[Show abstract][Hide abstract] ABSTRACT: Although antibody responses to dengue virus (DENV) in naturally infected individuals have been extensively studied, the functionality of DENV specific memory T cell responses in relation to clinical disease severity is incompletely understood.
Using ex vivo IFNγ ELISpot assays, and by determining cytokines produced in ELISpot supernatants, we investigated the functionality of DENV-specific memory T cell responses in a large cohort of individuals from Sri Lanka (n=338), who were naturally infected and were either hospitalized due to dengue or had mild or sub clinical dengue infection. We found that T cells of individuals with both past mild or sub clinical dengue infection and who were hospitalized produced multiple cytokines when stimulated with DENV-NS3 peptides. However, while DENV-NS3 specific T cells of those with mild/sub clinical dengue infection were more likely to produce only granzyme B (p=0.02), those who were hospitalized were more likely to produce both TNFα and IFNγ (p=0.03) or TNFα alone. We have also investigated the usefulness of a novel T cell based assay, which can be used to determine the past infecting DENV serotype. 92.4% of DENV seropositive individuals responded to at least one DENV serotype of this assay and none of the seronegatives responded. Individuals who were seronegative, but had received the Japanese encephalitis vaccine too made no responses, suggesting that the peptides used in this assay did not cross react with the Japanese encephalitis virus.
The types of cytokines produced by DENV-specific memory T cells appear to influence the outcome of clinical disease severity. The novel T cell based assay, is likely to be useful in determining the past infecting DENV serotype in immune-epidemiological studies and also in dengue vaccine trials.
[Show abstract][Hide abstract] ABSTRACT: The varicella-zoster virus (VZV) re-activation increases during ageing. Although the effects of VZV re-activation are observed in the skin (shingles) the number or functional capacity of cutaneous VZV specific T cells have not been investigated. The numbers of circulating IFN-γ secreting VZV specific CD4(+) T cells are significantly decreased in old subjects however other measures of VZV-specific CD4(+) T cells, including proliferative capacity to VZV antigen stimulation and identification of VZV-specific CD4(+) T cells with a MHC class II tetramer (epitope of IE-63 protein), were similar in both age groups. The majority of T cells in the skin of both age groups expressed CD69, a characteristic of skin resident T cells. VZV-specific CD4(+) T cells were significantly increased in the skin compared to the blood in young and old subjects and their function was similar in both age groups. In contrast the number of Foxp3(+) regulatory T cells (Tregs) and expression of the inhibitory receptor PD-1 on CD4(+) T cells were significantly increased in the skin of older humans. Therefore VZV-specific CD4(+) T cells in the skin of older individuals are functionally competent. However their activity may be restricted by multiple inhibitory influences in situ.Journal of Investigative Dermatology accepted article preview online, 03 March 2015. doi:10.1038/jid.2015.63.
[Show abstract][Hide abstract] ABSTRACT: Although plasma leakage is the hallmark of severe dengue infections, the factors that cause increased vascular permeability have not been identified. As platelet activating factor (PAF) is associated with an increase in vascular permeability in other diseases, we set out to investigate its role in acute dengue infection.
PAF levels were initially assessed in 25 patients with acute dengue infection to determine if they were increased in acute dengue. For investigation of the kinetics of PAF, serial PAF values were assessed in 36 patients. The effect of dengue serum on tight junction protein ZO-1 was determined by using human endothelial cell lines (HUVECs). The effect of dengue serum on and trans-endothelial resistance (TEER) was also measured on HUVECs.
PAF levels were significantly higher in patients with acute dengue (n = 25; p = 0.001) when compared to healthy individuals (n = 12). In further investigation of the kinetics of PAF in serial blood samples of patients (n = 36), PAF levels rose just before the onset of the critical phase. PAF levels were significantly higher in patients with evidence of vascular leak throughout the course of the illness when compared to those with milder disease. Serum from patients with dengue significantly down-regulated expression of tight junction protein, ZO-1 (p = 0.004), HUVECs. This was significantly inhibited (p = 0.004) by use of a PAF receptor (PAFR) blocker. Serum from dengue patients also significantly reduced TEER and this reduction was also significantly (p = 0.02) inhibited by prior incubation with the PAFR blocker.
Our results suggest the PAF is likely to be playing a significant role in inducing vascular leak in acute dengue infection which offers a potential target for therapeutic intervention.
[Show abstract][Hide abstract] ABSTRACT: Innate lymphoid cells are an emerging family of effector cells that contribute to lymphoid organogenesis, metabolism, tissue remodelling and protection against infections. They maintain homeostatic immunity at barrier surfaces such as lung, skin and gut (Nature 464:1367-1371, 2010, Nat Rev Immunol 13: 145-149, 2013). Several human and mouse studies suggest a role for innate lymphoid cells in inflammatory skin conditions including atopic eczema and psoriasis. Here we review the innate lymphoid cell family and discuss their function in the skin and during inflammation.
[Show abstract][Hide abstract] ABSTRACT: Vascular leak is the hallmark of severe dengue infections and leads to complications such as shock and multi-organ failure. Although many mediators have been implicated in the vascular leak in dengue, the role of sphingosine 1-phosphate (S1P) has not been investigated.
As S1P has been shown to be important in barrier integrity, we assessed the S1P levels in 28 patients with acute dengue and 12 healthy individuals. The S1P levels were significantly lower in patients with acute dengue (p = 0.002) and the levels in patients with grade IV dengue haemorrhagic fever (DHF) were significantly lower than those with dengue fever (p = 0.005). We then investigated the kinetics of S1P levels throughout the course of the illness in another 32 patients in serum samples obtained twice a day. We found that S1P levels were low throughout the course of illness and S1P levels were <0.5 µM in 12/23 patients with DHF when compared to 1/9 with DF.
As S1P has shown to be important in the endothelial barrier integrity and increases transendothelial resistance, low levels of S1P in acute dengue infection are likely to contribute to increased vascular permeability.
PLoS ONE 11/2014; 9(11):e113394. DOI:10.1371/journal.pone.0113394 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background
Early detection of complications significantly reduces dengue associated mortality and morbidity. We set out to determine if the NS1 rapid antigen detection test could be used as a point of care test to predict severe disease.Methods186 adult patients with confirmed dengue were enrolled during day 3¿8 of illness. Clinical and laboratory parameters were recorded during the course of the illness and NS1 antigen levels were determined using both the Panbio dengue early ELISA (Panbio, Australia) and a NS1 rapid antigen detection kit (SD Bioline, South Korea).Results59.1% of patients presented to hospital on day 5¿6 of illness when NS1 antigen positivity was significantly (p¿=¿0.008) associated with severe dengue (odds ratio 3.0, 95% CI 1.39 to 6.47) and the NS1 antigen levels were significantly higher (p¿=¿0.03) in those who went on to develop shock. Serum NS1 antigen levels significantly (p¿<¿0.0001) and inversely correlated with the total white cell counts and lymphocyte counts. The bedside NS1 test showed comparable sensitivity (97.4%) and specificity (93.7%) to the laboratory NS1 test in our setting and cohort.ConclusionNS1 antigen positivity is associated with a higher risk of developing severe dengue especially when positive beyond day 5 of illness in our cohort, and while further validation studies are required, the test can therefore potentially be used as a bedside point of care test as a warning sign of severe dengue.
[Show abstract][Hide abstract] ABSTRACT: Group 2 innate lymphoid cells (ILC2s) release interleukin-13 (IL-13) during protective immunity to helminth infection and detrimentally during allergy and asthma. Using two mouse models to deplete ILC2s in vivo, we demonstrate that T helper 2 (Th2) cell responses are impaired in the absence of ILC2s. We show that MHCII-expressing ILC2s interact with antigen-specific T cells to instigate a dialog in which IL-2 production from T cells promotes ILC2 proliferation and IL-13 production. Deletion of MHCII renders IL-13-expressing ILC2s incapable of efficiently inducing Nippostrongylus brasiliensis expulsion. Thus, during transition to adaptive T cell-mediated immunity, the ILC2 and T cell crosstalk contributes to their mutual maintenance, expansion and cytokine production. This interaction appears to augment dendritic-cell-induced T cell activation and identifies a previously unappreciated pathway in the regulation of type-2 immunity.
[Show abstract][Hide abstract] ABSTRACT: Background
Histamine is an abundant mediator accumulating in the skin of atopic patients, where it is thought to be derived from immune cells. While keratinocytes express histidine decarboxylase (HDC), levels of the enzyme in normal or diseased epidermis and factors that influence its expression in human keratinocytes are not known. Objectives
To assess levels of HDC in inflammatory skin diseases and factors influencing its expression. Methods
Normal and filaggrin-insufficient human keratinocytes, organotypic epidermal models and skin samples were investigated for the expression of HDC. The effect of cytokines, bacterial and allergen stimuli exposure and functional changes in differentiation were evaluated in vitro. ResultsWe detected abundant expression of the HDC protein in all models studied; expression was increased in atopic skin samples. Filaggrin-insufficient keratinocytes maintained HDC levels, but exposure of keratinocytes to thymic stromal lymphopoietin, tumour necrosis factor-, lipopolysaccharide (LPS) and house dust mite (HDM) extract increased HDC expression in vitro. Furthermore, filaggrin expression in cultured keratinocytes increased following histamine depletion. Conclusions
Keratinocytes express abundant HDC protein, and the levels increase in atopic skin. LPS, HDM and cytokines, which are implicated in allergic inflammation, promote the expression of the enzyme and upregulate histamine levels in keratinocytes. Actively produced histamine influences keratinocyte differentiation, suggesting functional relevance of the axis to atopic dermatitis. The findings therefore identify a new point of therapeutic intervention.
British Journal of Dermatology 06/2014; 171(4). DOI:10.1111/bjd.13199 · 4.10 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The role of the epidermis in the immune response is well known. While multiple cytokines are implicated in keratinocyte-mediated infection clearance and wound healing, little is known about the involvement of keratinocytes in promoting resolution of inflammation.
To assess effects of histamine stimulation on keratinocyte function.
We performed a combined microarray/Gene Ontology analysis of histamine-stimulated keratinocytes. Functional changes were tested by apoptosis assessment and scratch assays. Histamine receptor involvement was also assessed by blocking wound closure with specific antagonists.
Histamine treatment had extensive effects on keratinocytes, including effects on proinflammatory responses and cellular functions promoting wound healing. At the functional level, there was reduced apoptosis and enhancement of wound healing in vitro. At the receptor level, we identified involvement of all keratinocyte-expressed histamine receptors (HRHs), with HRH1 blockage resulting in the most prominent effect.
Histamine activates wound healing and infection clearance-related functions of keratinocytes. While enhancement of histamine-mediated wound healing is mediated predominantly via the HRH1 receptor, other keratinocyte-expressed receptors are also involved. These effects could promote resolution of skin inflammation caused by infection or superficial injury.
[Show abstract][Hide abstract] ABSTRACT: The role of epidermal barrier genes in the pathogenesis of atopic skin inflammation has recently been highlighted. Cytokines that are abundant in the skin during inflammation have been shown to exert various effects on the expression of barrier genes, although the role of histamine in this area of skin biology is not yet fully understood.
[Show abstract][Hide abstract] ABSTRACT: Activation of the group 2 innate lymphoid cell (ILC2) population leads to production of the classical type 2 cytokines, thus promoting type 2 immunity. Chemoattractant receptor-homologous molecule expressed on TH2 cells (CRTH2), a receptor for prostaglandin D2 (PGD2), is expressed by human ILC2s. However, the function of CRTH2 in these cells is unclear.
We sought to determine the role of PGD2 and CRTH2 in human ILC2s and compare it with that of the established ILC2 activators IL-25 and IL-33.
The effects of PGD2, IL-25, and IL-33 on the cell migration, cytokine production, gene regulation, and receptor expression of ILC2s were measured with chemotaxis, ELISA, Luminex, flow cytometry, quantitative RT-PCR, and QuantiGene assays. The effects of PGD2 under physiologic conditions were evaluated by using the supernatant from activated mast cells.
PGD2 binding to CRTH2 induced ILC2 migration and production of type 2 cytokines and many other cytokines. ILC2 activation through CRTH2 also upregulated the expression of IL-33 and IL-25 receptor subunits (ST2 and IL-17RA). The effects of PGD2 on ILC2s could be mimicked by the supernatant from activated human mast cells and inhibited by a CRTH2 antagonist.
PGD2 is an important and potent activator of ILC2s through CRTH2 mediating strong proallergic inflammatory responses. Through IgE-mediated mast cell degranulation, these innate cells can also contribute to adaptive type 2 immunity; thus CRTH2 bridges the innate and adaptive pathways in human ILC2s.
The Journal of allergy and clinical immunology 12/2013; 133(4). DOI:10.1016/j.jaci.2013.10.056 · 11.25 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Type 2 innate lymphoid cells (ILC2s, nuocytes, NHC) require RORA and GATA3 for their development. We show that human ILC2s express skin homing receptors and infiltrate the skin after allergen challenge, where they produce the type 2 cytokines IL-5 and IL-13. Skin-derived ILC2s express the IL-33 receptor ST2, which is up-regulated during activation, and are enriched in lesional skin biopsies from atopic patients. Signaling via IL-33 induces type 2 cytokine and amphiregulin expression, and increases ILC2 migration. Furthermore, we demonstrate that E-cadherin ligation on human ILC2 dramatically inhibits IL-5 and IL-13 production. Interestingly, down-regulation of E-cadherin is characteristic of filaggrin insufficiency, a cardinal feature of atopic dermatitis (AD). ILC2 may contribute to increases in type 2 cytokine production in the absence of the suppressive E-cadherin ligation through this novel mechanism of barrier sensing. Using Rag1(-/-) and RORα-deficient mice, we confirm that ILC2s are present in mouse skin and promote AD-like inflammation. IL-25 and IL-33 are the predominant ILC2-inducing cytokines in this model. The presence of ILC2s in skin, and their production of type 2 cytokines in response to IL-33, identifies a role for ILC2s in the pathogenesis of cutaneous atopic disease.
Journal of Experimental Medicine 12/2013; 210(13). DOI:10.1084/jem.20130351 · 13.91 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Comprehensive antigenic characterization of a T cell population of unknown specificity is challenging. Existing MHC class I expression systems are limited by the practical difficulty of probing cell populations with an MHC class I peptide library and the cross-reactivity of T cells that are able to recognise many variants of an index peptide. Using emulsion PCR and emulsion in vitro transcription/translation of a random library of peptides conjugated to CD8-null HLA-A*0201 on beads, we probed HLA-A*0201-restricted T cells with specificity for influenza, CMV and EBV. We observed significant enrichment for sequences containing HLA-A2 anchors and correct viral fragments for all T cell populations. HLA bead display provides a novel approach to identify the specificity of T cells.
[Show abstract][Hide abstract] ABSTRACT: Genotyping of wild type of varicella zoster virus (VZV) in Sri Lanka would help to distinguish the VZV wild type infection from varicella vaccine associated infections.
PCR-RFLP analysis of VZV ORF 38, 54 and 62 was used for genotyping in VZV from blood or vesicular fluid from 31 patients with chickenpox or herpes zoster. The PstI restriction site of ORF 38, BglI restriction site of ORF 54 and SmaI restriction site of ORF 62 were analyzed using RFLP to determine the genotype.
Except for one strain, all other VZV isolates had the genotype characteristic of the wild type VZV strain PstI+BglI+ SmaI-, which was characteristic of the Asian strain. None of the isolates had the American or the European VZV profile (PstI+BglI-) but were similar to isolates from Africa and Asia (PstI+BglI+). Interestingly, one of the VZV strains isolated from a patient with chickenpox had the characteristic genotype of the vaccine strain PstI- BglI+ SmaI+.
The genotype of the VZV in Sri Lanka is similar to the Asian VZV genotype and can be easily distinguished from the VZV vaccine strain by using the polymorphisms in ORF 38, ORF 54 and ORF 62.
Ceylon Medical Journal 12/2013; 58(4):153-6. DOI:10.4038/cmj.v58i4.6306