Plasma dynamics of neutrophil extracellular traps and cell-free DNA in septic and non-septic vasoplegic shock: a prospective comparative observational cohort study.

BACKGROUND: The association between neutrophil extracellular traps (NETs) and the requirement for vasopressor and inotropic support in vasoplegic shock is unclear. This study aimed to investigate the dynamics of plasma levels of NETs and cell-free DNA (cfDNA) up to 48 hours after the admission to the intensive care unit (ICU) for management of vasoplegic shock of infectious (SEPSIS) or non-infectious (following cardiac surgery, CARDIAC) origin. METHODS: Prospective, observational study of NETs and cfDNA plasma levels at 0H (admission) and then at 12H, 24H and 48H in SEPSIS and CARDIAC patients. The Vasopressor Inotropic Score (VIS), the Sequential Organ Failure Assessment (SOFA) score and time spent with invasive ventilation, in ICU and in hospital were recorded. Associations between NETs/cfDNA and VIS and SOFA were analysed by Spearman's correlation (rho), and between NETs/cfDNA and ventilation/ICU/hospitalisation times by generalised linear regression. RESULTS: Both NETs and cfDNA remained elevated over 48 hours in SEPSIS (n = 46) and CARDIAC (n = 30) patients, with time weighted average concentrations greatest in SEPSIS (NETs median difference 0.06 [0.02-0.11], p = 0.005; cfDNA median difference 0.48 [0.20-1.02], p < 0.001). The VIS correlated to NETs (rho = 0.3-0.60 in SEPSIS, p < 0.01, rho = 0.36-0.57 in CARDIAC, p ≤ 0.01) and cfDNA (rho = 0.40-0.56 in SEPSIS, p < 0.01, rho = 0.38-0.47 in CARDIAC, p < 0.05). NETs correlated with SOFA. Neither NETs nor cfDNA were independently associated with ventilator/ICU/hospitalisation times. CONCLUSION: Plasma levels of NETs and cfDNA correlated with the dose of vasopressors and inotropes administered over 48 hours in patients with vasoplegic shock from sepsis or following cardiac surgery. NETs levels also correlated with organ dysfunction. These findings suggest that similar mechanisms involving release of NETs are involved in the pathophysiology of vasoplegic shock irrespective of an infectious or non-infectious etiology.

IgG exacerbates genital chlamydial pathology in females by enhancing pathogenic CD8+ T cell responses.

Chlamydia trachomatis infections are an important sexually transmitted infection that can lead to inflammation, scarring and hydrosalpinx/infertility. However, infections are commonly clinically asymptomatic and do not receive treatment. The underlying cause of asymptomatic immunopathology remains unknown. Here, we demonstrate that IgG produced during male infection enhanced the incidence of immunopathology and infertility in females. Human endocervical cells expressing the neonatal Fc Receptor (FcRn) increased translocation of human IgG-opsonized C. trachomatis. Using total IgG purified from infected male mice, we opsonized C. muridarum and then infected female mice, mimicking sexual transmission. Following infection, IgG-opsonized Chlamydia was found to transcytose the epithelial barrier in the uterus, where it was phagocytosed by antigen-presenting cells (APCs) and trafficked to the draining lymph nodes. APCs then expanded both CD4+ and CD8+ T cell populations and caused significantly more infertility in female mice infected with non-opsonized Chlamydia. Enhanced phagocytosis of IgG-opsonized Chlamydia significantly increased pro-inflammatory signalling and T cell proliferation. As IgG is transcytosed by FcRn, we utilized FcRn-/- mice and observed that shedding kinetics of Chlamydia were only affected in FcRn-/- mice infected with IgG-opsonized Chlamydia. Depletion of CD8+ T cells in FcRn-/- mice lead to a significant reduction in the incidence of infertility. Taken together, these data demonstrate that IgG seroconversion during male infection can amplify female immunopathology, dependent on FcRn transcytosis, APC differentiation and enhanced CD8 T cell responses.

Targeted therapy, immunotherapy, and small molecules and peptidomimetics as emerging immunoregulatory agents for melanoma.

Primary cutaneous melanoma is the most lethal of all skin neoplasms and its incidence is increasing. Clinical management of advanced melanoma in the last decade has been revolutionised by the availability of immunotherapies and targeted therapies, used alone and in combination. This article summarizes advances in the treatment of late-stage melanoma including use of protein kinase inhibitors, antibody-based immune checkpoint inhibitors, adoptive immunotherapy, vaccines and more recently, small molecules and peptidomimetics as emerging immunoregulatory agents.

Antagonistic interactions safeguard mitotic propagation of genetic and epigenetic information in zebrafish.

The stability of cellular phenotypes in developing organisms depends on error-free transmission of epigenetic and genetic information during mitosis. Methylation of cytosine residues in genomic DNA is a key epigenetic mark that modulates gene expression and prevents genome instability. Here, we report on a genetic test of the relationship between DNA replication and methylation in the context of the developing vertebrate organism instead of cell lines. Our analysis is based on the identification of hypomorphic alleles of dnmt1, encoding the DNA maintenance methylase Dnmt1, and pole1, encoding the catalytic subunit of leading-strand DNA polymerase epsilon holoenzyme (Pole). Homozygous dnmt1 mutants exhibit genome-wide DNA hypomethylation, whereas the pole1 mutation is associated with increased DNA methylation levels. In dnmt1/pole1 double-mutant zebrafish larvae, DNA methylation levels are restored to near normal values, associated with partial rescue of mutant-associated transcriptional changes and phenotypes. Hence, a balancing antagonism between DNA replication and maintenance methylation buffers against replicative errors contributing to the robustness of vertebrate development.

Lymphocyte pathway analysis using naturally lymphocyte-deficient fish.

Comparative phylogenetic analyses are of potential value to establish the essential components of genetic networks underlying physiological traits. For species that naturally lack particular lymphocyte lineages, we show here that this strategy readily distinguishes trait-specific actors from pleiotropic components of the genetic network governing lymphocyte differentiation. Previously, three of the four members of the DNA polymerase X family have been implicated in the junctional diversification process during the somatic assembly of antigen receptors. Our phylogenetic analysis indicates that the presence of terminal deoxynucleotidyl transferase is strictly associated with the facility of V(D)J recombination, whereas PolL and PolM genes are retained even in species lacking Rag-mediated somatic diversification of antigen receptor genes.

Immune Checkpoint Inhibitors, Small-Molecule Immunotherapies and the Emerging Role of Neutrophil Extracellular Traps in Therapeutic Strategies for Head and Neck Cancer.

Immune checkpoint inhibitor (ICI) therapy has revolutionized the treatment of many cancer types, including head and neck cancers (HNC). When checkpoint and partner proteins bind, these send an "off" signal to T cells, which prevents the immune system from destroying tumor cells. However, in HNC, and indeed many other cancers, more people do not respond and/or suffer from toxic effects than those who do respond. Hence, newer, more effective approaches are needed. The challenge to durable therapy lies in a deeper understanding of the complex interactions between immune cells, tumor cells and the tumor microenvironment. This will help develop therapies that promote lasting tumorlysis by overcoming T-cell exhaustion. Here we explore the strengths and limitations of current ICI therapy in head and neck squamous cell carcinoma (HNSCC). We also review emerging small-molecule immunotherapies and the growing promise of neutrophil extracellular traps in controlling tumor progression and metastasis.

Flubendazole inhibits PD-1 and suppresses melanoma growth in immunocompetent mice.

BACKGROUND: Immune checkpoint inhibitor therapy has revolutionized the clinical management of a diverse range of cancer types, including advanced cutaneous melanoma. While immunotherapy targeting the PD-1/PD-L1 system has become standard of care, overall response rates remain unsatisfactory for most patients and there are no approved small molecule inhibitors of the PD-1/PD-L1 system. Flubendazole (FLU) is an anthelmintic that has been used to treat worm infections in humans and animals for decades. METHODS: Here we tested the anti-cancer activity of systemically delivered FLU with suppression of PD-1 in immunocompetent mice. RESULTS: In C57BL/6J mice bearing subcutaneous B16F10 melanoma, FLU reduced both tumor growth and PD-1 protein levels without affecting levels of PD-L1. FLU's suppression of PD-1 was accompanied by increased CD3+ T cell infiltration. Western blotting with extracts from human Jurkat T cells showed that FLU inhibited PD-1 protein expression, findings confirmed by flow cytometry. To gain mechanistic insights on FLU's ability to suppress PD-1 protein levels, we performed bulk RNA sequencing on extracts of Jurkat T cells exposed to the benzimidazole for 4 h. From a pool of 14,475 genes there were 1218 differentially-expressed genes; 687 with increased expression and 531 with decreased expression. Among the genes induced by FLU was the AP-1 family member, JUN and surprisingly, pdcd1. KEGG pathway analysis showed FLU up-regulated genes over-represented in multiple pathways (p < 0.01), the top hit being amoebiasis. FLU also affected the expression of genes in cancer-associated pathways, both through down-regulation and up-regulation. Gene set enrichment analysis revealed a large number of immunological signature gene sets correlated with FLU treatment, including gene sets associated with T cell differentiation, proliferation and function. The AP-1 inhibitor T5224 rescued PD-1 protein expression from inhibition by FLU. CONCLUSION: This study is the first to show that FLU can inhibit melanoma growth with PD-1 suppression in immunocompetent mice.

Origin and evolutionary malleability of T cell receptor α diversity.

Lymphocytes of vertebrate adaptive immune systems acquired the capability to assemble, from split genes in the germline, billions of functional antigen receptors1-3. These receptors show specificity; unlike the broadly tuned receptors of the innate system, antibodies (Ig) expressed by B cells, for instance, can accurately distinguish between the two enantiomers of organic acids4, whereas T cell receptors (TCRs) reliably recognize single amino acid replacements in their peptide antigens5. In developing lymphocytes, antigen receptor genes are assembled from a comparatively small set of germline-encoded genetic elements in a process referred to as V(D)J recombination6,7. Potential self-reactivity of some antigen receptors arising from the quasi-random somatic diversification is suppressed by several robust control mechanisms8-12. For decades, scientists have puzzled over the evolutionary origin of somatically diversifying antigen receptors13-16. It has remained unclear how, at the inception of this mechanism, immunologically beneficial expanded receptor diversity was traded against the emerging risk of destructive self-recognition. Here we explore the hypothesis that in early vertebrates, sequence microhomologies marking the ends of recombining elements became the crucial targets of selection determining the outcome of non-homologous end joining-based repair of DNA double-strand breaks generated during RAG-mediated recombination. We find that, across the main clades of jawed vertebrates, TCRα repertoire diversity is best explained by species-specific extents of such sequence microhomologies. Thus, selection of germline sequence composition of rearranging elements emerges as a major factor determining the degree of diversity of somatically generated antigen receptors.

Personalised Medicine and the Potential Role of Electrospinning for Targeted Immunotherapeutics in Head and Neck Cancer.

Advanced head and neck cancer (HNC) is functionally and aesthetically destructive, and despite significant advances in therapy, overall survival is poor, financial toxicity is high, and treatment commonly exacerbates tissue damage. Although response and durability concerns remain, antibody-based immunotherapies have heralded a paradigm shift in systemic treatment. To overcome limitations associated with antibody-based immunotherapies, exploration into de novo and repurposed small molecule immunotherapies is expanding at a rapid rate. Small molecule immunotherapies also have the capacity for chelation to biodegradable, bioadherent, electrospun scaffolds. This article focuses on the novel concept of targeted, sustained release immunotherapies and their potential to improve outcomes in poorly accessible and risk for positive margin HNC cases.

Stage-specific and cell type-specific requirements of ikzf1 during haematopoietic differentiation in zebrafish.

The zinc finger transcription factor Ikaros1 (Ikzf1) is required for lymphoid development in mammals. Four zinc fingers constitute its DNA binding domain and two zinc fingers are present in the C-terminal protein interaction module. We describe the phenotypes of zebrafish homozygous for two distinct mutant ikzf1 alleles. The IT325 variant lacks the C-terminal two zinc fingers, whereas the fr105 variant retains only the first zinc finger of the DNA binding domain. An intact ikzf1 gene is required for larval T cell development, whereas low levels of adult lymphoid development recover in the mutants. By contrast, the mutants exhibit a signature of increased myelopoiesis at larval and adult stages. Both mutations stimulate erythroid differentiation in larvae, indicating that the C-terminal zinc fingers negatively regulate the extent of red blood cell production. An unexpected differential effect of the two mutants on adult erythropoiesis suggests a direct requirement of an intact DNA binding domain for entry of progenitors into the red blood cell lineage. Collectively, our results reinforce the biological differences between larval and adult haematopoiesis, indicate a stage-specific function of ikzf1 in regulating the hierarchical bifurcations of differentiation, and assign distinct functions to the DNA binding domain and the C-terminal zinc fingers.

Synchronous sinonasal and respiratory papilloma: could long-term positive pressure ventilation be the cause? A rare case report.

This case report describes a rare presentation of synchronous pathologies-sinonasal inverted papilloma (SIP) and recurrent respiratory papillomatosis (RRP)-in a 47-year-old man using continuous positive airway pressure (CPAP) ventilation for progressive obstructive sleep apnoea. As far as we know, this is the first case of concurrent SIP and RRP disease described in the literature. The patient initially presented for management of chronic rhinosinusitis symptoms. He was found to have an extensive nasal lesion on flexible nasendoscopy, for which surgical management was recommended. However, during anaesthetic induction, he obstructed unexpectedly and was found to have an occlusive supraglottic lesion that required expedient ENT airway management. Diagnosis was made clinically and was supported with histopathology of excised tissue. Management involved multiple staged procedures for excision of sinonasal and glottic lesions and regular follow-up and imaging.

Genetic landscape of T cells identifies synthetic lethality for T-ALL.

To capture the global gene network regulating the differentiation of immature T cells in an unbiased manner, large-scale forward genetic screens in zebrafish were conducted and combined with genetic interaction analysis. After ENU mutagenesis, genetic lesions associated with failure of T cell development were identified by meiotic recombination mapping, positional cloning, and whole genome sequencing. Recessive genetic variants in 33 genes were identified and confirmed as causative by additional experiments. The mutations affected T cell development but did not perturb the development of an unrelated cell type, growth hormone-expressing somatotrophs, providing an important measure of cell-type specificity of the genetic variants. The structure of the genetic network encompassing the identified components was established by a subsequent genetic interaction analysis, which identified many instances of positive (alleviating) and negative (synthetic) genetic interactions. Several examples of synthetic lethality were subsequently phenocopied using combinations of small molecule inhibitors. These drugs not only interfered with normal T cell development, but also elicited remission in a model of T cell acute lymphoblastic leukaemia. Our findings illustrate how genetic interaction data obtained in the context of entire organisms can be exploited for targeted interference with specific cell types and their malignant derivatives.

Antigen receptor repertoires of one of the smallest known vertebrates.

The rules underlying the structure of antigen receptor repertoires are not yet fully defined, despite their enormous importance for the understanding of adaptive immunity. With current technology, the large antigen receptor repertoires of mice and humans cannot be comprehensively studied. To circumvent the problems associated with incomplete sampling, we have studied the immunogenetic features of one of the smallest known vertebrates, the cyprinid fish Paedocypris sp. "Singkep" ("minifish"). Despite its small size, minifish has the key genetic facilities characterizing the principal vertebrate lymphocyte lineages. As described for mammals, the frequency distributions of immunoglobulin and T cell receptor clonotypes exhibit the features of fractal systems, demonstrating that self-similarity is a fundamental property of antigen receptor repertoires of vertebrates, irrespective of body size. Hence, minifish achieve immunocompetence via a few thousand lymphocytes organized in robust scale-free networks, thereby ensuring immune reactivity even when cells are lost or clone sizes fluctuate during immune responses.

Beware the respiratory epithelial adenomatoid hamartoma-a malignant masquerador.

Respiratory epithelial adenomatoid hamartoma (REAH) is a rare benign tumour, which can masquerade as a sinonasal malignancy. Commonly arising from the posterior nasal septum, we present the second described case of a lateral nasal cavity wall REAH in a 68-year-old male with a 2-year history of progressive left nasal obstruction. Clinical and radiological assessment predicted malignancy; however, histopathology identified a benign pathology. He was subsequently treated with narrow local excision under general anaesthetic with no evidence of recurrence at post-operative intervals.

Transgenerational inheritance of impaired larval T cell development in zebrafish.

Evidence for transgenerational inheritance of epigenetic information in vertebrates is scarce. Aberrant patterns of DNA methylation in gametes may set the stage for transmission into future generations. Here, we describe a viable hypomorphic allele of dnmt1 in zebrafish that causes widespread demethylation of CpG dinucleotides in sperm and somatic tissues. We find that homozygous mutants are essentially normal, with the exception of drastically impaired lymphopoiesis, affecting both larval and adult phases of T cell development. The phenotype of impaired larval (but not adult) T cell development is transmitted to subsequent generations by genotypically wildtype fish. We further find that about 200 differentially methylated regions in sperm DNA of transmitting and non-transmitting males, including hypermethylated sites associated with runx3 and rptor genes, whose reduced activities are associated with impaired larval T cell development. Our results indicate a particular sensitivity of larval T cell development to transgenerationally inherited epimutations.

Pervasive changes of mRNA splicing in upf1-deficient zebrafish identify rpl10a as a regulator of T cell development.

The transcriptome of eukaryotic cells is constantly monitored for errors to avoid the production of undesired protein variants. The evolutionarily conserved nonsense-mediated mRNA decay (NMD) pathway degrades aberrant mRNAs, but also functions in the regulation of transcript abundance in response to changed physiological states. Here, we describe a zebrafish mutant of upf1, encoding the central component of the NMD machinery. Fish homozygous for the upf1t20450 allele (Y163X) survive until day 10 after fertilization, presenting with impaired T cell development as one of the most conspicuous features of the mutant phenotype. Analysis of differentially expressed genes identified dysregulation of the pre-mRNA splicing pathway, accompanied by perturbed autoregulation of canonical splicing activators (SRSF) and repressors (HNRNP). In upf1-deficient mutants, NMD-susceptible transcripts of ribosomal proteins that are known for their role as noncanonical splicing regulators were greatly increased, most notably, rpl10a When the levels of NMD-susceptible rpl10a transcripts were artificially increased in zebrafish larvae, T cell development was significantly impaired, suggesting that perturbed autoregulation of rpl10a splicing contributes to failing T cell development in upf1 deficiency. Our results identify an extraribosomal tissue-specific function to rpl10a in the immune system, and thus exemplify the advantages of the zebrafish model to study the effects of upf1-deficiency in the context of a vertebrate organism.

Lymphocyte-Specific Function of the DNA Polymerase Epsilon Subunit Pole3 Revealed by Neomorphic Alleles.

Immunodeficiencies are typically caused by loss-of-function mutations in lymphocyte-specific genes. Occasionally, mutations in ubiquitous general-purpose factors, including those affecting essential components of the DNA polymerase epsilon (POLE) holoenzyme, have cell-type-specific consequences. POLE3, one of the four components of the POLE holoenzyme, features a histone fold domain and a unique acidic C terminus, making it a particularly attractive candidate mediating cell type-specific activities of POLE. Mice lacking Pole3 survive up to late embryonic stages, indicating that this subunit is dispensable for DNA replication. The phenotypes of viable hypomorphic and neomorphic alleles are surprisingly tissue restricted and reveal a stage-specific function of the histone fold domain of Pole3 during T and B cell development. Gradual introduction of positively charged residues into the acidic C terminus leads to peripheral lymphopenia of increasing severity. Our findings serve as a paradigm to understand the molecular basis of cell-type-specific non-replicative functions of the ubiquitous POLE complex.

Cytidine deaminase 2 is required for VLRB antibody gene assembly in lampreys.

The antibodies of jawless vertebrates consist of leucine-rich repeat arrays encoded by somatically assembled VLRB genes. It is unknown how the incomplete germline VLRB loci are converted into functional antibody genes during B lymphocyte development in lampreys. In Lampetra planeri larvae lacking the cytidine deaminase CDA2 gene, VLRB assembly fails, whereas the T lineage-associated VLRA and VLRC antigen receptor gene assemblies occur normally. Thus, CDA2 acts in a B cell lineage-specific fashion to support the somatic diversification of VLRB antibody genes. CDA2 is closely related to activation-induced cytidine deaminase (AID), which is essential for the elaboration of immunoglobulin gene repertoires in jawed vertebrates. Our results thus identify a convergent mechanism of antigen receptor gene assembly and diversification that independently evolved in the two sister branches of vertebrates.