Comprehensive analysis of dengue virus-specific responses supports an HLA-linked protective role for CD8+ T cells.

The role of CD8(+) T cells in dengue virus infection and subsequent disease manifestations is not fully understood. According to the original antigenic sin theory, skewing of T-cell responses induced by primary infection with one serotype causes less effective response upon secondary infection with a different serotype, predisposing individuals to severe disease. A comprehensive analysis of CD8(+) responses in the general population from the Sri Lankan hyperendemic area, involving the measurement of ex vivo IFNγ responses associated with more than 400 epitopes, challenges the original antigenic sin theory. Although skewing of responses toward primary infecting viruses was detected, this was not associated with impairment of responses either qualitatively or quantitatively. Furthermore, we demonstrate higher magnitude and more polyfunctional responses for HLA alleles associated with decreased susceptibility to severe disease, suggesting that a vigorous response by multifunctional CD8(+) T cells is associated with protection from dengue virus disease.

Activated lymphocytes and high liver expression of IFN-γ are associated with fulminant hepatic failure in patients.

BACKGROUND & AIMS: To study immunological mechanisms of fulminant hepatic failure (FHF) derived from extensive liver lesions, 14 patients with FHF induced by different aetiologies were investigated by observance of both lymphocyte phenotyping and cytokine levels. METHODS: Five patients bearing benign acute hepatitis B (AHB) and seven healthy liver donors (HC) were used as controls. Samples of liver and blood from both FHF patients and HC were obtained during transplantation procedures. Plasma levels of IL-1ß, IL-4, IL-6, IL-8, IL-10, IFN-γ, TNF-α, MCP-1, RANTES and MIP-1α were quantified using a multiplex immunoassay. Cell characterization was carried out by flow cytometry. IFN-γ staining was performed on liver sections using immunofluorescence methods. RESULTS: An increase of peripheral frequency of natural killer (NK) cells expressing early activation markers (CD69, HLA-DR and CD38) and adhesion molecule CD44 was observed in FHF patients. Elevated frequency of T lymphocytes CD4(+) and CD8(+) expressing CD38 and adhesion molecules CD29 and CD44 was also observed in FHF. Additionally, an increase of natural killer T cells (NKT) was detected in FHF patients. High plasma cytokine levels were not statistically different between FHF and AHB patients. In comparison to HC, a strong liver expression of IFN-γ was detected in FHF patients. The increased frequency of CD4(+) CD44(+) and IL-8 cytokine was found in patients with poor prognosis. CONCLUSIONS: These findings indicate the involvement of NK and NKT cells as well as T lymphocytes CD4(+) and CD8(+) in the inflammatory process inducing FHF, confirmed by the high hepatic expression of IFN-γ.

Differential regulation of toll-like receptor-2, toll-like receptor-4, CD16 and human leucocyte antigen-DR on peripheral blood monocytes during mild and severe dengue fever.

Dengue fever (DF), a public health problem in tropical countries, may present severe clinical manifestations as result of increased vascular permeability and coagulation disorders. Dengue virus (DENV), detected in peripheral monocytes during acute disease and in in vitro infection, leads to cytokine production, indicating that virus-target cell interactions are relevant to pathogenesis. Here we investigated the in vitro and in vivo activation of human peripheral monocytes after DENV infection. The numbers of CD14(+) monocytes expressing the adhesion molecule intercellular adhesion molecule 1 (ICAM-1) were significantly increased during acute DF. A reduced number of CD14(+) human leucocyte antigen (HLA)-DR(+) monocytes was observed in patients with severe dengue when compared to those with mild dengue and controls; CD14(+) monocytes expressing toll-like receptor (TLR)2 and TLR4 were increased in peripheral blood from dengue patients with mild disease, but in vitro DENV-2 infection up-regulated only TLR2. Increased numbers of CD14(+) CD16(+) activated monocytes were found after in vitro and in vivo DENV-2 infection. The CD14(high) CD16(+) monocyte subset was significantly expanded in mild dengue, but not in severe dengue. Increased plasma levels of tumour necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma) and interleukin (IL)-18 in dengue patients were inversely associated with CD14(high) CD16(+), indicating that these cells might be involved in controlling exacerbated inflammatory responses, probably by IL-10 production. We showed here, for the first time, phenotypic changes on peripheral monocytes that were characteristic of cell activation. A sequential monocyte-activation model is proposed in which DENV infection triggers TLR2/4 expression and inflammatory cytokine production, leading eventually to haemorrhagic manifestations, thrombocytopenia, coagulation disorders, plasmatic leakage and shock development, but may also produce factors that act in order to control both intense immunoactivation and virus replication.

Dengue-2 infection and the induction of apoptosis in human primary monocytes.

Monocytes/macrophages are important targets for dengue virus (DENV) replication; they induce inflammatory mediators and are sources of viral dissemination in the initial phase of the disease. Apoptosis is an active process of cellular destruction genetically regulated, in which a complex enzymatic pathway is activated and may be trigged by many viral infections. Since the mechanisms of apoptotic induction in DENV-infected target cells are not yet defined, we investigated the virus-cell interaction using a model of primary human monocyte infection with DENV-2 with the aim of identifying apoptotic markers. Cultures analyzed by flow cytometry and confocal microscopy yielded DENV antigen positive cells with rates that peaked at the second day post infection (p.i.), decayed afterwards and produced the apoptosis-related cytokines TNF-alpha and IL-10. Phosphatidylserine, an early marker for apoptosis, was increased at the cell surface and the Fas death receptor was upregulated at the second day p.i. at significantly higher rates in DENV infected cell cultures than controls. However, no detectable changes were observed in the expression of the anti-apoptotic protein Bcl-2 in infected cultures. Our data support virus modulation of extrinsic apoptotic factors in the in vitro model of human monocyte DENV-2 infection. DENV may be interfering in activation and death mechanisms by inducing apoptosis in target cells.

Immunomodulating and antiviral activities of Uncaria tomentosa on human monocytes infected with Dengue Virus-2.

Uncaria tomentosa (Willd.) DC., a large woody vine native to the Amazon and Central American rainforests has been used medicinally by indigenous peoples since ancient times and has scientifically proven immunomodulating, anti-inflammatory, cytotoxic and antioxidant activities. Several inflammatory mediators that are implicated in vascular permeability and shock are produced after Dengue Virus (DENV) infection by monocytes, the primary targets for virus replication. Here we assessed the immunoregulatory and antiviral activities from U. tomentosa-derived samples, which were tested in an in vitro DENV infection model. DENV-2 infected human monocytes were incubated with U. tomentosa hydro-alcoholic extract or either its pentacyclic oxindole alkaloid-enriched or non-alkaloid fractions. The antiviral activity was determined by viral antigen (DENV-Ag) detection in monocytes by flow cytometry. Our results demonstrated an in vitro inhibitory activity by both extract and alkaloidal fraction, reducing DENV-Ag+ cell rates in treated monocytes. A multiple microbead immunoassay was applied for cytokine determination (TNF-alpha, IFN-alpha, IL-6 and IL-10) in infected monocyte culture supernatants. The alkaloidal fraction induced a strong immunomodulation: TNF-alpha and IFN-alpha levels were significantly decreased and there was a tendency towards IL-10 modulation. We conclude that the alkaloidal fraction was the most effective in reducing monocyte infection rates and cytokine levels. The antiviral and immunomodulating in vitro effects from U. tomentosa pentacyclic oxindole alkaloids displayed novel properties regarding therapeutic procedures in Dengue Fever and might be further investigated as a promising candidate for clinical application.

Multiplex cytokine profile from dengue patients: MIP-1beta and IFN-gamma as predictive factors for severity.

BACKGROUND: Dengue virus pathogenesis is not yet fully understood and the identification of patients at high risk for developing severe disease forms is still a great challenge in dengue patient care. During the present study, we evaluated prospectively the potential of cytokines present in plasma from patients with dengue in stratifying disease severity. METHODS: Seventeen-cytokine multiplex fluorescent microbead immunoassay was used for the simultaneous detection in 59 dengue patients. GLM models using bimodal or Gaussian family were determined in order to associate cytokines with clinical manifestations and laboratory diagnosis. RESULTS: IL-1beta, IFN-gamma, IL-4, IL-6, IL-13, IL-7 and GM-CSF were significantly increased in patients with severe clinical manifestations (severe dengue) when compared to mild disease forms (mild dengue). In contrast, increased MIP-1beta levels were observed in patients with mild dengue. MIP-1beta was also associated with CD56+NK cell circulating rates. IL-1beta, IL-8, TNF-alpha and MCP-1 were associated with marked thrombocytopenia. Increased MCP-1 and GM-CSF levels correlated with hypotension. Moreover, MIP-1beta and IFN-gamma were independently associated with both dengue severity and disease outcome. CONCLUSION: Our data demonstrated that the use of a multiple cytokine assay platform was suitable for identifying distinct cytokine profiles associated with the dengue clinical manifestations and severity. MIP-beta is indicated for the first time as a good prognostic marker in contrast to IFN-gamma that was associated with disease severity.

Timeline analysis of IgA and IgG levels in Covid-19 hospitalized patients according to the clinical outcome / Análise temporal das concentrações de IgA e IgG em pacientes hospitalizados com Covid-19 de acordo com o desfecho clínico

ABSTRACT Introduction: Studies suggest the association between antibody production and the severity of coronavirus disease 2019 (Covid-19). Objectives: To evaluate the concentrations of immunoglobulins class A (IgA) and class G (IgG) during the hospitalization period of Covid-19 patients according to the outcome (survival vs death). Materials and methods: Patients with severe acute respiratory syndrome of coronavirus 2 (Sars-CoV-2) infection confirmed by reverse transcriptase reaction followed by polymerase chain reaction (RT-PCR) were included in this prospective study. Samples were obtained weekly during the follow-up of individuals, considering symptom onset. Titers of anti-Sars-CoV-2 IgA and IgG were measured using a commercial immunoassay. Correlations between IgA/IgG and cycle threshold (Ct) values for N1 and N2 target genes were also assessed. Results: We studied 55 Covid-19 patients (59.7±16.2 years, 63.6% male), of which 28 (50.9%) died. We observed IgA and IgG positivity (IgA+ and IgG+) in 90.9% and 80% of patients, respectively. The highest IgA+ frequency was observed at weeks 2 and 3 and the highest IgG+ at weeks 3 and 4. It is important to note that patients who died presented lower IgA titers in the first two weeks (p < 0.05); however, a significant increase in IgA levels was observed in the subsequent weeks. Lastly, we identified that significant correlations between Ct values and immunoglobulins levels, both IgA and IgG were correlated with Ct N2 in patients who died. Conclusion: Our results suggest that lower IgA titers in early Covid-19, which is associated with lower Ct values, may indicate patients at higher risk for death.

RESUMEN Introducción: Los estudios sugieren una asociación entre la producción de anticuerpos y la gravedad de la enfermedad por coronavirus 2019 (Covid-19). Objetivos: Evaluar las concentraciones de inmunoglobulinas clase A (IgA) y clase G (IgG) durante la hospitalización de pacientes con Covid-19 según el desenlace (supervivencia vs muerte). Materiales y métodos: Se incluyeron en este estudio prospectivo pacientes con síndrome respiratorio agudo severo de infección por coronavirus 2 (Sars-CoV-2) confirmado por la reacción en cadena de la polimerasa con transcriptasa inversa (RT-PCR). Las muestras se obtuvieron semanalmente durante el seguimiento de los individuos, considerando la aparición de los síntomas. Los títulos de IgA e IgG anti-Sars-CoV-2 se midieron usando un inmunoensayo comercial. También se evaluaron las correlaciones entre IgA/IgG y los valores de los umbrales de ciclo [cycle threshold (Ct)] para los genes N1 y N2. Resultados: Se estudiaron 55 pacientes Covid-19 (59,7 ± 16,2 años, 63,6% varones), de los cuales 28 (50,9%) fallecieron. Observamos positividad de IgA e IgG (IgA+ e IgG+) en el 90,9% y el 80% de los pacientes, respectivamente. La frecuencia más alta de IgA+ se observó en las semanas dos y tres y la IgG + más alta en las semanas tres y cuatro. Es importante señalar que los pacientes que fallecieron presentaron títulos de IgA más bajos en las dos primeras semanas (p < 0,05); sin embargo, se observó un aumento significativo en los niveles de IgA en las semanas siguientes. Conclusión: Identificamos correlaciones significativas entre los valores de Ct y los niveles de Ig, tanto IgA como IgG se correlacionaron con Ct N2 en los pacientes que fallecieron. Nuestros resultados sugieren que los títulos de IgA más bajos en Covid-19 temprano, que se asocia con valores de Ct más bajos, pueden indicar que los pacientes tienen un mayor riesgo de muerte.

RESUMO Introdução: Estudos sugerem a associação entre a produção de anticorpos e a gravidade da coronavirus disease 2019 (Covid-19). Objetivos: Avaliar as concentrações de imunoglobulinas da classe A (IgA) e da classe G (IgG) durante a internação de pacientes com Covid-19 de acordo com o desfecho (sobrevida vs óbito). Materiais e métodos: Pacientes com infecção pela síndrome respiratória aguda grave do coronavírus 2 (Sars-CoV-2) confirmada por reação da transcriptase reversa seguida de reação em cadeia da polimerase (RT-PCR) foram incluídos neste estudo prospectivo. As amostras foram obtidas semanalmente durante o acompanhamento dos indivíduos, considerando o início dos sintomas. Os títulos de IgA e IgG anti-Sars-CoV-2 foram mensurados por meio de um imunoensaio comercial. Correlações entre IgA/IgG e valores de limiar de detecção [cycle thresholds (Ct)] para os genes alvos N1 e N2 também foram avaliadas. Resultados: Estudamos 55 pacientes com Covid-19 (59,7 ± 6,2 anos; 63,6% do sexo masculino); destes, 28 (50,9%) morreram. Observamos positividade para IgA e IgG (IgA+/IgG+) em 90,9% e 80% dos pacientes, respectivamente. A maior frequência de IgA+ foi verificada nas semanas 2 e 3, e a maior frequência de IgG+, nas semanas 3 e 4. É importante observar que os pacientes que morreram apresentaram títulos de IgA mais baixos nas primeiras duas semanas (p < 0,05); no entanto, um aumento significativo na concentração de IgA foi observado nas semanas subsequentes. Por fim, identificamos correlações significativas entre os valores de Ct e imunoglobulinas; tanto IgA quanto IgG foram correlacionadas com Ct N2 em pacientes que morreram. Conclusão: Nossos resultados sugerem que títulos mais baixos de IgA no início da Covid-19 - que estão associados a valores mais baixos de Ct - podem indicar pacientes com risco elevado de evoluir para óbito.

Inducible nitric oxide synthase (iNOS) expression in monocytes during acute Dengue Fever in patients and during in vitro infection.

UNLABELLED: Mononuclear phagocytes are considered to be main targets for Dengue Virus (DENV) replication. These cells are activated after infection, producing proinflammatory mediators, including tumour-necrosis factor-alpha, which has also been detected in vivo. Nitric oxide (NO), usually produced by activated mononuclear phagocytes, has antimicrobial and antiviral activities. METHODS: The expression of DENV antigens and inducible nitric oxide synthase (iNOS) in human blood isolated monocytes were analysed by flow cytometry using cells either from patients with acute Dengue Fever or after DENV-1 in vitro infection. DENV-1 susceptibility to iNOS inhibition and NO production was investigated using NG-methyl L-Arginine (NGMLA) as an iNOS inhibitor, which was added to DENV-1 infected human monocytes, and sodium nitroprussiate (SNP), a NO donor, added to infected C6/36 mosquito cell clone. Viral antigens after treatments were detected by flow cytometry analysis. RESULTS: INOS expression in activated monocytes was observed in 10 out of 21 patients with Dengue Fever and was absent in cells from ten healthy individuals. DENV antigens detected in 25 out of 35 patients, were observed early during in vitro infection (3 days), significantly diminished with time, indicating that virus replicated, however monocytes controlled the infection. On the other hand, the iNOS expression was detected at increasing frequency in in vitro infected monocytes from three to six days, exhibiting an inverse relationship to DENV antigen expression. We demonstrated that the detection of the DENV-1 antigen was enhanced during monocyte treatment with NGMLA. In the mosquito cell line C6/36, virus detection was significantly reduced in the presence of SNP, when compared to that of untreated cells. CONCLUSION: This study is the first to reveal the activation of DENV infected monocytes based on induction of iNOS both in vivo and in vitro, as well as the susceptibility of DENV-1 to a NO production.

Activated peripheral lymphocytes with increased expression of cell adhesion molecules and cytotoxic markers are associated with dengue fever disease.

The immune mechanisms involved in dengue fever and dengue hemorrhagic/dengue shock syndrome are not well understood. The ex vivo activation status of immune cells during the dengue disease in patients was examined. CD4 and CD8 T cells were reduced during the acute phase. Interestingly, CD8 T cells co-expressing activation marker HLA-DR, Q, P, and cytolytic granule protein-Tia-1 were significantly higher in dengue patients than in controls. Detection of adhesion molecules indicated that in dengue patients the majority of T cells (CD4 and CD8) express the activation/memory phenotype, characterized as CD44HIGH and lack the expression of the naïve cell marker, CD62L LOW. Also, the levels of T cells co-expressing ICAM-1 (CD54), VLA-4, and LFA-1 (CD11a) were significantly increased. CD8 T lymphocytes expressed predominantly low levels of anti-apoptotic molecule Bcl-2 in the acute phase, possibly leading to the exhibition of a phenotype of activated/effector cells. Circulating levels of IL-18, TGF-b1 and sICAM-1 were significantly elevated in dengue patients. Early activation events occur during acute dengue infection which might contribute to viral clearance. Differences in expression of adhesion molecules among CD4 and CD8 T cells might underlie the selective extravasation of these subsets from blood circulation into lymphoid organs and/or tissues. In addition, activated CD8 T cells would be more susceptible to apoptosis as shown by the alteration in Bcl-2 expression. Cytokines such as IL-18, TGF-b1, and sICAM-1 may be contributing by either stimulating or suppressing the adaptative immune response, during dengue infection, thereby perhaps establishing a relationship with disease severity.

Dengue-2 infection and the induction of apoptosis in human primary monocytes

Monocytes/macrophages are important targets for dengue virus (DENV) replication; they induce inflammatory mediators and are sources of viral dissemination in the initial phase of the disease. Apoptosis is an active process of cellular destruction genetically regulated, in which a complex enzymatic pathway is activated and may be trigged by many viral infections. Since the mechanisms of apoptotic induction in DENV-infected target cells are not yet defined, we investigated the virus-cell interaction using a model of primary human monocyte infection with DENV-2 with the aim of identifying apoptotic markers. Cultures analyzed by flow cytometry and confocal microscopy yielded DENV antigen positive cells with rates that peaked at the second day post infection (p.i.), decayed afterwards and produced the apoptosis-related cytokines TNF-á and IL-10. Phosphatidylserine, an early marker for apoptosis, was increased at the cell surface and the Fas death receptor was upregulated at the second day p.i. at significantly higher rates in DENV infected cell cultures than controls. However, no detectable changes were observed in the expression of the anti-apoptotic protein Bcl-2 in infected cultures. Our data support virus modulation of extrinsic apoptotic factors in the in vitro model of human monocyte DENV-2 infection. DENV may be interfering in activation and death mechanisms by inducing apoptosis in target cells.

Activated peripheral lymphocytes with increased expression of cell adhesion molecules and cytotoxic markers are associated with dengue fever disease

The immune mechanisms involved in dengue fever and dengue hemorrhagic/dengue shock syndrome are not well understood. The ex vivo activation status of immune cells during the dengue disease in patients was examined. CD4and CD8 T cells were reduced during the acute phase. Interestingly, CD8 T cells co-expressing activation marker HLA-DR, Q, P, and cytolytic granule protein-Tia-1 were significantly higher in dengue patients than in controls. Detection of adhesion molecules indicated that in dengue patients the majority of T cells (CD4 and CD8) express the activation/memory phenotype, characterized as CD44HIGH and lack the expression of the naïve cell marker, CD62L LOW. Also, the levels of T cells co-expressing ICAM-1 (CD54), VLA-4, and LFA-1 (CD11a) were significantly increased. CD8 T lymphocytes expressed predominantly low levels of anti-apoptotic molecule Bcl-2 in the acute phase, possibly leading to the exhibition of a phenotype of activated/effector cells. Circulating levels of IL-18, TGF-b1 and sICAM-1 were significantly elevated in dengue patients. Early activation events occur during acute dengue infection which might contribute to viral clearance. Differences in expression of adhesion molecules among CD4 and CD8 T cells might underlie the selective extravasation of these subsets from blood circulation into lymphoid organs and/or tissues. In addition, activated CD8 T cells would be more susceptible to apoptosis as shown by the alteration in Bcl-2 expression. Cytokines such as IL-18, TGF-b1, and sICAM-1 may be contributing by either stimulating or suppressing the adaptative immune response, during dengue infection, thereby perhaps establishing a relationship with disease severity.