Computational workflow for investigating highly variable genes in single-cell RNA-seq across multiple time points and cell types.

Here, we present a computational approach for investigating highly variable genes (HVGs) associated with biological pathways of interest, across multiple time points and cell types in single-cell RNA-sequencing (scRNA-seq) data. Using public dengue virus and COVID-19 datasets, we describe steps for using the framework to characterize the dynamic expression levels of HVGs related to common and cell-type-specific biological pathways over multiple immune cell types. For complete details on the use and execution of this protocol, please refer to Arora et al.1.

Intercellular crosstalk in adult dental pulp is mediated by heparin-binding growth factors Pleiotrophin and Midkine.

BACKGROUND: In-depth knowledge of the cellular and molecular composition of dental pulp (DP) and the crosstalk between DP cells that drive tissue homeostasis are not well understood. To address these questions, we performed a comparative analysis of publicly available single-cell transcriptomes of healthy adult human DP to 5 other reference tissues: peripheral blood mononuclear cells, bone marrow, adipose tissue, lung, and skin. RESULTS: Our analysis revealed that DP resident cells have a unique gene expression profile when compared to the reference tissues, and that DP fibroblasts are the main cell type contributing to this expression profile. Genes coding for pleiotrophin (PTN) and midkine (MDK), homologous heparin-binding growth-factors, possessed the highest differential expression levels in DP fibroblasts. In addition, we identified extensive crosstalk between DP fibroblasts and several other DP resident cells, including Schwann cells, mesenchymal stem cells and odontoblasts, mediated by PTN and MDK. CONCLUSIONS: DP fibroblasts emerge as unappreciated players in DP homeostasis, mainly through their crosstalk with glial cells. These findings suggest that fibroblast-derived growth factors possess major regulatory functions and thus have a potential role as dental therapeutic targets.

Early Activation of iNKT Cells Increased Survival Time of BALB/c Mice in a Murine Model of Melioidosis.

Melioidosis is an infectious disease caused by Burkholderia pseudomallei. High interferon gamma (IFN-γ) levels in naive mice were reported to mediate protection against B. pseudomallei infection. Invariant natural killer T (iNKT) cells can produce and secrete several cytokines, including IFN-γ. When iNKT cell-knockout (KO) BALB/c mice were infected with B. pseudomallei, their survival time was significantly shorter than wild-type mice. Naive BALB/c mice pretreated intraperitoneally with α-galactosylceramide (α-GalCer), an iNKT cell activator, 24 h before infection demonstrated 62.5% survival at the early stage, with prolonged survival time compared to nonpretreated infected control mice (14 ± 1 days versus 6 ± 1 days, respectively). At 4 h after injection with α-GalCer, treated mice showed significantly higher levels of serum IFN-γ, interleukin-4 (IL-4), IL-10, and IL-12 than control mice. Interestingly, the IFN-γ levels in the α-GalCer-pretreated group were decreased at 4, 24, and 48 h after infection, while they were highly increased in the control group. At 24 h postinfection in the α-GalCer group, bacterial loads were significantly lower in blood (no growth and 1,780.00 ± 51.21, P < 0.0001), spleens (no growth and 34,300 ± 1,106.04, P < 0.0001), and livers (1,550 ± 68.72 and 13,400 ± 1,066.67, P < 0.0001) than in the control group, but not in the lungs (15,300 ± 761.10 and 1,320 ± 41.63, P < 0.0001), and almost all were negative at 48 h postinfection. This study for the first time shows that early activation of iNKT cells by α-GalCer helps clearance of B. pseudomallei and prolongs mouse survival.

Single-cell temporal analysis of natural dengue infection reveals skin-homing lymphocyte expansion one day before defervescence.

Effective clinical management of acute dengue virus (DENV) infection relies on the timing of suitable treatments during the disease progression. We analyzed single-cell transcriptomic profiles of the peripheral blood mononuclear cell samples from two DENV patients, collected daily during acute phase and also at convalescence. Key immune cell types demonstrated different dynamic responses over the course of the infection. On the day before defervescence (Day -1), we observed the peak expression of several prominent genes in the adaptive immunological pathways. We also characterized unique effector T cell clusters that expressed skin-homing signature genes at Day -1, whereas upregulation of skin and gut homing genes was also observed in plasma cells and plasmablasts during the febrile period. This work provides an overview of unique molecular dynamics that signify the entry of the critical phase, and the findings could improve the patient management of DENV infection.

Zinc and vitamin C deficiencies associate with poor pulmonary function in children with persistent asthma.

BACKGROUND: One of the pathophysiologic mechanisms involved in asthma is the increase in oxidative stress. Zinc (Zn), vitamin C (VC), and vitamin E (VE) have antioxidant functions. However, the status of oxidative stress, Zn, VC, and VE in Thai asthmatic children have not been reported. OBJECTIVE: We aimed to evaluate the status of oxidative stress, Zn, VC, VE, pulmonary function tests, and airway inflammation in Thai asthmatic children with persistent asthma. METHODS: In this cross-sectional study, the data was collected from asthmatic children aged 7-17 years. The plasma PGF2α concentration as a marker of oxidative stress was measured using an ELISA kit. Plasma Zn concentration was measured through atomic absorption spectrophotometry. Plasma VC and VE concentrations were determined using HPLC. Pulmonary function tests were evaluated as forced expiratory volume in first second (FEV1) and forced vital capacity (FVC), using a spirometer. The status of airway inflammation was determined by measuring fractional exhaled nitric oxide. RESULTS: There were 76 asthmatic children in this study. Seventy-two participants had high oxidative stress. All participants had Zn deficiency. Nearly 40% of participants had VC deficiency. VC deficiency was associated with severe asthma and airway obstruction. Plasma Zn concentrations were positively correlated with FEV1 (r = 0.27) and FEV1/FVC ratio (r = 0.65). CONCLUSIONS: Deficiency of Zn and/or VC was related to severe asthma and decreased pulmonary function. Nutrition assessment and management should be considered to alleviate asthma burden.

Invariant Natural Killer T (iNKT) cells response in human melioidosis.

BACKGROUND: Melioidosis is an infectious disease caused by Burkholderia pseudomallei. In infected mice, IFN-γ can provide protection against B. pseudomallei infection. Invariant Natural Killer T (iNKT) cells are a subpopulation of T lymphocytes, activated by recognition of glycolipid ligands such as α-Galactosylceramide presented by CD1d, produce and secrete several cytokines, including IFN-γ and IL-4. The response of iNKT cells in human melioidosis was then investigated. OBJECTIVE: To determine the iNKT cells response in human melioidosis. METHODS: The number of human iNKT cells and its activation states were investigated in sepsis melioidosis patients compared with healthy controls using flow cytometry. The iNKT cells activation was confirmed in vitro using heatkilled B. pseudomallei with normal peripheral blood mononuclear cells. The components induced iNKT cell were also determined using different concentration of B. pseudomallei lipopolysaccharide (LPS), heat-killed B. pseudomallei treated with or without DNase, RNase, or proteinase. RESULTS: The number of human iNKT cells was significantly lower while the percentage of activated iNKT cells was higher in sepsis melioidosis when compared to control. In addition, B. pseudomallei can stimulate human iNKT cells in vitro. Heat-killed B. pseudomallei could activate iNKT cells but not relate to nucleic acid, proteins, or LPS. CONCLUSIONS: We found for the first time that the iNKT cells were activated during B. pseudomallei infection in human. However, the roles and the mechanism of iNKT cells during early state of infection needed to be further investigated.

Capturing tumour heterogeneity in pre- and post-chemotherapy colorectal cancer ascites-derived cells using single-cell RNA-sequencing.

Malignant ascites is an abnormal accumulation of fluid within the peritoneal cavity, caused by metastasis of several types of cancers, including colorectal cancer (CRC). Cancer cells in ascites reflect poor prognosis and serve as a good specimen to study tumour heterogeneity, as they represent a collection of multiple metastatic sites in the peritoneum. In the present study, we have employed single-cell RNA-sequencing (scRNA-seq) to explore and characterise ascites-derived cells from a CRC patient. The samples were prepared using mechanical and enzymatic dissociations, and obtained before and after a chemotherapy treatment. Unbiased clustering of 19,653 cells from four samples reveals 14 subclusters with unique transcriptomic patterns in four major cell types: epithelial cells, myeloid cells, fibroblasts, and lymphocytes. Interestingly, the percentages of cells recovered from different cell types appeared to be influenced by the preparation protocols, with more than 90% reduction in the number of myeloid cells recovered by enzymatic preparation. Analysis of epithelial cell subpopulations unveiled only three out of eleven subpopulations with clear contraction after the treatment, suggesting that the majority of the heterogeneous ascites-derived cells were resistant to the treatment, potentially reflecting the poor treatment outcome observed in the patient. Overall, our study showcases highly heterogeneous cancer subpopulations at single-cell resolution, which respond differently to a particular chemotherapy treatment. All in all, this work highlights the potential benefit of single-cell analyses in planning appropriate treatments and real-time monitoring of therapeutic response in cancer patients through routinely discarded ascites samples.

Innate Lymphoid Cells Activation and Transcriptomic Changes in Response to Human Dengue Infection.

Background: Dengue virus (DENV) infection has a global impact on public health. The clinical outcomes (of DENV) can vary from a flu-like illness called dengue fever (DF), to a more severe form, known as dengue hemorrhagic fever (DHF). The underlying innate immune mechanisms leading to protective or detrimental outcomes have not been fully elucidated. Helper innate lymphoid cells (hILCs), an innate lymphocyte recently discovered, functionally resemble T-helper cells and are important in inflammation and homeostasis. However, the role of hILCs in DENV infection had been unexplored. Methods: We performed flow cytometry to investigate the frequency and phenotype of hILCs in peripheral blood mononuclear cells from DENV-infected patients of different disease severities (DF and DHF), and at different phases (febrile and convalescence) of infection. Intracellular cytokine staining of hILCs from DF and DHF were also evaluated by flow cytometry after ex vivo stimulation. Further, the hILCs were sorted and subjected to transcriptome analysis using RNA sequencing. Differential gene expression analysis was performed to compare the febrile and convalescent phase samples in DF and DHF. Selected differentially expressed genes were then validated by quantitative PCR. Results: Phenotypic analysis showed marked activation of all three hILC subsets during the febrile phase as shown by higher CD69 expression when compared to paired convalescent samples, although the frequency of hILCs remained unchanged. Upon ex vivo stimulation, hILCs from febrile phase DHF produced significantly higher IFN-γ and IL-4 when compared to those of DF. Transcriptomic analysis showed unique hILCs gene expression in DF and DHF, suggesting that divergent functions of hILCs may be associated with different disease severities. Differential gene expression analysis indicated that hILCs function both in cytokine secretion and cytotoxicity during the febrile phase of DENV infection. Conclusions: Helper ILCs are activated in the febrile phase of DENV infection and display unique transcriptomic changes as well as cytokine production that correlate with severity. Targeting hILCs during early innate response to DENV might help shape subsequent immune responses and potentially lessen the disease severity in the future.

Inhibitory activity of FOXP3+ regulatory T cells reveals high specificity for displaying immune tolerance in remission state rheumatoid arthritis.

Immune regulation status may indicate immunological remission in rheumatoid arthritis (RA). This cross-sectional study aimed to determine the Regulatory T cell (Treg) properties, together with 14 plasma cytokines levels between active RA and clinical remission patients. Peripheral blood (PB) Foxp3+ Treg was collected from RA patients for determination of Treg inhibitory activity using a co-culture system. Other PB T cell types and plasma cytokines were determined by flow-cytometry. The Treg results were analyzed according to the disease activity score-28 (DAS28). Then sensitivity and specificity were calculated for the indication of the remission status. The number and inhibitory activity of Treg are higher in the clinical remission as compared to the active RA (p value < 0.0001). Also, Treg: CD4+CD25+CD127+ cell ratio demonstrates the similar result (p value < 0.05). Treg inhibitory activity is inversely correlated with the DAS28. Specificity and positive likelihood ratio of inhibitory activity for indicating remission status are 92.31% (95% CI 63.97-99.81) and 11.14 (95% CI 1.67-74.14), respectively. Treg inhibitory activity is a promising prognostic marker and probably represents the immunological remission status in RA.

Dengue viremia kinetics in asymptomatic and symptomatic infection.

BACKGROUND: Dengue infection is a global health threat. While symptomatic cases contribute to morbidity and mortality, the majority of infected people are asymptomatic but serve as an important reservoir. However, the kinetics of viremia in asymptomatic infections remains unknown. METHODS: We enrolled 279 hospital-based symptomatic index cases and quantified dengue virus (DENV) RNA at enrollment and at the day of defervescence. To identify asymptomatic cases, 175 household members of index cases were monitored for clinical symptoms during follow-up, and blood was taken twice weekly to test for and quantify DENV RNA until cleared. RESULTS: We detected DENV in thirteen asymptomatic household members (7.43%). Their DENV serotypes were primarily the same as those of their family index cases. The median peak DENV viremia in asymptomatic subjects was lower than that of symptomatic individuals during the febrile phase, and the viral decay rate was slower in asymptomatic infections. CONCLUSIONS: DENV level and kinetics in asymptomatic individuals differed significantly from those of symptomatic cases. Despite the lower viremia, the slower decay rate in asymptomatic infections could lead to their prolonging the infectious reservoir. The improvement of transmission control to prevent such long-lived asymptomatic infections from transmitting the DENV is needed.

Regulatory T Cell Suppressive Activity Predicts Disease Relapse During Disease-Modifying Anti-rheumatic Drug Dose Reduction in Rheumatoid Arthritis: A Prospective Cohort Study.

When the dose of conventional disease-modifying anti-rheumatic drugs (cDMARDs) is tapered in rheumatoid arthritis (RA) patients who achieve sustained remission, biomarkers for predicting disease relapse may be needed. A prospective, unblinded cohort study was conducted in nine RA patients with remission. Peripheral blood samples were collected at baseline and at 6, 12, and 24 weeks after cDMARD dose reduction (dose of combination regimens reduced to 50%) to determine the number of regulatory Foxp3+T cells (Tregs) and other T cell subpopulations as well as Treg suppressive activity. Additionally, plasma levels of 14 cytokines at each time-point were measured via flow cytometry. Univariate and multivariate analyses were performed to identify the factor(s) associated with RA relapse during the observational period. In univariate analysis, Treg suppression and DAS28 and VAS scores were associated with RA relapse after cDMARD dose tapering. However, in multivariate analysis, only Treg suppressive activity (<42%) was found to be an independent factor associated with RA relapse after cDMARD dose reduction to 50%. Of all patients who had ≥42% Treg suppressive activity during cDMAD reduction, three-fourth patients remained in the remission stage for 24 weeks. Treg suppressive activity (<42%) in RA patients with remission could be a potential biomarker for predicting RA relapse after cDMARD dose reduction, especially over a short-term period (24 weeks).

Novel salivary gland allergens from tropical mosquito species and IgE reactivity in allergic patients.

BACKGROUND: Mosquito allergy is common in tropical countries but remains under-diagnosed. This may be due to the lack of knowledge and diagnostic tools for tropical mosquito allergens. OBJECTIVE: We aimed to characterize allergens from tropical mosquito species and investigate IgE reactivity in mosquito-allergic patients to the salivary gland proteins from these mosquitoes. METHODS: Salivary gland extract (SGE) from 4 mosquito species, highly distributed in the tropics, including Aedes aegypti, Aedes albopictus, Culex quinquefasciatus, and Anopheles dirus b, were studied. SGE-specific IgE and IgG ELISA were developed, and serum from 64 mosquito-allergic and 22 non-allergic healthy control subjects was assayed. Further investigations using IgE-immunoblots followed by mass spectrometry analysis were performed to identify and characterize allergens from each species. RESULTS: Mosquito-allergic subjects have detectable serum IgE to SGE derived from local mosquito species, while the IgE levels to Aedes communis using commercially available ELISA were mostly minimal. IgE-immunoblot analysis and mass spectrometry identified 5 novel mosquito allergens from A. albopictus (Aed al 2, Aed al 3), C. quinquefasciatus (Cul q 2.01, Cul q 3), and A. dirus b (Ano d 2). Interestingly, 4 of the 5 new allergens belong to the D7 protein family. CONCLUSIONS & CLINICAL RELEVANCE: Five novel allergens from 3 tropical mosquito species were characterized. The majority of mosquito-allergic subjects who live in the tropics have IgE reactivity to these allergens. Our study paves the way for the development of diagnostic tests, component-resolved diagnostics, and future immunotherapy for mosquito allergy in tropical countries.

Efficient short-term expansion of human peripheral blood regulatory T cells for co-culture suppression assay.

Regulatory T cells (Tregs) are a small population of CD4+ lymphocytes and play a key role as suppressors of the immune system, a role that can be identified by employing a co-culture suppression assay. Conventional protocol requires a long period of in vitro expansion of Treg numbers; hence, this study describes an establishment of a co-culture suppression assay using a short-term expansion of peripheral blood (PB) Tregs and autologous T cells (Tconvs) IL-2-pre-cultured in parallel for the same length of time, thereby obviating the need of freeze/thawed autologous Tconvs. Tregs and Tconvs were isolated from PB mononuclear cells employing magnetic bead-aided depletion of CD8+ cells followed by cell sorting of CD4+ CD25high+CD127low- (Treg) and CD4+ CD25-CD127+ (Tconv) cell populations. Following a 3-day co-cultivation period under optimized conditions, Treg suppression activity was monitored by comparing using flow cytometry the number of carboxyfluorescein succinimidyl ester-labeled Tconvs to that of Treg-minus control. The assay allowed significant differentiation between Treg suppression activity of patients with active rheumatoid arthritis and those in remission. This method should be more convenient and time-saving than the conventional Treg suppression assay in current use.

Neutrophil Activation and Early Features of NET Formation Are Associated With Dengue Virus Infection in Human.

The involvement of the immune system in the protection and pathology of natural dengue virus (DENV) has been extensively studied. However, despite studies that have referred to activation of neutrophils in DENV infections, the exact roles of neutrophils remain elusive. Here, we explored the phenotypic and functional responses of neutrophils in a cohort of adult dengue patients. Results indicated that during an acute DENV infection, neutrophils up-regulate CD66b expression, and produce a more robust respiratory response as compared with that in convalescent or healthy individuals; this confirmed in vivo neutrophil activation during DENV infection. Spontaneous decondensation of nuclei, an early event of neutrophil extracellular trap (NET) formation, was also markedly increased in cells isolated from DENV-infected patients during the acute phase of the infection. In vitro incubation of NETs with DENV-2 virus significantly decreased DENV infectivity. Interestingly, increased levels of NET components were found in the serum of patients with more severe disease form-dengue hemorrhagic fever (DHF), but not uncomplicated dengue fever, during the acute phase of the infection. Levels of pro-inflammatory cytokines IL-8 and TNFα were also increased in DHF patients as compared with those in healthy and DF subjects. This suggested that NETs may play dual roles during DENV infection. The increased ability for NET formation during acute DENV infection appeared to be independent of PAD4-mediated histone H3 hyper-citrullination. Our study suggests that neutrophils are involved in immunological responses to DENV infection.

Mosquito allergy in children: Clinical features and limitation of commercially-available diagnostic tests.

OBJECTIVE: To determine the clinical features of mosquito allergy in children and the ability of commercially available mosquito allergy tests to detect children with mosquito allergy in Thailand. METHODS: Patients with mosquito allergy aged 1 month to 18 years were recruited. Demographic data, history of mosquito allergy (onset of the reaction, reaction type) and clinical features were recorded. A skin prick test using a commercially available whole body allergen extract from Culex pipiens was performed, and serum was tested for specific IgE antibodies to Aedes communis whole body extract. RESULTS: A total of 50 patients with mosquito allergy were enrolled. The median age of enrolled children was 6.2 years with an average age of onset of 2 years [interquartile range (IQR) 1-6]. Half of the children were female. The most common skin lesion from mosquito allergy was erythematous papules (n = 45, 76.3%). The majority of children (58%) were in stage 3 (immediate and delayed type of reactions). One child (2%) was in the desensitization stage after 4.6 years of symptoms. The causative mosquito species could be identified only in 26 (52%) children: 16 (32%) children were positive for Aedes communis, 17 (34%) children were positive for Culex pipiens and 7 (14%) children were positive for both Aedes communis and Culex pipiens. Having positive IgE antibodies against Aedes communis was significantly more common in boys (n = 13, 48.1%) than girls (n = 3, 13%) (p < 0.01). CONCLUSIONS: Immediate and delayed skin reaction is the most common manifestation in mosquito allergy children. Commercially available tests for mosquito allergy can detect only 30-50% of children with mosquito allergy.

Detection of ß-lactoglobulin in human breast-milk 7 days after cow milk ingestion.

BACKGROUND: ß-lactoglobulin (BLG), a major allergen in cow milk (CM) can be detected in human breast-milk (BM) and is associated with exacerbation of symptoms in breastfed infants with cow milk protein allergy (CMPA). Currently, it is not known how long lactating mothers who consume dairy products need to withhold breastfeeding. OBJECTIVE: To elucidate the kinetics of BLG in BM after maternal ingestion of a single dose of CM. METHODS: Nineteen lactating mothers, four of whom had infants with CMPA, were instructed to avoid CM for 7 days before ingesting a single dose of CM and to continue to withhold CM thereafter throughout the study period. BLG was measured by ELISA in BM from 15 mothers of healthy infants before and at 3, 6 and 24 h, and 3 and 7 days after CM ingestion. Four pairs of mothers and CMPA infants were enrolled for BM challenge after the mothers had ingested CM. RESULTS: After CM ingestion, the level of BLG in BM increased significantly from 0.58 ng/ml (0.58 g/L) (IQR 0.38-0.88) to a peak level of 1.23 ng/ml (IQR 1.03-2.29), p < 0.001. The BLG level on day 3 (1.15 ng/ml, IQR 0.89-1.45) and day 7 (1.08 ng/ml (IQR 0.86-1.25) after CM ingestion was significantly higher than baseline (p = 0.01 and p = 0.001, respectively). BLG was detected in all BM samples from the four mothers of CMPA infants after CM ingestion, and the level was not different from that in the mothers of the 15 healthy infants. Three of the four CMPA infants developed symptoms such as maculopapular rash and hypersecretion in the airways after BM challenge. CONCLUSIONS: BLG can be detected in BM up to 7 days after CM ingestion. Lactating mothers should suspend breastfeeding to CMPA infants more than 7 days after CM ingestion.

Development of viable TAP-tagged dengue virus for investigation of host-virus interactions in viral replication.

Dengue virus (DENV) is a mosquito-borne flavivirus responsible for life-threatening dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS). The viral replication machinery containing the core non-structural protein 5 (NS5) is implicated in severe dengue symptoms but molecular details remain obscure. To date, studies seeking to catalogue and characterize interaction networks between viral NS5 and host proteins have been limited to the yeast two-hybrid system, computational prediction and co-immunoprecipitation (IP) of ectopically expressed NS5. However, these traditional approaches do not reproduce a natural course of infection in which a number of DENV NS proteins colocalize and tightly associate during the replication process. Here, we demonstrate the development of a recombinant DENV that harbours a TAP tag in NS5 to study host-virus interactions in vivo. We show that our engineered DENV was infective in several human cell lines and that the tags were stable over multiple viral passages, suggesting negligible structural and functional disturbance of NS5. We further provide proof-of-concept for the use of rationally tagged virus by revealing a high confidence NS5 interaction network in human hepatic cells. Our analysis uncovered previously unrecognized hnRNP complexes and several low-abundance fatty acid metabolism genes, which have been implicated in the viral life cycle. This study sets a new standard for investigation of host-flavivirus interactions.

iNKT Cells and Their Potential Lipid Ligands during Viral Infection.

Invariant natural killer T (iNKT) cells are a unique population of lipid-reactive CD1d-restricted innate-like T lymphocytes. Despite being a minor population, they serve as an early source of cytokines and promote immunological crosstalk thus bridging innate and adaptive immunity. Diseases ranging from allergy, autoimmunity, and cancer, as well as infectious diseases, including viral infection, have been reported to be influenced by iNKT cells. However, it remains unclear how iNKT cells are activated during viral infection, as virus-derived lipid antigens have not been reported. Cytokines may activate iNKT cells during infections from influenza and murine cytomegalovirus, although CD1d-dependent activation is evident in other viral infections. Several viruses, such as dengue virus, induce CD1d upregulation, which correlates with iNKT cell activation. In contrast, herpes simplex virus type 1 (HSV-1), human immunodeficiency virus (HIV), Epstein-Barr virus, and human papilloma virus promote CD1d downregulation as a strategy to evade iNKT cell recognition. These observations suggest the participation of a CD1d-dependent process in the activation of iNKT cells in response to viral infection. Endogenous lipid ligands, including phospholipids as well as glycosphingolipids, such as glucosylceramide, have been proposed to mediate iNKT cell activation. Pro-inflammatory signals produced during viral infection may stimulate iNKT cells through enhanced CD1d-dependent endogenous lipid presentation. Furthermore, viral infection may alter lipid composition and inhibit endogenous lipid degradation. Recent advances in this field are reviewed.

Recombinase Polymerase Amplification Assay for Rapid Diagnostics of Dengue Infection.

BACKGROUND: Over 2.5 billion people are exposed to the risk of contracting dengue fever (DF). Early diagnosis of DF helps to diminish its burden on public health. Real-time reverse transcription polymerase amplification assays (RT-PCR) are the standard method for molecular detection of the dengue virus (DENV). Real-time RT-PCR analysis is not suitable for on-site screening since mobile devices are large, expensive, and complex. In this study, two RT-recombinase polymerase amplification (RT-RPA) assays were developed to detect DENV1-4. METHODOLOGY/PRINCIPAL FINDINGS: Using two quantitative RNA molecular standards, the analytical sensitivity of a RT-RPA targeting the 3´non-translated region of DENV1-4 was found to range from 14 (DENV4) to 241 (DENV1-3) RNA molecules detected. The assay was specific and did not cross detect other Flaviviruses. The RT-RPA assay was tested in a mobile laboratory combining magnetic-bead based total nucleic acid extraction and a portable detection device in Kedougou (Senegal) and in Bangkok (Thailand). In Kedougou, the RT-RPA was operated at an ambient temperature of 38 °C with auxiliary electricity tapped from a motor vehicle and yielded a clinical sensitivity and specificity of 98% (n=31) and 100% (n=23), respectively. While in the field trial in Bangkok, the clinical sensitivity and specificity were 72% (n=90) and 100%(n=41), respectively. CONCLUSIONS/SIGNIFICANCE: During the first 5 days of infection, the developed DENV1-4 RT-RPA assays constitute a suitable accurate and rapid assay for DENV diagnosis. Moreover, the use of a portable fluorescence-reading device broadens its application potential to the point-of-care for outbreak investigations.

Invariant NKT cell response to dengue virus infection in human.

BACKGROUND: Dengue viral infection is a global health threat without vaccine or specific treatment. The clinical outcome varies from asymptomatic, mild dengue fever (DF) to severe dengue hemorrhagic fever (DHF). While adaptive immune responses were found to be detrimental in the dengue pathogenesis, the roles of earlier innate events remain largely uninvestigated. Invariant natural killer T (iNKT) cells represent innate-like T cells that could dictate subsequent adaptive response but their role in human dengue virus infection is not known. We hypothesized that iNKT cells play a role in human dengue infection. METHODS: Blood samples from a well-characterized cohort of children with DF, DHF, in comparison to non-dengue febrile illness (OFI) and healthy controls at various time points were studied. iNKT cells activation were analyzed by the expression of CD69 by flow cytometry. Their cytokine production was then analyzed after α-GalCer stimulation. Further, the CD1d expression on monocytes, and CD69 expression on conventional T cells were measured. RESULTS: iNKT cells were activated during acute dengue infection. The level of iNKT cell activation associates with the disease severity. Furthermore, these iNKT cells had altered functional response to subsequent ex vivo stimulation with α-GalCer. Moreover, during acute dengue infection, monocytic CD1d expression was also upregulated and conventional T cells also became activated. CONCLUSION: iNKT cells might play an early and critical role in the pathogenesis of severe dengue viral infection in human. Targeting iNKT cells and CD1d serve as a potential therapeutic strategy for severe dengue infection in the future.