Design of a targeted blood transcriptional panel for monitoring immunological changes accompanying pregnancy.

Background: Immunomodulatory processes exert steering functions throughout pregnancy. Detecting diversions from this physiologic immune clock may help identify pregnant women at risk for pregnancy-associated complications. We present results from a data-driven selection process to develop a targeted panel of mRNAs that may prove effective in detecting pregnancies diverting from the norm. Methods: Based on a de novo dataset from a resource-constrained setting and a dataset from a resource-rich area readily available in the public domain, whole blood gene expression profiles of uneventful pregnancies were captured at multiple time points during pregnancy. BloodGen3, a fixed blood transcriptional module repertoire, was employed to analyze and visualize gene expression patterns in the two datasets. Differentially expressed genes were identified by comparing their abundance to non-pregnant postpartum controls. The selection process for a targeted gene panel considered (i) transcript abundance in whole blood; (ii) degree of correlation with the BloodGen3 module; and (iii) pregnancy biology. Results: We identified 176 transcripts that were complemented with eight housekeeping genes. Changes in transcript abundance were seen in the early stages of pregnancy and similar patterns were observed in both datasets. Functional gene annotation suggested significant changes in the lymphoid, prostaglandin and inflammation-associated compartments, when compared to the postpartum controls. Conclusion: The gene panel presented here holds promise for the development of predictive, targeted, transcriptional profiling assays. Such assays might become useful for monitoring of pregnant women, specifically to detect potential adverse events early. Prospective validation of this targeted assay, in-depth investigation of functional annotations of differentially expressed genes, and assessment of common pregnancy-associated complications with the aim to identify these early in pregnancy to improve pregnancy outcomes are the next steps.

Abundance of ACVR1B transcript is elevated during septic conditions: Perspectives obtained from a hands-on reductionist investigation.

Sepsis is a complex heterogeneous condition, and the current lack of effective risk and outcome predictors hinders the improvement of its management. Using a reductionist approach leveraging publicly available transcriptomic data, we describe a knowledge gap for the role of ACVR1B (activin A receptor type 1B) in sepsis. ACVR1B, a member of the transforming growth factor-beta (TGF-beta) superfamily, was selected based on the following: 1) induction upon in vitro exposure of neutrophils from healthy subjects with the serum of septic patients (GSE49755), and 2) absence or minimal overlap between ACVR1B, sepsis, inflammation, or neutrophil in published literature. Moreover, ACVR1B expression is upregulated in septic melioidosis, a widespread cause of fatal sepsis in the tropics. Key biological concepts extracted from a series of PubMed queries established indirect links between ACVR1B and "cancer", "TGF-beta superfamily", "cell proliferation", "inhibitors of activin", and "apoptosis". We confirmed our observations by measuring ACVR1B transcript abundance in buffy coat samples obtained from healthy individuals (n=3) exposed to septic plasma (n = 26 melioidosis sepsis cases)ex vivo. Based on our re-investigation of publicly available transcriptomic data and newly generated ex vivo data, we provide perspective on the role of ACVR1B during sepsis. Additional experiments for addressing this knowledge gap are discussed.

Development of a fixed module repertoire for the analysis and interpretation of blood transcriptome data.

As the capacity for generating large-scale molecular profiling data continues to grow, the ability to extract meaningful biological knowledge from it remains a limitation. Here, we describe the development of a new fixed repertoire of transcriptional modules, BloodGen3, that is designed to serve as a stable reusable framework for the analysis and interpretation of blood transcriptome data. The construction of this repertoire is based on co-clustering patterns observed across sixteen immunological and physiological states encompassing 985 blood transcriptome profiles. Interpretation is supported by customized resources, including module-level analysis workflows, fingerprint grid plot visualizations, interactive web applications and an extensive annotation framework comprising functional profiling reports and reference transcriptional profiles. Taken together, this well-characterized and well-supported transcriptional module repertoire can be employed for the interpretation and benchmarking of blood transcriptome profiles within and across patient cohorts. Blood transcriptome fingerprints for the 16 reference cohorts can be accessed interactively via:  https://drinchai.shinyapps.io/BloodGen3Module/ .

Organizing training workshops on gene literature retrieval, profiling, and visualization for early career researchers.

Early-career researchers must acquire the skills necessary to effectively search and extract information from biomedical literature. This ability is for instance crucial for evaluating the novelty of experimental results, and assessing potential publishing opportunities. Given the rapidly growing volume of publications in the field of biomedical research, new systematic approaches need to be devised and adopted for the retrieval and curation of literature relevant to a specific theme. In this context, we present a hands-on training curriculum aimed at retrieval, profiling, and visualization of literature associated with a given topic. The curriculum was implemented in a workshop in January 2021. Here we provide supporting material and step-by-step implementation guidelines with the ISG15 gene literature serving as an illustrative use case. Workshop participants can learn several skills, including: 1) building and troubleshoot PubMed queries in order to retrieve the literature associated with a gene of interest; 2) identifying key concepts relevant to given themes (such as cell types, diseases, and biological processes); 3) measuring the prevalence of these concepts in the gene literature; 4) extracting key information from relevant articles, and 5) developing a background section or summary on the basis of this information. Finally, trainees can learn to consolidate the structured information captured through this process for presentation via an interactive web application.

Cohort profile: molecular signature in pregnancy (MSP): longitudinal high-frequency sampling to characterise cross-omic trajectories in pregnancy in a resource-constrained setting.

PURPOSE: A successful pregnancy relies on the interplay of various biological systems. Deviations from the norm within a system or intersystemic interactions may result in pregnancy-associated complications and adverse pregnancy outcomes. Systems biology approaches provide an avenue of unbiased, in-depth phenotyping in health and disease. The molecular signature in pregnancy (MSP) cohort was established to characterise longitudinal, cross-omic trajectories in pregnant women from a resource constrained setting. Downstream analysis will focus on characterising physiological perturbations in uneventful pregnancies, pregnancy-associated complications and adverse outcomes. PARTICIPANTS: First trimester pregnant women of Karen or Burman ethnicity were followed prospectively throughout pregnancy, at delivery and until 3 months post partum. Serial high-frequency sampling to assess whole blood transcriptomics and microbiome composition of the gut, vagina and oral cavity, in conjunction with assessment of gene expression and microbial colonisation of gestational tissue, was done for all cohort participants. FINDINGS TO DATE: 381 women with live born singletons averaged 16 (IQR 15-18) antenatal visits (13 094 biological samples were collected). At 5% (19/381) the preterm birth rate was low. Other adverse events such as maternal febrile illness 7.1% (27/381), gestational diabetes 13.1% (50/381), maternal anaemia 16.3% (62/381), maternal underweight 19.2% (73/381) and a neonate born small for gestational age 20.2% (77/381) were more often observed than preterm birth. FUTURE PLANS: Results from the MSP cohort will enable in-depth characterisation of cross-omic molecular trajectories in pregnancies from a population in a resource-constrained setting. Moreover, pregnancy-associated complications and unfavourable pregnancy outcomes will be investigated at the same granular level, with a particular focus on population relevant needs such as effect of tropical infections on pregnancy. More detailed knowledge on multiomic perturbations will ideally result in the development of diagnostic tools and ultimately lead to targeted interventions that may disproportionally benefit pregnant women from this resource-limited population. TRIAL REGISTRATION NUMBER: NCT02797327.

A modular framework for the development of targeted Covid-19 blood transcript profiling panels.

BACKGROUND: Covid-19 morbidity and mortality are associated with a dysregulated immune response. Tools are needed to enhance existing immune profiling capabilities in affected patients. Here we aimed to develop an approach to support the design of targeted blood transcriptome panels for profiling the immune response to SARS-CoV-2 infection. METHODS: We designed a pool of candidates based on a pre-existing and well-characterized repertoire of blood transcriptional modules. Available Covid-19 blood transcriptome data was also used to guide this process. Further selection steps relied on expert curation. Additionally, we developed several custom web applications to support the evaluation of candidates. RESULTS: As a proof of principle, we designed three targeted blood transcript panels, each with a different translational connotation: immunological relevance, therapeutic development relevance and SARS biology relevance. CONCLUSION: Altogether the work presented here may contribute to the future expansion of immune profiling capabilities via targeted profiling of blood transcript abundance in Covid-19 patients.

Annexin A3 in sepsis: novel perspectives from an exploration of public transcriptome data.

According to publicly available transcriptome datasets, the abundance of Annexin A3 (ANXA3) is robustly increased during the course of sepsis; however, no studies have examined the biological significance or clinical relevance of ANXA3 in this pathology. Here we explored this interpretation gap and identified possible directions for future research. Based on reference transcriptome datasets, we found that ANXA3 expression is restricted to neutrophils, is upregulated in vitro after exposure to plasma obtained from septic patients, and is associated with adverse clinical outcomes. Secondly, an increase in ANXA3 transcript abundance was also observed in vivo, in the blood of septic patients in multiple independent studies. ANXA3 is known to mediate calcium-dependent granules-phagosome fusion in support of microbicidal activity in neutrophils. More recent work has also shown that ANXA3 enhances proliferation and survival of tumour cells via a Caspase-3-dependent mechanism. And this same molecule is also known to play a critical role in regulation of apoptotic events in neutrophils. Thus, we posit that during sepsis ANXA3 might either play a beneficial role, by facilitating microbial clearance and resolution of the infection; or a detrimental role, by prolonging neutrophil survival, which is known to contribute to sepsis-mediated organ damage.

Influence of storage conditions of small volumes of blood on immune transcriptomic profiles.

OBJECTIVE: Transcriptome analysis of human whole blood is used to discover biomarkers of diseases and to assess phenotypic traits. Here we have collected small volumes of blood in Tempus solution and tested whether different storage conditions have an impact on transcriptomic profiling. Fifty µl of blood were collected in 100µl of Tempus solutions, freezed at - 20 °C for 1 day and eventually thawed, stored and processed under five different conditions: (i) - 20 °C for 1 week; (ii) +4 °C for 1 week; (iii) room temperature for 1 week; (iv) room temperature for 1 day, - 20 °C for 1 day, room temperature until testing at day 7, (v) - 20 °C for 1 week, RNA was isolated and stored in GenTegra solution. We used 272 immune transcript specific assays to test the expression profiling using qPCR based Fluidigm BioMark HD dynamic array. RESULTS: RNA yield ranged between 0.17 and 1.39µg. Except for one sample, RIN values were > 7. Using Principal Component Analysis, we saw that the storage conditions did not drive sample distribution. The condition that showed larger variability was the RT-FR-RT (room temperature-freezing-room temperature), suggesting that freezing-thawing cycles may have a worse effect on data reproducibility than keeping the samples at room temperature.

A prospective cohort for the investigation of alteration in temporal transcriptional and microbiome trajectories preceding preterm birth: a study protocol.

INTRODUCTION: Preterm birth (PTB) results from heterogeneous influences and is a major contributor to neonatal mortality and morbidity that continues to have adverse effects on infants beyond the neonatal period. This protocol describes the procedures to determine molecular signatures predictive of PTB through high-frequency sampling during pregnancy, at delivery and the postpartum period. METHODS AND ANALYSIS: Four hundred first trimester pregnant women from either Myanmar or Thailand of either Karen or Burman ethnicity, with a viable, singleton pregnancy will be enrolled in this non-interventional, prospective pregnancy birth cohort study and will be followed through to the postpartum period. Fortnightly finger prick capillary blood sampling will allow the monitoring of genome-wide transcript abundance in whole blood. Collection of stool samples and vaginal swabs each trimester, at delivery and postpartum will allow monitoring of intestinal and vaginal microbial composition. In a nested case-control analysis, perturbations of transcript abundance in capillary blood as well as longitudinal changes of the gut, vaginal and oral microbiome will be compared between mothers giving birth to preterm and matched cases giving birth to term neonates. Placenta tissue of preterm and term neonates will be used to determine bacterial colonisation as well as for the establishment of coding and non-coding RNA profiles. In addition, RNA profiles of circulating, non-coding RNA in cord blood serum will be compared with those of maternal peripheral blood serum at time of delivery. ETHICS AND DISSEMINATION: This research protocol that aims to detect perturbations in molecular trajectories preceding adverse pregnancy outcomes was approved by the ethics committee of the Faculty of Tropical Medicine, Mahidol University in Bangkok, Thailand (Ethics Reference: TMEC 15-062), the Oxford Tropical Research Ethics Committee (Ethics Reference: OxTREC: 33-15) and the local Tak Province Community Ethics Advisory Board. The results of this cooperative project will be disseminated in multiple publications staggered over time in international peer-reviewed scientific journals. TRIAL REGISTRATION NUMBER: NCT02797327; Pre-results.

Interferon gamma and interferon gamma inducible protein-10 in detecting tuberculosis infection.

OBJECTIVE: This study aimed to compare the levels of TB-antigen specific Interferon gamma (IFN-γ) and IFN-γ inducible protein (IP)-10 in culture of whole blood samples from healthy controls (HC) and healthy household contacts (HHC). METHODOLOGY: A total of 386 study subjects, which included 186 HC and 200 HHC, were recruited. QuantiFERON-TB Gold in-tube (QFT-IT) assay was employed to measure IFN-γ levels. IP-10 levels were measured in the supernatants collected from QFT-IT tubes. Tuberculin skin test was also performed. RESULTS: The levels of TB antigen specific IFN-γ and IP-10 were significantly higher in HHC compared to HC. There was no significant difference observed between positivity of QFT-IT and IP-10 in HC and HHC. The positivity of TST was significantly lower in subjects <17 year, when compared to IP-10 (p<0.005). The reduced cut-off point 0.22IU/ml significantly increased the positivity of QFT-IT among children with high risk for latent TB infection (LTBI). CONCLUSIONS: Measurement of TB antigen specific IFN-γ and IP-10 can be potential markers for the detection of LTBI.

Is IP-10 an accurate marker for detecting M. tuberculosis-specific response in HIV-infected persons?

BACKGROUND: The suboptimal sensitivity of Interferon (IFN)-γ-based in-vitro assays, especially in immunocompromised individuals, emphasizes the need for alternative markers for diagnosing tuberculosis (TB). The objective of this study was to evaluate whether interferon-inducible protein (IP)-10, monocyte chemotactic protein (MCP)-2 and interleukin (IL)-2 can be useful biomarkers for evaluating a specific response to RD1 antigens associated to active TB disease in HIV-infected individuals. METHODOLOGY/PRINCIPAL FINDINGS: The study was carried out in India, the country with the highest TB burden in the world. Sixty-six HIV-infected individuals were prospectively enrolled, 28 with active-pulmonary-TB and 38 without. The whole blood assay based on RD1-selected peptides (experimental test) and QuantiFERON-TB Gold In tube (QFT-IT) was performed. Plasma was harvested at day-1-post-culture and soluble factors were evaluated by ELISA. The results indicate that by detecting IP-10, the sensitivity of the experimental test and QFT-antigen (75% and 85.7% respectively) for active TB was higher compared to the same assays based on IFN-γ (42.9% and 60.7% respectively) and was not influenced by the ability to respond to the mitogen. By detecting IP-10, the specificity of the experimental test and QFT-antigen (57.9% and 13.2% respectively) for active TB was lower than what was reported for the same assays using IFN-γ-detection (78.9% and 68.4% respectively). On the other side, in vitro IL-2 and MCP-2 responses were not significantly associated with active TB. CONCLUSIONS: HIV infection does not impair RD1-specific response detected by IP-10, while it significantly decreases IFN-γ-mediated responses. At the moment it is unclear whether higher detection is related to higher sensitivity or lower specificity of the assay. Further studies in high and low TB endemic countries are needed to elucidate this.

Role of QuantiFERON-TB gold, interferon gamma inducible protein-10 and tuberculin skin test in active tuberculosis diagnosis.

BACKGROUND: The measurement of Interferon gamma or Interferon gamma inducible protein (IP)-10 in antigen stimulated blood samples is suggested as an alternative method for latent tuberculosis (TB) diagnosis. Nonetheless, their role in active TB diagnosis, particularly in TB endemic settings is yet to be defined. In this study, the sensitivities and specificities of Interferon gamma release assay (IGRA), IP-10 assay and tuberculin skin test (TST) in detecting active TB cases were assessed in human immunodeficiency virus (HIV) sero-negative TB patients and healthy controls respectively. METHODS/PRINCIPAL FINDINGS: A total of 177 adult TB patients and 100 healthy controls were included for this study. QuantiFERON-TB Gold In-tube (QFT-IT) method was used to analyze the sensitivity and specificity of IGRA. QFT-IT, IP-10 and TST yielded the diagnostic sensitivities of 90.6% (95%CI: 86.3%-94.9%), 92.5% (95%CI: 88.6%-96.4%) and 68.9% (95%CI: 60.6%-77.2%) and specificities of 55% (95% CI: 35.2%-54.8%), 48% (95% CI: 38.2%-57.8%) and 75.5% (95% CI: 66.8%-84.2%), respectively. The extent of pulmonary involvement or presence of diabetes mellitus did not appear to influence the sensitivities of any of these tests. The combination of any of the two tests among QFT-IT, IP-10 and TST showed >98% sensitivity among smear negative cases and particularly the combination of IP-10, TST and smear microscopy showed 100% sensitivity, however, the specificity was decreased to 44.8%. CONCLUSIONS/SIGNIFICANCE: QFT-IT and IP-10 were highly sensitive in detecting active TB cases. The combination with TST improved the sensitivity of QFT-IT and IP-10 significantly. Although the higher sensitivity of combination of QFT-IT/IP-10 and TST may be useful in active TB diagnosis, they are limited by their poor specificity due to the high prevalence of latent TB in our settings.

Role of interferon gamma release assay in active TB diagnosis among HIV infected individuals.

BACKGROUND: A rapid and specific test is urgently needed for tuberculosis (TB) diagnosis especially among human immunodeficiency virus (HIV) infected individuals. In this study, we assessed the sensitivity of Interferon gamma release assay (IGRA) in active tuberculosis patients who were positive for HIV infection and compared it with that of tuberculin skin test (TST). METHODOLOGY/PRINCIPAL FINDINGS: A total of 105 HIV-TB patients who were naïve for anti tuberculosis and anti retroviral therapy were included for this study out of which 53 (50%) were culture positive. Of 105 tested, QuantiFERON-TB Gold in-tube (QFT-G) was positive in 65% (95% CI: 56% to 74%), negative in 18% (95% CI: 11% to 25%) and indeterminate in 17% (95% CI: 10% to 24%) of patients. The sensitivity of QFT-G remained similar in pulmonary TB and extra-pulmonary TB patients. The QFT-G positivity was not affected by low CD4 count, but it often gave indeterminate results especially in individuals with CD4 count < 200 cells/microl. All of the QFT-G indeterminate patients whose sputum culture were positive, showed < or = 0.25 IU/ml of IFN-gamma response to phytohemagglutinin (PHA). TST was performed in all the 105 patients and yielded the sensitivity of 31% (95% CI: 40% to 22%). All the TST positives were QFT-G positives. The sensitivity of TST was decreased, when CD4 cell counts declined. CONCLUSIONS/SIGNIFICANCE: Our study shows neither QFT-G alone or in combination with TST can be used to exclude the suspicion of active TB disease. However, unlike TST, QFT-G yielded fewer false negative results even in individuals with low CD4 count. The low PHA cut-off point for indeterminate results suggested in this study (< or = 0.25 IU/ml) may improve the proportion of valid QFT-G results.