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Proteomics of Extracellular Vesicles in mild and severe dengue patients’ plasma

ABSTRACT: We have isolated extracellular vesicles from healthy donors' plasma, other febrile illness plasma, mild dengue patients (Platelet counts= >100,000 per mm3 of blood) , and severe dengue patients' plasma (Platelet counts= <30,000 per mm3 of blood) by ultracentrifugation. For understanding, the differences in protein cargo among the EVs isolated from these groups were subjected to SWATH analysis. We have used three biological replicates of each group of EVs. A- EVS DERIVED FROM SEVERE DENGUE PLASMA B- EVS DERIVED FROM MILD DENGUE PLASMA C- EVS DERIVED FROM OTHER FEBRILE ILLNESS PLASMA D- EVS DERIVED FROM HEALTHY DONORS PLASMA

INSTRUMENT(S): ZenoTOF 7600

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Blood Plasma

DISEASE(S): Dengue Disease

SUBMITTER: Sharda Kumari

LAB HEAD: Professor Arup Banerjee

PROVIDER: PXD058037 | Pride | 2024-12-25

REPOSITORIES: Pride

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Project description:Background: We report the detailed development of biomarkers to predict the clinical outcome under dengue infection. Transcriptional signatures from purified peripheral blood mononuclear cells were derived from whole-genome gene-expression microarray data and validated by quantitative PCR and tested in independent samples. Methodology/Principal Findings: The study was performed on patients of a well-characterized dengue cohort from Recife, Brazil. The samples analyzed were collected prospectively from acute febrile dengue patients who evolved with different degrees of disease severity, classic dengue fever or dengue hemorrhagic fever (DHF) and compared with similar samples from other non-dengue febrile illnesses. The DHF samples were collected 2-3 days before the presentation of the plasma leakage symptoms. Differentially-expressed genes were selected by univariate statistical tests as well as multivariate classification techniques. The results showed that at early stages of dengue infection, the genes involved in effector mechanisms of innate immune response presented a weaker activation on patients who later developed hemorrhagic fever, whereas the genes involved in apoptosis were expressed in higher levels. Conclusions/Significance: Some of the gene expression signatures displayed estimated accuracy rates of more than 95%, indicating that expression profiling with these signatures may provide a useful means of DHF prognosis at early stages of infection The samples correspond to blood samples from 26 patients from a cohort in Brazil, divided into three classes, DHF, DF, ND, as follows: 18 were confirmed dengue 3, genotype III cases, among which 10 were diagnosed as dengue hemorrhagic fever (DHF) and 8 as classical dengue fever (DF), and 8 control samples (ND) from febrile patients confirmed to be not infected with dengue. None of the DHF patients presented vasculopathy signs and symptoms at the time the samples used in the functional genomic characterization were collected. At the time of collection the patients referred approximately 5 days of disease and the absence of fever was reported two to three days after enrollment. The samples from the DF, DHF and ND patients were matched to avoid significant differences regarding patient age, gender, dengue infection history and days of symptoms among the groups.

Project description: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-g 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.

			Action	DRS
	2023_08_09_Plasma_Exosome_AB.sne	Other
	A1.1.zip	Other
	A1.2.zip	Other
	A1.3.zip	Other
	A2.1.wiff.zip	Wiff

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Proteomics of Extracellular Vesicles in mild and severe dengue patients’ plasma

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