A Novel Application of Spinning Disk Technology to Collect Plasma from Whole Blood Prior to Quantifying Plasma RNA.

The spinning disk technology has previously been utilized to isolate bacterial components from blood in hours instead of days. We hypothesized that this platform could be applied as an alternative approach to isolating plasma RNA from a whole blood sample. We consequently tested the efficacy of the spinning disk technology to extract plasma from whole blood upstream of RNA isolation and analysis. To do so, we collected plasma using either the spinning disk or the typical two-spin centrifuge method. We found that the spinning disk method results in significantly more hemolysis during collection than the conventional two-spin centrifuge method. However, when plasma RNA recovered from both collection methods was quantified using quantitative Real-Time Polymerase Chain Reaction (qRT-PCR), we found that the spinning disk method yielded a higher plasma RNA concentration than the two-spin centrifuge method. This suggests that the spinning disk may be an efficient alternative method to recover plasma RNA. Further work is needed to determine whether red blood cell RNA contamination is present in the plasma RNA extracted from spinning disk-processed plasma.

The Role of Atypical Chemokine Receptors in Neuroinflammation and Neurodegenerative Disorders.

Neuroinflammation is associated with several neurodegenerative disorders, including Alzheimer's disease (AD), Parkinson's disease (PD), and multiple sclerosis (MS). Neuroinflammation provides protection in acute situations but results in significant damage to the nervous system if chronic. Overexpression of chemokines within the brain results in the recruitment and activation of glial and peripheral immune cells which can propagate a cascading inflammatory response, resulting in neurodegeneration and the onset of neurodegenerative disorders. Recent work has identified the role of atypical chemokine receptors (ACKRs) in neurodegenerative conditions. ACKRs are seven-transmembrane domain receptors that do not follow canonical G protein signaling, but regulate inflammatory responses by modulating chemokine abundance, location, and availability. This review summarizes what is known about the four ACKRs and three putative ACKRs within the brain, highlighting their known expression and discussing the current understanding of each ACKR in the context of neurodegeneration. The ability of ACKRs to alter levels of chemokines makes them an appealing therapeutic target for neurodegenerative conditions. However, further work is necessary to understand the expression of several ACKRs within the neuroimmune system and the effectiveness of targeted drug therapies in the prevention and treatment of neurodegenerative conditions.

Characterizing the Interplay of Lymphocytes in Graves' Disease.

Graves' disease (GD) is a thyroid-specific autoimmune disease with a high prevalence worldwide. The disease is primarily mediated by B cells, which produce autoantibodies against the thyroid-stimulating hormone receptor (TSHR), chronically stimulating it and leading to high levels of thyroid hormones in the body. Interest in characterizing the immune response in GD has motivated many phenotyping studies. The immunophenotype of the cells involved and the interplay between them and their secreted factors are crucial to understanding disease progression and future treatment options. T cell populations are markedly distinct, including increased levels of Th17 and follicular helper T cells (Tfh), while Treg cells appear to be impaired. Some B cells subsets are autoreactive, and anti-TSHR antibodies are the key disease-causing outcome of this interplay. Though some consensus across phenotyping studies will be discussed here, there are also complexities that are yet to be resolved. A better understanding of the immunophenotype of Graves' disease can lead to improved treatment strategies and novel drug targets.

Immunomodulatory role of oral microbiota in inflammatory diseases and allergic conditions.

In recent years, the interplay between oral microbiota and systemic disease has gained attention as poor oral health is associated with several pathologies. The oral microbiota plays a role in the maintenance of overall health, and its dysbiosis influences chronic inflammation and the pathogenesis of gum diseases. Periodontitis has also been associated with other diseases and health complications such as cancer, neurogenerative and autoimmune disorders, chronic kidney disease, cardiovascular diseases, rheumatic arthritis, respiratory health, and adverse pregnancy outcomes. The host microbiota can influence immune cell development and immune responses, and recent evidence suggests that changes in oral microbiota composition may also contribute to sensitization and the development of allergic reactions, including asthma and peanut allergies. Conversely, there is also evidence that allergic reactions within the gut may contribute to alterations in oral microbiota composition. Here we review the current evidence of the role of the oral microbiota in inflammatory diseases and health complications, as well as its future relevance in improving health and ameliorating allergic disease.

A Chromosome-length Assembly of the Black Petaltail (Tanypteryx hageni) Dragonfly.

We present a chromosome-length genome assembly and annotation of the Black Petaltail dragonfly (Tanypteryx hageni). This habitat specialist diverged from its sister species over 70 million years ago, and separated from the most closely related Odonata with a reference genome 150 million years ago. Using PacBio HiFi reads and Hi-C data for scaffolding we produce one of the most high-quality Odonata genomes to date. A scaffold N50 of 206.6 Mb and a single copy BUSCO score of 96.2% indicate high contiguity and completeness.

Bioaerosol and microbial exposures from residential evaporative coolers and their potential health outcomes: A review.

Evaporative cooling is an energy efficient form of air conditioning in dry climates that functions by pulling hot, dry outdoor air across a wet evaporative pad. While evaporative coolers can help save energy, they also have the potential to influence human health. Studies have shown residential evaporative coolers may pull outdoor air pollutants into the home or contribute to elevated levels of indoor bioaerosols that may be harmful to health. There is also evidence that evaporative coolers can enable a diverse microbial environment that may confer early-life immunological protection against the development of allergies and asthma or exacerbate these same hypersensitivities. This review summarizes the current knowledge of bioaerosol and microbiological studies associated with evaporative coolers, focusing on harmful and potentially helpful outcomes from their use. We evaluate the effects of evaporative coolers on indoor bacterial endotoxins, fungal ß-(1 → 3)-D-glucans, dust mite antigens, residential microbial communities, and Legionella pneumophila. To our knowledge, this is the first review to summarize and evaluate studies on the influence that evaporative coolers have on the bioaerosol and microbiological profile of homes. This brings to light a gap in the literature on evaporative coolers, which is the lack of data on health effects associated with their use.

Alternative CAR Therapies: Recent Approaches in Engineering Chimeric Antigen Receptor Immune Cells to Combat Cancer.

For nearly three decades, chimeric antigen receptors (CARs) have captivated the interest of researchers seeking to find novel immunotherapies to treat cancer. CARs were first designed to work with T cells, and the first CAR T cell therapy was approved to treat B cell lymphoma in 2017. Recent advancements in CAR technology have led to the development of modified CARs, including multi-specific CARs and logic gated CARs. Other immune cell types, including natural killer (NK) cells and macrophages, have also been engineered to express CARs to treat cancer. Additionally, CAR technology has been adapted in novel approaches to treating autoimmune disease and other conditions and diseases. In this article, we review these recent advancements in alternative CAR therapies and design, as well as their mechanisms of action, challenges in application, and potential future directions.

Meta-Analysis of Two Human RNA-seq Datasets to Determine Periodontitis Diagnostic Biomarkers and Drug Target Candidates.

Periodontitis is a chronic inflammatory oral disease that affects approximately 42% of adults 30 years of age or older in the United States. In response to microbial dysbiosis within the periodontal pockets surrounding teeth, the host immune system generates an inflammatory environment in which soft tissue and alveolar bone destruction occur. The objective of this study was to identify diagnostic biomarkers and the mechanistic drivers of inflammation in periodontitis to identify drugs that may be repurposed to treat chronic inflammation. A meta-analysis comprised of two independent RNA-seq datasets was performed. RNA-seq analysis, signal pathway impact analysis, protein-protein interaction analysis, and drug target analysis were performed to identify the critical pathways and key players that initiate inflammation in periodontitis as well as to predict potential drug targets. Seventy-eight differentially expressed genes, 10 significantly impacted signaling pathways, and 10 hub proteins in periodontal gingival tissue were identified. The top 10 drugs that may be repurposed for treating periodontitis were then predicted from the gene expression and pathway data. The efficacy of these drugs in treating periodontitis has yet to be investigated. However, this analysis indicates that these drugs may serve as potential therapeutics to treat inflammation in gingival tissue affected by periodontitis.

CD5 Deficiency Alters Helper T Cell Metabolic Function and Shifts the Systemic Metabolome.

Metabolic function plays a key role in immune cell activation, destruction of foreign pathogens, and memory cell generation. As T cells are activated, their metabolic profile is significantly changed due to signaling cascades mediated by the T cell receptor (TCR) and co-receptors found on their surface. CD5 is a T cell co-receptor that regulates thymocyte selection and peripheral T cell activation. The removal of CD5 enhances T cell activation and proliferation, but how this is accomplished is not well understood. We examined how CD5 specifically affects CD4+ T cell metabolic function and systemic metabolome by analyzing serum and T cell metabolites from CD5WT and CD5KO mice. We found that CD5 removal depletes certain serum metabolites, and CD5KO T cells have higher levels of several metabolites. Transcriptomic analysis identified several upregulated metabolic genes in CD5KO T cells. Bioinformatic analysis identified glycolysis and the TCA cycle as metabolic pathways promoted by CD5 removal. Functional metabolic analysis demonstrated that CD5KO T cells have higher oxygen consumption rates (OCR) and higher extracellular acidification rates (ECAR). Together, these findings suggest that the loss of CD5 is linked to CD4+ T cell metabolism changes in metabolic gene expression and metabolite concentration.

Alphaviruses: Host pathogenesis, immune response, and vaccine & treatment updates.

Human pathogens belonging to the Alphavirus genus, in the Togaviridae family, are transmitted primarily by mosquitoes. The signs and symptoms associated with these viruses include fever and polyarthralgia, defined as joint pain and inflammation, as well as encephalitis. In the last decade, our understanding of the interactions between members of the alphavirus genus and the human host has increased due to the re-appearance of the chikungunya virus (CHIKV) in Asia and Europe, as well as its emergence in the Americas. Alphaviruses affect host immunity through cytokines and the interferon response. Understanding alphavirus interactions with both the innate immune system as well as the various cells in the adaptive immune systems is critical to developing effective therapeutics. In this review, we summarize the latest research on alphavirus-host cell interactions, underlying infection mechanisms, and possible treatments.

Exposome and Immunity Training: How Pathogen Exposure Order Influences Innate Immune Cell Lineage Commitment and Function.

Immune memory is a defining characteristic of adaptive immunity, but recent work has shown that the activation of innate immunity can also improve responsiveness in subsequent exposures. This has been coined "trained immunity" and diverges with the perception that the innate immune system is primitive, non-specific, and reacts to novel and recurrent antigen exposures similarly. The "exposome" is the cumulative exposures (diet, exercise, environmental exposure, vaccination, genetics, etc.) an individual has experienced and provides a mechanism for the establishment of immune training or immunotolerance. It is becoming increasingly clear that trained immunity constitutes a delicate balance between the dose, duration, and order of exposures. Upon innate stimuli, trained immunity or tolerance is shaped by epigenetic and metabolic changes that alter hematopoietic stem cell lineage commitment and responses to infection. Due to the immunomodulatory role of the exposome, understanding innate immune training is critical for understanding why some individuals exhibit protective phenotypes while closely related individuals may experience immunotolerant effects (e.g., the order of exposure can result in completely divergent immune responses). Research on the exposome and trained immunity may be leveraged to identify key factors for improving vaccination development, altering inflammatory disease development, and introducing potential new prophylactic treatments, especially for diseases such as COVID-19, which is currently a major health issue for the world. Furthermore, continued exposome research may prevent many deleterious effects caused by immunotolerance that frequently result in host morbidity or mortality.

Atypical chemokine receptor ACKR2-V41A has decreased CCL2 binding, scavenging, and activation, supporting sustained inflammation and increased Alzheimer's disease risk.

A recent genome-wide association study (GWAS) of 59 cerebrospinal fluid (CSF) proteins with a connection to Alzheimer's disease (AD) demonstrated an association between increased levels of chemokine ligand 2 (CCL2) with an atypical chemokine receptor chemokine-binding protein 2 variant V41A (ACKR2-V41A; rs2228467). High levels of CCL2 are associated with increased risk of AD development as well as other inflammatory diseases. In this study we characterized the biological function of the ACKR2-V41A receptor compared to the wild type allele by measuring its ligand binding affinity, CCL2 scavenging efficiency, and cell activation sensitivity. We transfected Chinese hamster ovary cells with plasmids carrying wild type ACKR2 (ACKR2-WT) or the mutant ACKR2-V41A receptor. Binding affinity assays showed that ACKR2-V41A has a lower binding affinity for CCL2 and CCL4 than ACKR2-WT. CCL2 scavenging results aligned with binding affinity assays, with ACKR2-V41A cells scavenging CCL2 with a lower efficiency than ACKR2-WT. Cell activation assays also showed that ACKR2-V41A cells had significantly lower receptor upregulation (ß-Arrestin-dependent signaling pathway) upon stimulation compared to ACKR2-WT cells. These findings provide molecular and biological mechanistic insights into the GWAS association of ACKR2-V41A with increased levels of CCL2 in CSF and possibly other chemokine ligands. Increased CCL2 levels are associated with accelerated cognitive decline and increased risk of AD. Understanding how this atypical chemokine receptor allele increases serum markers of inflammation could lead to novel therapeutic solutions for AD.

Novel monoclonal antibodies against thymidine kinase 1 and their potential use for the immunotargeting of lung, breast and colon cancer cells.

BACKGROUND: Thymidine kinase 1 (TK1) is a pyrimidine salvage pathway enzyme that is up-regulated in malignant tissues and elevated in the serum of cancer patients. While TK1 has been well established as a tumor biomarker, little has been done to explore its potential as a tumor target. Recently, we reported the membrane expression of TK1 on malignant cells, but not on normal cells. This study explores the possible use of monoclonal antibodies for the targeting of membrane associated TK1 in lung, breast, colon and prostate cancer cells. METHODS: We generated and evaluated a panel of monoclonal antibodies against six different epitopes exposed in the tetrameric form of TK1. Antibodies were developed with hybridoma technology and validated with Western blot, siRNA TK1 knockdown, enzyme-linked immunosorbent assay (ELISA) and flow cytometry. The therapeutic potential of the antibodies was evaluated in vitro in antibody-dependent cell-mediated-cytotoxicity (ADCC) experiments. RESULTS: Binding of the antibodies to TK1 was confirmed by Western blot in purified recombinant protein, cancer serum, and cell lysate. After a TK1 knockdown was performed, a reduction of TK1 expression was observed with five antibodies. Using indirect ELISA, we identified 3B2E11, 9C10, 7H2, 3B4, 8G2 among the most sensitive antibodies (LOD = 10.73-66.9 pg/ml). Surface expression of TK1 on the membrane of various cancer cell lines was analyzed with flow cytometry. Antibodies 8G2, 3B4, 7HD and 5F7G11 detected TK1 on the membrane of various cancer cell lines, including lung, prostate, colon and breast. No significant binding was detected on normal lymphocytes. Increased cytolysis of lung (~ 70%. p = 0.0001), breast (~ 70%, p = 0.0461) and colon (~ 50% p = 0.0216) cancer cells by effector cells was observed when anti-TK1 antibodies were added during ADCC experiments. CONCLUSIONS: The antibodies developed showed potential to be used to detect and target TK1 on the membrane of various tumor cells. The targeting of TK1 in malignant cells using monoclonal antibodies may be a feasible approach for the elimination of high TK1 expressing tumor cells.

Hypoxanthine Guanine Phosphoribosyltransferase expression is negatively correlated with immune activity through its regulation of purine synthesis.

INTRODUCTION: The purpose of this study was to examine the effects of elevated Hypoxanthine Guanine Phosphoribosyltransferase (HPRT) on the immune response in the tumor microenvironment. METHODOLOGY: HPRT expression was evaluated in cancer patients and correlated with cytokine expression, survival, and immune cell infiltration. An HPRT knockdown cell line was created to evaluate HPRT impact on purine expression and subsequent purine treatment was administered to immune cells to determine their influence on cell activation. RESULTS: HPRT expression was negatively correlated with the general expression of both pro-inflammatory and anti-inflammatory cytokines. Additionally, HPRT expression was also negatively correlated with the infiltration of immune cell subsets: B-cells, CD4 + T cells, macrophages, neutrophils, and dendritic cells (p < 0.001) and CD8 + T-cells (p < 0.01). When HPRT was knocked down in a Raji cell line, the levels of adenosine were reduced significantly compared to the wild type. When examining the level of Ca2+ influx of Raji compared to the HPRT Raji knockdown cell, there was a significant decrease in calcium influx in the knockdown cells when compared to the wild type cells. This demonstrates that HPRT had a significant impact on overall cell activation and the ability of the cells to properly influx calcium needed for their activation. CONCLUSIONS: We conclude that purine levels significantly reduce immune cell activation in cancer and the upregulation of HPRT in malignant tissue is a contributing factors to the immunosuppressive microenvironment.

Obesity and CD8 T cell metabolism: Implications for anti-tumor immunity and cancer immunotherapy outcomes.

Obesity is an established risk factor for many cancers and has recently been found to alter the efficacy of T cell-based immunotherapies. Currently, however, the effects of obesity on immunometabolism remain unclear. Understanding these associations is critical, given the fact that T cell metabolism is tightly linked to effector function. Thus, any obesity-associated changes in T cell bioenergetics are likely to drive functional changes at the cellular level, alter the metabolome and cytokine/chemokine milieu, and impact cancer immunotherapy outcomes. Here, we provide a brief overview of T cell metabolism in the presence and absence of solid tumor growth and summarize current literature regarding obesity-associated changes in T cell function and bioenergetics. We also discuss recent findings related to the impact of host obesity on cancer immunotherapy outcomes and present potential mechanisms by which T cell metabolism may influence therapeutic efficacy. Finally, we describe promising pharmaceutical therapies that are being investigated for their ability to improve CD8 T cell metabolism and enhance cancer immunotherapy outcomes in patients, regardless of their obesity status.

Genomics Education in the Era of Personal Genomics: Academic, Professional, and Public Considerations.

Since the completion of the Human Genome Project in 2003, genomic sequencing has become a prominent tool used by diverse disciplines in modern science. In the past 20 years, the cost of genomic sequencing has decreased exponentially, making it affordable and accessible. Bioinformatic and biological studies have produced significant scientific breakthroughs using the wealth of genomic information now available. Alongside the scientific benefit of genomics, companies offer direct-to-consumer genetic testing which provide health, trait, and ancestry information to the public. A key area that must be addressed is education about what conclusions can be made from this genomic information and integrating genomic education with foundational genetic principles already taught in academic settings. The promise of personal genomics providing disease treatment is exciting, but many challenges remain to validate genomic predictions and diagnostic correlations. Ethical and societal concerns must also be addressed regarding how personal genomic information is used. This genomics revolution provides a powerful opportunity to educate students, clinicians, and the public on scientific and ethical issues in a personal way to increase learning. In this review, we discuss the influence of personal genomics in society and focus on the importance and benefits of genomics education in the classroom, clinics, and the public and explore the potential consequences of personal genomic education.

CD4 Inhibits Helper T Cell Activation at Lower Affinity Threshold for Full-Length T Cell Receptors Than Single Chain Signaling Constructs.

CD4+ T cells are crucial for effective repression and elimination of cancer cells. Despite a paucity of CD4+ T cell receptor (TCR) clinical studies, CD4+ T cells are primed to become important therapeutics as they help circumvent tumor antigen escape and guide multifactorial immune responses. However, because CD8+ T cells directly kill tumor cells, most research has focused on the attributes of CD8+ TCRs. Less is known about how TCR affinity and CD4 expression affect CD4+ T cell activation in full length TCR (flTCR) and TCR single chain signaling (TCR-SCS) formats. Here, we generated an affinity panel of TCRs from CD4+ T cells and expressed them in flTCR and three TCR-SCS formats modeled after chimeric antigen receptors (CARs) to understand the contributions of TCR-pMHCII affinity, TCR format, and coreceptor CD4 interactions on CD4+ T cell activation. Strikingly, the coreceptor CD4 inhibited intermediate and high affinity TCR-construct activation by Lck-dependent and -independent mechanisms. These inhibition mechanisms had unique affinity thresholds dependent on the TCR format. Intracellular construct formats affected the tetramer staining for each TCR as well as IL-2 production. IL-2 production was promoted by increased TCR-pMHCII affinity and the flTCR format. Thus, CD4+ T cell therapy development should consider TCR affinity, CD4 expression, and construct format.

A full semester flow cytometry course improves graduate and undergraduate student confidence.

Flow cytometry is a versatile and high throughput technique for rapid and efficient biological testing. It requires a high level of conceptual, technical, and analytical skills to properly design experiments, effectively operate flow cytometry machines, and analyze the data. A lack of training and development of any of these three skills can result in underutilization and improper use of flow cytometric machines that can impede research progress. Often students develop these conceptual, technical, and analysis skills from trial and error, but many students either do not use this powerful flow cytometry technology, use it improperly or ineffectively, or give up using it without proper training and support. Here we report on a course which teaches flow cytometry skills to undergraduate and graduate students. The design of this course is unique in that it teaches conceptual, technical, and analytical skills related to flow cytometry in a full semester format. Undergraduate and graduate students reported significant increases in their confidence levels over the course of the semester. Here we provide our findings and resources for others who may want to implement a similar course.

Associations between evaporative cooling and dust-mite allergens, endotoxins, and ß-(1 → 3)-d-glucans in house dust: A study of low-income homes.

Recent work suggests that evaporative coolers increase the level and diversity of bioaerosols, but this association remains understudied in low-income homes. We conducted a cross-sectional study of metropolitan, low-income homes in Utah with evaporative coolers (n = 20) and central air conditioners (n = 28). Dust samples (N = 147) were collected from four locations in each home and analyzed for dust-mite allergens Der p1 and Der f1, endotoxins, and ß-(1 â†’ 3)-d-glucans. In all sample locations combined, Der p1 or Der f1 was significantly higher in evaporative cooler versus central air conditioning homes (OR = 2.29, 95% CI = 1.05-4.98). Endotoxin concentration was significantly higher in evaporative cooler versus central air conditioning homes in furniture (geometric mean (GM) = 8.05 vs 2.85 EU/mg, P < .01) and all samples combined (GM = 3.60 vs 1.29 EU/mg, P = .03). ß-(1 â†’ 3)-d-glucan concentration and surface loads were significantly higher in evaporative cooler versus central air conditioning homes in all four sample locations and all samples combined (P < .01). Our study suggests that low-income, evaporative cooled homes have higher levels of immunologically important bioaerosols than central air-conditioned homes in dry climates, warranting studies on health implications and other exposed populations.

Correction to: Membrane expression of thymidine kinase 1 and potential clinical relevance in lung, breast, and colorectal malignancies.

[This corrects the article DOI: 10.1186/s12935-018-0633-9.].