Specific Salivary Neuropeptides Shift Synchronously during Acute Stress in Fire Recruits.

Once thought of as an immune-privileged site, we now know that the nervous system communicates in a bidirectional manner with the immune system via the neuroimmune axis. Neuropeptides constitute a component of this axis, playing critical roles in the brain and periphery. The function of salivary neuropeptides in the acute stress response is not well understood. The purpose of this study is to investigate salivary neuropeptide levels during acute stress. Salivary samples were collected from fire recruits engaged in a stress training exercise previously shown to induce acute stress, at three separate timepoints during the exercise and levels of oxytocin, neurotensin, Substance P, α-MSH, and ß-Endorphin were measured using the Human Neuropeptide 5-Plex Custom Assay Eve Technologies. All neuropeptides increased throughout the acute stress simulation and during the recovery phase. Exploratory factor analysis (EFA) identified one factor contributing to baseline values across five neuropeptides and Pairwise Pearson Correlation Coefficient analysis showed positive correlations >0.9 for almost all neuropeptide combinations at the pre-stress timepoint. Further analysis identified negative and positive correlations between past-life trauma and self-assessed hardiness, respectively. Calculated neuropeptide scores showed an overall positive correlation to self-assessed hardiness. Altogether, our results suggest that salivary neuropeptides increase synchronously during acute stress and higher levels correlate with an increase in self-assessed hardiness. Further study is required to determine if interventions designed to enhance neuropeptide activity can increase stress resilience, especially in high-stress occupations such as firefighting.

A distinct immune cytokine profile is associated with morning cortisol and repeated stress.

OBJECTIVE: The objective of this study was to investigate possible immune cytokine trends throughout a week-long surgical simulation mass-casualty training session in order to determine the effects of stress inoculation on the immune system. METHODS: Thirty-seven military medical students participated in a hyper-realistic surgical simulation training event conducted at Strategic Operations site in San Diego, California. Salivary samples were collected every morning of the stress training exercise for 4 consecutive days. Cortisol, along with a panel of 42 immune cytokines, was measured using multiplex enzyme-linked immunosorbent assays from Eve Technologies. The determined concentrations were averaged and plotted on a scatter plot, and then points were fit to a second-order polynomial trendline of best fit to measure. RESULTS: The cytokines epidermal growth factor, growth-related oncogene-α, interleukin (IL)-1α, and platelet-derived growth factor-AA followed a noted pattern of cortisol decrease throughout the week. In addition, cytokines IL-27, granulocyte colony stimulating factor, IL-10, and IL-13 demonstrated a late peak, followed by a return to baseline at the conclusion of training. Finally, the cytokine monocyte chemoattractant protein-1 displayed a decline throughout the week followed by an increase on the last day of stress training. CONCLUSIONS: Altogether, these results help to identify important biomarkers that may help to improve long-term stress adaptation and prevent post-traumatic stress disorder following exposure to repeated stress.

Correlation analysis of salivary cytokines and hormones with resiliency.

BACKGROUND: Frequent exposure to acute stress increases risk of suicide, posttraumatic stress disorder, and other stress-related disorders. Neuroendocrine and immunologic dysregulation associated with stress may underlie predispositions to psychological disorders and inflammatory disease processes in individuals, such as first-responders and other healthcare professionals, who function in high stress situations. The Hardiness Resilience Gauge (HRG) can be used to psychometrically measure resilience, a psychological modifier of the stress response. Using the HRG alongside salivary biomarker profiling, may help to identify low resilience phenotypes and allow mitigation and early therapeutic interventions. There is a paucity of knowledge regarding biomarkers of resilience. This study aims to evaluate the relationship between factors of resilience with salivary biomarker levels and fluctuations during and following acute stress. METHODS: Sixty-three first responders underwent a standardized stress-inducing training exercise, providing salivary samples before (prestress), immediately after (post-stress), and 1 hour after the event (recovery). The HRG was administered before (initial) and after (final) the event. Multiplex ELISA panels quantified 42 cytokines and 6 hormones from the samples, which were analyzed for relationships to psychometric factors of resilience measured by the HRG. RESULTS: Several biomarkers correlated with psychological resilience following the acute stress event. The HRG scores correlated ( p < 0.05) with a select set of biomarkers with moderate-to-strong correlations (|r| > 0.3). These included EGF, GROα, PDGFAA, TGFα, VEGFA, interleukin (IL)1Ra, TNFα, IL18, cortisol, FGF2, IL13, IL15, and IL6. Interestingly, fluctuations of EGF, GROα, and PDGFAA in post-stress compared with recovery were positively correlated with factors of resilience, which were negatively correlated from the pre-stress to post-stress period. CONCLUSION: This exploratory analysis discovered a small subset of salivary biomarkers that are significantly correlated with acute stress and resilience. Further investigation of their specific roles in acute stress and associations with resiliency phenotypes is warranted.

Principal component analysis of salivary cytokines and hormones in the acute stress response.

The acute stress response is characterized by activation of multiple interconnected systems in the body, resulting in the release of a flood of hormones and immune mediators into circulation. In addition to detection of these molecules in the serum, saliva can serve as a source of these markers as well and can be collected in a non-invasive way. The complete profile of salivary biomarkers associated with the hypothalamic pituitary adrenal/gonadal axes and the immune system during the acute stress response has not been fully elucidated. In a cohort of 62 first responders engaged in a stress training exercise, we set out to determine patterns of cytokine, chemokine and hormone shifts during the acute stress response. Salivary samples were collected immediately before (pre-stress), immediately after (post-stress) and 1 h after the stress test (recovery). Multiplex ELISA panels of 42 cytokines and 6 steroid and thyroid hormones were used to determine concentrations of these biomarkers during the three aforementioned time points. Principal components analysis was conducted to determine patterns in the large data sets collected. In our ≥0.3 loading principal components analysis, for pre-stress vs. post, post-stress vs. recovery and pre-stress vs. recovery, a total of three, four and three factors accounted for 56.6, 68.34, and 61.70% of the biomarker variation for each phase respectively. In the ≥0.7 loading principal components analysis, three, four and three factors were found for pre-stress vs. post, post-stress vs. recovery and pre-stress vs. recovery stages, respectively. Of note, in our ≥0.3 loading principal components analysis, MCP1 was present in all three factors from pre-stress to post-stress, and fractalkine was found to be in all four factors post-stress vs. recovery and pre vs. recovery from stress. Additionally, hormones testosterone, estradiol, T4 and T3 grouped together consistently in the same factor for all phases of acute stress in both ≥0.3 and ≥0.7 principal components analysis. Overall, our results identified specific patterns of immune markers and hormones that shift during acute stress and warrant further investigation to understand their mechanistic role in regulating the stress response.

Moving beyond Titers.

Vaccination to prevent and even eliminate disease is amongst the greatest achievements of modern medicine. Opportunities remain in vaccine development to improve protection across the whole population. A next step in vaccine development is the detailed molecular characterization of individual humoral immune responses against a pathogen, especially the rapidly evolving pathogens. New technologies such as sequencing the immune repertoire in response to disease, immunogenomics/vaccinomics, particularly the individual HLA variants, and high-throughput epitope characterization offer new insights into disease protection. Here, we highlight the emerging technologies that could be used to identify variation within the human population, facilitate vaccine discovery, improve vaccine safety and efficacy, and identify mechanisms of generating immunological memory. In today's vaccine-hesitant climate, these techniques used individually or especially together have the potential to improve vaccine effectiveness and safety and thus vaccine uptake rates. We highlight the importance of using these techniques in combination to understand the humoral immune response as a whole after vaccination to move beyond neutralizing titers as the standard for immunogenicity and vaccine efficacy, especially in clinical trials.

Thygeson Superficial Punctate Keratitis: A Clinical and Immunologic Review.

ABSTRACT: Thygeson superficial punctate keratitis (TSPK) is clinically characterized by exacerbations and remissions of gray-white opacities within the corneal epithelium, most often bilateral but may be asymmetric. Symptoms typically include photophobia, tearing, blurring, and eye irritation. Although disease progression and prognosis are well described, the exact cause is unknown. Hypotheses exist implicating virus-mediated immunity as the cause of TSPK following cases of viral keratitis; however, several polymerase chain reaction studies refute the infectious process concurrently with symptomatic TSPK. This is further supported by the consistent lack of response to antiviral and antibacterial treatment. A subset of dendritic cells known as Langerhans cells (LC) found within the corneal epithelium has been positively correlated with exacerbations of TSPK. Langerhans cells proliferate to protect and mitigate the cornea's inflammatory response, but the inflammatory triggers and relapses associated with TSPK are not well understood. Several topical drugs exist to treat inflammation related to TSPK; however, drug delivery is a major barrier to treatment because of the tear film and epithelial barrier. Drug-eluting contact lenses that target intermediates of inflammation could serve as a more effective treatment modality because of the increased bioavailability of the drugs. This review is an in-depth survey of the literature regarding the relationship between the origin and pathophysiology of LC and TSPK at the immunologic level. We also discuss potential pharmacotherapeutic interventions for TSPK prevention and treatment.

Potential Therapeutic Targets for Combination Antibody Therapy against Pseudomonas aeruginosa Infections.

Despite advances in antimicrobial therapy and even the advent of some effective vaccines, Pseudomonas aeruginosa (P. aeruginosa) remains a significant cause of infectious disease, primarily due to antibiotic resistance. Although P. aeruginosa is commonly treatable with readily available therapeutics, these therapies are not always efficacious, particularly for certain classes of patients (e.g., cystic fibrosis (CF)) and for drug-resistant strains. Multi-drug resistant P. aeruginosa infections are listed on both the CDC's and WHO's list of serious worldwide threats. This increasing emergence of drug resistance and prevalence of P. aeruginosa highlights the need to identify new therapeutic strategies. Combinations of monoclonal antibodies against different targets and epitopes have demonstrated synergistic efficacy with each other as well as in combination with antimicrobial agents typically used to treat these infections. Such a strategy has reduced the ability of infectious agents to develop resistance. This manuscript details the development of potential therapeutic targets for polyclonal antibody therapies to combat the emergence of multidrug-resistant P. aeruginosa infections. In particular, potential drug targets for combinational immunotherapy against P. aeruginosa are identified to combat current and future drug resistance.

Epitope characterization of anti-drug antibodies-a tool for discovery and health: an overview of the necessity of early epitope characterization to avoid anti-drug antibodies and promote patient health.

Introduction: The market for monoclonal antibody (mAb) therapies is growing rapidly as the pharmaceutical industry expands its development across a broad spectrum of diseases. Unfortunately, as shown in the recent failure of bococizumab by Pfizer, these treatments often stimulate the formation of problematic anti-drug antibodies (ADAs). ADAs can cause side effects and limit efficacy for many patients. To increase efficacy and decrease safety concerns from ADAs, immunogenicity characterization is needed early in the drug development process. Here, we present emerging techniques that hold promise to improve ADA assays and their potential applications to pharmaceutical development and personalized medicine.Areas covered: This manuscript outlines the importance of epitope characterization to better understand immunogenicity and describes a strategy for using this information in treating patients taking mAb therapies.Expert opinion: We propose using high-information assays to characterize epitopes to help mAb therapy engineering and potentially improve individual patient outcomes. To understand this, we will discuss three different aspects of ADAs: (1) the problem of ADAs and what is currently being done about them, (2) the current state of epitope characterization and how it is being utilized, and (3) how early epitope characterization can advance drug discovery and improve outcomes for patients taking mAb therapies.

The channel-kinase TRPM7 regulates antigen gathering and internalization in B cells.

Members of the transient receptor potential (TRP) family of ion channels are cellular sensors involved in numerous physiological and pathological processes. We identified the TRP subfamily M member 7 (TRPM7) channel-kinase as a previously uncharacterized regulator of B cell activation. We showed that TRPM7 played a critical role in the early events of B cell activation through both its ion channel and kinase functions. DT40 B cells deficient in TRPM7 or expressing a kinase-deficient mutant of TRPM7 showed defective gathering of antigen and prolonged B cell receptor (BCR) signaling. We showed that lipid metabolism was altered in TRPM7-deficient cells and in cells expressing a kinase-deficient mutant of TRPM7 and suggest that PLC-γ2 may be a target of the kinase activity of TRPM7. Primary B cells that expressed less TRPM7 or were treated with a pharmacological inhibitor of TRPM7 also displayed defective antigen gathering and increased BCR signaling. Finally, we demonstrated that blocking TRPM7 function compromised antigen internalization and presentation to T cells. These data suggest that TRPM7 controls an essential process required for B cell affinity maturation and the production of high-affinity antibodies.

TRPM6 kinase activity regulates TRPM7 trafficking and inhibits cellular growth under hypomagnesic conditions.

The channel kinases TRPM6 and TRPM7 are both members of the melastatin-related transient receptor potential (TRPM) subfamily of ion channels and the only known fusions of an ion channel pore with a kinase domain. TRPM6 and TRPM7 form functional, tetrameric channel complexes at the plasma membrane by heteromerization. TRPM6 was previously shown to cross-phosphorylate TRPM7 on threonine residues, but not vice versa. Genetic studies demonstrated that TRPM6 and TRPM7 fulfill non-redundant functions and that each channel contributes uniquely to the regulation of Mg(2+) homeostasis. Although there are indications that TRPM6 and TRPM7 can influence each other's cellular distribution and activity, little is known about the functional relationship between these two channel-kinases. In the present study, we examined how TRPM6 kinase activity influences TRPM7 serine phosphorylation, intracellular trafficking, and cell surface expression of TRPM7, as well as Mg(2+)-dependent cellular growth. We found TRPM7 serine phosphorylation via the TRPM6 kinase, but no TRPM6 serine phosphorylation via the TRPM7 kinase. Intracellular trafficking of TRPM7 was altered in HEK-293 epithelial kidney cells and DT40 B cells in the presence of TRPM6 with intact kinase activity, independently of the availability of extracellular Mg(2+), but TRPM6/7 surface labeling experiments indicate comparable levels of the TRPM6/7 channels at the plasma membrane. Furthermore, using a complementation approach in TRPM7-deficient DT40 B-cells, we demonstrated that wild-type TRPM6 inhibited cell growth under hypomagnesic cell culture conditions in cells co-expressing TRPM6 and TRPM7; however, co-expression of a TRPM6 kinase dead mutant had no effect-a similar phenotype was also observed in TRPM6/7 co-expressing HEK-293 cells. Our results provide first clues about how heteromer formation between TRPM6 and TRPM7 influences the biological activity of these ion channels. We show that TRPM6 regulates TRPM7 intracellular trafficking and TRPM7-dependent cell growth. All these effects are dependent upon the presence of an active TRPM6 kinase domain. Dysregulated Mg(2+)-homeostasis causes or exacerbates many pathologies. As TRPM6 and TRPM7 are expressed simultaneously in numerous cell types, understanding how their relationship impacts regulation of Mg(2+)-uptake is thus important knowledge.

Regulation of T cell receptor complex-mediated signaling by ubiquitin and ubiquitin-like modifications.

Post-translational protein modifications are a dynamic method of regulating protein function in response to environmental signals. As with any cellular process, T cell receptor (TCR) complex-mediated signaling is highly regulated, since the strength and duration of TCR-generated signals governs T cell development and activation. While regulation of TCR complex-mediated signaling by phosphorylation has been well studied, regulation by ubiquitin and ubiquitin-like modifiers is still an emerging area of investigation. This review will examine how ubiquitin, E3 ubiquitin ligases, and other ubiquitin-like modifications such as SUMO and NEDD8 regulate TCR complex-mediated signaling.

The role of Mg2+ in immune cells.

The physiological and clinical relevance of Mg(2+) has evolved over the last decades. The molecular identification of multiple Mg(2+) transporters (Acdp2, MagT1, Mrs2, Paracellin-1, SLC41A1, SLC41A2, TRPM6 and TRPM7) and their biophysical characterization in recent years has improved our understanding of Mg(2+) homeostasis regulation and has provided a basis for investigating the role of Mg(2+) in the immune system. Deletions and mutations of Mg(2+) transporters produce severe phenotypes with more systemic symptoms than those seen with Ca(2+) channel deletions, which tend to be more specific and less profound. Deficiency of the Mg(2+) permeable ion channels TRPM6 or TRPM7 in mice is lethal at embryonic day 12.5 or at day 6.5, respectively, and, even more surprisingly, chicken DT40 B cells lacking TRPM7 die after 24-48 h. Recent progress made in Mg(2+) research has helped to define underlying mechanisms of two hereditary diseases, human Hypomagnesemia (TRPM6 deletion) and X-chromosomal immunodeficiency (MagT1 deletion), and has revealed a potential new role for Mg(2+) as a second messenger. Future elucidation of human Mg(2+) transporters (Mrs2, SLC41A1, SLC41A2, TRPM7) expressed in immunocytes, beyond MagT1 and TRPM6, will widen our knowledge about the potential role of Mg(2+) in the activation of the immune response.

Identification of Ser/Thr phosphorylation sites in the C2-domain of phospholipase C γ2 (PLCγ2) using TRPM7-kinase.

PLC-isozymes are central elements of cellular signaling downstream of numerous receptors. PLCγ2 is a pivotal component of B cell receptor (BCR) signaling. The regulation of PLCγ2-dependent signaling functions by Tyr-phosphorylation is well characterized, however, the potential role of Ser/Thr phosphorylation events remains undefined. TRPM7 is the fusion of a Ser/Thr kinase with an ion channel, and an essential component of Mg(2+)-homeostasis regulation. Although the interaction between the C2 domain of several PLC-isozymes and TRPM7 is well established, previous studies have focused on the effect of PLC-activity on TRPM7. Here, we investigated whether Ser/Thr phosphorylation sites in the C2 domain of PLCγ2 could be identified using TRPM7-kinase. We show that TRPM7-kinase phosphorylates PLCγ2 in its C2-domain at position Ser1164 and in the linker region preceding the C2-domain at position Thr1045. Using a complementation approach in PLCγ2(-/-) DT40 cells, we found that the PLCγ2-S1164A mutant fully restores BCR mediated Ca(2+)-responses under standard growth conditions. However, under hypomagnesic conditions, PLCγ2-S1164A fails to reach Ca(2+)-levels seen in cells expressing PLCγ2 wildtype. These results suggest that Mg(2+)-sensitivity of the BCR signaling pathway may be regulated by Ser/Thr phosphorylation of PLCγ2.

The Mg2+ transporter MagT1 partially rescues cell growth and Mg2+ uptake in cells lacking the channel-kinase TRPM7.

Magnesium (Mg(2+)) transport across membranes plays an essential role in cellular growth and survival. TRPM7 is the unique fusion of a Mg(2+) permeable pore with an active cytosolic kinase domain, and is considered a master regulator of cellular Mg(2+) homeostasis. We previously found that the genetic deletion of TRPM7 in DT40 B cells results in Mg(2+) deficiency and severe growth impairment, which can be rescued by supplementation with excess extracellular Mg(2+). Here, we show that gene expression of the Mg(2+) selective transporter MagT1 is upregulated in TRPM7(-/-) cells. Furthermore, overexpression of MagT1 in TRPM7(-/-) cells augments their capacity to uptake Mg(2+), and improves their growth behavior in the absence of excess Mg(2+).