Distinct, age-dependent TLR7/8 signaling responses in porcine gamma-delta T cells.

Gamma-Delta T cells are a prominent subset of T cells in pigs. However, developmental changes, antigen recognition, cell migration, and their contributions to pathogen clearance remain largely unknown. We have recently shown that porcine γδ T cells express Toll-like receptors (TLRs), and that TLR7/8 stimulation can function as a co-stimulatory signal that complements cytokine-induced signals to enhance INFγ production. Nonetheless, the signaling pathways behind this increased cytokine responsiveness remained unclear. Here, we analyzed the signaling pathways by measuring cellular kinase activity and selective inhibition, confirming that the TLR7/8 expression by γδ T cells is indeed functional. Moreover, TLR downstream signaling responses showed a distinct age-dependency, emphasizing the importance of age in immune function. While the TLR7/8 co-stimulation depended on activation of IRAK1/4, p38 and JNK in adult-derived γδ T cells, γδ T cells from young pigs utilized only p38, indicating the existence of an alternative signaling pathway in young pigs. Overall, this data suggests that porcine γδ T cells could be able to recognize viral RNA through TLR7/8 and subsequently support the survival and activation of the adaptive immune response by cytokine production.

Oral vaccination as a potential strategy to manage chronic wasting disease in wild cervid populations.

Prion diseases are a novel class of infectious disease based in the misfolding of the cellular prion protein (PrPC) into a pathological, self-propagating isoform (PrPSc). These fatal, untreatable neurodegenerative disorders affect a variety of species causing scrapie in sheep and goats, bovine spongiform encephalopathy (BSE) in cattle, chronic wasting disease (CWD) in cervids, and Creutzfeldt-Jacob disease (CJD) in humans. Of the animal prion diseases, CWD is currently regarded as the most significant threat due its ongoing geographical spread, environmental persistence, uptake into plants, unpredictable evolution, and emerging evidence of zoonotic potential. The extensive efforts to manage CWD have been largely ineffective, highlighting the need for new disease management tools, including vaccines. Development of an effective CWD vaccine is challenged by the unique biology of these diseases, including the necessity, and associated dangers, of overcoming immune tolerance, as well the logistical challenges of vaccinating wild animals. Despite these obstacles, there has been encouraging progress towards the identification of safe, protective antigens as well as effective strategies of formulation and delivery that would enable oral delivery to wild cervids. In this review we highlight recent strategies for antigen selection and optimization, as well as considerations of various platforms for oral delivery, that will enable researchers to accelerate the rate at which candidate CWD vaccines are developed and evaluated.

Longitudinal analysis of SARS-CoV-2 reinfection reveals distinct kinetics and emergence of cross-neutralizing antibodies to variants of concern.

The ongoing evolution of SARS-CoV-2 continues to raise new questions regarding the duration of immunity to reinfection with emerging variants. To address these knowledge gaps, controlled investigations in established animal models are needed to assess duration of immunity induced by each SARS-CoV-2 lineage and precisely evaluate the extent of cross-reactivity and cross-protection afforded. Using the Syrian hamster model, we specifically investigated duration of infection acquired immunity to SARS-CoV-2 ancestral Wuhan strain over 12 months. Plasma spike- and RBD-specific IgG titers against ancestral SARS-CoV-2 peaked at 4 months post-infection and showed a modest decline by 12 months. Similar kinetics were observed with plasma virus neutralizing antibody titers which peaked at 2 months post-infection and showed a modest decline by 12 months. Reinfection with ancestral SARS-CoV-2 at regular intervals demonstrated that prior infection provides long-lasting immunity as hamsters were protected against severe disease when rechallenged at 2, 4, 6, and 12 months after primary infection, and this coincided with the induction of high virus neutralizing antibody titers. Cross-neutralizing antibody titers against the B.1.617.2 variant (Delta) progressively waned in blood over 12 months, however, re-infection boosted these titers to levels equivalent to ancestral SARS-CoV-2. Conversely, cross-neutralizing antibodies to the BA.1 variant (Omicron) were virtually undetectable at all time-points after primary infection and were only detected following reinfection at 6 and 12 months. Collectively, these data demonstrate that infection with ancestral SARS-CoV-2 strains generates antibody responses that continue to evolve long after resolution of infection with distinct kinetics and emergence of cross-reactive and cross-neutralizing antibodies to Delta and Omicron variants and their specific spike antigens.

Vaccines for prion diseases: a realistic goal?

Prion diseases are fatal infectious neurodegenerative disorders and prototypic conformational diseases, caused by the conformational conversion of the normal cellular prion protein (PrPC) into the pathological PrPSc isoform. Examples are scrapie in sheep and goat, bovine spongiform encephalopathy (BSE) in cattle, chronic wasting disease (CWD) in cervids, and Creutzfeldt-Jacob disease (CJD) in humans. There are no therapies available, and animal prion diseases like BSE and CWD can negatively affect the economy, ecology, animal health, and possibly human health. BSE is a confirmed threat to human health, and mounting evidence supports the zoonotic potential of CWD. CWD is continuously expanding in North America in numbers and distribution and was recently identified in Scandinavian countries. CWD is the only prion disease occurring both in wild and farmed animals, which, together with extensive shedding of infectivity into the environment, impedes containment strategies. There is currently a strong push to develop vaccines against CWD, including ones that can be used in wildlife. The immune system does not develop a bona fide immune response against prion infection, as PrPC and PrPSc share an identical protein primary structure, and prions seem not to represent a trigger for immune responses. This asks for alternative vaccine strategies, which focus on PrPC-directed self-antibodies or exposure of disease-specific structures and epitopes. Several groups have established a proof-of-concept that such vaccine candidates can induce some levels of protective immunity in cervid and rodent models without inducing unwanted side effects. This review will highlight the most recent developments and discuss progress and challenges remaining.

Offspring affected with in utero Zika virus infection retain molecular footprints in the bone marrow and blood cells.

Congenital virus infections, for example cytomegalovirus and rubella virus infections, commonly affect the central nervous and hematological systems in fetuses and offspring. However, interactions between emerging congenital Zika virus and hematological system-bone marrow and blood-in fetuses and offspring are mainly unknown. Our overall goal was to determine whether silent in utero Zika virus infection can cause functional and molecular footprints in the bone marrow and blood of fetuses and offspring. We specifically focused on silent fetal infection because delayed health complications in initially asymptomatic offspring were previously demonstrated in animal and human studies. Using a well-established porcine model for Zika virus infection and a set of cellular and molecular experimental tools, we showed that silent in utero infection causes multi-organ inflammation in fetuses and local inflammation in the fetal bone marrow. In utero infection also caused footprints in the offspring bone marrow and PBMCs. These findings should be considered in a broader clinical context because of growing concerns about health sequelae in cohorts of children affected with congenital Zika virus infection in the Americas. Understanding virus-induced molecular mechanisms of immune activation and inflammation in fetuses may provide targets for early in utero interventions. Also, identifying early biomarkers of in utero-acquired immunopathology in offspring may help to alleviate long-term sequelae.

Plasma Cytokines and Birth Weight as Biomarkers of Vaccine-Induced Humoral Responses in Piglets.

Failure to mount an effective immune response to vaccination leaves individuals at risk for infection and can compromise herd immunity. Vaccine unresponsiveness can range from poor responses "low responders" to a failure to seroconvert "non-responders." Biomarkers of vaccine unresponsiveness, particularly those measured at the time of vaccination, could facilitate more strategic vaccination programs. We previously reported that pro-inflammatory cytokine signaling within peripheral blood mononuclear cells, elevated plasma interferon-gamma (IFNγ), and low birth weight correlated with vaccine-induced serum IgG titers in piglets that were below the threshold of detectable seroconversion (vaccine non-responders). These observations suggested that plasma IFNγ concentration and birth weight might serve as pre-vaccination biomarkers of vaccine unresponsiveness. To test this hypothesis, piglets (n = 67) from a different production facility were vaccinated with the same commercial Mycoplasma hyopneumoniae bacterin (RespiSure-One) to determine if there was a consistent and significant association between vaccine-induced serum IgG titers and either plasma cytokine concentrations or birth weight. All piglets seroconverted following vaccination with significantly less variability in vaccine-induced serum IgG titers than observed in the previous vaccine trial. Piglets exhibited highly variable birth weights and plasma cytokine concentrations prior to vaccination, but there were no significant associations (p > 0.05) between these variables and vaccine-induced serum IgG titers. There were significant (p < 0.001) differences in plasma IFNγ concentrations among individual litters (n = 6), and plasma IFNγ concentrations decreased in all pigs from birth to 63-days of age. One of the six litters (n = 11 piglets) exhibited significantly elevated plasma IFNγ concentrations during the first 3 weeks of life (p < 0.001) and at the time of vaccination (p < 0.01). This litter, however, had similar vaccine-induced serum IgG titers when compared to the other piglets in this study. Collectively the two studies indicate that while plasma cytokines and birth weight can be associated with vaccine non-responsiveness, their temporal and individual variation, as well as the complexity of the vaccine responsiveness phenotype, make them inconsistent biomarkers for predicting the less extreme phenotype of vaccine low responders.

Signaling differences in peripheral blood mononuclear cells of high and low vaccine responders prior to, and following, vaccination in piglets.

Individual variability in responses to vaccination can result in vaccinated subjects failing to develop a protective immune response. Vaccine non-responders can remain susceptible to infection and may compromise efforts to achieve herd immunity. Biomarkers of vaccine unresponsiveness could aid vaccine research and development as well as strategically improve vaccine administration programs. We previously vaccinated piglets (n = 117) against a commercial Mycoplasma hyopneumoniae vaccine (RespiSure-One) and observed in low vaccine responder piglets, as defined by serum IgG antibody titers, differential phosphorylation of peptides involved in pro-inflammatory cytokine signaling within peripheral blood mononuclear cells (PBMCs) prior to vaccination, elevated plasma interferon-gamma concentrations, and lower birth weight compared to high vaccine responder piglets. In the current study, we use kinome analysis to investigate signaling events within PBMCs collected from the same high and low vaccine responders at 2 and 6 days post-vaccination. Furthermore, we evaluate the use of inflammatory plasma cytokines, birthweight, and signaling events as biomarkers of vaccine unresponsiveness in a validation cohort of high and low vaccine responders. Differential phosphorylation events (FDR < 0.05) within PBMCs are established between high and low responders at the time of vaccination and at six days post-vaccination. A subset of these phosphorylation events were determined to be consistently differentially phosphorylated (p < 0.05) in the validation cohort of high and low vaccine responders. In contrast, there were no differences in birth weight (p > 0.5) and plasma IFNγ concentrations at the time of vaccination (p > 0.6) between high and low responders within the validation cohort. The results in this study suggest, at least within this study population, phosphorylation biomarkers are more robust predictors of vaccine responsiveness than other physiological markers.

Kinome Analysis to Define Mechanisms of Adjuvant Action: PCEP Induces Unique Signaling at the Injection Site and Lymph Nodes.

Understanding the mechanism of action of adjuvants through systems biology enables rationale criteria for their selection, optimization, and application. As kinome analysis has proven valuable for defining responses to infectious agents and providing biomarkers of vaccine responsiveness, it is a logical candidate to define molecular responses to adjuvants. Signaling responses to the adjuvant poly[di(sodiumcarboxylatoethylphenoxy)phosphazene] (PCEP) were defined at the site of injection and draining lymph node at 24 h post-vaccination. Kinome analysis indicates that PCEP induces a proinflammatory environment at the injection site, including activation of interferon and IL-6 signaling events. This is supported by the elevated expression of proinflammatory genes (IFNγ, IL-6 and TNFα) and the recruitment of myeloid (neutrophils, macrophages, monocytes and dendritic cells) and lymphoid (CD4+, CD8+ and B) cells. Kinome analysis also indicates that PCEP's mechanism of action is not limited to the injection site. Strong signaling responses to PCEP, but not alum, are observed at the draining lymph node where, in addition to proinflammatory signaling, PCEP activates responses associated with growth factor and erythropoietin stimulation. Coupled with the significant (p < 0.0001) recruitment of macrophages and dendritic cells to the lymph node by PCEP (but not alum) supports the systemic consequences of the adjuvant. Collectively, these results indicate that PCEP utilizes a complex, multi-faceted MOA and support the utility of kinome analysis to define cellular responses to adjuvants.

High-resolution analysis of long-term serum antibodies in humans following convalescence of SARS-CoV-2 infection.

Long-term antibody responses to SARS-CoV-2 have focused on responses to full-length spike protein, specific domains within spike, or nucleoprotein. In this study, we used high-density peptide microarrays representing the complete proteome of SARS-CoV-2 to identify binding sites (epitopes) targeted by antibodies present in the blood of COVID-19 resolved cases at 5 months post-diagnosis. Compared to previous studies that evaluated epitope-specific responses early post-diagnosis (< 60 days), we found that epitope-specific responses to nucleoprotein and spike protein have contracted, and that responses to membrane protein have expanded. Although antibody titers to full-length spike and nucleoprotein remain steady over months, taken together our data suggest that the population of epitope-specific antibodies that contribute to this reactivity is dynamic and evolves over time. Further, the spike epitopes bound by polyclonal antibodies in COVID-19 convalescent serum samples aligned with known target sites that can neutralize viral activity suggesting that the maintenance of these antibodies might provide rapid serological immunity. Finally, the most dominant epitopes for membrane protein and spike showed high diagnostic accuracy providing novel biomarkers to refine blood-based antibody tests. This study provides new insights into the specific regions of SARS-CoV-2 targeted by serum antibodies long after infection.

PIIKA 2.5: Enhanced quality control of peptide microarrays for kinome analysis.

Peptide microarrays consisting of defined phosphorylation target sites are an effective approach for high throughput analysis of cellular kinase (kinome) activity. Kinome peptide arrays are highly customizable and do not require species-specific reagents to measure kinase activity, making them amenable for kinome analysis in any species. Our group developed software, Platform for Integrated, Intelligent Kinome Analysis (PIIKA), to enable more effective extraction of meaningful biological information from kinome peptide array data. A subsequent version, PIIKA2, unveiled new statistical tools and data visualization options. Here we introduce PIIKA 2.5 to provide two essential quality control metrics and a new background correction technique to increase the accuracy and consistency of kinome results. The first metric alerts users to improper spot size and informs them of the need to perform manual resizing to enhance the quality of the raw intensity data. The second metric uses inter-array comparisons to identify outlier arrays that sometimes emerge as a consequence of technical issues. In addition, a new background correction method, background scaling, can sharply reduce spatial biases within a single array in comparison to background subtraction alone. Collectively, the modifications of PIIKA 2.5 enable identification and correction of technical issues inherent to the technology and better facilitate the extraction of meaningful biological information. We show that these metrics demonstrably enhance kinome analysis by identifying low quality data and reducing batch effects, and ultimately improve clustering of treatment groups and enhance reproducibility. The web-based and stand-alone versions of PIIKA 2.5 are freely accessible at via http://saphire.usask.ca.

Identifying kinase targets of PPARγ in human breast cancer.

Breast cancer is the most common cancer in women. Despite advances in screening women for genetic predisposition to breast cancer and risk stratification, a majority of women carriers remain undetected until they become affected. Thus, there is a need to develop a cost-effective, rapid, sensitive and non-invasive early-stage diagnostic method. Kinases are involved in all fundamental cellular processes and mutations in kinases have been reported as drivers of cancer. PPARγ is a ligand-activated transcription factor that plays important roles in cell proliferation and metabolism. However, the complete set of kinases modulated by PPARγ is still unknown. In this study, we identified human kinases that are potential PPARγ targets and evaluated their differential expression and gene pair correlations in human breast cancer patient dataset TCGA-BRCA. We further confirmed the findings in human breast cancer cell lines MCF7 and SK-BR-3 using a kinome array. We observed that gene pair correlations are lost in tumours as compared to healthy controls and could be used as a supplement strategy for diagnosis and prognosis of breast cancer.

EPIphany-A Platform for Analysis and Visualization of Peptide Immunoarray Data.

Antibodies are critical effector molecules of the humoral immune system. Upon infection or vaccination, populations of antibodies are generated which bind to various regions of the invading pathogen or exogenous agent. Defining the reactivity and breadth of this antibody response provides an understanding of the antigenic determinants and enables the rational development and assessment of vaccine candidates. High-resolution analysis of these populations typically requires advanced techniques such as B cell receptor repertoire sequencing, mass spectrometry of isolated immunoglobulins, or phage display libraries that are dependent upon equipment and expertise which are prohibitive for many labs. High-density peptide microarrays representing diverse populations of putative linear epitopes (immunoarrays) are an effective alternative for high-throughput examination of antibody reactivity and diversity. While a promising technology, widespread adoption of immunoarrays has been limited by the need for, and relative absence of, user-friendly tools for consideration and visualization of the emerging data. To address this limitation, we developed EPIphany, a software platform with a simple web-based user interface, aimed at biological users, that provides access to important analysis parameters, data normalization options, and a variety of unique data visualization options. This platform provides researchers the greatest opportunity to extract biologically meaningful information from the immunoarray data, thereby facilitating the discovery and development of novel immuno-therapeutics.

A Bovine Enteric Mycobacterium Infection Model to Analyze Parenteral Vaccine-Induced Mucosal Immunity and Accelerate Vaccine Discovery.

Mycobacterial diseases of cattle are responsible for considerable production losses worldwide. In addition to their importance in animals, these infections offer a nuanced approach to understanding persistent mycobacterial infection in native host species. Mycobacteriumavium ssp. paratuberculosis (MAP) is an enteric pathogen that establishes a persistent, asymptomatic infection in the small intestine. Difficulty in reproducing infection in surrogate animal models and limited understanding of mucosal immune responses that control enteric infection in the natural host have been major barriers to MAP vaccine development. We previously developed a reproducible challenge model to establish a consistent MAP infection using surgically isolated intestinal segments prepared in neonatal calves. In the current study, we evaluated whether intestinal segments could be used to screen parenteral vaccines that alter mucosal immune responses to MAP infection. Using Silirum® - a commercial MAP bacterin - we demonstrate that intestinal segments provide a platform for assessing vaccine efficacy within a relatively rapid period of 28 days post-infection. Significant differences between vaccinates and non-vaccinates could be detected using quantitative metrics including bacterial burden in intestinal tissue, MAP shedding into the intestinal lumen, and vaccine-induced mucosal immune responses. Comparing vaccine-induced responses in mucosal leukocytes isolated from the site of enteric infection versus blood leukocytes revealed substantial inconsistences between these immune compartments. Moreover, parenteral vaccination with Silirum did not induce equal levels of protection throughout the small intestine. Significant control of MAP infection was observed in the continuous but not the discrete Peyer's patches. Analysis of these regional mucosal immune responses revealed novel correlates of immune protection associated with reduced infection that included an increased frequency of CD335+ innate lymphoid cells, and increased expression of IL21 and IL27. Thus, intestinal segments provide a novel model to accelerate vaccine screening and discovery by testing vaccines directly in the natural host and provides a unique opportunity to interrogate mucosal immune responses to mycobacterial infections.

Kinome profiling of peripheral blood mononuclear cells collected prior to vaccination reveals biomarkers and potential mechanisms of vaccine unresponsiveness in pigs.

Inter-individual variance in host immune responses following vaccination can result in failure to develop protective immunity leaving individuals at risk for infection in addition to compromising herd immunity. While developing more efficacious vaccines is one strategy to mitigate this problem, predicting vaccine responsiveness prior to vaccination could inform which individuals require adjunct disease management strategies. To identify biomarkers of vaccine responsiveness, a cohort of pigs (n = 120) were vaccinated and pigs representing the high (n = 6; 90th percentile) and low (n = 6; 10th percentile) responders based on vaccine-specific antibody responses following vaccination were further analyzed. Kinase-mediated phosphorylation events within peripheral blood mononuclear cells collected prior to vaccination identified 53 differentially phosphorylated peptides when comparing low responders with high responders. Functional enrichment analysis revealed pro-inflammatory cytokine signaling pathways as dysregulated, and this was further substantiated by detection of higher (p < 0.01) concentrations of interferon-gamma in plasma of low responders compared to high responders prior to vaccination. In addition, low responder pigs with high plasma interferon-gamma showed lower (p < 0.01) birth weights than high responder pigs. These associations between vaccine responsiveness, cytokine signaling within peripheral immune cells, and body weight in pigs provide both evidence and insight into potential biomarkers for identifying low responders to vaccination.

Regional Dichotomy in Enteric Mucosal Immune Responses to a Persistent Mycobacterium avium ssp. paratuberculosis Infection.

Chronic enteric Mycobacterium avium ssp. paratuberculosis (MAP) infections are endemic in ruminants globally resulting in significant production losses. The mucosal immune responses occurring at the site of infection, specifically in Peyer's patches (PP), are not well-understood. The ruminant small intestine possesses two functionally distinct PPs. Discrete PPs function as mucosal immune induction sites and a single continuous PP, in the terminal small intestine, functions as a primary lymphoid tissue for B cell repertoire diversification. We investigated whether MAP infection of discrete vs. continuous PPs resulted in the induction of significantly different pathogen-specific immune responses and persistence of MAP infection. Surgically isolated intestinal segments in neonatal calves were used to target MAP infection to individual PPs. At 12 months post-infection, MAP persisted in continuous PP (n = 4), but was significantly reduced (p = 0.046) in discrete PP (n = 5). RNA-seq analysis revealed control of MAP infection in discrete PP was associated with extensive transcriptomic changes (1,707 differentially expressed genes) but MAP persistent in continuous PP elicited few host responses (4 differentially expressed genes). Cytokine gene expression in tissue and MAP-specific recall responses by mucosal immune cells isolated from PP, lamina propria and mesenteric lymph node revealed interleukin (IL)22 and IL27 as unique correlates of protection associated with decreased MAP infection in discrete PP. This study provides the first description of mucosal immune responses occurring in bovine discrete jejunal PPs and reveals that a significant reduction in MAP infection is associated with specific cytokine responses. Conversely, MAP infection persists in the continuous ileal PP with minimal perturbation of host immune responses. These data reveal a marked dichotomy in host-MAP interactions within the two functionally distinct PPs of the small intestine and identifies mucosal immune responses associated with the control of a mycobacterial infection in the natural host.

From Beef to Bees: High-Throughput Kinome Analysis to Understand Host Responses of Livestock Species to Infectious Diseases and Industry-Associated Stress.

Within human health research, the remarkable utility of kinase inhibitors as therapeutics has motivated efforts to understand biology at the level of global cellular kinase activity (the kinome). In contrast, the diminished potential for using kinase inhibitors in food animals has dampened efforts to translate this research approach to livestock species. This, in our opinion, was a lost opportunity for livestock researchers given the unique potential of kinome analysis to offer insight into complex biology. To remedy this situation, our lab developed user-friendly, cost-effective approaches for kinome analysis that can be readily incorporated into most research programs but with a specific priority to enable the technology to livestock researchers. These contributions include the development of custom software programs for the creation of species-specific kinome arrays as well as comprehensive deconvolution and analysis of kinome array data. Presented in this review are examples of the application of kinome analysis to highlight the utility of the technology to further our understanding of two key complex biological events of priority to the livestock industry: host immune responses to infectious diseases and animal stress responses. These advances and examples of application aim to provide both mechanisms and motivation for researchers, particularly livestock researchers, to incorporate kinome analysis into their research programs.

Attachment impacts cortisol awakening response in chronically depressed individuals.

BACKGROUND: Early life experiences shape individual attachment, creating a template for regulating emotions in interpersonal situations, likely to persist across the lifespan. Research has shown that individual attachment creates vulnerability for depression, and also impacts the Hypothalamic-Pituitary-Adrenal (HPA) axis. Still, the relationship between attachment and the HPA axis in depressed individuals is unclear. Cortisol awakening response (CAR) has been recently investigated as a possibly useful physiological marker related to attachment insecurity and depression risk. However, research exploring the relationship between the CAR and attachment in individuals with chronic depression in either the presence or the absence of comorbid anxiety is lacking. The purpose of the current study was to fill this gap, by comparing the CAR in individuals with chronic depression with/without comorbid anxieties and controls. In addition, we also wanted to explore the relationship between attachment and the CAR in this group and to explore their predictive role for later depression severity. METHODS: Individuals experiencing a current depressive episode at least six months in length (cMDD; n = 63) and healthy controls (HC; n = 57) were enrolled in the study (total n = 120). Participants completed a structured clinical diagnostic interview (SCID-I) as well as measures of depression severity (Beck Depression Inventory-II (BDI-II) and Hamilton Rating Scale for Depression) and attachment dimensions (Experiences in Close Relationships scale; ECR) at baseline. In addition, participants provided salivary samples at four time points (i.e. 0 (S1), 30, 45 and 60 min) following awakening on two consecutive days. S1 cortisol, the area under the curve with respect to ground (AUCg) and increase (AUCi) were calculated based on the average values across both days. The HC and cMDD groups were compared on all measures. The CAR for individuals with cMDD alone (n = 14) and individuals with cMDD with two or more comorbid anxiety disorders (cMDD ≥ 2Anx; n = 30) were also compared. A subset of participants (n = 59) agreed to return for follow up one year later. Participants returning for follow up repeated the BDI-II and ECR. No salivary samples were collected at follow-up. RESULTS: The cMDD group had significantly lower S1 cortisol and AUCg compared to the HC group (both p ≤ 0.02). cMDD and cMDD ≥ 2Anx groups did not differ in their CAR. Regression analyses revealed that depression severity and the attachment interaction term was associated with lower S1 and AUCg cortisol (p < 0.01). Greater attachment avoidance was positively associated with S1 cortisol (p = 0.02), while mean awakening time on sample days was negatively associated with S1 cortisol. We also found a significant interaction between the attachment dimensions such that at low levels of attachment anxiety, attachment avoidance had a positive relationship with S1 cortisol and AUCg. The opposite relationship existed when attachment anxiety was high. Higher baseline BDI-II score and higher baseline attachment anxiety were predictive of higher scores on the BDI-II one-year later (both p < 0.05). CONCLUSIONS: The current findings bring evidence that depression severity is associated with blunting of the CAR irrespective of the comorbid status with anxiety disorders. In addition, attachment avoidance may protect against the CAR blunting in individuals with low attachment anxiety. However, individuals with high attachment anxiety and avoidance might have additional CAR blunting. Attachment anxiety might be a good predictor of future depression severity.

Kinome Analysis of Honeybee (Apis mellifera L.) Dark-Eyed Pupae Identifies Biomarkers and Mechanisms of Tolerance to Varroa Mite Infestation.

The mite Varroa destructor is a serious threat to honeybee populations. Selective breeding for Varroa mite tolerance could be accelerated by biomarkers within individual bees that could be applied to evaluate a colony phenotype. Previously, we demonstrated differences in kinase-mediated signaling between bees from colonies of extreme phenotypes of mite susceptibility. We expand these findings by defining a panel of 19 phosphorylation events that differ significantly between individual pupae from multiple colonies with distinct Varroa mite tolerant phenotypes. The predictive capacity of these biomarkers was evaluated by analyzing uninfested pupae from eight colonies representing a spectrum of mite tolerance. The pool of biomarkers effectively discriminated individual pupae on the basis of colony susceptibility to mite infestation. Kinome analysis of uninfested pupae from mite tolerant colonies highlighted an increased innate immune response capacity. The implication that differences in innate immunity contribute to mite susceptibility is supported by the observation that induction of innate immune signaling responses to infestation is compromised in pupae of the susceptible colonies. Collectively, biomarkers within individual pupae that are predictive of the susceptibility of colonies to mite infestation could provide a molecular tool for selective breeding of tolerant colonies.

Ibrutinib as a potential therapeutic option for HER2 overexpressing breast cancer - the role of STAT3 and p21.

Treatment response rates to current anticancer therapies for HER2 overexpressing breast cancer are limited and are associated with severe adverse drug reactions. Tyrosine kinases perform crucial roles in cellular processes by mediating cell signalling cascades. Ibrutinib is a recently approved Tyrosine Kinase Inhibitor (TKI) that has been shown be an effective therapeutic option for HER2 overexpressing breast cancer. The molecular mechanisms, pathways, or genes that are modulated by ibrutinib and the mechanism of action of ibrutinib in HER2 overexpressing breast cancer remain obscure. In this study, we have performed a kinome array analysis of ibrutinib treatment in two HER2 overexpressing breast cancer cell lines. Our analysis shows that ibrutinib induces changes in nuclear morphology and causes apoptosis via caspase-dependent extrinsic apoptosis pathway with the activation of caspases-8, caspase-3, and cleavage of PARP1. We further show that phosphorylated STAT3Y705 is upregulated and phosphorylated p21T145 is downregulated upon ibrutinib treatment. We propose that STAT3 upregulation is a passive response as a result of induction of DNA damage and downregulation of phosphorylated p21 is promoting cell cycle arrest and apoptosis in the two HER2 overexpressing cell lines. These results suggest that inhibitors of STAT3 phosphorylation may be potential options for combination therapy to help increase the efficacy of ibrutinib against HER2-overexpressing tumors.

Therapeutic vaccines for amyotrophic lateral sclerosis directed against disease specific epitopes of superoxide dismutase 1.

Emerging evidence suggests seeding and prion-like propagation of mutant Superoxide Dismutase 1 (SOD1) misfolding to be a potential mechanism for ALS pathogenesis and progression. Immuno-targeting of misfolded SOD1 has shown positive clinical outcomes in mutant SOD1 transgenic mice. However, a major challenge in developing active immunotherapies for proteinopathies such as ALS is the design of immunogens enabling exclusive recognition of pathogenic species of a self-protein. Ideally, one would achieve a robust antibody response against the disease-misfolded protein while sparing the natively folded conformer to avoid inducing deleterious autoimmune complications, or inhibiting its normal function. Using a motor neuron disease mouse model expressing human SOD1-G37R, we herein report the immunogenicity and therapeutic efficacy of two ALS vaccines, tgG-DSE2lim and tgG-DSE5b, based on the notion that native SOD1 would undergo early unfolding in disease to present "disease specific epitopes" (DSE). Both vaccines elicited rapid, robust, and well-sustained epitope-specific antibody responses with a desirable Th2-biased immune response. Both vaccines significantly extended the life expectancy of hSOD1G37R mice, with tgG-DSE2lim displaying greater protection than tgG-DSE5b at earlier pre-symptomatic stage. tgG-DSE5b, but not tgG-DSE2lim, significantly delayed disease onset and appreciably slowed disease progression. This implies that conformationally distinct species of misfolded SOD1 may derive from the same mutation, thereby modifying disease phenotypes in a different fashion. Our results validate the rationale for conformation-based immuno-targeting of misfolded SOD1 as a promising therapeutic strategy to slow or even halt disease progression in familial ALS associated with SOD1 mutations, as well as a prophylactic intervention for carriers of SOD1 mutations. Our study not only provides important proof-of-principle data for the development of a safe and effective human therapeutic/prophylactic ALS vaccine against misfolded SOD1, but also predicts a great potential to extend our DSE-based vaccination approach to other types of ALS, such as those associated with TDP-43 proteinopathies.