneficial effect of insulin on cognition is blunted in APOE4 carriers. To investigate how APOE impacts insulin response, we assessed the effects of an acute insulin injection in APOE3- and APOE4-targeted replacement mice that respectively express the human APOE3 or APOE4 isoform instead of the endogenous murine ApoE protein. We evaluated cognition, insulin signaling and proteins implicated in Aβ transport and tau phosphorylation in the cortex and brain capillaries. We found that a single acute insulin injection increased Akt pSer473 in APOE4 compared to APOE3 mice (+113% versus +78.5%), indicating that APOE4 carriage potentiates activation of insulin upstream signaling pathway in the brain. Insulin also led to decreased concentrations of the receptor for advanced glycation endproducts (RAGE) in brain capillaries in both groups of mice. Moreover, higher phosphorylation of tau at Ser202, one of the key markers of AD neuropathology, was observed in insulin-injected APOE4 mice (+44%), consistent with findings in human APOE4 carriers (+400% compared to non-carriers). Therefore, our data suggest that APOE4 carriage leads to an increased insulin-induced activation of cerebral Akt pathway, associated with higher AD-like tau neuropathology. Our results provide evidence of altered insulin signaling in APOE4 carriers as well as a possible mechanism to explain the absence of cognitive benefit from insulin therapy in these individuals.

Accelerated cardiac remodeling in desmoplakin transgenic mice in response to endurance exercise is associated with perturbed Wnt/β-catenin signaling.

Arrhythmogenic ventricular cardiomyopathy (AVC) is a frequent underlying cause for arrhythmias and sudden cardiac death especially during intense exercise. The mechanisms involved remain largely unknown. The purpose of this study was to investigate how chronic endurance exercise contributes to desmoplakin (DSP) mutation-induced AVC pathogenesis. Transgenic mice with overexpression of desmoplakin, wild-type (Tg-DSP(WT)), or the R2834H mutant (Tg-DSP(R2834H)) along with control nontransgenic (NTg) littermates were kept sedentary or exposed to a daily running regimen for 12 wk. Cardiac function and morphology were analyzed using echocardiography, electrocardiography, histology, immunohistochemistry, RNA, and protein analysis. At baseline, 4-wk-old mice from all groups displayed normal cardiac function. When subjected to exercise, all mice retained normal cardiac function and left ventricular morphology; however, Tg-DSP(R2834H) mutants displayed right ventricular (RV) dilation and wall thinning, unlike NTg and Tg-DSP(WT). The Tg-DSP(R2834H) hearts demonstrated focal fat infiltrations in RV and cytoplasmic aggregations consisting of desmoplakin, plakoglobin, and connexin 43. These aggregates coincided with disruption of the intercalated disks, intermediate filaments, and microtubules. Although Tg-DSP(R2834H) mice already displayed high levels of p-GSK3-β(Ser9) and p-AKT1(Ser473) under sedentary conditions, decrease of nuclear GSK3-β and AKT1 levels with reduced p-GSK3-β(Ser9), p-AKT1(Ser473), and p-AKT1(Ser308) and loss of nuclear junctional plakoglobin was apparent after exercise. In contrast, Tg-DSP(WT) showed upregulation of p-AKT1(Ser473), p-AKT1(Ser308), and p-GSK3-β(Ser9) in response to exercise. Our data suggest that endurance exercise accelerates AVC pathogenesis in Tg-DSP(R2834H) mice and this event is associated with perturbed AKT1 and GSK3-β signaling. Our study suggests a potential mechanism-based approach to exercise management in patients with AVC.

Early overexpression of FAS in beta cells protects NOD mice from autoimmune diabetes (BA6P.119)

Abstract Fas receptor signaling is essential for the induction of pancreatic β cell death in autoimmune diabetes, upon being engaged by its ligand FasL (CD95L). We found that early overexpression of this proapoptotic receptor on pancreatic β cells delayed diabetes onset and reduced diabetes incidence in the NOD mice, and its late overexpression in β cells does not have any protective effect on the disease onset. We have further explored whether early Fas expression on β cells promotes the immunodeviation of the autorreactive CD4+T repertoire to the T regulatory phenotype, at the expense of the proinflammatory, diabetes-causing Th1-Th17 phenotypes. We did not find an increased frequency of CD4+Treg cells in the peripheral lymphoid organs or increased FoxP3 expression in islets from NOD mice overexpressing Fas early in life. Diabetes protection was improved by neutralizing the Th17 response in transgenic mice, which suggests that protection mediated by Fas overerexpression is not due to the abrogation of the Th17 response, at least not exclusively.

Identification of Mycobacterium tuberculosis PPE68-specific HLA-A*0201-restricted epitopes for tuberculosis diagnosis.

PPE68 is a Mycobacterium tuberculosis-specific protein which is absent from the vaccine strains of BCG. A panel of 14 PPE68-derived peptides predicted to bind to HLA-A*0201 was synthesized. The HLA-A*0201 restriction of these peptides was determined in T2 cell line and HLA-A*0201 transgenic mice. The specificity of peptides was assessed in pulmonary tuberculosis (TB) patients using IFN-γ enzyme-linked immunospot (ELISPOT) assay, and immunodominant peptides were further used to evaluate their diagnostic potential in HLA-A*0201-positive pulmonary TB patients. 13 out of 14 peptides were identified as high-affinity binders. Of these peptides, 12 peptides induced significant IFN-γ-secreting T cell response in transgenic mice and 9 peptides were efficiently recognized by peripheral blood mononuclear cells of 10 HLA-A*0201-positive TB patients. Four immunodominant HLA-A*0201-restricted epitopes (PPE68126-134, PPE68133-141, PPE68140-148, and PPE68148-156) were recognized by the most of 80 HLA-A*0201-positive TB patients (81, 86, 74, and 84 %, respectively). These epitopes may be used for a potential diagnosis of M. tuberculosis infection.

Distinct conformational and functional effects of two adjacent pathogenic mutations in cardiac troponin I at the interface with troponin T

The α-helix in troponin I (TnI) at the interface with troponin T (TnT) is a highly conserved structure. A point mutation in this region, A116G, was found in human cardiac TnI in a case of cardiomyopathy. An adjacent dominantly negative mutation found in turkey cardiac TnI (R111C, equivalent to K117C in human and K118C in mouse) decreased diastolic function and blunted beta-adrenergic response in transgenic mice. To investigate the functional importance of the TnI–TnT interface and pathological impact of the cardiac TnI mutations, we engineered K118C and A117G mutations in mouse cardiac TnI for functional studies. Despite their adjacent locations, A117G substitution results in faster mobility of cardiac TnI in SDS–PAGE whereas K118C decreases gel mobility, indicating significant and distinct changes in overall protein conformation. Consistently, monoclonal antibody epitope analysis demonstrated distinct local and remote conformational alterations in the two mutant proteins. Protein binding assays showed that K118C, but not A117G, decreased the relative binding affinity of cardiac TnI for TnT. K118C mutation decreased binding affinity for troponin C in a Ca2+-dependent manner, whereas A117G had a similar but less profound effect. Protein kinase A phosphorylation or truncation to remove the cardiac specific N-terminal extension of cardiac TnI resulted in similar conformational changes in the region interfacing with TnT and minimized the functional impacts of the mutations. The data demonstrate potent conformational and functional impacts of the TnT-interfacing helix in TnI and suggest a role of the N-terminal extension of cardiac TnI in modulating TnI–TnT interface functions.

Regulation of fibroblast growth factor 2 expression in oxygen-induced retinopathy.

Fibroblast growth factor (FGF) 2 is a potent endothelial cell mitogen and survival factor that is postulated to participate in the pathogenesis of retinopathy of prematurity (ROP). The purpose of the current study was to determine the transcriptional and translational regulation of FGF2 expression in oxygen-induced retinopathy (OIR), the animal model of ROP. We examined FGF2 protein and mRNA expression and optokinetic visual responses in transgenic mice possessing a dual-luciferase bicistronic transgene containing a 5'-internal ribosome entry site (IRES) of FGF2. We found that retinal FGF2 protein isoform expression varies with age but not in response to OIR. Analysis of luciferase, protein, and mRNA data indicate that FGF2 protein expression is translationally repressed during the vaso-obliterative phase of OIR, possibly by inhibiting elongation. At the transition from vaso-obliteration to neovascularization, heightened FGF2 protein expression corresponds to maintenance of IRES activity and diminished cap-dependent translational activity. During neovascularization, FGF2 expression is primarily regulated by transcription. Mice recovering from OIR display alterations in visual optokinetic responses and increased FGF2 protein expression at 6 weeks of age. In total, these findings illustrate the complexity of translational and transcriptional regulation of FGF2 protein expression in OIR. The augmentation of FGF2 expression and reduced optokinetic responses during the resolution of surface vasculopathy may indicate a role for FGF2 in the maintenance of neuroretinal function in OIR/ROP.

Molecular rationale for the use of PI3K/AKT/mTOR pathway inhibitors in combination with crizotinib in ALK-mutated neuroblastoma

Mutations in the ALK tyrosine kinase receptor gene represent important therapeutic targets in neuroblastoma, yet their clinical translation has been challenging. The ALK(F1174L) mutation is sensitive to the ALK inhibitor crizotinib only at high doses and mediates acquired resistance to crizotinib in ALK-translocated cancers. We have shown that the combination of crizotinib and an inhibitor of downstream signaling induces a favorable response in transgenic mice bearing ALK(F1174L)/MYCN-positive neuroblastoma. Here, we investigated the molecular basis of this effect and assessed whether a similar strategy would be effective in ALK-mutated tumors lacking MYCN overexpression. We show that in ALK-mutated, MYCN-amplified neuroblastoma cells, crizotinib alone does not affect mTORC1 activity as indicated by persistent RPS6 phosphorylation. Combined treatment with crizotinib and an ATP-competitive mTOR inhibitor abrogated RPS6 phosphorylation, leading to reduced tumor growth and prolonged survival in ALK(F1174L)/MYCN-positive models compared to single agent treatment. By contrast, this combination, while inducing mTORC1 downregulation, caused reciprocal upregulation of PI3K activity in ALK-mutated cells expressing wild-type MYCN. Here, an inhibitor with potency against both mTOR and PI3K was more effective in promoting cytotoxicity when combined with crizotinib. Our findings should enable a more precise selection of molecularly targeted agents for patients with ALK-mutated tumors.