Adipokine levels and their association with clinical disease severity in patients with dengue.

Adipokines have not been studied in acute dengue, despite their emerging role in inducing and regulating inflammation. Therefore, we sought to identify adipokine levels in patients with varying severities of acute dengue to understand their role in disease pathogenesis. We determined the levels of leptin, resistin, omentin, adiponectin, as well as IFNß, and NS1 using quantitative ELISA in patients with dengue fever (DF = 49) and dengue haemorrhagic fever (DHF = 22) at admission (febrile phase) and at the time of discharge (recovery phase). The viral loads and serotypes of all samples were quantified using quantitative real-time RT-PCR. Resistin levels (p = 0.04) and omentin (p = 0.006) levels were significantly higher in patients who developed DHF. Omentin levels in the febrile phase also correlated with the AST (Spearman's r = 0.38, p = 0.001) and ALT levels (Spearman's r = 0.24, p = 0.04); as well as serum leptin levels with both AST (Spearman's r = 0.27, p = 0.02) and ALT (Spearman's r = 0.28, p = 0.02). Serum adiponectin levels in the febrile phase did not correlate with any of the other adipokines or with liver enzymes, but inversely correlated with CRP levels (Spearman's r = -0.31, p = 0.008). Although not significant (p = 0.14) serum IFNß levels were lower in the febrile phase in those who progressed to develop DHF (median 0, IQR 0 to 39.4 pg/ml), compared to those who had DF (median 37.1, IQR 0 to 65.6 pg.ml). The data suggest that adipokines are likely to play a role in the pathogenesis of dengue, which should be further explored for the potential to be used as prognostic markers and as therapeutic targets.

Identification of allergens in coconut milk and oil with patients sensitized to coconut milk in Sri Lanka.

BACKGROUND: Despite the low prevalence of IgE sensitivity to fresh or boiled coconut milk and coconut oil, those may contain allergens of which the clinical significance remains undetermined. This study aimed to identify and compare allergens in fresh coconut milk (FCM), boiled coconut milk (BCM), unrefined wet-processed coconut oil (WPCO), and dry-processed coconut oil (DPCO) using sera from patients with allergy to coconut milk. METHODS: The study included 18 patients with immediate hypersensitivity to coconut milk, including five who developed anaphylaxis. Sensitization was assessed by skin prick test and ImmunoCAPs using commercially available coconut extracts. Immunoblotting was performed to identify and compare allergen profiles. RESULTS: Total sIgE levels and overall IgE reactivity of patients with anaphylaxis were higher compared to patients with allergy. Twelve allergens ranging from 5 to 128 kDa including six novel allergens with 5, 12, 47, 87, 110, and 128 kDa were visualized in immunoblots with FCM. Similarly, nine allergens of 5, 12, 17, 32, 35, 47, 87, 110, and 128 kDa were detected in BCM. One allergen (110 kDa) was discerned in all four extracts. Higher IgE prevalence was detected with three allergens of 55, 87, and 110 kDa. CONCLUSIONS: Allergens of BCM and unrefined coconut oil (WPCO and DPCO) were determined for the first time. Novel allergens of 87 and 110 kDa and the 55 kDa allergen have the highest potential to be used in Component Resolved Diagnostics. Further, these findings demonstrate that, patients who have an allergy to coconut milk could also react to boiled coconut milk and unrefined coconut oil.

Urinary leukotrienes and histamine in patients with varying severity of acute dengue.

BACKGROUND: Vascular leak is a hallmark of severe dengue, and high leukotriene levels have been observed in dengue mouse models, suggesting a role in disease pathogenesis. We sought to explore their role in acute dengue, by assessing levels of urinary LTE4 and urinary histamine in patients with varying severity of acute dengue. METHODS: Urinary LTE4, histamine and creatinine were measured by a quantitative ELISA, in healthy individuals (n = 19), patients with dengue fever (DF = 72) and dengue haemorrhagic fever DHF (n = 48). The kinetics of LTE4 and histamine and diurnal variations were assessed in a subset of patients. RESULTS: Urinary LTE4 levels were significantly higher (p = 0.004) in patients who proceed to develop DHF when compared to patients with DF during early illness (≤ 4 days) and during the critical phase (p = 0.02), which continued to rise in patients who developed DHF during the course of illness. However, LTE4 is unlikely to be a good biomarker as ROCs gave an AUC value of 0.67 (95% CI 0.57 and 0.76), which was nevertheless significant (p = 0.002). Urinary LTE4 levels did not associate with the degree of viraemia, infecting virus serotype and was not different in those with primary vs secondary dengue. Urinary histamine levels were significantly high in patients with acute dengue although no difference was observed between patients with DF and DHF and again did not associate with the viraemia. Interestingly, LTE4, histamine and the viral loads showed a marked diurnal variation in both patients with DF and DHF. CONCLUSIONS: Our data suggest that LTE4 could play a role in disease pathogenesis and since there are safe and effective cysteinyl leukotriene receptor blockers, it would be important to assess their efficacy in reducing dengue disease severity.

SARS-CoV-2 neutralizing antibodies in patients with varying severity of acute COVID-19 illness.

In order to support vaccine development, and to aid convalescent plasma therapy, it would be important to understand the kinetics, timing and persistence of SARS-CoV-2 neutralizing antibodies (NAbs), and their association with clinical disease severity. Therefore, we used a surrogate viral neutralization test to evaluate their levels in patients with varying severity of illness, in those with prolonged shedding and those with mild/asymptomatic illness at various time points. Patients with severe or moderate COVID-19 illness had earlier appearance of NAbs at higher levels compared to those with mild or asymptomatic illness. Furthermore, those who had prolonged shedding of the virus, had NAbs appearing faster and at higher levels than those who cleared the virus earlier. During the first week of illness the NAb levels of those with mild illness was significantly less (p = 0.01), compared to those with moderate and severe illness. At the end of 4 weeks (28 days), although 89% had NAbs, 38/76 (50%) in those with > 90 days had a negative result for the presence of NAbs. The Ab levels significantly declined during convalescence (> 90 days since onset of illness), compared to 4 to 8 weeks since onset of illness. Our data show that high levels of NAbs during early illness associated with clinical disease severity and that these antibodies declined in 50% of individuals after 3 months since onset of illness.

Similarities and differences between the 'cytokine storms' in acute dengue and COVID-19.

Severe pneumonia and multiorgan dysfunction in COVID-19 and dengue haemorrhagic fever (DHF) are two diseases that can associate with an altered immune response to the infecting virus. To determine the similarities and differences in the cytokine and chemokine responses in these two infections, we compared responses in patients with varying severity of COVID-19 and acute dengue at different time points of illness. During early disease, patients who proceeded to develop COVID-19 severe pneumonia (SP) and DHF had significantly higher levels of IL-6, IL-10 and MIP3α than those who developed mild illness. The lowest levels of IFNγ in early illness were seen in those who succumbed to their illness due to COVID-19. Levels of serum IL-10 (p = 0.0001), IL-6 (p = 0.002), MIP-3α (p = 0.02) and CD40-L levels (p = 0.002) significantly increased from 5 to 9 day of illness to 10-21 day of illness in patients with moderate-to-severe COVID-19, but not in those with mild illness. In contrast, these cytokine/chemokine levels remained unchanged in those with DHF or dengue fever (DF) during febrile and critical phases. Although IL-10 levels were significantly higher in COVID-19 patients with SP, patients with DHF had 25-fold higher levels, whereas IL-6 levels were 11-fold higher in those with COVID-19 SP. IL-10 and other cytokines were evaluated in a larger cohort of patients during early illness (≤ 4 days) who proceeded to develop DF (n = 71) or DHF (n = 64). Of the cytokines evaluated, IL-10 was significantly higher (p < 0.0001) in those who went on to develop DHF compared to DF. Low IFNγ response to the SARS-CoV2 and high levels of immunosuppressive IL-10 in both COVID-19 and dengue during early illness are indicators of an altered antiviral response potentially contributing to disease severity.

Role of NS1 antibodies in the pathogenesis of acute secondary dengue infection.

The role of NS1-specific antibodies in the pathogenesis of dengue virus infection is poorly understood. Here we investigate the immunoglobulin responses of patients with dengue fever (DF) and dengue hemorrhagic fever (DHF) to NS1. Antibody responses to recombinant-NS1 are assessed in serum samples throughout illness of patients with acute secondary DENV1 and DENV2 infection by ELISA. NS1 antibody titres are significantly higher in patients with DHF compared to those with DF for both serotypes, during the critical phase of illness. Furthermore, during both acute secondary DENV1 and DENV2 infection, the antibody repertoire of DF and DHF patients is directed towards distinct regions of the NS1 protein. In addition, healthy individuals, with past non-severe dengue infection have a similar antibody repertoire as those with mild acute infection (DF). Therefore, antibodies that target specific NS1 epitopes could predict disease severity and be of potential benefit in aiding vaccine and treatment design.

Fabrication of 6-gingerol, doxorubicin and alginate hydroxyapatite into a bio-compatible formulation: enhanced anti-proliferative effect on breast and liver cancer cells.

Ample attention has been devoted to the construction of anti-cancer drug delivery systems with increased stability, and controlled and targeted delivery, minimizing toxic effects. In this study we have designed a magnetically attractive hydroxyapatite (m-HAP) based alginate polymer bound nanocarrier to perform targeted, controlled and pH sensitive drug release of 6-gingerol, doxorubicin, and their combination, preferably at low pH environments (pH 5.3). They have exhibited higher encapsulation efficiency which is in the range of 97.4-98.9% for both 6-gingerol and doxorubicin molecules whereas the co-loading has accounted for a value of 81.87 ± 0.32%. Cell proliferation assays, fluorescence imaging and flow cytometric analysis, demonstrated the remarkable time and dose responsive anti-proliferative effect of drug loaded nanoparticles on MCF-7 cells and HEpG2 cells compared with their neat counter parts. Also, these systems have exhibited significantly reduced toxic effects on non-targeted, non-cancerous cells in contrast to the excellent ability to selectively kill cancerous cells. This study has suggested that this HAP based system is a versatile carrier capable of loading various drug molecules, ultimately producing a profound anti-proliferative effect.

Cultured ELISpot Assay to Investigate Dengue Virus Specific T-Cell Responses.

The cultured Enzyme-Linked ImmunoSpot (ELISpot) assay is a functional T cell assay, which is commonly used to assess virus-specific T cell responses. The use of an in vitro expansion step before the ELISpot distinguishes such "cultured" ELISpots from "ex vivo" ELISpots. Cultured ELISpots have the advantage that lower frequency responses can be analyzed compared to ex vivo ELISpots, but do carry the associated potential distortions of the expansion phase. Cultured ELISpot assays are of value to determine silent and symptomatic transmission of the Dengue virus (DENV) in the community and to identify the correlates of a DENV-specific protective immune response. We have evaluated T cell responses to the DENV using cultured ELISpot assays with serotype-specific T cell epitopes to determine past infecting dengue virus (DENV) serotypes. The peptides used in this assay do not cross react with the Japanese encephalitis virus nor other flaviviruses. Therefore, this assay is likely to be useful in determining the past infecting DENV serotype in immune-epidemiological studies and in dengue vaccine trials.

Effective delivery of hydrophobic drugs to breast and liver cancer cells using a hybrid inorganic nanocarrier: A detailed investigation using cytotoxicity assays, fluorescence imaging and flow cytometry.

This study was focused on developing a drug carrier system composed of a polymer containing hydroxyapatite (HAp) shell and a magnetic core of iron oxide nanoparticles. Doxorubicin and/or curcumin were loaded into the carrier via a simple diffusion deposition approach, with encapsulation efficiencies (EE) for curcumin and doxorubicin of 93.03 ±â€¯0.3% and 97.37 ±â€¯0.12% respectively. The co-loading of curcumin and doxorubicin led to a total EE of 76.02 ±â€¯0.48%. Release studies were carried out at pH 7.4 and 5.3, and revealed a greater extent of release at pH 5.3, showing the formulations to have potential applications in tumor microenvironments. Cytotoxicity assays, fluorescence imaging and flow cytometry demonstrated that the formulations could effectively inhibit the growth of MCF-7 (breast) and HEpG2 (liver) cancer cells, being more potent than the free drug molecules both in terms of dose and duration of action. Additionally, hemolysis tests and cytotoxicity evaluations determined the drug-loaded carriers to be non-toxic towards non-cancerous cells. These formulations thus have great potential in the development of new cancer therapeutics.

Pathogenesis of vascular leak in dengue virus infection.

Endothelial dysfunction leading to vascular leak is the hallmark of severe dengue. Vascular leak typically becomes clinically evident 3-6 days after the onset of illness, which is known as the critical phase. This critical phase follows the period of peak viraemia, and lasts for 24-48 hr and usually shows rapid and complete reversal, suggesting that it is likely to occur as a result of inflammatory mediators, rather than infection of the endothelium. Cytokines such as tumour necrosis factor-α, which are known to be elevated in the critical phase of dengue, are likely to be contributing factors. Dengue NS1, a soluble viral protein, has also been shown to disrupt the endothelial glycocalyx and thus contribute to vascular leak, although there appears to be a discordance between the timing of NS1 antigenaemia and occurrence of vascular leak. In addition, many inflammatory lipid mediators are elevated in acute dengue viral infection such as platelet activating factor (PAF) and leukotrienes. Furthermore, many other inflammatory mediators such as vascular endothelial growth factor and angiopoietin-2 have been shown to be elevated in patients with dengue haemorrhagic fever, exerting their action in part by inducing the activity of phospholipases, which have diverse inflammatory effects including generation of PAF. Platelets have also been shown to significantly contribute to endothelial dysfunction by production of interleukin-1ß through activation of the NLRP3 inflammasome and also by inducing production of inflammatory cytokines by monocytes. Drugs that block down-stream immunological mediator pathways such as PAF may also be beneficial in the treatment of severe disease.

Lipopolysaccharide acts synergistically with the dengue virus to induce monocyte production of platelet activating factor and other inflammatory mediators.

BACKGROUND: Platelet Activating Factor (PAF) has been shown to be an important mediator of vascular leak in acute dengue. Antibody dependent enhancement (ADE) and microbial translocation has also shown to contribute to severe dengue. Since monocytes are one of the primary targets of the dengue virus (DENV) we sought to investigate if monocytes were a source of PAF, and the effect of ADE and microbial endotoxin (LPS) on DENV infected monocytes. METHODS: PAF and cytokine levels were evaluated in serial blood samples, in patients with acute dengue infection. The effect of ADE and LPS in production of PAF and cytokines from DENV infected primary human monocytes derived macrophages (MDMθ) was assessed. Gene expression analysis was undertaken to investigate mechanisms by which LPS potentiates PAF and cytokine production by DENV infected MDMθ. RESULTS: Serum PAF levels significantly correlated with both TNF-α (p < 0.0001) and IL-1ß (p < 0.0001) in patients with acute DENV infection. Although primary human MDMθ produced inflammatory cytokines following infection with the DENV, they did not produce PAF following in vitro DENV infection alone, or in the presence of dengue immune serum. Levels of PAF produced by DENV infected MDMθ co-cultured with LPS was significantly higher than uninfected MDMθs co-cultured with LPS. Although TLR-4 was upregulated in uninfected MDMθs co-cultured with LPS, this upregulation was not significant in DENV infected MDMθ. Only expression of RIG-I was significantly up regulated (p < 0.05) when DENV infected MDMθ were co-cultured with LPS. CONCLUSION: LPS acts synergistically with the DENV to induce production of PAF and other inflammatory cytokines, which suggests that microbial translocation that has shown to occur in acute dengue, could contribute to dengue disease severity.

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.

Polymorphisms of Transporter Associated with Antigen Presentation, Tumor Necrosis Factor-α and Interleukin-10 and their Implications for Protection and Susceptibility to Severe Forms of Dengue Fever in Patients in Sri Lanka.

CONTEXT: To date, a clear understanding of dengue disease pathogenesis remains elusive. Some infected individuals display no symptoms while others develop severe life-threatening forms of the disease. It is widely believed that host genetic factors influence dengue severity. AIMS: This study evaluates the relationship between certain polymorphisms and dengue severity in Sri Lankan patients. SETTINGS AND DESIGN: Polymorphism studies are carried out on genes for; transporter associated with antigen presentation (TAP), promoter of tumor necrosis factor-α (TNF-α), and promoter of interleukin-10 (IL-10). In other populations, TAP1 (333), TAP2 (379), TNF-α (-308), and IL-10 (-1082, -819, -592) have been associated with dengue and a number of different diseases. Data have not been collected previously for these polymorphisms for dengue patients in Sri Lanka. MATERIALS AND METHODS: The polymorphisms were typed by amplification refractory mutation system polymerase chain reaction in 107 dengue hemorrhagic fever (DHF) patients together with 62 healthy controls. STATISTICAL ANALYSIS USED: Pearson's Chi-square contingency table analysis with Yates' correction. RESULTS: Neither the TAP nor the IL-10 polymorphisms considered individually can define dengue disease outcome with regard to severity. However, the genotype combination, IL-10 (-592/-819/-1082) CCA/ATA was significantly associated with development of severe dengue in these patients, suggesting a risk factor to developing DHF. Also, identified is the genotype combination IL-10 (-592/-819/-1082) ATA/ATG which suggested a possibility for protection from DHF. The TNF-α (-308) GG genotype was also significantly associated with severe dengue, suggesting a significant risk factor. CONCLUSIONS: The results reported here are specific to the Sri Lankan population. Comparisons with previous reports imply that data may vary from population to population.

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.

Rapid effector function of varicella-zoster virus glycoprotein I-specific CD4+ T cells many decades after primary infection.

Glycoprotein I (gI) of varicella-zoster virus (VZV) contributes to viral virulence and is therefore a potentially important target for T cell control of viral replication. Persisting effector function of gI-specific T cells after primary infection has not been previously examined. We have shown that, many decades after infection, relatively high frequencies gI-specific interferon- gamma responses are detectable ex vivo and are dominated by CD4(+) T cells. We characterized the optimal peptide of the strongest response in our cohort showing restriction through DRB4*01. These findings are consistent with gI-specific CD4(+) T cell involvement in the control of VZV replication.