Novel Approach for Pancreas Transcriptomics Reveals the Cellular Landscape in Homeostasis and Acute Pancreatitis.

BACKGROUND & AIMS: Acinar cells produce digestive enzymes that impede transcriptomic characterization of the exocrine pancreas. Thus, single-cell RNA-sequencing studies of the pancreas underrepresent acinar cells relative to histological expectations, and a robust approach to capture pancreatic cell responses in disease states is needed. We sought to innovate a method that overcomes these challenges to accelerate study of the pancreas in health and disease. METHODS: We leverage FixNCut, a single-cell RNA-sequencing approach in which tissue is reversibly fixed with dithiobis(succinimidyl propionate) before dissociation and single-cell preparation. We apply FixNCut to an established mouse model of acute pancreatitis, validate findings using GeoMx whole transcriptome atlas profiling, and integrate our data with prior studies to compare our method in both mouse and human pancreas datasets. RESULTS: FixNCut achieves unprecedented definition of challenging pancreatic cells, including acinar and immune populations in homeostasis and acute pancreatitis, and identifies changes in all major cell types during injury and recovery. We define the acinar transcriptome during homeostasis and acinar-to-ductal metaplasia and establish a unique gene set to measure deviation from normal acinar identity. We characterize pancreatic immune cells, and analysis of T-cell subsets reveals a polarization of the homeostatic pancreas toward type-2 immunity. We report immune responses during acute pancreatitis and recovery, including early neutrophil infiltration, expansion of dendritic cell subsets, and a substantial shift in the transcriptome of macrophages due to both resident macrophage activation and monocyte infiltration. CONCLUSIONS: FixNCut preserves pancreatic transcriptomes to uncover novel cell states during homeostasis and following pancreatitis, establishing a broadly applicable approach and reference atlas for study of pancreas biology and disease.

Malaria epidemiology in low-endemicity areas of the northern coast of Ecuador: high prevalence of asymptomatic infections.

BACKGROUND: The recent scale-up in malaria control measures in Latin America has resulted in a significant decrease in the number of reported cases in several countries including Ecuador, where it presented a low malaria incidence in recent years (558 reported cases in 2015) with occasional outbreaks of both Plasmodium falciparum and Plasmodium vivax in the coastal and Amazonian regions. This success in malaria control in recent years has led Ecuador to transition its malaria policy from control to elimination. RESULTS: This study evaluated the general knowledge, attitude and practices (KAP) about malaria, as well as its prevalence in four communities of an endemic area in northwest Ecuador. A total of 258 interviews to assess KAP in the community indicated that most people in the study area have a basic knowledge about the disease but did not use to contribute to its control. Six hundred and forty-eight blood samples were collected and analysed by thick blood smear and real-time PCR. In addition, the distribution of the infections was mapped in the study communities. Although, no parasites were found by microscopy, by PCR the total malaria prevalence was 7.5% (6.9% P. vivax and 0.6% P. falciparum), much higher than expected and comparable to that reported in endemic areas of neighbouring countries with higher malaria transmission. Serology using ELISA and immunofluorescence indicated 27% respondents for P. vivax and 22% respondents for P. falciparum. CONCLUSIONS: Results suggest that despite a great malaria reduction in Ecuador, transition from control to elimination would demand further improvement in malaria diagnostics, including active case detection to identify and treat parasite asymptomatic carriers, as well as community participation in its elimination.

Plasmodium vivax gametocyte infectivity in sub-microscopic infections.

BACKGROUND: The use of molecular techniques has put in the spotlight the existence of a large mass of malaria sub-microscopic infections among apparently healthy populations. These sub-microscopic infections are considered an important pool for maintained malaria transmission. METHODS: In order to assess the appearance of Plasmodium vivax gametocytes in circulation, gametocyte density and the parasite infectivity to Anopheles mosquitoes, a study was designed to compare three groups of volunteers either experimentally infected with P. vivax sporozoites (early infections; n = 16) or naturally infected patients (acute malaria, n = 16 and asymptomatic, n = 14). In order to determine gametocyte stage, a quantitative reverse transcriptase PCR (RT-qPCR) assay targeting two sexual stage-specific molecular markers was used. Parasite infectivity was assessed by membrane feeding assays (MFA). RESULTS: In early infections P. vivax gametocytes could be detected starting at day 7 without giving rise to infected mosquitoes during 13 days of follow-up. Asymptomatic carriers, with presumably long-lasting infections, presented the highest proportion of mature gametocytes and were as infective as acute patients. CONCLUSIONS: This study shows the potential role of P. vivax asymptomatic carriers in malaria transmission should be considered when new policies are envisioned to redirect malaria control strategies towards targeting asymptomatic infections as a tool for malaria elimination.

Global genetic diversity of the Plasmodium vivax transmission-blocking vaccine candidate Pvs48/45.

BACKGROUND: Plasmodium vivax 48/45 protein is expressed on the surface of gametocytes/gametes and plays a key role in gamete fusion during fertilization. This protein was recently expressed in Escherichia coli host as a recombinant product that was highly immunogenic in mice and monkeys and induced antibodies with high transmission-blocking activity, suggesting its potential as a P. vivax transmission-blocking vaccine candidate. To determine sequence polymorphism of natural parasite isolates and its potential influence on the protein structure, all pvs48/45 sequences reported in databases from around the world as well as those from low-transmission settings of Latin America were compared. METHODS: Plasmodium vivax parasite isolates from malaria-endemic regions of Colombia, Brazil and Honduras (n = 60) were used to sequence the Pvs48/45 gene, and compared to those previously reported to GenBank and PlasmoDB (n = 222). Pvs48/45 gene haplotypes were analysed to determine the functional significance of genetic variation in protein structure and vaccine potential. RESULTS: Nine non-synonymous substitutions (E35K, Y196H, H211N, K250N, D335Y, E353Q, A376T, K390T, K418R) and three synonymous substitutions (I73, T149, C156) that define seven different haplotypes were found among the 282 isolates from nine countries when compared with the Sal I reference sequence. Nucleotide diversity (π) was 0.00173 for worldwide samples (range 0.00033-0.00216), resulting in relatively high diversity in Myanmar and Colombia, and low diversity in Mexico, Peru and South Korea. The two most frequent substitutions (E353Q: 41.9 %, K250N: 39.5 %) were predicted to be located in antigenic regions without affecting putative B cell epitopes or the tertiary protein structure. CONCLUSIONS: There is limited sequence polymorphism in pvs48/45 with noted geographical clustering among Asian and American isolates. The low genetic diversity of the protein does not influence the predicted antigenicity or protein structure and, therefore, supports its further development as transmission-blocking vaccine candidate.

Consistent prevalence of asymptomatic infections in malaria endemic populations in Colombia over time.

BACKGROUND: Malaria control programmes rely on confirmation of parasite presence in patients' blood prior to treatment administration. Plasmodium parasites are detected mostly by microscopy or rapid diagnostic test (RDT). Although these methods contribute significantly to malaria control/elimination, they are not suitable for detecting the significant proportion of asymptomatic subjects harbouring low levels of parasitaemia, which endure untreated as potential reservoirs for transmission. Malaria prevalence was assessed in endemic regions of Colombia over a 4-year follow-up. METHODS: A series of cross-sectional surveys were conducted between 2011 and 2014 in low to moderate malaria transmission sentinel sites (SS) of Tumaco, Buenaventura and Tierralta municipalities of Colombia. A census was performed and a random sample of houses was selected from each SS prior to each survey. Inhabitants were asked to answer a questionnaire on clinical, epidemiological and demographic aspects, and to provide a blood sample for malaria diagnosis using microscopy and quantitative real time polymerase chain reaction (qPCR). RESULTS: A total of 3059 blood samples were obtained from all SS, 58.5 % of which were from women and displayed a malaria prevalence ranging from 4 % (95 % CI 3-5 %) to 10 % (95 % CI 8-12 %) in the 4 years' study period. Almost all malaria cases (n = 220, 97 %) were sub-microscopic and only detectable by qPCR; 90 % of the cases were asymptomatic at the time of blood collection. While Buenaventura and Tierralta had a decreasing tendency during the follow-up, Tumaco had a rise in 2013 and then a decrease in 2014. Plasmodium vivax accounted for the majority (66-100 %) of cases in Tierralta and Buenaventura and for 25-50 % of the cases in Tumaco. CONCLUSIONS: This study demonstrates an important prevalence of asymptomatic malaria cases not detectable by microscopy, which therefore remain untreated representing a parasite pool for malaria transmission. This demands the introduction of alternative strategies for diagnosis and treatment, especially for areas of low transmission to reduce it to appropriate levels for malaria pre-elimination efforts to start.

Malaria elimination challenges in Mesoamerica: evidence of submicroscopic malaria reservoirs in Guatemala.

BACKGROUND: Even though malaria incidence has decreased substantially in Guatemala since 2000, Guatemala remains one of the countries with the highest malaria transmission in Mesoamerica. Guatemala is committed to eliminating malaria as part of the initiative 'Elimination of Malaria in Mesoamerica and the Island of Hispaniola' (EMMIE); however, it is still in the control phase. During the past decade, the government strengthened malaria control activities including mass distribution of long-lasting insecticide-impregnated bed nets, early diagnosis and prompt treatment. This study aimed to determine the prevalence of malaria, including gametocytes, in three areas of Guatemala using active case detection (ACD) and quantitative polymerase chain reaction (qPCR). METHODS: Cross-sectional surveys were conducted in three departments with varying transmission intensities: Escuintla, Alta Verapaz and Zacapa. Blood samples from 706 volunteers were screened for malaria using microscopy and qPCR which was also used to determine the prevalence of gametocytes among infected individuals. Results were collected and analysed using REDCap and R Project, respectively. RESULTS: Malaria was diagnosed by microscopy in only 2.8 % (4/141) of the volunteers from Escuintla. By contrast, qPCR detected a prevalence of 7.1 % (10/141) in the same volunteers, 8.4 % (36/429) in Alta Verapaz, and 5.9 % (8/136) in Zacapa. Overall, 7.6 % (54/706) of the screened individuals were positive, with an average parasitaemia level of 40.2 parasites/µL (range 1-1133 parasites/µL) and 27.8 % carried mature gametocytes. Fifty-seven percent (31/54) of qPCR positive volunteers were asymptomatic and out of the 42.6 % of symptomatic individuals, only one had a positive microscopy result. CONCLUSIONS: This study found a considerable number of asymptomatic P. vivax infections that were mostly submicroscopic, of which, approximately one-quarter harboured mature gametocytes. This pattern is likely to contribute to maintaining transmission across the region. Robust surveillance systems, molecular diagnostic tests and tailored malaria detection activities for each endemic site may prove to be imperative in accelerating malaria elimination in Guatemala and possibly across all of Mesoamerica.

High prevalence of sub-microscopic infections in Colombia.

BACKGROUND: Malaria transmission in Latin America is typically characterized as hypo-endemic and unstable with ~170 million inhabitants at risk of malaria infection. Although Colombia has witnessed an important decrease in malaria transmission, the disease remains a public health problem with an estimated ~10 million people currently living in areas with malaria risk and ~61,000 cases reported in 2012. This study aimed to establish the malaria prevalence in three endemic regions of Colombia to aid in designing new interventions for malaria elimination. METHODS: A cross-sectional survey was conducted in three regions of Colombia with different malaria epidemiological profiles: Tierralta (Ta), Tumaco (Tu) and Buenaventura (Bv). The Annual Parasite Index (API) was 10.7, 6.9 and 3.1, respectively. Participants were asked to respond to a sociodemographic questionnaire and then were bled to determine the Duffy genotype and the prevalence of malaria infection by microscopy and quantitative real-time PCR (qPCR). RESULTS: The study was conducted between October 2011 and January 2012. Eight sentinel sites with 1,169 subjects from 267 households were included. The overall prevalence of sub-microscopic infections measured by thick blood smear (TBS) was 0.3% (n=4) whereas by qPCR it was 9.7% (n=113), with a greater proportion (13%) in 40-50 years old individuals. Furthermore, different regions displayed different prevalence of sub-microscopic infections: Bv 12%, Ta 15%, and Tu 4%. From these 113 samples (qPCR), 74% were positive for P. vivax and 22% for P. falciparum, and 4% were mixed infections, which correlates to the overall parasite prevalence in Colombia. This study showed that in the southern Pacific coast of Colombia (Bv and Tu), around 56% of the population have a Duffy-negative genotype, compared to the northern region (Ta) where the percentage of Duffy-negative genotype is around 3%. CONCLUSIONS: Sub-microscopic infections are prevalent across different regions in Colombia, particularly in areas with relatively low transmission intensity. The poor microscopy results suggest the need for more sensitive diagnostic tools for detection of sub-microscopic infections. This study underscores the importance of conducting active case surveillance to more accurately determine malaria incidence, and highlights the need for updating the malaria guidelines to track and treat sub-microscopic malaria infections.

Field evaluation of an automated RDT reader and data management device for Plasmodium falciparum/Plasmodium vivax malaria in endemic areas of Colombia.

BACKGROUND: Massive implementation of malaria diagnostics in low-resource countries is regarded as a pivotal strategy in control and elimination efforts. Although malaria rapid diagnostic tests (RDTs) are considered a viable alternative, there are still obstacles to the widespread implementation of this strategy, such as reporting constraints and lack of proper quality assurance of RDT-based programmes at point-of-care (POC). METHODS: A prospective cohort of patients, seeking routine care for febrile episodes at health centres in malaria-endemic areas of Colombia, was used to assess the diagnostic performance of a device based on smartphone technology (Deki ReaderTM, former codename "GenZero"), that assists users at POC to process RDTs. After informed consent, patients were enrolled into the study and blood samples were collected for thick blood smear (TBS) and RDT. The RDT results were interpreted by both visual inspection and Deki Reader device and concordance between visual and device interpretation was measured. Microscopy corrected by real-time polymerase chain reaction (PCR) and microscopy were "gold standard" tests to assess the diagnostic performance. RESULTS: In total, 1,807 patients were enrolled at seven health centres in malaria-endemic areas of Colombia. Thirty-three Plasmodium falciparum and 100 Plasmodium vivax cases were positive by corrected microscopy. Both sensitivity and specificity were 93.9% (95% CI 69.7-95.2) and 98.7% (95% CI 98.5-99.4) for P. falciparum, and 98.0% (95% CI 90.3-98.9) and 97.9% (95% CI 97.1-98.5) for P. vivax. Percentage concordance between visual and device interpretation of RDT was 98.5% and 99.0% for P. vivax and P. falciparum, respectively.The RDT, when compared to TBS, showed high sensitivity and specificity for P. falciparum in both visual and device interpretation, and good overall diagnostic performance for P. vivax. Comparison between PCR as gold standard and visual and device interpretation showed acceptable overall performance for both species. CONCLUSIONS: The diagnostic performance of the Deki Reader was comparable to visual interpretation of RDTs (without significant differences) for both malaria species. Providing standardized automated interpretation of RDTs at POC in remote areas, in addition to almost real-time reporting of cases and enabling quality control would greatly benefit large-scale implementation of RDT-based malaria diagnostic programmes.

Knowledge, attitudes and practices of malaria in Colombia.

BACKGROUND: Although Colombia has witnessed an important decrease in malaria transmission, the disease remains a public health problem with an estimated ~10 million people currently living in areas with malaria risk and ~61,000 cases reported in 2012. This study aimed to determine and compare the level of knowledge, attitudes and practices (KAP) about malaria in three endemic communities of Colombia to provide the knowledge framework for development of new intervention strategies for malaria elimination. METHODS: A cross-sectional KAP survey was conducted in the municipalities of Tierralta, Buenaventura and Tumaco, categorized according to high risk (HR) and moderate risk (MR) based on the annual parasite index (API). Surveys were managed using REDCap and analysed using MATLAB and GraphPad Prism. RESULTS: A total of 267 residents, mostly women (74%) were surveyed. Although no differences were observed on the knowledge of classical malaria symptoms between HR and MR regions, significant differences were found in knowledge and attitudes about transmission mechanisms, anti-malarial use and malaria diagnosis. Most responders in both regions (93.5% in MR, and 94.3% in HR areas) indicated use of insecticide-treated nets (ITNs) to protect themselves from malaria, and 75.5% of responders in HR indicated they did nothing to prevent malaria transmission outdoors. Despite a high level of knowledge in the study regions, significant gaps persisted relating to practices. Self-medication and poor adherence to treatment, as well as lack of both indoor and outdoor vector control measures, were significantly associated with higher malaria risk. CONCLUSIONS: Although significant efforts are currently being made by the Ministry of Health to use community education as one of the main components of the control strategy, these generic education programmes may not be applicable to all endemic regions of Colombia given the substantial geographic, ethnic and cultural diversity.

Integrated plasma proteomics identifies tuberculosis-specific diagnostic biomarkers.

BACKGROUNDNovel biomarkers to identify infectious patients transmitting Mycobacterium tuberculosis are urgently needed to control the global tuberculosis (TB) pandemic. We hypothesized that proteins released into the plasma in active pulmonary TB are clinically useful biomarkers to distinguish TB cases from healthy individuals and patients with other respiratory infections.METHODSWe applied a highly sensitive non-depletion tandem mass spectrometry discovery approach to investigate plasma protein expression in pulmonary TB cases compared to healthy controls in South African and Peruvian cohorts. Bioinformatic analysis using linear modeling and network correlation analyses identified 118 differentially expressed proteins, significant through 3 complementary analytical pipelines. Candidate biomarkers were subsequently analyzed in 2 validation cohorts of differing ethnicity using antibody-based proximity extension assays.RESULTSTB-specific host biomarkers were confirmed. A 6-protein diagnostic panel, comprising FETUB, FCGR3B, LRG1, SELL, CD14, and ADA2, differentiated patients with pulmonary TB from healthy controls and patients with other respiratory infections with high sensitivity and specificity in both cohorts.CONCLUSIONThis biomarker panel exceeds the World Health Organization Target Product Profile specificity criteria for a triage test for TB. The new biomarkers have potential for further development as near-patient TB screening assays, thereby helping to close the case-detection gap that fuels the global pandemic.FUNDINGMedical Research Council (MRC) (MR/R001065/1, MR/S024220/1, MR/P023754/1, and MR/W025728/1); the MRC and the UK Foreign Commonwealth and Development Office; the UK National Institute for Health Research (NIHR); the Wellcome Trust (094000, 203135, and CC2112); Starter Grant for Clinical Lecturers (Academy of Medical Sciences UK); the British Infection Association; the Program for Advanced Research Capacities for AIDS in Peru at Universidad Peruana Cayetano Heredia (D43TW00976301) from the Fogarty International Center at the US NIH; the UK Technology Strategy Board/Innovate UK (101556); the Francis Crick Institute, which receives funding from UKRI-MRC (CC2112); Cancer Research UK (CC2112); and the NIHR Biomedical Research Centre of Imperial College NHS.

Characterization of a malaria outbreak in Colombia in 2010.

BACKGROUND: Although malaria has presented a significant reduction in morbidity and mortality worldwide during the last decade, it remains a serious global public health problem. In Colombia, during this period, many factors have contributed to sustained disease transmission, with significant fluctuations in an overall downward trend in the number of reported malaria cases. Despite its epidemiological importance, few studies have used surveillance data to describe the malaria situation in Colombia. This study aims to describe the characteristics of malaria cases reported during 2010 to the Public Health Surveillance System (SIVIGILA) of the National Institute of Health (INS) of Colombia. METHODS: A descriptive study was conducted using malaria information from SIVIGILA 2010. Cases, frequencies, proportions, ratio and measures of central tendency and data dispersion were calculated. In addition, the annual parasite index (API) and the differences between the variables reported in 2009 and 2010 were estimated. RESULTS: A total of 117,108 cases were recorded by SIVIGILA in 2010 for a national API of 10.5/1,000 habitants, with a greater number of cases occurring during the first half of the year. More than 90% of cases were reported in seven departments (=states): Antioquia: 46,476 (39.7%); Chocó: 22,493 (19.2%); Cordoba: 20,182 (17.2%); Valle: 6,360 (5.4%); Guaviare: 5,876 (5.0%); Nariño: 4,085 (3.5%); and Bolivar: 3,590 (3.1%). Plasmodium vivax represented ~71% of the cases; Plasmodium falciparum ~28%; and few infrequent cases caused by Plasmodium malariae. CONCLUSIONS: Overall, a greater incidence was found in men (65%) than in women (35%). Although about a third of cases occurred in children <15 years, most of these cases occurred in children >5 years of age. The ethnic distribution indicated that about 68% of the cases occurred in mestizos and whites, followed by 23% in Afro-descendants, and the remainder (9%) in indigenous communities. In over half of the cases, consultation occurred early, with 623 complicated and 23 fatal cases. However, the overall incidence increased, corresponding to an epidemic burst and indicating the need to strengthen prevention and control activities as well as surveillance to reduce the risk of outbreaks and the consequent economic and social impact.

Impaired expression of metallothioneins contributes to allergen-induced inflammation in patients with atopic dermatitis.

Regulation of cutaneous immunity is severely compromised in inflammatory skin disease. To investigate the molecular crosstalk underpinning tolerance versus inflammation in atopic dermatitis, we utilise a human in vivo allergen challenge study, exposing atopic dermatitis patients to house dust mite. Here we analyse transcriptional programmes at the population and single cell levels in parallel with immunophenotyping of cutaneous immunocytes revealed a distinct dichotomy in atopic dermatitis patient responsiveness to house dust mite challenge. Our study shows that reactivity to house dust mite was associated with high basal levels of TNF-expressing cutaneous Th17 T cells, and documents the presence of hub structures where Langerhans cells and T cells co-localised. Mechanistically, we identify expression of metallothioneins and transcriptional programmes encoding antioxidant defences across all skin cell types, that appear to protect against allergen-induced inflammation. Furthermore, single nucleotide polymorphisms in the MTIX gene are associated with patients who did not react to house dust mite, opening up possibilities for therapeutic interventions modulating metallothionein expression in atopic dermatitis.

Loss of T cell tolerance in the skin following immunopathology is linked to failed restoration of the dermal niche by recruited macrophages.

T cell pathology in the skin leads to monocyte influx, but we have little understanding of the fate of recruited cells within the diseased niche, or the long-term impact on cutaneous immune homeostasis. By combining a murine model of acute graft-versus-host disease (aGVHD) with analysis of patient samples, we demonstrate that pathology initiates dermis-specific macrophage differentiation and show that aGVHD-primed macrophages continue to dominate the dermal compartment at the relative expense of quiescent MHCIIint cells. Exposure of the altered dermal niche to topical haptens after disease resolution results in hyper-activation of regulatory T cells (Treg), but local breakdown in tolerance. Disease-imprinted macrophages express increased IL-1ß and are predicted to elicit altered TNF superfamily interactions with cutaneous Treg, and we demonstrate the direct loss of T cell regulation within the resolved skin. Thus, T cell pathology leaves an immunological scar in the skin marked by failure to re-set immune homeostasis.

Transcriptional programming of immunoregulatory responses in human Langerhans cells.

Human epidermal Langerhans cells (LCs) maintain immune homeostasis in the skin. To examine transcriptional programming of human primary LCs during homeostasis, we performed scRNA-seq analysis of LCs before and after migration from the epidermis, coupled with functional assessment of their regulatory T cell priming capabilities. The analysis revealed that steady-state LCs exist in a continuum of maturation states and upregulate antigen presentation genes along with an immunoregulatory module including the genes IDO1, LGALS1, LAMTOR1, IL4I, upon their migration. The migration-induced transition in genomic state is accompanied by the ability of LCs to more efficiently prime regulatory T cell responses in co-culture assays. Computational analyses of the scRNAseq datasets using SCENIC and Partial Information Decomposition in Context identified a set of migration-induced transcription factors including IRF4, KLF6 and RelB as key nodes within a immunoregulatory gene regulatory network. These findings support a model in which efficient priming of immunoregulatory responses by LCs is dependent on coordinated upregulation of a migration-coupled maturation program with a immunoregulation-promoting genomic module.

Evaluating the Immune Response in Treatment-Naive Hospitalised Patients With Influenza and COVID-19.

The worldwide COVID-19 pandemic has claimed millions of lives and has had a profound effect on global life. Understanding the body's immune response to SARS-CoV-2 infection is crucial in improving patient management and prognosis. In this study we compared influenza and SARS-CoV-2 infected patient cohorts to identify distinct blood transcript abundances and cellular composition to better understand the natural immune response associated with COVID-19, compared to another viral infection being influenza, and identify a prognostic signature of COVID-19 patient outcome. Clinical characteristics and peripheral blood were acquired upon hospital admission from two well characterised cohorts, a cohort of 88 patients infected with influenza and a cohort of 80 patients infected with SARS-CoV-2 during the first wave of the pandemic and prior to availability of COVID-19 treatments and vaccines. Gene transcript abundances, enriched pathways and cellular composition were compared between cohorts using RNA-seq. A genetic signature between COVID-19 survivors and non-survivors was assessed as a prognostic predictor of COVID-19 outcome. Contrasting immune responses were detected with an innate response elevated in influenza and an adaptive response elevated in COVID-19. Additionally ribosomal, mitochondrial oxidative stress and interferon signalling pathways differentiated the cohorts. An adaptive immune response was associated with COVID-19 survival, while an inflammatory response predicted death. A prognostic transcript signature, associated with circulating immunoglobulins, nucleosome assembly, cytokine production and T cell activation, was able to stratify COVID-19 patients likely to survive or die. This study provides a unique insight into the immune responses of treatment naïve patients with influenza or COVID-19. The comparison of immune response between COVID-19 survivors and non-survivors enables prognostication of COVID-19 patients and may suggest potential therapeutic strategies to improve survival.

Blood gene expression predicts intensive care unit admission in hospitalised patients with COVID-19.

Background: The COVID-19 pandemic has created pressure on healthcare systems worldwide. Tools that can stratify individuals according to prognosis could allow for more efficient allocation of healthcare resources and thus improved patient outcomes. It is currently unclear if blood gene expression signatures derived from patients at the point of admission to hospital could provide useful prognostic information. Methods: Gene expression of whole blood obtained at the point of admission from a cohort of 78 patients hospitalised with COVID-19 during the first wave was measured by high resolution RNA sequencing. Gene signatures predictive of admission to Intensive Care Unit were identified and tested using machine learning and topological data analysis, TopMD. Results: The best gene expression signature predictive of ICU admission was defined using topological data analysis with an accuracy: 0.72 and ROC AUC: 0.76. The gene signature was primarily based on differentially activated pathways controlling epidermal growth factor receptor (EGFR) presentation, Peroxisome proliferator-activated receptor alpha (PPAR-α) signalling and Transforming growth factor beta (TGF-ß) signalling. Conclusions: Gene expression signatures from blood taken at the point of admission to hospital predicted ICU admission of treatment naïve patients with COVID-19.

Resolving cellular systems by ultra-sensitive and economical single-cell transcriptome filtering.

Single-cell transcriptomics suffer from sensitivity limits that restrict low abundance transcript identification, affects clustering and can hamper downstream analyses. Here, we describe Constellation sequencing (Constellation-Seq), a molecular transcriptome filter that delivers two orders of magnitude sensitivity gains by maximizing read utility while reducing the data sparsity and sequencing costs. The technique reliably measures changes in gene expression and was demonstrated by resolving rare dendritic cell populations from a peripheral blood mononuclear cell sample sample and exploring their biology with extreme resolution. The simple and powerful method is fully compatible with standard scRNA-Seq library preparation protocols and can be used for hypothesis testing, marker validation or investigating pathways.

An IRF1-IRF4 Toggle-Switch Controls Tolerogenic and Immunogenic Transcriptional Programming in Human Langerhans Cells.

Langerhans cells (LCs) reside in the epidermis as a dense network of immune system sentinels, coordinating both immunogenic and tolerogenic immune responses. To determine molecular switches directing induction of LC immune activation, we performed mathematical modelling of gene regulatory networks identified by single cell RNA sequencing of LCs exposed to TNF-alpha, a key pro-inflammatory signal produced by the skin. Our approach delineated three programmes of LC phenotypic activation (immunogenic, tolerogenic or ambivalent), and confirmed that TNF-alpha enhanced LC immunogenic programming. Through regulon analysis followed by mutual information modelling, we identified IRF1 as the key transcription factor for the regulation of immunogenicity in LCs. Application of a mathematical toggle switch model, coupling IRF1 with tolerance-inducing transcription factors, determined the key set of transcription factors regulating the switch between tolerance and immunogenicity, and correctly predicted LC behaviour in LCs derived from different body sites. Our findings provide a mechanistic explanation of how combinatorial interactions between different transcription factors can coordinate specific transcriptional programmes in human LCs, interpreting the microenvironmental context of the local tissue microenvironments.

Peptide: MHC-based DNA vaccination strategy to activate natural killer cells by targeting killer cell immunoglobulin-like receptors.

BACKGROUND: Natural killer (NK) cells are increasingly being recognized as agents for cancer immunotherapy. The killer cell immunoglobulin-like receptors (KIRs) are expressed by NK cells and are immunogenetic determinants of the outcome of cancer. In particular, KIR2DS2 is associated with protective responses to several cancers and also direct recognition of cancer targets in vitro. Due to the high homology between activating and inhibitory KIR genes to date, it has been challenging to target individual KIR for therapeutic benefit. METHODS: A novel KIR2DS2-targeting therapeutic peptide:MHC DNA vaccine was designed and used to immunize mice transgenic for KIR genes (KIR-Tg). NK cells were isolated from the livers and spleens of vaccinated mice and then analyzed for activation by flow cytometry, RNA profiling and cytotoxicity assays. In vivo assays of NK cell function using a syngeneic cancer model (B16 melanoma) and an adoptive transfer model for human hepatocellular carcinoma (Huh7) were performed. RESULTS: Injecting KIR-Tg mice with the vaccine construct activated NK cells in both liver and spleens of mice, with preferential activation of KIR2DS2-positive NK cells. KIR-specific activation was most marked on the CD11b+CD27+ mature subset of NK cells. RNA profiling indicated that the DNA vaccine upregulated genes associated with cellular metabolism and downregulated genes related to histone H3 methylation, which are associated with immune cell maturation and NK cell function. Vaccination led to canonical and cross-reactive peptide:MHC-specific NK cell responses. In vivo, DNA vaccination led to enhanced antitumor responses against B16F10 melanoma cells and also enhanced responses against a tumor model expressing the KIR2DS2 ligand HLA-C*0102. CONCLUSION: We show the feasibility of a peptide-based KIR-targeting vaccine strategy to activate NK cells and hence generate functional antitumor responses. This approach does not require detailed knowledge of the tumor peptidomes nor HLA matching with the patient. It therefore offers a novel opportunity for targeting NK cells for cancer immunotherapy.

Dual dean entrainment with volume ratio modulation for efficient droplet co-encapsulation: extreme single-cell indexing.

The future of single cell diversity screens involves ever-larger sample sizes, dictating the need for higher throughput methods with low analytical noise to accurately describe the nature of the cellular system. Current approaches are limited by the Poisson statistic, requiring dilute cell suspensions and associated losses in throughput. In this contribution, we apply Dean entrainment to both cell and bead inputs, defining different volume packets to effect efficient co-encapsulation. Volume ratio scaling was explored to identify optimal conditions. This enabled the co-encapsulation of single cells with reporter beads at rates of ∼1 million cells per hour, while increasing assay signal-to-noise with cell multiplet rates of ∼2.5% and capturing ∼70% of cells. The method, called Pirouette coupling, extends our capacity to investigate biological systems.