Laksiri Gomes

Cellular and Cytokine Correlates of Severe Dengue Infection

Background The occurrence of dengue haemorrhagic fever (DHF) is thought to result from a complex interplay between the virus, host genetics and host immune factors. Existing published data are not consistent, in part related to relatively small sample sizes. We set out to determine possible associations between dengue virus (DEN-V) NS3 specific T cells and cytokine and chemokine levels and the pathogenesis of severe disease in a large cohort of individuals with DHF. Methodology/Principal Findings By using ex vivo IFNγ ELISpot assays we determined DENV-NS3 specific responses in patients with varying severity of DHF. Other cytokines produced by DENV-NS3 specific T cells were determined by using multiple bead array analysis (MBAA). We also determined the serum cytokine levels using MBAA, lymphocyte subsets and Annexin V expression of lymphocytes in patients with varying severity of DHF. Of the 112 DHF patients studied, 29 developed shock. Serum IL-10 and IP-10 levels positively and significantly correlated with T cell apoptosis while IL-10 levels inversely correlated with T cell numbers. In contrast, TGFß showed a very significant (P<0.0001) and positive correlation (Spearman’s R = 0.65) with the platelet counts, consistent with platelet release. We found that whilst patients with severe dengue had lower total T cell numbers, the DV-NS3 specific T cells persisted and produced high levels of IFNγ but not TNFα, IL-3, IL-13, IL-2, IL-10 or IL-17. Conclusions/Significance Our data suggest that serum IL-10, TNFα and TGFβ differentially associate with dengue disease severity.

Serum IL-10 as a marker of severe dengue infection

BackgroundSeveral studies have shown that serum IL-10, IFNγ and MIF are elevated in patients in severe dengue (SD) and could be used as potential biomarkers. We proceeded to determine if these cytokines could be used as biomarkers in a large cohort of adult dengue patients with varying severity of dengue infection.MethodsSerum IL-10 levels were determined in 259 of whom 40 had severe dengue infection. Serum IFNγ and IFNα levels were done in 78 and MIF levels were done in 65 patients with acute dengue infection. Clinical features and laboratory investigations were undertaken during the febrile and critical phase.ResultsWe found that serum IL-10 levels were significantly higher (p = 0.001) in patients with SD, when compared to those with non SD. Serum IL-10 levels significantly and inversely correlated with white cell counts (R = −0.23, p = 0.0002) and lymphocyte counts (R = −0.29, p < 0.0001) but significantly and positively correlated with aspartate tranaminase levels (R = 0.16, p = 0.01) and alanine transaminase levels (R = 0.22, p = 0.007). However, IL-10 levels did not have a good predictive value in discriminating those who were likely to develop SD (AUC = 0.66). Serum IFNγ levels were also significantly higher (p = 0.04) in patients with SD when compared to non SD. There was no difference (p = 0.34) in serum IFNα levels and serum MIF levels (p = 0.15) in patients with SD and non SD.ConclusionAlthough serum IL-10 was significantly elevated in patients with SD it had a poor discriminatory value in identifying those with SD and non SD and therefore, is unsuitable to be used as a robust biomarker in this cohort.

Suppression of virus specific immune responses by IL-10 in acute dengue infection.

Background Elevated IL-10 has been shown to be associated with severe dengue infection (DI). We proceeded to investigate the role of IL-10 in the pathogenesis of acute DI. Materials and methods Ex vivo and cultured IFNγ ELISpot assays for dengue virus (DENV) NS3 protein and non dengue viral proteins were carried out in 26 patients with acute DI (16 with dengue haemorrhagic fever) and 12 healthy dengue seropositive individuals from Sri Lanka. DENV serotype specific (SS) responses were determined by using a panel of SS peptides. Results Serum IL-10 level were significantly higher (p = 0.02) in those who did not have in vitro responses to DENV-SS peptides (mean 144.2 pg/ml) when compared to those who responded (mean 75.7 pg/ml). DENV-NS3 specific ex vivo IFNγ ELISpot responses were also significantly lower (p = 0.0001) in those who did not respond to DENV-SS peptides (mean 42 SFU/million PBMCs) when compared to those who responded to DENV-SS peptides (mean 1024 SFU/million PBMCs). Serum IL-10 levels correlated significantly (p = 0.03) and inversely (Spearmans R = −0.45) with ex vivo DENV-NS3 specific responses but not with ex vivo non DENV specific responses (Spearmans R = −014, p = 0.52). Blockage of IL-10 in vitro significantly increased (p = 0.04) the ex vivo IFNγ ELISpot DENV-NS3 specific responses but had no effect on responses to non DENV proteins. Conclusion IL-10 appears to contribute to the pathogenesis of acute dengue infections by inhibiting DENV-specific T cell responses, which can be restored by blocking IL-10.

Evaluation of the WHO revised criteria for classification of clinical disease severity in acute adult dengue infection

BackgroundThe WHO guidelines were revised recently to identify patients with severe dengue (SD) early. We proceeded to determine the usefulness of the warning signs in the new WHO guidelines in predicting SD and we have also attempted to define other simple laboratory parameters that could be useful in predicting SD.MethodsClinical and laboratory parameters were recorded in 184 patients in 2011, with confirmed dengue viral infections, admitted to a medical ward in two tertiary care hospitals in Colombo, Sri Lanka.ResultsWe found that the presence of 5 or more dengue warning signs were significantly (p = 0.02) associated with the development of SD (odds ratio 5.14, 95% CI = 1.312 to 20.16). The AST levels were significantly higher (p = 0.0001) in patients with abdominal pain (mean 243.5, SD ± 200.7), when compared to those who did not have abdominal pain (mean 148.5, SD ± 218.6). Lymphocyte counts <1,500 cells/mm3 were significantly (p = 0.005) associated with SD (odds ratio 3.367, 95% CI 1.396 to 8.123). High AST levels were also significantly associated (p < 0.0001) with SD (odds ratio 27.26, 95% CI 1.632 to 455.2). Platelet counts <20,000cells/mm3, were again significantly associated (p < 0.001) with severe disease (odds ratio 1.632 to 455.2, 95% CI 3.089 to 14.71). The PCR was positive in 26/84 of the patients and we found that the infecting serotype was DEN-1 in all 26 patients.ConclusionsThe presence of 5 or more warning signs appears to be a predictor of SD. Lymphocyte counts <1,500 cells/mm3, platelet counts <20,000/mm3 and raised AST levels were associated with SD and could be used to help identify patients who are likely to develop SD.

Sphingosine 1-phosphate in acute dengue infection.

Background 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. Metholodology/Principal findings 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. Conclusions/Significance 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.

Dengue NS1 antigen as a marker of severe clinical disease

BackgroundEarly 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.

Platelet Activating Factor Contributes to Vascular Leak in Acute Dengue Infection

Background 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. Materials and Methods 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. Results 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. Conclusion 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.

Functionality of Dengue Virus Specific Memory T Cell Responses in Individuals Who Were Hospitalized or Who Had Mild or Subclinical Dengue Infection

Background 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. Methodology/Principal findings 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. Conclusions/significance 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.

Dengue NS1 antigen contributes to disease severity by inducing interleukin (IL)-10 by monocytes

Both dengue NS1 antigen and serum interleukin (IL)‐10 levels have been shown to associate with severe clinical disease in acute dengue infection, and IL‐10 has also been shown to suppress dengue‐specific T cell responses. Therefore, we proceeded to investigate the mechanisms by which dengue NS1 contributes to disease pathogenesis and if it is associated with altered IL‐10 production. Serum IL‐10 and dengue NS1 antigen levels were assessed serially in 36 adult Sri Lankan individuals with acute dengue infection. We found that the serum IL‐10 levels correlated positively with dengue NS1 antigen levels (Spearmans r = 0·47, P < 0·0001), and NS1 also correlated with annexin V expression by T cells in acute dengue (Spearmans r = 0·63, P = 0·001). However, NS1 levels did not associate with the functionality of T cell responses or with expression of co‐stimulatory molecules. Therefore, we further assessed the effect of dengue NS1 on monocytes and T cells by co‐culturing primary monocytes and peripheral blood mononuclear cells (PBMC), with varying concentrations of NS1 for up to 96 h. Monocytes co‐cultured with NS1 produced high levels of IL‐10, with the highest levels seen at 24 h, and then declined gradually. Therefore, our data show that dengue NS1 appears to contribute to pathogenesis of dengue infection by inducing IL‐10 production by monocytes.

Dengue NS1 antigen contributes to disease severity by inducing IL‐10 by monocytes

Both dengue NS1 antigen and serum interleukin (IL)‐10 levels have been shown to associate with severe clinical disease in acute dengue infection, and IL‐10 has also been shown to suppress dengue‐specific T cell responses. Therefore, we proceeded to investigate the mechanisms by which dengue NS1 contributes to disease pathogenesis and if it is associated with altered IL‐10 production. Serum IL‐10 and dengue NS1 antigen levels were assessed serially in 36 adult Sri Lankan individuals with acute dengue infection. We found that the serum IL‐10 levels correlated positively with dengue NS1 antigen levels (Spearmans r = 0·47, P < 0·0001), and NS1 also correlated with annexin V expression by T cells in acute dengue (Spearmans r = 0·63, P = 0·001). However, NS1 levels did not associate with the functionality of T cell responses or with expression of co‐stimulatory molecules. Therefore, we further assessed the effect of dengue NS1 on monocytes and T cells by co‐culturing primary monocytes and peripheral blood mononuclear cells (PBMC), with varying concentrations of NS1 for up to 96 h. Monocytes co‐cultured with NS1 produced high levels of IL‐10, with the highest levels seen at 24 h, and then declined gradually. Therefore, our data show that dengue NS1 appears to contribute to pathogenesis of dengue infection by inducing IL‐10 production by monocytes.