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.     

Viral Load,Clinical Disease Severity and Cellular Immune Responses in Primary Varicella Zoster Virus Infection in Sri Lanka

Background

In Sri Lanka, varicella zoster virus (VZV) is typically acquired during adulthood with significant associated disease morbidity and mortality. T cells are believed to be important in the control of VZV replication and in the prevention of reactivation. The relationship between viral load, disease severity and cellular immune responses in primary VZV infection has not been well studied.

Methodology

We used IFNγ ELISpot assays and MHC class II tetramers based on VZV gE and IE63 epitopes, together with quantitative real time PCR assays to compare the frequency and phenotype of specific T cells with virological and clinical outcomes in 34 adult Sri Lankan individuals with primary VZV infection.

Principal Findings

Viral loads were found to be significantly higher in patients with moderate to severe infection compared to those with mild infection (p<0.001) and were significantly higher in those over 25 years of age (P<0.01). A significant inverse correlation was seen between the viral loads and the ex vivo IFNγ ELISpot responses of patients (P<0.001, r = −0.85). VZV-specific CD4+ T cells expressed markers of intermediate differentiation and activation.

Conclusions

Overall, these data show that increased clinical severity in Sri Lankan adults with primary VZV infection associates with higher viral load and reduced viral specific T cell responses.     

Biofunctionalization of biomaterials for accelerated in situ endothelialization: a review

The higher patency rates of cardiovascular implants, including vascular bypass grafts, stents, and heart valves are related to their ability to inhibit thrombosis, intimal hyperplasia, and calcification. In native tissue, the endothelium plays a major role in inhibiting these processes. Various bioengineering research strategies thereby aspire to induce endothelialization of graft surfaces either prior to implantation or by accelerating in situ graft endothelialization. This article reviews potential bioresponsive molecular components that can be incorporated into (and/or released from) biomaterial surfaces to obtain accelerated in situ endothelialization of vascular grafts. These molecules could promote in situ endothelialization by the mobilization of endothelial progenitor cells (EPC) from the bone marrow, encouraging cell-specific adhesion (endothelial cells (EC) and/or EPC) to the graft and, once attached, by controlling the proliferation and differentiation of these cells. EC and EPC interactions with the extracellular matrix continue to be a principal source of inspiration for material biofunctionalization, and therefore, the latest developments in understanding these interactions will be discussed.     

Activation tagging in indica rice identifies ribosomal proteins as potential targets for manipulation of water‐use efficiency and abiotic stress tolerance in plants

We have generated 3900 enhancer‐based activation‐tagged plants, in addition to 1030 stable Dissociator‐enhancer plants in a widely cultivated indica rice variety, BPT‐5204. Of them, 3000 were screened for water‐use efficiency (WUE) by analysing photosynthetic quantum efficiency and yield‐related attributes under water‐limiting conditions that identified 200 activation‐tagged mutants, which were analysed for flanking sequences at the site of enhancer integration in the genome. We have further selected five plants with low Δ¹³C, high quantum efficiency and increased plant yield compared with wild type for a detailed investigation. Expression studies of 18 genes in these mutants revealed that in four plants one of the three to four tagged genes became activated, while two genes were concurrently up‐regulated in the fifth plant. Two genes coding for proteins involved in 60S ribosomal assembly, RPL6 and RPL23A, were among those that became activated by enhancers. Quantitative expression analysis of these two genes also corroborated the results on activating–tagging. The high up‐regulation of RPL6 and RPL23A in various stress treatments and the presence of significant cis‐regulatory elements in their promoter regions along with the high up‐regulation of several of RPL genes in various stress treatments indicate that they are potential targets for manipulating WUE/abiotic stress tolerance.     

In vivo study of a model tissue-engineered small-diameter vascular bypass graft

Conventionally used vascular grafts such as polyester (Dacron) or expanded polytetrafluoroethylene perform inadequately as small-diameter vascular bypass grafts (SDBGs). SDBGs, which can maintain long-term patency and those that could potentially evolve with the somatic growth, are highly desirable in vascular surgery and thus research into tissue-engineered blood vessels (TEBVs) is of keen interest. A TEBV was developed by seeding endothelial cells onto a collagen matrix that was cross-linked and contracted by smooth muscle cells (SMCs). A polyester graft served as a scaffold. Recovery studies (12 TEBVs and seven controls) were carried out to assess in vivo endothelialization and long-term patency of TEBVs. Hemodynamic observations indicated para-anastomotic turbulences and high shear stress at anastomosis. Recovery studies demonstrated confluent endothelialization, thrombus-free surfaces, and patent TEBVs in all cases. Graft incorporation and neovascularization of the scaffold occurred in both hybrid and control grafts. However, thickened neointima formation occurred in TEBV grafts, which was most likely caused by the rigidity of polyester scaffold. Significant perigraft inflammatory changes could be observed in both TEBVs and control grafts at 1, 4, and 8 weeks. In conclusion, the TEBVs demonstrated satisfactory performance as an infra-renal-aortic graft in a porcine model. The TEBV serves as a promising model and facilitates the development of a TEBV in a clinical setting, potentially with human stem cells and with more biocompatible, biodegradable scaffolds that are mechanically more compliant with natural vessels.     

Orchestrating cell/material interactions for tissue engineering of surgical implants

Research groups are currently recognising a critical clinical need for innovative approaches to organ failure and agenesis. Allografting, autologous reconstruction and prosthetics are hampered with severe limitations. Pertinently, readily available 'laboratory-grown' organs and implants are becoming a reality. Tissue engineering constructs vary in their design complexity depending on the specific structural and functional demands. Expeditious methods on integrating autologous stem cells onto nanoarchitectured 3D nanocomposites, are being transferred from lab to patients with a number of successful first-in-man experiences. Despite the need for a complete understanding of cell/material interactions tissue engineering is offering a plethora of exciting possibilities in regenerative medicine.     

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.