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 TGFß differentially associate with dengue disease severity.

Identification of serotype-specific T cell responses to highly conserved regions of the dengue viruses.

Determining previous infecting dengue virus (DENV) serotypes has been difficult due to highly cross-reactive immune responses from previous DENV infections. Determining the correlates of serotype-specific immune responses would be crucial in understanding dengue transmission in the community and would also help to determine the correlates of protective immune responses. Therefore, we set out to define highly conserved, serotype-specific regions of the DENVs. Serotype-specific and highly conserved regions of the four DENV serotypes were identified using Basic Local Alignment Search Tool (BLAST) searches and custom perl scripts. Using ex-vivo and cultured enzyme-linked immunospot (ELISPOT) assays, we identified serotype-specific T cell epitopes within the four DENV serotypes in healthy adult donors from Sri Lanka. We identified T cell responses to 19 regions of the four DENV serotypes. Six peptides were from the NS2A region and four peptides were from the NS4A region. All immune donors responded to peptides of at least two DENV serotypes, suggesting that heterologous infection is common in Sri Lanka. Eight of 20 individuals responded to at least two peptides of DENV-4, despite this serotype not being implicated previously in any of the epidemics in Sri Lanka. The use of these regions to determine past and current infecting DENV serotypes will be of value to characterize further the dynamics of silent dengue transmission in the community. In addition, these T cell responses to these regions could be used to characterize DENV serotype-specific immune responses and thus possibly help us to understand the immune correlates of a protective immune response.

Banding of oesophageal varices using locally improvised materials.

Bleeding from oesophageal varices is the commonest cause of significant upper gastrointestinal bleeding in Sri Lanka. Endoscopic band ligation is an effective method in the management of varices. But the cost of banding equipment is high, unaffordable for a majority of our patients. We have devised a cheap method to carry out banding of varices. Banding of varices using this technique was carried out in 235 patients. In the patients who were followed up, there was a reduction in the size of varices across all grades of varices. No complications due to banding were noted, and only 10 patients developed re-bleeding. Since the initial experience on efficacy and safety of this technique is encouraging, we believe that its widespread adoption in Sri Lanka would be cost effective and life-saving.