Ocular surface immune transcriptome and tear cytokines in corneal infection patients.

Background: Microbial keratitis is one of the leading causes of blindness globally. An overactive immune response during an infection can exacerbate damage, causing corneal opacities and vision loss. This study aimed to identify the differentially expressed genes between corneal infection patients and healthy volunteers within the cornea and conjunctiva and elucidate the contributing pathways to these conditions' pathogenesis. Moreover, it compared the corneal and conjunctival transcriptomes in corneal-infected patients to cytokine levels in tears. Methods: Corneal and conjunctival swabs were collected from seven corneal infection patients and three healthy controls under topical anesthesia. RNA from seven corneal infection patients and three healthy volunteers were analyzed by RNA sequencing (RNA-Seq). Tear proteins were extracted from Schirmer strips via acetone precipitation from 38 cases of corneal infection and 14 healthy controls. The cytokines and chemokines IL-1ß, IL-6, CXCL8 (IL-8), CX3CL1, IL-10, IL-12 (p70), IL-17A, and IL-23 were measured using an antibody bead assay. Results: A total of 512 genes were found to be differentially expressed in infected corneas compared to healthy corneas, with 508 being upregulated and four downregulated (fold-change (FC) <-2 or > 2 and adjusted p <0.01). For the conjunctiva, 477 were upregulated, and 3 were downregulated (FC <-3 or ≥ 3 and adjusted p <0.01). There was a significant overlap in cornea and conjunctiva gene expression in patients with corneal infections. The genes were predominantly associated with immune response, regulation of angiogenesis, and apoptotic signaling pathways. The most highly upregulated gene was CXCL8 (which codes for IL-8 protein). In patients with corneal infections, the concentration of IL-8 protein in tears was relatively higher in patients compared to healthy controls but did not show statistical significance. Conclusions: During corneal infection, many genes were upregulated, with most of them being associated with immune response, regulation of angiogenesis, and apoptotic signaling. The findings may facilitate the development of treatments for corneal infections that can dampen specific aspects of the immune response to reduce scarring and preserve sight.

The interaction between Epstein-Barr virus and multiple sclerosis genetic risk loci: insights into disease pathogenesis and therapeutic opportunities.

Multiple sclerosis (MS) is a chronic neurodegenerative autoimmune disease, characterised by the demyelination of neurons in the central nervous system. Whilst it is unclear what precisely leads to MS, it is believed that genetic predisposition combined with environmental factors plays a pivotal role. It is estimated that close to half the disease risk is determined by genetic factors. However, the risk of developing MS cannot be attributed to genetic factors alone, and environmental factors are likely to play a significant role by themselves or in concert with host genetics. Epstein-Barr virus (EBV) infection is the strongest known environmental risk factor for MS. There has been increasing evidence that leaves little doubt that EBV is necessary, but not sufficient, for developing MS. One plausible explanation is EBV may alter the host immune response in the presence of MS risk alleles and this contributes to the pathogenesis of MS. In this review, we discuss recent findings regarding how EBV infection may contribute to MS pathogenesis via interactions with genetic risk loci and discuss possible therapeutic interventions.

Differential gene expression of the healthy conjunctiva during the day.

PURPOSE: To determine if there is diurnal variation in gene expression in normal healthy conjunctival cells. METHODS: Bulbar conjunctival swab samples were collected from four healthy subjects in the morning and evening of the same day. The two swab samples were taken from one eye of each participant, with a minimum of five hours gap between the two samples. RNA was extracted and analysed using RNA sequencing (RNA-Seq). RESULTS: A total of 121 genes were differentially expressed between the morning and the evening conjunctival samples, of which 94 genes were upregulated in the morning, and 27 genes were upregulated in the evening. Many of the genes that were upregulated in the morning were involved in defence, cell turnover and regulation of gene expression, while the genes upregulated in the evening were involved in signalling and mucin production. CONCLUSIONS: This study has identified several genes whose expression changes over the course of the day. Knowledge of diurnal variations of conjunctival gene expression provides an insight into the regulatory status of the healthy eye and provides a baseline for examining changes during ocular surface disease.

Genetic and transcriptomic analyses support a switch to lytic phase in Epstein Barr virus infection as an important driver in developing Systemic Lupus Erythematosus.

To investigate the molecular mechanisms through which Epstein-Barr virus (EBV) may contribute to Systemic Lupus Erythematosus (SLE) pathogenesis, we interrogated SLE genetic risk loci for signatures of EBV infection. We first compared the gene expression profile of SLE risk genes across 459 different cell/tissue types. EBV-infected B cells (LCLs) had the strongest representation of highly expressed SLE risk genes. By determining an SLE risk allele effect on gene expression (expression quantitative trait loci, eQTL) in LCLs and 16 other immune cell types, we identified 79 SLE risk locus:gene pairs putatively interacting with EBV infection. A total of 10 SLE risk genes from this list (CD40, LYST, JAZF1, IRF5, BLK, IKZF2, IL12RB2, FAM167A, PTPRC and SLC15A) were targeted by the EBV transcription factor, EBNA2, differentially expressed between LCLs and B cells, and the majority were also associated with EBV DNA copy number, and expression level of EBV encoded genes. Our final gene network model based on these genes is suggestive of a nexus involving SLE risk loci and EBV latency III and B cell proliferation signalling pathways. Collectively, our findings provide further evidence to support the interaction between SLE risk loci and EBV infection that is in part mediated by EBNA2. This interplay may increase the tendency towards EBV lytic switching dependent on the presence of SLE risk alleles. These results support further investigation into targeting EBV as a therapeutic strategy for SLE.

Gender and the Sex Hormone Estradiol Affect Multiple Sclerosis Risk Gene Expression in Epstein-Barr Virus-Infected B Cells.

Multiple Sclerosis (MS) is a complex immune-mediated disease of the central nervous system. Treatment is based on immunomodulation, including specifically targeting B cells. B cells are the main host for the Epstein-Barr Virus (EBV), which has been described as necessary for MS development. Over 200 genetic loci have been identified as increasing susceptibility to MS. Many MS risk genes have altered expression in EBV infected B cells, dependent on the risk genotype, and are themselves regulated by the EBV transcription factor EBNA2. Females are 2-3 times more likely to develop MS than males. We investigated if MS risk loci might mediate the gender imbalance in MS. From a large public dataset, we identified gender-specific associations with EBV traits, and MS risk SNP/gene pairs with gender differences in their associations with gene expression. Some of these genes also showed gender differences in correlation of gene expression level with Estrogen Receptor 2. To test if estrogens may drive these gender specific differences, we cultured EBV infected B cells (lymphoblastoid cell lines, LCLs), in medium depleted of serum to remove the effects of sex hormones as well as the estrogenic effect of phenol red, and then supplemented with estrogen (100 nM estradiol). Estradiol treatment altered MS risk gene expression, LCL proliferation rate, EBV DNA copy number and EBNA2 expression in a sex-dependent manner. Together, these data indicate that there are estrogen-mediated gender-specific differences in MS risk gene expression and EBV functions. This may in turn contribute to gender differences in host response to EBV and to MS susceptibility.

The interaction of Epstein-Barr virus encoded transcription factor EBNA2 with multiple sclerosis risk loci is dependent on the risk genotype.

BACKGROUND: Epstein-Barr virus (EBV) infection may be necessary for the development of Multiple sclerosis (MS). Earlier we had identified six MS risk loci that are co-located with binding sites for the EBV transcription factor Epstein-Barr Nuclear Antigen 2 (EBNA2) in EBV-infected B cells (lymphoblastoid cell lines - LCLs). METHODS: We used an allele-specific chromatin immunoprecipitation PCR assay to assess EBNA2 allelic preference. We treated LCLs with a peptide inhibitor of EBNA2 (EBNA2-TAT), reasoning that inhibiting EBNA2 function would alter gene expression at these loci if it was mediated by EBNA2. FINDINGS: We found that EBNA2 binding was dependent on the risk allele for five of these six MS risk loci (p < 0·05). Treatment with EBNA2-TAT significantly altered the expression of TRAF3 (p < 0·05), CD40 (p < 0·001), CLECL1 (p <0·0001), TNFAIP8 (p < 0·001) and TNFRSF1A (p < 0·001). INTERPRETATION: These data suggest that EBNA2 can enhance or reduce expression of the gene depending on the risk allele, likely promoting EBV infection. This is consistent with the concept that these MS risk loci affect MS risk through altering the response to EBNA2. Together with the extensive data indicating a pathogenic role for EBV in MS, this study supports targeting EBV and EBNA2 to reduce their effect on MS pathogenesis. FUNDING: Funding was provided by grants from MS Research Australia, National Health and Medical Research Council of Australia, Australian Government Research Training Program, Multiple Sclerosis International Federation, Trish Multiple Sclerosis Research Foundation.

Expression of CYP24A1 and other multiple sclerosis risk genes in peripheral blood indicates response to vitamin D in homeostatic and inflammatory conditions.

Although genetic and epidemiological evidence indicates vitamin D insufficiency contributes to multiple sclerosis (MS), and serum levels of vitamin D increase on treatment with cholecalciferol, recent metanalyses indicate that this vitamin D form does not ameliorate disease. Genetic variation in genes regulating vitamin D, and regulated by vitamin D, affect MS risk. We evaluated if the expression of vitamin D responsive MS risk genes could be used to assess vitamin D response in immune cells. Peripheral blood mononuclear cells (PBMCs) were isolated from healthy controls and people with MS treated with dimethyl fumarate. We assayed changes in expression of vitamin D responsive MS risk (VDRMS) genes in response to treatment with 25 hydroxy vitamin D in the presence or absence of inflammatory stimuli. Expression of CYP24A1 and other VDRMS genes was significantly altered in PBMCs treated with vitamin D in the homeostatic and inflammatory models. Gene expression in MS samples had similar responses to controls, but lower initial expression of the risk genes. Vitamin D treatment abrogated these differences. Expression of CYP24A1 and other MS risk genes in blood immune cells indicate vitamin D response and could enable assessment of immunological response to vitamin D in clinical trials and on therapy.

Human anogenital monocyte-derived dendritic cells and langerin+cDC2 are major HIV target cells.

Tissue mononuclear phagocytes (MNP) are specialised in pathogen detection and antigen presentation. As such they deliver HIV to its primary target cells; CD4 T cells. Most MNP HIV transmission studies have focused on epithelial MNPs. However, as mucosal trauma and inflammation are now known to be strongly associated with HIV transmission, here we examine the role of sub-epithelial MNPs which are present in a diverse array of subsets. We show that HIV can penetrate the epithelial surface to interact with sub-epithelial resident MNPs in anogenital explants and define the full array of subsets that are present in the human anogenital and colorectal tissues that HIV may encounter during sexual transmission. In doing so we identify two subsets that preferentially take up HIV, become infected and transmit the virus to CD4 T cells; CD14+CD1c+ monocyte-derived dendritic cells and langerin-expressing conventional dendritic cells 2 (cDC2).

The Interaction of Human and Epstein-Barr Virus miRNAs with Multiple Sclerosis Risk Loci.

Although the causes of Multiple Sclerosis (MS) still remain largely unknown, multiple lines of evidence suggest that Epstein-Barr virus (EBV) infection may contribute to the development of MS. Here, we aimed to identify the potential contribution of EBV-encoded and host cellular miRNAs to MS pathogenesis. We identified differentially expressed host miRNAs in EBV infected B cells (LCLs) and putative host/EBV miRNA interactions with MS risk loci. We estimated the genotype effect of MS risk loci on the identified putative miRNA:mRNA interactions in silico. We found that the protective allele of MS risk SNP rs4808760 reduces the expression of hsa-mir-3188-3p. In addition, our analysis suggests that hsa-let-7b-5p may interact with ZC3HAV1 differently in LCLs compared to B cells. In vitro assays indicated that the protective allele of MS risk SNP rs10271373 increases ZC3HAV1 expression in LCLs, but not in B cells. The higher expression for the protective allele in LCLs is consistent with increased IFN response via ZC3HAV1 and so decreased immune evasion by EBV. Taken together, this provides evidence that EBV infection dysregulates the B cell miRNA machinery, including MS risk miRNAs, which may contribute to MS pathogenesis via interaction with MS risk genes either directly or indirectly.

Age-dependent VDR peak DNA methylation as a mechanism for latitude-dependent multiple sclerosis risk.

BACKGROUND: The mechanisms linking UV radiation and vitamin D exposure to the risk of acquiring the latitude and critical period-dependent autoimmune disease, multiple sclerosis, is unclear. We examined the effect of vitamin D on DNA methylation and DNA methylation at vitamin D receptor binding sites in adult and paediatric myeloid cells. This was accomplished through differentiating CD34+ haematopoietic progenitors into CD14+ mononuclear phagocytes, in the presence and absence of calcitriol. RESULTS: Few DNA methylation changes occurred in cells treated with calcitriol. However, several VDR-binding sites demonstrated increased DNA methylation in cells of adult origin when compared to cells of paediatric origin. This phenomenon was not observed at other transcription factor binding sites. Genes associated with these sites were enriched for intracellular signalling and cell activation pathways involved in myeloid cell differentiation and adaptive immune system regulation. CONCLUSION: These results suggest vitamin D exposure at critical periods during development may contribute to latitude-related differences in autoimmune disease incidence.

HBV vaccination and HBV infection induces HBV-specific natural killer cell memory.

OBJECTIVE: Vaccination against hepatitis B virus (HBV) confers protection from subsequent infection through immunological memory that is traditionally considered the domain of the adaptive immune system. This view has been challenged following the identification of antigen-specific memory natural killer cells (mNKs) in mice and non-human primates. While the presence of mNKs has been suggested in humans based on the expansion of NK cells following pathogen exposure, evidence regarding antigen-specificity is lacking. Here, we demonstrate the existence of HBV-specific mNKs in humans after vaccination and in chronic HBV infection. DESIGN: NK cell responses were evaluated by flow cytometry and ELISA following challenge with HBV antigens in HBV vaccinated, non-vaccinated and chronic HBV-infected individuals. RESULTS: NK cells from vaccinated subjects demonstrated higher cytotoxic and proliferative responses against autologous hepatitis B surface antigen (HBsAg)-pulsed monocyte-derived dendritic cells (moDCs) compared with unvaccinated subjects. Moreover, NK cell lysis of HBsAg-pulsed moDCs was significantly higher than that of hepatitis B core antigen (HBcAg)-pulsed moDCs (non-vaccine antigen) or tumour necrosis factor α-activated moDCs in a NKG2D-dependent manner. The mNKs response was mediated by CD56dim NK cells coexpressing CD57, CD69 and KLRG1. Further, mNKs from chronic hepatitis B patients exhibited greater degranulation against HBcAg-pulsed moDCs compared with unvaccinated or vaccinated patients. Notably, mNK activity was negatively correlated with HBV DNA levels. CONCLUSIONS: Our data support the presence of a mature mNKs following HBV antigen exposure either through vaccination or infection. Harnessing these antigen specific, functionally active mNKs provides an opportunity to develop novel treatments targeting HBV in chronic infection.

Regulation of the methylome in differentiation from adult stem cells may underpin vitamin D risk in MS.

Multiple lines of evidence indicate Multiple Sclerosis (MS) is affected by vitamin D. This effect may be mediated by methylation in immune cell progenitors. We aimed to determine (1) if haematopoietic stem cell methylation constrains methylation in daughter cells and is variable between individuals, and (2) the interaction of methylation with the vitamin D receptor binding sites. We interrogated genomic methylation levels from matching purified CD34+ haematopoietic stem cells and progeny CD14+ monocytes and CD56+ NK cells from 11 individuals using modified reduced representation bisulfite sequencing. Differential methylation of Vitamin D Receptor binding sites and MS risk genes was assessed from this and using pyrosequencing for the vitamin D regulated MS risk gene ZMIZ1. Although DNA methylation states at CpG islands and other sites are almost entirely recapitulated between progenitor and progeny immune cells, significant variation was detected at some regions between cell subsets and individuals; including around the MS risk genes HLA DRB1 and the vitamin D repressor NCOR2. Methylation of the vitamin D responsive MS risk gene ZMIZ1 was associated with risk SNP and disease. We conclude that DNA methylation settings in adult haematopoietic stem cells may contribute to individual variation in vitamin D responses in immune cells.

Vitamin D and its Effects on DNA Methylation in Development, Aging, and Disease.

DNA methylation is increasingly being recognized as a mechanism through which environmental exposures confer disease risk. Several studies have examined the association between vitamin D and changes in DNA methylation in areas as diverse as human and animal development, genomic stability, chronic disease risk, and malignancy. In many cases, they have demonstrated clear associations between vitamin D and DNA methylation in candidate disease pathways. Despite this, a clear understanding of the mechanisms by which these factors interact is unclear. This paper reviews the current understanding of the effects of vitamin D on DNA methylation. In light of current knowledge in the field, the potential mechanisms mediating vitamin D effects on DNA methylation are discussed, as are the limiting factors and future avenues for research into this exciting area.

The interaction of Multiple Sclerosis risk loci with Epstein-Barr virus phenotypes implicates the virus in pathogenesis.

Translating the findings of genome wide association studies (GWAS) to new therapies requires identification of the relevant immunological contexts to interrogate for genetic effects. In one of the largest GWAS, more than 200 risk loci have been identified for Multiple Sclerosis (MS) susceptibility. Infection with Epstein-Barr virus (EBV) appears to be necessary for the development of Multiple Sclerosis (MS). Many MS risk loci are associated with altered gene expression in EBV infected B cells (LCLs). We have interrogated this immunological context to identify interaction between MS risk loci and EBV DNA copy number, intrinsic growth rate and EBV encoded miRNA expression. The EBV DNA copy number was associated with significantly more risk alleles for MS than for other diseases or traits. EBV miRNAs BART4-3p and BART3-5p were highly associated with EBV DNA copy number and MS risk loci. The poliovirus receptor (PVR) risk SNP was associated with EBV DNA copy number, PVR and miRNA expression. Targeting EBV miRNAs BART4-3p and BART3-5p, and the gene PVR, may provide therapeutic benefit in MS. This study also indicates how immunological context and risk loci interactions can be exploited to validate and develop novel therapeutic approaches.

Transcribed B lymphocyte genes and multiple sclerosis risk genes are underrepresented in Epstein-Barr Virus hypomethylated regions.

Epstein-Barr Virus (EBV) infection appears to be necessary for the development of Multiple Sclerosis (MS), although the specific mechanisms are unknown. More than 200 single-nucleotide polymorphisms (SNPs) are known to be associated with the risk of developing MS. About a quarter of these are also highly associated with proximal gene expression in B cells infected with EBV (lymphoblastoid cell lines-LCLs). The DNA of LCLs is hypomethylated compared with both uninfected and activated B cells. Since methylation can affect gene expression, and so cell differentiation and immune evasion, we hypothesised that EBV-driven hypomethylation may affect the interaction between EBV infection and MS. We interrogated an existing dataset comprising three individuals with whole-genome bisulfite sequencing data from EBV transformed B cells and CD40L-activated B cells. DNA methylation surrounding MS risk SNPs associated with gene expression in LCLs (LCLeQTL) was less likely to be hypomethylated than randomly selected chromosomal regions. Differential methylation was independent of genomic features such as promoter regions, but genes preferentially expressed in EBV-infected B cells, including the LCLeQTL genes, were underrepresented in the hypomethylated regions. Our data does not indicate MS genetic risk is affected by EBV hypomethylation.

Neutrophils-related host factors associated with severe disease and fatality in patients with influenza infection.

Severe influenza infection has no effective treatment available. One of the key barriers to developing host-directed therapy is a lack of reliable prognostic factors needed to guide such therapy. Here, we use a network analysis approach to identify host factors associated with severe influenza and fatal outcome. In influenza patients with moderate-to-severe diseases, we uncover a complex landscape of immunological pathways, with the main changes occurring in pathways related to circulating neutrophils. Patients with severe disease display excessive neutrophil extracellular traps formation, neutrophil-inflammation and delayed apoptosis, all of which have been associated with fatal outcome in animal models. Excessive neutrophil activation correlates with worsening oxygenation impairment and predicted fatal outcome (AUROC 0.817-0.898). These findings provide new evidence that neutrophil-dominated host response is associated with poor outcomes. Measuring neutrophil-related changes may improve risk stratification and patient selection, a critical first step in developing host-directed immune therapy.

Evidence from genome wide association studies implicates reduced control of Epstein-Barr virus infection in multiple sclerosis susceptibility.

BACKGROUND: Genome wide association studies have identified > 200 susceptibility loci accounting for much of the heritability of multiple sclerosis (MS). Epstein-Barr virus (EBV), a memory B cell tropic virus, has been identified as necessary but not sufficient for development of MS. The molecular and immunological basis for this has not been established. Infected B cell proliferation is driven by signalling through the EBV produced cell surface protein LMP1, a homologue of the MS risk gene CD40. METHODS: We have investigated transcriptomes of B cells and EBV-infected B cells at Latency III (LCLs) and identified MS risk genes with altered expression on infection and with expression levels associated with the MS risk genotype (LCLeQTLs). The association of LCLeQTL genomic burden with EBV phenotypes in vitro and in vivo was examined. The risk genotype effect on LCL proliferation with CD40 stimulation was assessed. RESULTS: These LCLeQTL MS risk SNP:gene pairs (47 identified) were over-represented in genes dysregulated between B and LCLs (p < 1.53 × 10-4), and as target loci of the EBV transcription factor EBNA2 (p < 3.17 × 10-16). Overall genetic burden of LCLeQTLs was associated with some EBV phenotypes but not others. Stimulation of the CD40 pathway by CD40L reduced LCL proliferation (p < 0.001), dependent on CD40 and TRAF3 MS risk genotypes. Both CD40 and TRAF3 risk SNPs are in binding sites for the EBV transcription factor EBNA2, with expression of each correlated with EBNA2 expression dependent on genotype. CONCLUSIONS: These data indicate targeting EBV may be of therapeutic benefit in MS.

The latitude-dependent autoimmune disease risk genes ZMIZ1 and IRF8 regulate mononuclear phagocytic cell differentiation in response to vitamin D.

Epidemiological, molecular and genetic studies have indicated that high serum vitamin D levels are associated with lower risk of several autoimmune diseases. The vitamin D receptor (VDR) binding sites in monocytes and dendritic cells (DCs) are more common in risk genes for diseases with latitude dependence than in risk genes for other diseases. The transcription factor genes Zinc finger MIZ domain-containing protein 1 (ZMIZ1) and interferon regulatory factor 8 (IRF8)-risk genes for many of these diseases-have VDR binding peaks co-incident with the risk single nucleotide polymorphisms (SNPs). We show these genes are responsive to vitamin D: ZMIZ1 expression increased and IRF8 expression decreased, and this response was affected by genotype in different cell subsets. The IL10/IL12 ratio in tolerogenic DCs increased with vitamin D. These data indicate that vitamin D regulation of ZMIZ1 and IRF8 in DCs and monocytes contribute to latitude-dependent autoimmune disease risk.

Unsupervised Analysis of Transcriptomics in Bacterial Sepsis Across Multiple Datasets Reveals Three Robust Clusters.

OBJECTIVES: To find and validate generalizable sepsis subtypes using data-driven clustering. DESIGN: We used advanced informatics techniques to pool data from 14 bacterial sepsis transcriptomic datasets from eight different countries (n = 700). SETTING: Retrospective analysis. SUBJECTS: Persons admitted to the hospital with bacterial sepsis. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: A unified clustering analysis across 14 discovery datasets revealed three subtypes, which, based on functional analysis, we termed "Inflammopathic, Adaptive, and Coagulopathic." We then validated these subtypes in nine independent datasets from five different countries (n = 600). In both discovery and validation data, the Adaptive subtype is associated with a lower clinical severity and lower mortality rate, and the Coagulopathic subtype is associated with higher mortality and clinical coagulopathy. Further, these clusters are statistically associated with clusters derived by others in independent single sepsis cohorts. CONCLUSIONS: The three sepsis subtypes may represent a unifying framework for understanding the molecular heterogeneity of the sepsis syndrome. Further study could potentially enable a precision medicine approach of matching novel immunomodulatory therapies with septic patients most likely to benefit.

A community approach to mortality prediction in sepsis via gene expression analysis.

Improved risk stratification and prognosis prediction in sepsis is a critical unmet need. Clinical severity scores and available assays such as blood lactate reflect global illness severity with suboptimal performance, and do not specifically reveal the underlying dysregulation of sepsis. Here, we present prognostic models for 30-day mortality generated independently by three scientific groups by using 12 discovery cohorts containing transcriptomic data collected from primarily community-onset sepsis patients. Predictive performance is validated in five cohorts of community-onset sepsis patients in which the models show summary AUROCs ranging from 0.765-0.89. Similar performance is observed in four cohorts of hospital-acquired sepsis. Combining the new gene-expression-based prognostic models with prior clinical severity scores leads to significant improvement in prediction of 30-day mortality as measured via AUROC and net reclassification improvement index These models provide an opportunity to develop molecular bedside tests that may improve risk stratification and mortality prediction in patients with sepsis.