Karyotypic stasis and swarming influenced the evolution of viral tolerance in a species-rich bat radiation.

The emergence of COVID-19 and severe acute respiratory syndrome (SARS) has prioritized understanding bats' viral tolerance. Myotis bats are exceptionally species rich and have evolved viral tolerance. They also exhibit swarming, a cryptic behavior where large, multi-species assemblages gather for mating, which has been hypothesized to promote interspecific hybridization. To resolve the coevolution of genome architecture and their unusual antiviral tolerance, we undertook a phylogenomic analysis of 60 Old World Myotis genomes. We demonstrate an extensive history of introgressive hybridization that has replaced the species phylogeny across 17%-93% of the genome except for pericentromeric regions of macrochromosomes. Introgression tracts were enriched on microchromosome regions containing key antiviral pathway genes overexpressed during viral challenge experiments. Together, these results suggest that the unusual Myotis karyotype may have evolved to selectively position immune-related genes in high recombining genomic regions prone to introgression of divergent alleles, including a diversity of interleukin loci responsible for the release of pro-inflammatory cytokines.

Immunonutrition: facilitating mucosal immune response in teleost intestine with amino acids through oxidant-antioxidant balance.

Comparative animal models generate fundamental scientific knowledge of immune responses. However, these studies typically are conducted in mammals because of their biochemical and physiological similarity to humans. Presently, there has been an interest in using teleost fish models to study intestinal immunology, particularly intestinal mucosa immune response. Instead of targeting the pathogen itself, a preferred approach for managing fish health is through nutrient supplementation, as it is noninvasive and less labor intensive than vaccine administrations while still modulating immune properties. Amino acids (AAs) regulate metabolic processes, oxidant-antioxidant balance, and physiological requirements to improve immune response. Thus, nutritionists can develop sustainable aquafeeds through AA supplementation to promote specific immune responses, including the intestinal mucosa immune system. We propose the use of dietary supplementation with functional AAs to improve immune response by discussing teleost fish immunology within the intestine and explore how oxidative burst is used as an immune defense mechanism. We evaluate immune components and immune responses in the intestine that use oxidant-antioxidant balance through potential selection of AAs and their metabolites to improve mucosal immune capacity and gut integrity. AAs are effective modulators of teleost gut immunity through oxidant-antioxidant balance. To incorporate nutrition as an immunoregulatory means in teleost, we must obtain more tools including genomic, proteomic, nutrition, immunology, and macrobiotic and metabonomic analyses, so that future studies can provide a more holistic understanding of the mucosal immune system in fish.

Functional interferon-epsilon gene polymorphisms and sexually transmitted infections of the endometrium.

PROBLEM: Interferon-epsilon (IFNε) is the only type I IFN constitutively expressed in the female reproductive tract and fluctuates across the menstrual cycle in humans. Mouse models show that IFNε protects against Chlamydia trachomatis, Herpes Simplex Virus, HIV, and Zika in mice, but human studies are limited. Bacterial sexually transmitted infections (STI) can ascend to the upper genital tract and cause pelvic inflammatory disease (PID) and subsequent infertility. However, the host immunological mechanisms that play a role in the ascension and infection of the endometrium in individuals with clinically suspected PID are not elucidated. METHOD OF STUDY: This pilot investigation determined if IFNε gene variants are associated with bacterial vaginosis (BV) and endometrial infection with C. trachomatis, Neisseria gonorrhoeae, and Mycoplasma genitalium using biospecimens from 154 self-report Black individuals who participated in the PID Evaluation and Clinical Health (PEACH) study. RESULTS: The T allele for rs2039381 was associated with endometrial STI infection (OR 2.7, 95% CI: 1.0-7.1) and the C allele for rs1125488 was inversely associated with BV (OR: .2, 95% CI: .05-.8). CONCLUSIONS: Few studies have examined IFNε gene variants, our study raises the possibility that IFNε gene variants may be a potential host contributor to STI pathogenesis.

Analysis of shark NCR3 family genes reveals primordial features of vertebrate NKp30.

Natural killer (NK) cells play major roles in innate immunity against viruses and cancer. Natural killer receptors (NKR) expressed by NK cells recognize foreign- or self-ligands on infected and transformed cells as well as healthy cells. NKR genes are the most rapidly evolving loci in vertebrates, and it is generally difficult to detect orthologues in different taxa. The unique exception is NKp30, an activating NKR in mammals that binds to the self-ligand B7H6. The NKp30-encoding gene, NCR3, has been found in most vertebrates including sharks, the oldest vertebrates with human-type adaptive immunity. NCR3 has a special, non-rearranging VJ-type immunoglobulin superfamily (IgSF) domain that predates the emergence of the rearranging antigen receptors. Herein we show that NCR3 loci are linked to the shark major histocompatibility complex (MHC), proving NCR3's primordial association with the MHC. We identified eight subtypes of differentially expressed highly divergent shark NCR3 family genes. Using in situ hybridization, we detected one subtype, NS344823, to be expressed by predominantly single cells outside of splenic B cell zones. The expression by non-B cells was also confirmed by PCR in peripheral blood lymphocytes. Surprisingly, high expression of NS344823 was detected in the thymic cortex, demonstrating NS344823 expression in developing T cells. Finally, we show for the first time that shark T cells are found as single cells or in small clusters in the splenic red pulp, also unassociated with the large B cell follicles we previously identified.

Using PacBio SMRT data for identification of class I MHC alleles in a wildlife species, Zalophus californianus (California sea lion).

High allelic polymorphism and association with disease susceptibility has made the genes encoding major histocompatibility complex (MHC) antigen presentation molecules in humans, domesticated animals, and wildlife species of wide interest to ecologists, evolutionary biologists, and health specialists. The often multifaceted polygenism and extreme polymorphism of this immunogenetic system have made it especially difficult to characterize in non-model species. Here we compare and contrast the workflows of traditional Sanger sequencing of plasmid-cloned amplicons to Pacific Biosciences SMRT circular consensus sequencing (CCS) in their ability to capture alleles of MHC class I in a wildlife species where characterization of these genes was absent. We assessed two California sea lions (Zalophus californianus), a species suffering from a high prevalence of an aggressive cancer associated with a sexually transmitted gamma herpesvirus. In this pilot study, SMRT CCS proved superior in identifying more alleles from each animal than the more laborious plasmid cloning/Sanger workflow (12:7, 10:7), and no alleles were identified with the cloning/Sanger approach that were not identified by SMRT CCS. We discuss the advantages and disadvantages of each approach including cost, allele rarefaction, and sequence fidelity.

Molecular characterization and expression analysis of the chicken-type and goose-type lysozymes from totoaba (Totoaba macdonaldi).

Lysozymes play a key role in innate immune response to bacterial pathogens, catalyzing the hydrolysis of the peptidoglycan layer of bacterial cell walls. In this study, the genes encoding the c-type (TmLyzc) and g-type (TmLyzg) lysozymes from Totoaba macdonaldi were cloned and characterized. The cDNA sequences of TmLyzg and TmLyzc were 582 and 432 bp, encoding polypeptides of 193 and 143 amino acids, respectively. Amino acid sequences of these lysozymes shared high identity (60-90%) with their counterparts of other teleosts and showed conserved functional-structural signatures of the lysozyme superfamily. Phylogenetic analysis indicated a close relationship with their vertebrate homologues but distinct evolutionary paths for each lysozyme. Expression analysis by qRT-PCR revealed that TmLyzc was expressed in stomach and pyloric caeca, while TmLyzg was highly expressed in stomach and heart. These results suggest that both lysozymes play important roles in defense of totoaba against bacterial infections or as digestive enzyme.

Deimination Protein Profiles in Alligator mississippiensis Reveal Plasma and Extracellular Vesicle-Specific Signatures Relating to Immunity, Metabolic Function, and Gene Regulation.

Alligators are crocodilians and among few species that endured the Cretaceous-Paleogene extinction event. With long life spans, low metabolic rates, unusual immunological characteristics, including strong antibacterial and antiviral ability, and cancer resistance, crocodilians may hold information for molecular pathways underlying such physiological traits. Peptidylarginine deiminases (PADs) are a group of calcium-activated enzymes that cause posttranslational protein deimination/citrullination in a range of target proteins contributing to protein moonlighting functions in health and disease. PADs are phylogenetically conserved and are also a key regulator of extracellular vesicle (EV) release, a critical part of cellular communication. As little is known about PAD-mediated mechanisms in reptile immunology, this study was aimed at profiling EVs and protein deimination in Alligator mississippiensis. Alligator plasma EVs were found to be polydispersed in a 50-400-nm size range. Key immune, metabolic, and gene regulatory proteins were identified to be posttranslationally deiminated in plasma and plasma EVs, with some overlapping hits, while some were unique to either plasma or plasma EVs. In whole plasma, 112 target proteins were identified to be deiminated, while 77 proteins were found as deiminated protein hits in plasma EVs, whereof 31 were specific for EVs only, including proteins specific for gene regulatory functions (e.g., histones). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed KEGG pathways specific to deiminated proteins in whole plasma related to adipocytokine signaling, while KEGG pathways of deiminated proteins specific to EVs included ribosome, biosynthesis of amino acids, and glycolysis/gluconeogenesis pathways as well as core histones. This highlights roles for EV-mediated export of deiminated protein cargo with roles in metabolism and gene regulation, also related to cancer. The identification of posttranslational deimination and EV-mediated communication in alligator plasma revealed here contributes to current understanding of protein moonlighting functions and EV-mediated communication in these ancient reptiles, providing novel insight into their unusual immune systems and physiological traits. In addition, our findings may shed light on pathways underlying cancer resistance, antibacterial and antiviral resistance, with translatable value to human pathologies.

Post-Translational Protein Deimination Signatures in Serum and Serum-Extracellular Vesicles of Bos taurus Reveal Immune, Anti-Pathogenic, Anti-Viral, Metabolic and Cancer-Related Pathways for Deimination.

The bovine immune system is known for its unusual traits relating to immunoglobulin and antiviral responses. Peptidylarginine deiminases (PADs) are phylogenetically conserved enzymes that cause post-translational deimination, contributing to protein moonlighting in health and disease. PADs also regulate extracellular vesicle (EV) release, forming a critical part of cellular communication. As PAD-mediated mechanisms in bovine immunology and physiology remain to be investigated, this study profiled deimination signatures in serum and serum-EVs in Bos taurus. Bos EVs were poly-dispersed in a 70-500 nm size range and showed differences in deiminated protein cargo, compared with whole sera. Key immune, metabolic and gene regulatory proteins were identified to be post-translationally deiminated with some overlapping hits in sera and EVs (e.g., immunoglobulins), while some were unique to either serum or serum-EVs (e.g., histones). Protein-protein interaction network analysis of deiminated proteins revealed KEGG pathways common for serum and serum-EVs, including complement and coagulation cascades, viral infection (enveloped viruses), viral myocarditis, bacterial and parasitic infections, autoimmune disease, immunodeficiency intestinal IgA production, B-cell receptor signalling, natural killer cell mediated cytotoxicity, platelet activation and hematopoiesis, alongside metabolic pathways including ferroptosis, vitamin digestion and absorption, cholesterol metabolism and mineral absorption. KEGG pathways specific to EVs related to HIF-1 signalling, oestrogen signalling and biosynthesis of amino acids. KEGG pathways specific for serum only, related to Epstein-Barr virus infection, transcription mis-regulation in cancer, bladder cancer, Rap1 signalling pathway, calcium signalling pathway and ECM-receptor interaction. This indicates differences in physiological and pathological pathways for deiminated proteins in serum-EVs, compared with serum. Our findings may shed light on pathways underlying a number of pathological and anti-pathogenic (viral, bacterial, parasitic) pathways, with putative translatable value to human pathologies, zoonotic diseases and development of therapies for infections, including anti-viral therapies.

A Broad Role for Cysteines in Bovine Antibody Diversity.

Ab diversity in most vertebrates results from the assortment of amino acid side chains on CDR loops formed through V(D)J recombination. Cows (Bos taurus) have a low combinatorial diversity potential because of a small number of highly homologous V, D, and J gene segments. Despite this, a subset of the Ab repertoire (∼10%) contains exceptionally long CDR H chain (HC) 3 (H3) regions with a rich diversity of cysteines and disulfide-bonded loops that diversify through a single V-D-J recombination event followed by massive somatic hypermutation. However, the much larger portion of the repertoire, encoding shorter CDR H3s, has not been examined in detail. Analysis of germline gene segments reveals noncanonical cysteines in the HC V regions and significant cysteine content in the HC D regions. Deep sequencing analysis of naturally occurring shorter CDR H3 (<40 aa) Ab genes shows that HC V and HC D regions preferentially combine to form a functional gene with an even number of total cysteines in the final V region, suggesting that disulfide bonds contribute to diversity not only in ultralong CDR H3 bovine Abs but in shorter CDR H3 bovine Abs as well. In addition to germline "hard-coded" cysteines, the bovine Ab repertoire can produce additional cysteine codons through somatic hypermutation, further diversifying the repertoire. Given the limited combinatorial diversity at the bovine Ig loci, this helps to explain how diversity is created in shorter CDR H3 Abs and potentially provides novel structural paratopes in bovine Ab combining sites.

Refining In Vitro Toxicity Models: Comparing Baseline Characteristics of Lung Cell Types.

There is an ever-evolving need in the field of in vitro toxicology to improve the quality of experimental design, ie, from ill-defined cell cultures to well-characterized cytotoxicological models. This evolution is especially important as environmental health scientists begin to rely more heavily on cell culture models in pulmonary toxicology studies. The research presented in this study analyzes the differences and similarities of cells derived from two different depths of the human lung with varying phenotypes. We compared cell cycle and antioxidant-related mRNA and protein concentrations of primary, transformed, and cancer-derived cell lines from the upper and lower airways. In all, six of the most commonly used cell lines reported in in vitro toxicology research papers were included in this study (ie, PTBE, BEAS-2B, A549, PSAE, Met-5A, and Calu-3). Comparison of cell characteristics was accomplished through molecular biology (q-PCR, ELISA, and flow cytometry) and microscopy (phase and fluorescence) techniques as well as cellular oxidative stress endpoint analyses. After comparing the responses of each cell type using statistical analyses, results confirmed significant differences in background levels of cell cycle regulators, inherent antioxidant capacity, pro-inflammatory status, and differential toxicological responses. The analyzed data improve our understanding of the cell characteristics, and in turn, aids in more accurate interpretation of toxicological results. Our conclusions suggest that in vitro toxicology studies should include a detailed cell characterization component in published papers.

Immunogenetic factors driving formation of ultralong VH CDR3 in Bos taurus antibodies.

The antibody repertoire of Bos taurus is characterized by a subset of variable heavy (VH) chain regions with ultralong third complementarity determining regions (CDR3) which, compared to other species, can provide a potent response to challenging antigens like HIV env. These unusual CDR3 can range to over seventy highly diverse amino acids in length and form unique ß-ribbon 'stalk' and disulfide bonded 'knob' structures, far from the typical antigen binding site. The genetic components and processes for forming these unusual cattle antibody VH CDR3 are not well understood. Here we analyze sequences of Bos taurus antibody VH domains and find that the subset with ultralong CDR3 exclusively uses a single variable gene, IGHV1-7 (VHBUL) rearranged to the longest diversity gene, IGHD8-2. An eight nucleotide duplication at the 3' end of IGHV1-7 encodes a longer V-region producing an extended F ß-strand that contributes to the stalk in a rearranged CDR3. A low amino acid variability was observed in CDR1 and CDR2, suggesting that antigen binding for this subset most likely only depends on the CDR3. Importantly a novel, potentially AID mediated, deletional diversification mechanism of the B. taurus VH ultralong CDR3 knob was discovered, in which interior codons of the IGHD8-2 region are removed while maintaining integral structural components of the knob and descending strand of the stalk in place. These deletions serve to further diversify cysteine positions, and thus disulfide bonded loops. Hence, both germline and somatic genetic factors and processes appear to be involved in diversification of this structurally unusual cattle VH ultralong CDR3 repertoire.

"Double-duty" conventional dendritic cells in the amphibian Xenopus as the prototype for antigen presentation to B cells.

Two populations of dendritic cells (DCs) are found in mammals, one derived from hematopoietic precursors (conventional/cDC), and another derived from mesenchymal precursors, the follicular DC (FDC); the latter is specialized for antigen presentation to B cells, and has only been definitively demonstrated in mammals. Both cDC and FDC are necessary for induction of germinal centers (GC) and GC-dependent class switch recombination (CSR) and somatic hypermutation (SHM). We demonstrate that in Xenopus, an amphibian in which immunoglobulin CSR and SHM occur without GC formation, a single type of DC has properties of both cDC and FDC, including high expression of MHC class II for the former and display of native antigen at the cell surface for the latter. Our data confirm that the advent of FDC functionality preceded emergence of bona fide FDC, which was in turn crucial for the development of GC formation and efficient affinity maturation in mammals.

Rapid elicitation of broadly neutralizing antibodies to HIV by immunization in cows.

No immunogen to date has reliably elicited broadly neutralizing antibodies to HIV in humans or animal models. Advances in the design of immunogens that antigenically mimic the HIV envelope glycoprotein (Env), such as the soluble cleaved trimer BG505 SOSIP, have improved the elicitation of potent isolate-specific antibody responses in rabbits and macaques, but so far failed to induce broadly neutralizing antibodies. One possible reason for this failure is that the relevant antibody repertoires are poorly suited to target the conserved epitope regions on Env, which are somewhat occluded relative to the exposed variable epitopes. Here, to test this hypothesis, we immunized four cows with BG505 SOSIP. The antibody repertoire of cows contains long third heavy chain complementary determining regions (HCDR3) with an ultralong subset that can reach more than 70 amino acids in length. Remarkably, BG505 SOSIP immunization resulted in rapid elicitation of broad and potent serum antibody responses in all four cows. Longitudinal serum analysis for one cow showed the development of neutralization breadth (20%, n = 117 cross-clade isolates) in 42 days and 96% breadth (n = 117) at 381 days. A monoclonal antibody isolated from this cow harboured an ultralong HCDR3 of 60 amino acids and neutralized 72% of cross-clade isolates (n = 117) with a potent median IC50 of 0.028 µg ml-1. Breadth was elicited with a single trimer immunogen and did not require additional envelope diversity. Immunization of cows may provide an avenue to rapidly generate antibody prophylactics and therapeutics to address disease agents that have evolved to avoid human antibody responses.

What the shark immune system can and cannot provide for the expanding design landscape of immunotherapy.

INTRODUCTION: Sharks have successfully lived in marine ecosystems, often atop food chains as apex predators, for nearly one and a half billion years. Throughout this period they have benefitted from an immune system with the same fundamental components found in terrestrial vertebrates like man. Additionally, sharks have some rather extraordinary immune mechanisms which mammals lack. AREAS COVERED: In this review the author briefly orients the reader to sharks, their adaptive immunity, and their important phylogenetic position in comparative immunology. The author also differentiates some of the myths from facts concerning these animals, their cartilage, and cancer. From thereon, the author explores some of the more remarkable capabilities and products of shark lymphocytes. Sharks have an isotype of light chain-less antibodies that are useful tools in molecular biology and are moving towards translational use in the clinic. These special antibodies are just one of the several tricks of shark lymphocyte antigen receptor systems. EXPERT OPINION: While shark cartilage has not helped oncology patients, shark immunoglobulins and T cell receptors do offer exciting novel possibilities for immunotherapeutics. Much of the clinical immunology developmental pipeline has turned from traditional vaccines to passively delivered monoclonal antibody-based drugs for targeted depletion, activation, blocking and immunomodulation. The immunogenetic tools of shark lymphocytes, battle-tested since the dawn of our adaptive immune system, are well poised to expand the design landscape for the next generation of immunotherapy products.

Reshaping antibody diversity.

Some species mount a robust antibody response despite having limited genome-encoded combinatorial diversity potential. Cows are unusual in having exceptionally long CDR H3 loops and few V regions, but the mechanism for creating diversity is not understood. Deep sequencing reveals that ultralong CDR H3s contain a remarkable complexity of cysteines, suggesting that disulfide-bonded minidomains may arise during repertoire development. Indeed, crystal structures of two cow antibodies reveal that these CDR H3s form a very unusual architecture composed of a ß strand "stalk" that supports a structurally diverse, disulfide-bonded "knob" domain. Diversity arises from somatic hypermutation of an ultralong DH with a severe codon bias toward mutation to cysteine. These unusual antibodies can be elicited to recognize defined antigens through the knob domain. Thus, the bovine immune system produces an antibody repertoire composed of ultralong CDR H3s that fold into a diversity of minidomains generated through combinations of somatically generated disulfides.

Distinct immunomodulatory effects of a panel of nanomaterials in human dermal fibroblasts.

There are many efforts in understanding the effects of nanoparticles on cell viability and metabolism, however, not much is known regarding the distinct molecular mechanisms of inflammation and cellular stress using low dosing concentrations. To address this gap in the literature, we utilized a novel experimental design that specifically probes the effects of a panel of commonly studied engineered nanomaterials along immunomodulatory pathways, including NF-κB. The panel of particles selected for this study included quantum dot nanocrystals, titanium dioxide, hydroxylated fullerenes, and silver nanoparticles. Cell viability, antioxidant activity, select messenger RNA, and protein modulation were studied in primary human dermal fibroblasts (HDF) and NF-κB knockdown HDF cells. Inflammatory and non-inflammatory immune responses were measured using protein and real-time PCR array analysis from HDF cells exposed to sub-lethal concentrations of nanoparticles. Differences in cellular response to nanoparticles in protein and antioxidant experiments were evident in NF-κB knockdown cells. The methods used in the study, along with the resultant data sets, serve as a potential model for studying the complex pathway-specific biochemical responses in cell and tissue systems associated with nanoparticle exposures.

Expression of the inflammatory chemokines CCL2, CCL5 and CXCL2 and the receptors CCR1-3 and CXCR2 in T lymphocytes from mammary tumor-bearing mice.

Chemokines and their receptors have been studied in several solid tumor models as mediators of inflammation. In turn, inflammation has been implicated in the promotion and progression of tumors, and as such, chemokines have been proposed as novel molecular targets for chemotherapy. While the expression of these molecules has been described in tumor cells, endothelial cells, macrophages and neutrophils, less attention has been paid to the expression profile of these molecules by T lymphocytes in the periphery or infiltrating the tumor. Using the D1-DMBA-3 murine mammary adenocarcinoma model, we aimed to better characterize the differential expression of chemokines and/or their receptors in the host and in the tumor microenvironment, and specifically, in the T cells of tumor-bearing mice compared to normal control animals. We found that T lymphocytes from tumor-bearing mice express the pro-inflammatory chemokines, CCL2, CCL5 and CXCL2, as well as the chemokine receptors, CCR1, CCR2, CCR3 and CXCR2.

Proteasome, transporter associated with antigen processing, and class I genes in the nurse shark Ginglymostoma cirratum: evidence for a stable class I region and MHC haplotype lineages.

Cartilaginous fish (e.g., sharks) are derived from the oldest vertebrate ancestor having an adaptive immune system, and thus are key models for examining MHC evolution. Previously, family studies in two shark species showed that classical class I (UAA) and class II genes are genetically linked. In this study, we show that proteasome genes LMP2 and LMP7, shark-specific LMP7-like, and the TAP1/2 genes are linked to class I/II. Functional LMP7 and LMP7-like genes, as well as multiple LMP2 genes or gene fragments, are found only in some sharks, suggesting that different sets of peptides might be generated depending upon inherited MHC haplotypes. Cosmid clones bearing the MHC-linked classical class I genes were isolated and shown to contain proteasome gene fragments. A non-MHC-linked LMP7 gene also was identified on another cosmid, but only two exons of this gene were detected, closely linked to a class I pseudogene (UAA-NC2); this region probably resulted from a recent duplication and translocation from the functional MHC. Tight linkage of proteasome and class I genes, in comparison with gene organizations of other vertebrates, suggests a primordial MHC organization. Another nonclassical class I gene (UAA-NC1) was detected that is linked neither to MHC nor to UAA-NC2; its high level of sequence similarity to UAA suggests that UAA-NC1 also was recently derived from UAA and translocated from MHC. These data further support the principle of a primordial class I region with few class I genes. Finally, multiple paternities in one family were demonstrated, with potential segregation distortions.