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Background: Respiratory syncytial virus (RSV) is the leading cause of viral lower respiratory tract infection (LRTI) and hospitalization in infants. Mostly because of the incomplete understanding of the disease pathogenesis, there is no licensed vaccine, and treatment remains symptomatic. We analyzed whole blood transcriptional profiles to characterize the global host immune response to acute RSV LRTI in infants, to characterize its specificity compared with influenza and human rhinovirus (HRV) LRTI, and to identify biomarkers that can objectively assess RSV disease severity. Methods and Findings: This was a prospective observational study over six respiratory seasons including a cohort of infants hospitalized with RSV (n = 135), HRV (n = 30), and influenza (n =16) LRTI, and healthy age- and sex-matched controls (n = 39). A specific RSV transcriptional profile was identified in whole blood (training cohort, n = 45 infants; Dallas, Texas, US) and validated in three different cohorts (test cohort, n = 46, Dallas, Texas, US; validation cohort A, n = 16, Turku, Finland; validation cohort B, n = 28, Columbus, Ohio, US) with high sensitivity (94% [95% CI 87%-98%]) and specificity (98% [95% CI 88%-99%]). It classified infants with RSV LRTI versus HRV or influenza LRTI with 95% accuracy. The immune dysregulation induced by RSV (overexpression of neutrophil, inflammation, and interferon genes, and suppression of T and B cell genes) persisted beyond the acute disease, and immune dysregulation was greatly impaired in younger infants ( Conclusions: Blood RNA profiles of infants with RSV LRTI allow specific diagnosis, better understanding of disease pathogenesis, and assessment of disease severity. This study opens new avenues for biomarker discovery and identification of potential therapeutic or preventive targets, and demonstrates that large microarray datasets can be translated into a biologically meaningful context and applied to the clinical setting. Please see later in the article for the Editors' Summary., Introduction Lower respiratory tract infections (LRTIs) are associated with significant morbidity and mortality in children worldwide [1]. The implementation of vaccination with conjugate vaccines against the most common causes of [...]

Neutrophil extracellular traps (NETs) are a recently identified, web-like, extracellular structure composed of decondensed nuclear DNA and associated antimicrobial granules. NETs are extruded into the extracellular environment via the reactive oxygen species (ROS)-dependent cell death pathway participating in inflammation and autoimmune diseases. Transketolase (TKT) is a thiamine pyrophosphate (vitamin B1)-dependent enzyme that links the pentose phosphate pathway with the glycolytic pathway by feeding excess sugar phosphates into the main carbohydrate metabolic pathways to generate biosynthetic reducing capacity in the form of NADPH as a substrate for ROS generation. In this work, TKT was selected as a lead candidate from 24 NET-associated proteins obtained by literature screening and knowledge gap assessment. Consequently, we determined whether TKT influenced NET formation in vitro. We firstly established that the release of ROS-dependent NETs was significantly decreased after purified human PMNs were pretreated with oxythiamine, a TKT inhibitor, and in a concentration dependent manner. As a cofactor for TKT reaction, we evaluated the release of NET formation either in vitamin B1 treatment or in combined use of oxythiamine and vitamin B1, and found that those treatments also exerted a significant suppressive effect on the amount of NET-DNA and ROS production. The regulation of TKT by oxythiamine and/or vitamin B1 may therefore be associated with response to the modulation of NET formation by preventing generation of excessive NETs in inflammatory diseases. [ABSTRACT FROM AUTHOR]

Background: Early identification of children with Kawasaki Disease (KD) is key for timely initiation of intravenous immunoglobulin (IVIG) therapy. However, the diagnosis of the disease remains challenging, especially in children with an incomplete presentation (inKD). Moreover, we currently lack objective tools for identification of non-response (NR) to IVIG. Methods: Children with KD were enrolled and samples obtained before IVIG treatment and sequentially at 24 h and 4–6 weeks post-IVIG in a subset of patients. We also enrolled children with other febrile illnesses [adenovirus (AdV); group A streptococcus (GAS)] and healthy controls (HC) for comparative analyses. Blood transcriptional profiles were analyzed to define: a) the cKD and inKD biosignature, b) compare the KD signature with other febrile illnesses and, c) identify biomarkers predictive of clinical outcomes. Results: We identified a cKD biosignature (n = 39; HC, n = 16) that was validated in two additional cohorts of children with cKD (n = 37; HC, n = 20) and inKD (n = 13; HC, n = 8) and was characterized by overexpression of inflammation, platelets, apoptosis and neutrophil genes, and underexpression of T and NK cell genes. Classifier genes discriminated KD from adenovirus with higher sensitivity and specificity (92% and 100%, respectively) than for GAS (75% and 87%, respectively). We identified a genomic score (MDTH) that was higher at baseline in IVIG-NR [median 12,290 vs. 5,572 in responders, p = 0.009] and independently predicted IVIG-NR. Conclusion: A reproducible biosignature from KD patients was identified, and was similar in children with cKD and inKD. A genomic score allowed early identification of children at higher risk for non-response to IVIG. [ABSTRACT FROM AUTHOR]

Globally, diarrheal diseases are a leading cause of death in children under five and disproportionately affect children in developing countries. Children who contract diarrheal diseases are rarely screened to identify the etiologic agent due to time and cost considerations associated with pathogen-specific screening and hence pathogen-directed therapy is uncommon. The development of biomarkers to rapidly identify underlying pathogens could improve treatment options and clinical outcomes in childhood diarrheal diseases. Here, we perform RNA sequencing on blood samples collected from children evaluated in an emergency room setting with diarrheal disease where the pathogen(s) present are known. We determine host response gene signatures specific to Salmonella, Shigella and rotavirus, but not E. coli, infections that distinguish them from each other and from healthy controls. Specifically, we observed differential expression of genes related to chemokine receptors or inflammasome signaling in Shigella cases, such as CCR3, CXCR8, and NLRC4, and interferon response genes, such as IFI44 and OASL, in rotavirus cases. Our findings add insight into the host peripheral immune response to these pathogens, and suggest strategies and limitations for the use host response transcript signatures for diagnosing the etiologic agent of childhood diarrheal diseases. [ABSTRACT FROM AUTHOR]

Background: Mycobacterium tuberculosis infection is a leading cause of infectious death worldwide. Gene-expression microarray studies profiling the blood transcriptional response of tuberculosis (TB) patients have been undertaken in order to better understand the host immune response as well as to identify potential biomarkers of disease. To date most of these studies have focused on pulmonary TB patients with gene-expression profiles of extra-pulmonary TB patients yet to be compared to those of patients with pulmonary TB or sarcoidosis. Methods: A novel cohort of patients with extra-pulmonary TB and sarcoidosis was recruited and the transcriptional response of these patients compared to those with pulmonary TB using a variety of transcriptomic approaches including testing a previously defined 380 gene meta-signature of active TB. Results: The 380 meta-signature broadly differentiated active TB from healthy controls in this new dataset consisting of pulmonary and extra-pulmonary TB. The top 15 genes from this meta-signature had a lower sensitivity for differentiating extra-pulmonary TB from healthy controls as compared to pulmonary TB. We found the blood transcriptional responses in pulmonary and extra-pulmonary TB to be heterogeneous and to reflect the extent of symptoms of disease. Conclusions: The transcriptional signature in extra-pulmonary TB demonstrated heterogeneity of gene expression reflective of symptom status, while the signature of pulmonary TB was distinct, based on a higher proportion of symptomatic individuals. These findings are of importance for the rational design and implementation of mRNA based TB diagnostics. [ABSTRACT FROM AUTHOR]

Analysis of the mouse transcriptional response to Listeria monocytogenes infection reveals that a large set of genes are perturbed in both blood and tissue and that these transcriptional responses are enriched for pathways of the immune response. Further we identified enrichment for both type I and type II interferon (IFN) signaling molecules in the blood and tissues upon infection. Since type I IFN signaling has been reported widely to impair bacterial clearance we examined gene expression from blood and tissues of wild type (WT) and type I IFNαβ receptor-deficient (Ifnar1-/-) mice at the basal level and upon infection with L. monocytogenes. Measurement of the fold change response upon infection in the absence of type I IFN signaling demonstrated an upregulation of specific genes at day 1 post infection. A less marked reduction of the global gene expression signature in blood or tissues from infected Ifnar1-/- as compared to WT mice was observed at days 2 and 3 after infection, with marked reduction in key genes such as Oasg1 and Stat2. Moreover, on in depth analysis, changes in gene expression in uninfected mice of key IFN regulatory genes including Irf9, Irf7, Stat1 and others were identified, and although induced by an equivalent degree upon infection this resulted in significantly lower final gene expression levels upon infection of Ifnar1-/- mice. These data highlight how dysregulation of this network in the steady state and temporally upon infection may determine the outcome of this bacterial infection and how basal levels of type I IFN-inducible genes may perturb an optimal host immune response to control intracellular bacterial infections such as L. monocytogenes. [ABSTRACT FROM AUTHOR]

Most West Nile virus (WNV) infections are asymptomatic, but some lead to neuroinvasive disease with symptoms ranging from disorientation to paralysis and death. Evidence from animal models suggests that neuroinvasive infections may arise as a consequence of impaired immune protection. However, other data suggest that neurologic symptoms may arise as a consequence of immune mediated damage. We demonstrate that elevated immune responses are present in neuroinvasive disease by directly characterizing WNV-specific T cells in subjects with laboratory documented infections using human histocompatibility leukocyte antigen (HLA) class II tetramers. Subjects with neuroinvasive infections had higher overall numbers of WNV-specific T cells than those with asymptomatic infections. Independent of this, we also observed age related increases in WNV-specific T cell responses. Further analysis revealed that WNV-specific T cell responses included a population of atypically polarized CXCR3+CCR4+CCR6- T cells, whose presence was highly correlated with neuroinvasive disease. Moreover, a higher proportion of WNV-specific T cells in these subjects co-produced interferon-γ and interleukin 4 than those from asymptomatic subjects. More globally, subjects with neuroinvasive infections had reduced numbers of CD4+FoxP3+ Tregs that were CTLA4 positive and exhibited a distinct upregulated transcript profile that was absent in subjects with asymptomatic infections. Thus, subjects with neuroinvasive WNV infections exhibited elevated, dysregulated, and atypically polarized responses, suggesting that immune mediated damage may indeed contribute to pathogenic outcomes. [ABSTRACT FROM AUTHOR]

Enhancing pre-existing anti-tumor immunity leads to therapeutic benefit for some patients, but why some tumors are more immunogenic than others remains unresolved. We took a unique systems approach to relate patient survival to immune gene expression in >3,500 tumor RNAseq profiles from a dozen tumor types. We found significant links between immune gene expression and patient survival in 8/12 tumor types, with tumors partitioned by gene expression comprising distinct molecular subtypes. T/NK cell genes were most clearly survival-related for melanoma, head and neck, and bladder tumors, whereas myeloid cell genes were most clearly survival-related with kidney and breast tumors. T/NK or myeloid cell gene expression was linked to poor prognosis in bladder and kidney tumors, respectively, suggesting tumor-specific immunosuppressive checkpoints. Our results suggest new biomarkers for existing cancer immunotherapies and identify targets for new immunotherapies. [ABSTRACT FROM AUTHOR]

Diarrheagenic Escherichia coli (DEC) cause acute and persistent diarrhoea worldwide, but little is known about their epidemiology in Mexico. We determined the prevalence of bacterial enteropathogens in 831 children with acute diarrhoea over a four-year period in Yucatan, Mexico. Six DEC supplementary virulence genes (SVG), mainly associated with enteroaggregative E. coli (EAEC), were sought in 3100 E. coli isolates. DEC was the most common bacterial enteropathogen (28%), surpassing Salmonella (12%) and Shigella (9%). Predominant DEC groups were diffusely adherent E. coli (DAEC) (35%), EAEC (24%), and enteropathogenic E. coli (EPEC) (19%). Among children with DEC infections, 14% had severe illness mainly caused by EPEC (26%) and DAEC (18%); 30% had moderate diarrhoea mainly caused by DAEC (36%), mixed DEC infections (33%) and EAEC (32%). DAEC was most prevalent during spring, while ETEC, EAEC and EPEC predominated in summer. EAEC was more frequent in children 6–24 months old than in those younger than 6 months of age (P = 0.008, OR = 4.2, 95% CI, 1.3–13.9). The presence of SVG dispersin, (aatA), dispersin-translocator (aatA), enteroaggregative heat-stable toxin 1 (astA), plasmid encoded toxin (pet), cytolethal distending toxin (cdt) was higher in DEC than non-DEC strains, (36% vs 26%, P <0.0001, OR = 1.5, 95% CI, 1.3–1.8). 98% of EAEC-infected children harboured strains with SVG; 85% carried the aap-aatA gene combination, and 33% of these also carried astA. 28% of both EPEC and ETEC, and 6% of DAEC patients had strains with SVG. 54% of EPEC patients carried pet-positive strains alone or in combination with astA; only this DEC group harboured cdt-positive isolates. All ETEC patients carried astA- or astA-aap-positive strains. astA and aap were the most common SVG in DAEC (3% and 2%) and non-DEC strains (21% and 13%). DEC carrying SVG are an important cause of moderate to severe bacterial diarrhoea in Mexican children. Author Summary: Diarrhoea is an important cause of illness and death among young children in low- and middle-income countries. Nonetheless, very few epidemiological studies of diarrhoea have been conducted in Mexico during the last two decades. In recent years, a group of bacteria known as diarrheagenic Escherichia coli (DEC) have been recognized as a major cause of diarrheal illness worldwide. This group cannot be identified by the conventional biochemical methods used for other diarrhoeal pathogens such as Salmonella or Shigella, which limits its identification. We conducted a study in Yucatan, Mexico, to identify the bacterial causes of acute diarrhoea in children less than five years of age who required hospitalization. All DEC strains were further characterized for the presence of six E. coli supplementary virulence genes (SVG). Of the 831 children with acute diarrhoea, a bacterial pathogen was found in 56%. DEC was the most prevalent (28%) pathogen, surpassing Salmonella and Shigella. Among children with DEC diarrhoea, 44% were moderately or severely ill. Thirty-six percent of the DEC strains had one or more SVG, which were more common in older children. Our results strongly suggest that DEC carrying SVG are an important cause of moderate to severe bacterial diarrhoeal disease in Mexico. [ABSTRACT FROM AUTHOR]

While immunotherapies are rapidly becoming mainstays of cancer treatment, significant gaps remain in our understanding of how to optimally target them, alone or in combination. Here we describe a novel method to monitor levels of immune cells and pathways in expression data from solid tumors using pre-defined groups or modules of co-regulated immune genes. We show that expression of an interconnected sub-network of type I interferon-stimulated genes (ISGs) in melanomas at the time of diagnosis significantly predicted patient survival, as did, to a lesser extent, sub-networks of T helper/T regulatory and NK/T Cytotoxic cell genes. As a group, poor prognosis tumors with reduced ISG and immune gene levels exhibited significant copy number loss of the interferon gene cluster located at chromosome 9p21.3. Our studies demonstrate a link between type I interferon action and immune cell levels in melanomas, and suggest that therapeutic approaches augmenting both activities may be most beneficial. [ABSTRACT FROM AUTHOR]

The use of human whole blood for transcriptomic analysis has potential advantages over the use of isolated immune cells for studying the transcriptional response to pathogens and their products. Whole blood stimulation can be carried out in a laboratory without the expertise or equipment to isolate immune cells from blood, with the added advantage of being able to undertake experiments using very small volumes of blood. Toll like receptors (TLRs) are a family of pattern recognition receptors which recognise highly conserved microbial products. Using the TLR2 ligand (Pam3CSK4) and the TLR4 ligand (LPS), human whole blood was stimulated for 0, 1, 3, 6, 12 or 24 hours at which times mRNA was isolated and a comparative microarray was undertaken. A common NFκB transcriptional programme was identified following both TLR2 and TLR4 ligation which peaked at between 3 to 6 hours including upregulation of many of the NFκB family members. In contrast an interferon transcriptional response was observed following TLR4 but not TLR2 ligation as early as 1 hour post stimulation and peaking at 6 hours. These results recapitulate the findings observed in previously published studies using isolated murine and human myeloid cells indicating that in vitro stimulated human whole blood can be used to interrogate the early transcriptional kinetic response of innate cells to TLR ligands. Our study demonstrates that a transcriptomic analysis of mRNA isolated from human whole blood can delineate both the temporal response and the key transcriptional differences following TLR2 and TLR4 ligation. [ABSTRACT FROM AUTHOR]

Determination of antigen-specific T cell repertoires in human blood has been a challenge. Here, we show a novel integrated approach that permits determination of multiple parameters of antigen-specific T cell repertoires. The approach consists of two assays: the Direct assay and the Cytokine-driven assay. Briefly, human PBMCs are first stimulated with overlapping peptides encoding a given antigen for 48 hours to measure cytokine secretion (Direct assay). Peptide-reactive T cells are further expanded by IL-2 for 5 days; and after overnight starvation, expanded cells are stimulated with the same peptides from the initial culture to analyze cytokine secretion (Cytokine-driven assay). We first applied this integrated approach to determine the type of islet-antigen-specific T cells in healthy adults. Out of ten donors, the Direct assay identified GAD65-specific CD4+ T cells in three adults and zinc transporter 8 (ZnT8)-specific CD4+ T cells in five adults. The intracytoplasmic cytokine staining assay showed that these islet-antigen-specific CD4+ T cells belonged to the CD45RO+ memory compartment. The Cytokine-driven assay further revealed that islet-antigen-specific CD4+ T cells in healthy adults were capable of secreting various types of cytokines including type 1 and type 2 cytokines as well as IL-10. We next applied our integrated assay to determine whether the type of ZnT8-specific CD4+ T cells is different between Type 1 diabetes patients and age/gender/HLA-matched healthy adults. We found that ZnT8-specific CD4+ T cells were skewed towards Th1 cells in T1D patients, while Th2 and IL-10-producing cells were prevalent in healthy adults. In conclusion, the Direct assay and the Cytokine-driven assay complement each other, and the combination of the two assays provides information of antigen-specific T cell repertoires on the breadth, type, and avidity. This strategy is applicable to determine the differences in the quality of antigen-specific T cells between health and disease. [ABSTRACT FROM AUTHOR]

Rationale: Globally there are approximately 9 million new active tuberculosis cases and 1.4 million deaths annually. Effective antituberculosis treatment monitoring is difficult as there are no existing biomarkers of poor adherence or inadequate treatment earlier than 2 months after treatment initiation. Inadequate treatment leads to worsening disease, disease transmission and drug resistance. Objectives: To determine if blood transcriptional signatures change in response to antituberculosis treatment and could act as early biomarkers of a successful response. Methods: Blood transcriptional profiles of untreated active tuberculosis patients in South Africa were analysed before, during (2 weeks and 2 months), at the end of (6 months) and after (12 months) antituberculosis treatment, and compared to individuals with latent tuberculosis. An active-tuberculosis transcriptional signature and a specific treatment-response transcriptional signature were derived. The specific treatment response transcriptional signature was tested in two independent cohorts. Two quantitative scoring algorithms were applied to measure the changes in the transcriptional response. The most significantly represented pathways were determined using Ingenuity Pathway Analysis. Results: An active tuberculosis 664-transcript signature and a treatment specific 320-transcript signature significantly diminished after 2 weeks of treatment in all cohorts, and continued to diminish until 6 months. The transcriptional response to treatment could be individually measured in each patient. Conclusions: Significant changes in the transcriptional signatures measured by blood tests were readily detectable just 2 weeks after treatment initiation. These findings suggest that blood transcriptional signatures could be used as early surrogate biomarkers of successful treatment response. [ABSTRACT FROM AUTHOR]

Staphylococcus aureus infections are associated with diverse clinical manifestations leading to significant morbidity and mortality. To define the role of the host response in the clinical manifestations of the disease, we characterized whole blood transcriptional profiles of children hospitalized with community-acquired S. aureus infection and phenotyped the bacterial strains isolated. The overall transcriptional response to S. aureus infection was characterized by over-expression of innate immunity and hematopoiesis related genes and under-expression of genes related to adaptive immunity. We assessed individual profiles using modular fingerprints combined with the molecular distance to health (MDTH), a numerical score of transcriptional perturbation as compared to healthy controls. We observed significant heterogeneity in the host signatures and MDTH, as they were influenced by the type of clinical presentation, the extent of bacterial dissemination, and time of blood sampling in the course of the infection, but not by the bacterial isolate. System analysis approaches provide a new understanding of disease pathogenesis and the relation/interaction between host response and clinical disease manifestations. [ABSTRACT FROM AUTHOR]

Human T-lymphotropic virus type 1 (HTLV-1) is a retrovirus that persists lifelong in the host. In ~4% of infected people, HTLV-1 causes a chronic disabling neuroinflammatory disease known as HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The pathogenesis of HAM/TSP is unknown and treatment remains ineffective. We used gene expression microarrays followed by flow cytometric and functional assays to investigate global changes in blood transcriptional profiles of HTLV-1-infected and seronegative individuals. We found that perturbations of the p53 signaling pathway were a hallmark of HTLV-1 infection. In contrast, a subset of interferon (IFN)-stimulated genes was over-expressed in patients with HAM/TSP but not in asymptomatic HTLV-1 carriers or patients with the clinically similar disease multiple sclerosis. The IFN-inducible signature was present in all circulating leukocytes and its intensity correlated with the clinical severity of HAM/TSP. Leukocytes from patients with HAM/TSP were primed to respond strongly to stimulation with exogenous IFN. However, while type I IFN suppressed expression of the HTLV-1 structural protein Gag it failed to suppress the highly immunogenic viral transcriptional transactivator Tax. We conclude that over-expression of a subset of IFN-stimulated genes in chronic HTLV-1 infection does not constitute an efficient host response but instead contributes to the development of HAM/TSP. [ABSTRACT FROM AUTHOR]

Staphylococcus aureus has emerged as a significant pathogen causing severe invasive disease in otherwise healthy people. Despite considerable advances in understanding the epidemiology, resistance mechanisms, and virulence factors produced by the bacteria, there is limited knowledge of the in vivo host immune response to acute, invasive S. aureus infections. Herein, we report that peripheral blood mononuclear cells from patients with severe S. aureus infections demonstrate a distinctive and robust gene expression profile which is validated in a distinct group of patients and on a different microarray platform. Application of a systems-wide modular analysis framework reveals significant over-expression of innate immunity genes and under-expression of genes related to adaptive immunity. Simultaneous flow cytometry analyses demonstrated marked alterations in immune cell numbers, with decreased central memory CD4 and CD8 T cells and increased numbers of monocytes. CD14+ monocyte numbers significantly correlated with the gene expression levels of genes related to the innate immune response. These results demonstrate the value of applying a systems biology approach that reveals the significant alterations in the components of circulating blood lymphocytes and monocytes in invasive S. aureus infections. [ABSTRACT FROM AUTHOR]

About half of all subjects with common variable immune deficiency (CVID) are afflicted with inflammatory complications including hematologic autoimmunity, granulomatous infiltrations, interstitial lung disease, lymphoid hyperplasia and/or gastrointestinal inflammatory disease. The pathogenesis of these conditions is poorly understood but singly and in aggregate, these lead to significantly increased (11 fold) morbidity and mortality, not experienced by CVID subjects without these complications. To explore the dysregulated networks in these subjects, we applied whole blood transcriptional profiling to 91 CVID subjects, 47 with inflammatory conditions and 44 without, in comparison to subjects with XLA and healthy controls. As compared to other CVID subjects, males with XLA or healthy controls, the signature of CVID subjects with inflammatory complications was distinguished by a marked up-regulation of IFN responsive genes. Chronic up-regulation of IFN pathways is known to occur in autoimmune disease due to activation of TLRs and other still unclarified cytoplasmic sensors. As subjects with inflammatory complications were also more likely to be lymphopenic, have reduced B cell numbers, and a greater reduction of B, T and plasma cell networks, we suggest that more impaired adaptive immunity in these subjects may lead to chronic activation of innate IFN pathways in response to environmental antigens. The unbiased use of whole blood transcriptome analysis may provides a tool for distinguishing CVID subjects who are at risk for increased morbidity and earlier mortality. As more effective therapeutic options are developed, whole blood transcriptome analyses could also provide an efficient means of monitoring the effects of treatment of the inflammatory phenotype. [ABSTRACT FROM AUTHOR]

The use of human whole blood for transcriptomic analysis has potential advantages over the use of isolated immune cells for studying the transcriptional response to pathogens and their products. Whole blood stimulation can be carried out in a laboratory without the expertise or equipment to isolate immune cells from blood, with the added advantage of being able to undertake experiments using very small volumes of blood. Toll like receptors (TLRs) are a family of pattern recognition receptors which recognise highly conserved microbial products. Using the TLR2 ligand (Pam3CSK4) and the TLR4 ligand (LPS), human whole blood was stimulated for 0, 1, 3, 6, 12 or 24 hours at which times mRNA was isolated and a comparative microarray was undertaken. A common NFκB transcriptional programme was identified following both TLR2 and TLR4 ligation which peaked at between 3 to 6 hours including upregulation of many of the NFκB family members. In contrast an interferon transcriptional response was observed following TLR4 but not TLR2 ligation as early as 1 hour post stimulation and peaking at 6 hours. These results recapitulate the findings observed in previously published studies using isolated murine and human myeloid cells indicating that in vitro stimulated human whole blood can be used to interrogate the early transcriptional kinetic response of innate cells to TLR ligands. Our study demonstrates that a transcriptomic analysis of mRNA isolated from human whole blood can delineate both the temporal response and the key transcriptional differences following TLR2 and TLR4 ligation. [ABSTRACT FROM AUTHOR]

The use of human whole blood for transcriptomic analysis has potential advantages over the use of isolated immune cells for studying the transcriptional response to pathogens and their products. Whole blood stimulation can be carried out in a laboratory without the expertise or equipment to isolate immune cells from blood, with the added advantage of being able to undertake experiments using very small volumes of blood. Toll like receptors (TLRs) are a family of pattern recognition receptors which recognise highly conserved microbial products. Using the TLR2 ligand (Pam3CSK4) and the TLR4 ligand (LPS), human whole blood was stimulated for 0, 1, 3, 6, 12 or 24 hours at which times mRNA was isolated and a comparative microarray was undertaken. A common NFκB transcriptional programme was identified following both TLR2 and TLR4 ligation which peaked at between 3 to 6 hours including upregulation of many of the NFκB family members. In contrast an interferon transcriptional response was observed following TLR4 but not TLR2 ligation as early as 1 hour post stimulation and peaking at 6 hours. These results recapitulate the findings observed in previously published studies using isolated murine and human myeloid cells indicating that in vitro stimulated human whole blood can be used to interrogate the early transcriptional kinetic response of innate cells to TLR ligands. Our study demonstrates that a transcriptomic analysis of mRNA isolated from human whole blood can delineate both the temporal response and the key transcriptional differences following TLR2 and TLR4 ligation.

Rationale: New approaches to define factors underlying the immunopathogenesis of pulmonary diseases including sarcoidosis and tuberculosis are needed to develop new treatments and biomarkers. Comparing the blood transcriptional response of tuberculosis to other similar pulmonary diseases will advance knowledge of disease pathways and help distinguish diseases with similar clinical presentations., Objectives: To determine the factors underlying the immunopathogenesis of the granulomatous diseases, sarcoidosis and tuberculosis, by comparing the blood transcriptional responses in these and other pulmonary diseases., Methods: We compared whole blood genome-wide transcriptional profiles in pulmonary sarcoidosis, pulmonary tuberculosis, to community acquired pneumonia and primary lung cancer and healthy controls, before and after treatment, and in purified leucocyte populations., Measurements and Main Results: An Interferon-inducible neutrophil-driven blood transcriptional signature was present in both sarcoidosis and tuberculosis, with a higher abundance and expression in tuberculosis. Heterogeneity of the sarcoidosis signature correlated significantly with disease activity. Transcriptional profiles in pneumonia and lung cancer revealed an over-abundance of inflammatory transcripts. After successful treatment the transcriptional activity in tuberculosis and pneumonia patients was significantly reduced. However the glucocorticoid-responsive sarcoidosis patients showed a significant increase in transcriptional activity. 144-blood transcripts were able to distinguish tuberculosis from other lung diseases and controls., Conclusions: Tuberculosis and sarcoidosis revealed similar blood transcriptional profiles, dominated by interferon-inducible transcripts, while pneumonia and lung cancer showed distinct signatures, dominated by inflammatory genes. There were also significant differences between tuberculosis and sarcoidosis in the degree of their transcriptional activity, the heterogeneity of their profiles and their transcriptional response to treatment.

Determination of antigen-specific T cell repertoires in human blood has been a challenge. Here, we show a novel integrated approach that permits determination of multiple parameters of antigen-specific T cell repertoires. The approach consists of two assays: the Direct assay and the Cytokine-driven assay. Briefly, human PBMCs are first stimulated with overlapping peptides encoding a given antigen for 48 hours to measure cytokine secretion (Direct assay). Peptide-reactive T cells are further expanded by IL-2 for 5 days; and after overnight starvation, expanded cells are stimulated with the same peptides from the initial culture to analyze cytokine secretion (Cytokine-driven assay). We first applied this integrated approach to determine the type of islet-antigen-specific T cells in healthy adults. Out of ten donors, the Direct assay identified GAD65-specific CD4(+) T cells in three adults and zinc transporter 8 (ZnT8)-specific CD4(+) T cells in five adults. The intracytoplasmic cytokine staining assay showed that these islet-antigen-specific CD4(+) T cells belonged to the CD45RO(+) memory compartment. The Cytokine-driven assay further revealed that islet-antigen-specific CD4(+) T cells in healthy adults were capable of secreting various types of cytokines including type 1 and type 2 cytokines as well as IL-10. We next applied our integrated assay to determine whether the type of ZnT8-specific CD4(+) T cells is different between Type 1 diabetes patients and age/gender/HLA-matched healthy adults. We found that ZnT8-specific CD4(+) T cells were skewed towards Th1 cells in T1D patients, while Th2 and IL-10-producing cells were prevalent in healthy adults. In conclusion, the Direct assay and the Cytokine-driven assay complement each other, and the combination of the two assays provides information of antigen-specific T cell repertoires on the breadth, type, and avidity. This strategy is applicable to determine the differences in the quality of antigen-specific T cells between health and disease.