Skip Navigation

Cellular Immune Activation in Children with Acute Dengue Virus Infections Is Modulated by Apoptosis

  1. Khin S. Myint1,
  2. Timothy P. Endy1,a,
  3. Duangrat Mongkolsirichaikul1,
  4. Choompun Manomuth1,
  5. Siripen Kalayanarooj2,
  6. David W. Vaughn3,a,
  7. Ananda Nisalak1,
  8. Sharone Green4,
  9. Alan L. Rothman4,
  10. Francis A. Ennis4 and
  11. Daniel H. Libraty4
  1. 1Department of Virology, Armed Forces Research Institute of Medical Sciences, and
  2. 2Department of Pediatrics, Queen Sirikit National Institute of Child Health, Bangkok, Thailand;
  3. 3Department of Virus Diseases, Walter Reed Army Institute of Research, Silver Spring, Maryland;
  4. 4Center for Infectious Disease and Vaccine Research, University of Massachusetts Medical School, Worcester, Massachusetts
  1. Reprints or correspondence: Dr. Daniel H. Libraty, Center for Infectious Disease and Vaccine Research, University of Massachusetts Medical School, Rm. S5-326, 55 Lake Ave. N, Worcester, MA 01655 (daniel.libraty{at}umassmed.edu)
 
Next Section

Abstract

Apoptosis is an important modulator of cellular immune responses during systemic viral infections. Peripheral-blood mononuclear cell (PBMC) apoptosis and plasma soluble levels of CD95, a mediator of apoptosis, were determined in sequential samples from children participating in a prospective study of dengue virus (DV) infections. During the period of defervescence, levels of PBMC apoptosis were higher in children developing dengue hemorrhagic fever (DHF), the most severe form of illness, than in those with dengue fever (DF) and other, nondengue, febrile illnesses. CD8+ T lymphocytes made up approximately half of the peak circulating apoptotic PBMCs in DHF and DF. Maximum plasma levels of soluble CD95 were also higher in children with DHF than in those with DF. The level of PBMC apoptosis correlated with dengue disease severity. Apoptosis appears to be involved in modulation of the innate and adaptive immune responses to DV infection and is likely involved in the evolution of immune responses in other viral hemorrhagic fevers

Dengue is an emerging arboviral disease caused by infection with one of the dengue viruses (DVs), a group of 4 antigenically related mosquitoborne flaviviruses [1]. It remains a major cause of morbidity throughout tropical and subtropical regions of the world [2, 3]. Classic dengue fever (DF) is characterized by high fevers, retroorbital headache, severe myalgias, and a rash. Dengue hemorrhagic fever (DHF) is characterized by the development of plasma leakage and a hemorrhagic diathesis around the time of defervescence. In severe cases, hypotension and shock ensue and are the principal causes of morbidity and mortality. Several studies have demonstrated that sequential heterotypic DV infections (secondary infections) are more likely to produce DHF [4–6]. Host factors that may increase the total viral burden [7] or exaggerate the cellular immune response to DV infection [8] have been implicated in this immune enhancement of disease severity

Apoptosis, or programmed cell death, is a fundamental regulatory feature of the immune system. In the innate immune response to viral infection, apoptosis may occur as a pathogen-directed mechanism of viral dissemination and immune escape or may represent an appropriate host response for limiting virus replication [9]. Apoptosis is also the primary mechanism for homeostatic control of antigen-reactive lymphocyte expansion in the adaptive immune response [10]. Apoptosis can be antigen dependent and initiated by signaling through death receptors, such as CD95 (Fas) [11]. Apoptosis may also occur by neglect when antigen-specific receptors are not stimulated or when soluble survival factors are withdrawn from the cellular environment [12]

Modulation of apoptotic cell death plays a role in the pathogenesis of several viral infectious diseases. Dysregulation of apoptosis is a factor in the CD4+ T lymphocyte depletion seen in HIV infection [13], in the anergy associated with measles virus and varicella zoster virus infections in infancy [14], and in the hepatocyte cell death seen in viral hepatitis [15]. DV infection can trigger or modify apoptosis in a variety of cell types in vitro [16–20]. In vivo, apoptosis of hepatocytes has been seen on histopathologic analysis of the liver in a few fatal cases of dengue [21]. Apoptosis of circulating CD8+ T lymphoctyes has also been reported in a small number of dengue cases [22]. Cells of the innate immune response that are also targets for DV infection (monocytes, macrophages, and dendritic cells) and effector cells of the adaptive immune response to DV (lymphocytes) make up the majority of peripheral-blood mononuclear cells (PBMCs)

The goal of the present study was to examine apoptosis in ex vivo PBMCs, particularly CD8+ T lymphoctyes, during acute DV infections to elucidate the role that cellular apoptosis plays in evolution of the immune response. Levels of PBMC apoptosis, CD8+ T lymphoctye apoptosis, and plasma levels of soluble CD95 (sCD95) were examined on consecutive days around the time of defervescence (and around the time of plasma leakage in children with DHF) in children enrolled in a prospective, hospital-based study of dengue. The study included children with febrile illnesses found to have DHF, DF, or other, nondengue, febrile illnesses (OFIs)

Previous SectionNext Section

Methods

Study designDetails of the investigational protocol have been published elsewhere [23]. The investigational protocol was approved by the Institutional Review Boards of the Thai Ministry of Public Health, the Office of the US Army Surgeon General, and the University of Massachusetts Medical School. Parents or guardians of all study subjects provided written, informed consent. Enrollment criteria were an age of 6 months to 14 years, a febrile illness with symptoms of a duration of <72 h, no hypotension or shock, and no other obvious source of infection. Children were observed in the hospital until at least 1 day after defervescence. Venous blood samples were drawn daily up to the day after defervescence or for a maximum of 5 consecutive days and for 8–13 days after enrollment (early convalescence). A complete blood count (T540 counter; Coulter) was obtained daily. Serial hematocrit determinations were obtained daily and every 6 h on defervescence, and a right lateral decubitus chest radiograph was obtained on the day after defervescence

Study definitionsStudy day 1 was the day a child was enrolled in the study. Fever day 0 was the day of defervescence, when the temperature dropped below 38°C without a subsequent elevation. Days before fever day 0 were designated fever days −1, −2, and so on. Primary or secondary DV infections were identified using previously established serologic criteria for IgM/IgG ELISAs and hemagglutination-inhibition assays to DV serotypes 1–4 and Japanese encephalitis virus in paired specimens [24–26]. DV serotypes were identified by virus isolation in Toxorhynchites splendens mosquitoes [23] or a serotype-specific reverse-transcriptase polymerase chain reaction assay [27]. In those identified with a DV infection, a clinical diagnosis of DF or DHF and severity grades (I–IV) were assigned in accordance with World Health Organization criteria [26]. Those without evidence of DV infection were defined as having OFIs

Sample processingBlood was drawn into EDTA tubes (Becton Dickinson), immediately placed on ice, and transported to a nearby blood-processing laboratory. PBMCs were isolated by Histopaque (Sigma) density-gradient centrifugation. PBMCs (1× 105) were placed onto poly-l-lysine–coated (Sigma Chemical) glass microscope slides and centrifuged at 800 rpm for 2 min in a cytocentrifuge (Cytospin 3; Shandon). After air drying for 30 min, slides were individually wrapped in foil and frozen at −70°C for future staining. Plasma was divided into aliquots and frozen at −70°C until analysis

Single-color staining for detection of apoptosisApoptotic cells were identified by DNA fragmentation, using a terminal deoxynucleotidyltransferase (TdT)–mediated digoxigenin-11-dUTP nick end–labeling assay followed by antidigoxigenin immunoperoxidase staining (TUNEL method) (ApopTag Peroxidase In Situ Apoptosis Detection Kit; Intergen). The staining was performed on 4% paraformaldehyde–fixed cytospin slides in accordance with the manufacturer’s instructions. Positive and negative control slides provided by the manufacturer and a negative control slide made by omitting the TdT enzyme were included in all assays. All slides were read in a blinded fashion. The number of TUNEL positive–staining cells on each slide was determined at ×200 magnification over the entire cytospin area. The total number of PBMCs on each slide was estimated by counting all cells within a square ocular grid in 4 quadrants of the cytospin area at ×400 magnification. The total PBMC count per slide was calculated as follows: (average cell count in grid determined in 4 quadrants)×(πr2/l2), where r equals the radius of the cytospin area and l equals the length of the ocular grid at ×400 magnification

Dual-color staining for apoptosis and CD8 expression Immunofluorescent dual-color staining for simultaneous detection of apoptosis and CD8 expression was performed on cytospin slides fixed with 1% formaldehyde followed by 1% Nonidet P40. First, staining for apoptotic cells was performed using the TUNEL method described above, except fluorescein isothiocyanate (FITC)–conjugated antidigoxigenin antibody was used in the final step (ApopTag Fluorescein In Situ Apoptosis Detection Kit; Intergen). Staining for CD8 expression was performed using a phycoerythrin (PE)–conjugated anti-human CD8 monoclonal antibody (RPA-T8; Pharmingen). Positive control slides used for CD8 staining were fixed cytospin slides of healthy donor PBMCs. Negative control slides were fixed cytospin slides of CD8− Jurkat T lymphoctyes (gift from V. Polonis, Department of Retrovirology, Armed Forces Research Institute of Medical Sciences, Bangkok, Thailand) induced to undergo apoptosis with 2 μmol/L campothecin (Sigma). Dual-color-stained slides were read under a Leitz epifluorescent microscope equipped with FITC and PE filters. All slides were read in a blinded fashion. Quantification of positive-staining cells and total PBMCs was performed as described above

Assay for sCD95Plasma levels of sCD95 were determined using a commercial ELISA kit in accordance with the manufacturer’s instructions (OptEIA Human Fas Kit; Pharmingen). All samples were assayed in a blinded fashion and in duplicate

Statistical analysisStudent’s t test and analysis of variance were used for comparisons between normally distributed continuous variables; the Mann-Whitney U test was used for comparisons between continuous variables not normally distributed. χ2 analysis or Fisher’s exact test was used for comparisons among proportional data. Pearson’s correlation test was used to examine associations between continuous variables. P<.05 was considered significant; .05⩽P⩽.10 was considered a nonsignificant trend. All values are presented as mean±SE, unless otherwise stated. The statistical software package SPSS (version 10.0; SPSS) was used for all statistical analyses

Previous SectionNext Section

Results

Study population characteristicsThe study population consisted of 185 children (DF group, n=58; DHF group, n=59; and OFI group, n=68); their characteristics are summarized in table 1. The mean ages of the children in the DF and DHF groups were higher than in the OFI group. Overall, DV-1 or DV-3 infections accounted for 74% of the dengue cases. Among the 117 children with dengue, the majority were secondary infections (77%). The proportion of children with secondary DV infections who developed DHF was greater than in primary infections (59% vs. 23%, respectively; P=.003). In statistical analyses, we examined trends in all dengue cases and within secondary DV infections

Figure 1
View larger version:
Figure 1

A–D Apoptosis in peripheral-blood mononuclear cells detected by in situ immunoperoxidase staining of DNA fragmentation by use a terminal deoxynucleotidyltransferase-mediated dUTP nick end–labeling assay (TUNEL method). A Apoptotic cell demonstrating chromatin margination. B Apoptotic cell demonstrating nuclear clumping, condensation, and segmentation. C and D Apoptotic cells engulfed by phagocytes. E In situ immunofluorescent staining (phycoerythrin) of CD8 in a cell engulfed by a phagocyte. F Concurrent in situ immunofluorescent staining of apoptotic DNA fragmentation in the CD8+ T lymphoctye in panel E. Scale bar, 10 μm

Figure 2
View larger version:
Figure 2

Peripheral-blood mononuclear cell (PBMC) apoptosis determined on sequential days in children with dengue hemorrhagic fever (DHF) (□), dengue fever (DF) (◊), and other, nondengue, febrile illnesses (OFIs) (○). Fever day 0 is the day of defervescence. The early convalescent time point is 8–13 days after hospital admission; all children had recovered and were afebrile. Values are mean±SE. *P<.001, for DF or DHF vs. OFIs; †P⩽.05, for DHF vs. DF

Figure 3
View larger version:
Figure 3

Plasma soluble CD95 levels determined on sequential days in children with dengue hemorrhagic fever (DHF) (□), dengue fever (DF) (◊), and other, nondengue, febrile illnesses (OFIs) (○). Fever day 0 is the day of defervescence. The early convalescent time point is 8–13 days after hospital admission; all children had recovered and were afebrile. Values are mean±SE. *P⩽.002, for OFIs vs. DF or DHF; †P=.05, for DHF vs. DF; ‡P⩽.02, for DHF vs. DF

Table 1
View larger version:
Table 1

Study population characteristics

PBMC apoptosisWe used the TUNEL method to evaluate apoptosis in PBMCs from sequential blood samples in children with DHF, DF, and OFIs. To insure that TUNEL positivity represented apoptosis, in preliminary experiments, we demonstrated that PBMCs killed by heat treatment (a method that does not induce apoptosis) and fixed to slides did not stain TUNEL positive (data not shown). In all samples from the children with dengue or OFIs, TUNEL-positive–staining PBMCs displayed focal nuclear staining and the morphologic characteristics of apoptosis. The cytologic findings included shrunken cells, chromatin margination, nuclear clumping, nuclear condensation, and segmentation. Many apoptotic cells were being phagocytosed by circulating mononuclear phagocytes (figure 1A–1D)

The mean daily proportion of PBMCs undergoing apoptosis was significantly higher in children with dengue than in those with OFIs from 2 days before defervescence until the day after defervescence (all days, P<.001). The mean daily proportion of PBMCs undergoing apoptosis was significantly higher in children with DHF than in those with DF on the same days (fever day −2, P=.05; fever days −1 and 0, P=.01; fever day +1, P=.001 ) (figure 2). When only secondary DV infections were examined, PBMC apoptosis levels remained higher in children with DHF than in those with DF during fever days −1 to +1 (fever day −1, P=.05; fever day 0, P=.06; fever day +1, P=.003; data not shown)

Peak levels of PBMC apoptosis were higher in children with DHF than in those with DF in all subjects with dengue (DHF vs. DF, 24±3 vs. 13±1 apoptotic PBMCs/105 PBMCs; P<.001) and in the subsets selected for CD8 staining (DHF vs. DF, 44±9 vs. 20±4 apoptotic PBMCs/105 PBMCs; P=.02) and plasma sCD95 measurements (DHF vs. DF, 21±3 vs. 13±2 apoptotic PBMCs/105 PBMCs; P=.02)

CD8+ T lymphocytes and apoptosisActivated DV antigen-specific CD8+ T lymphoctyes have been observed during the acute phase of dengue [22], and, in an earlier study, we found that the frequency of circulating activated CD8+ T lymphoctyes was higher in children with DHF than in those with DF and higher in those with DF than in those with OFIs [28]. Thirty-two subjects were included in the present study and in the earlier study examining CD8+ T lymphoctye activation. Among these subjects (DHF, n=14; DF, n=14; OFIs, n=4), the frequency of activated (CD69+) CD8+ T lymphoctyes was also higher in children with DHF than in those with DF and higher in those with DF than in those with OFIs (CD3+CD8+ T lymphoctyes expressing CD69: DHF, 10.3%±1.6%; DF, 8.6%±1.0%; OFIs, 2.0%±0.3%; P=.007). We stained for apoptosis and CD8 expression on the day that PBMC apoptosis level peaked between fever days −3 and +1 in selected subjects with acute DV infection (DHF, n=9; DF, n=9) (figure 1E–1F). At peak levels of PBMC apoptosis, CD8+ T lymphocytes made up approximately half of the apoptotic PBMCs in both the DHF and DF groups (DHF, 48%±7%; DF, 61%±6%; P=.2). When only secondary DV infections were examined (DHF, n=8; DF, n=7), the proportion of apoptotic CD8+ T lymphocytes was also not found to be significantly different between the 2 groups (DHF, 44%±7%; DF, 58%±7%; P=.2)

sCD95 levelsActivation of the CD95/CD95 ligand (CD95L) pathway is an important mediator of apoptosis [29]. In subjects with acute DV infections (DHF, n=39; DF, n=34), mean plasma sCD95 levels increased throughout acute illness and defervescence to the early convalescent time point. Plasma sCD95 levels were higher in children with DF or DHF than in those with OFIs (n=13) during fever days −2 to 0. Mean plasma sCD95 levels were higher in children with DHF than in those with DF beginning on fever day −1 and extending to early convalescence (figure 3). In secondary DV infections (DHF, n=35; DF, n=18), mean plasma sCD95 levels remained significantly higher in children with DHF than in those with DF on fever day 0 and early convalescence (P=.05 and .03, respectively; data not shown). In all cases, maximum plasma levels of sCD95 correlated with maximum levels of PBMC apoptosis in children with DF and DHF (Pearson’s correlation, r=0.4; P=.005)

Previous SectionNext Section

Discussion

In the course of a DV infection, 2 challenges are placed on the host immune response: (1) elimination of DV and DV-infected cells and (2) modulation of the adaptive immune response activated by the infecting virus. The ability of the host immune response to meet these challenges is directly reflected in the clinical severity of dengue illness. Earlier studies have implicated higher viral burdens and levels of T lymphocyte activation in the pathogenesis of DHF [28, 30–32]. Apoptosis is a potentially important mechanism for modulation of the cellular immune responses in dengue and, by extension, in other viral hemorrhagic fevers

We found an increased frequency of circulating apoptotic PBMCs in children with DHF compared with those with DF and OFIs. Prior studies, some of which have included children in the present study, have shown that DHF is characterized by a greater viral burden and higher state of immune activation than is DF. Early in illness, circulating levels of viral RNA [31, 33], soluble tumor necrosis factor (TNF) receptor type II [32], and interleukin (IL)–10 [34] are higher in children with DHF than in those with DF. When DV is present, the balance between levels of the proapoptotic cytokine TNF-α [10] and the antiapoptotic cytokine IL-10 [35] may affect the degree of death receptor–mediated apoptosis in PBMCs. While defervescence occurs, extracellular DV is rapidly disappearing [23, 31], and, as in other acute systemic viral infections, there is expansion and activation of CD8+ T lymphocytes [36, 37]. CD8+ T lymphocyte activation is greater in subjects with DHF than in those with DF [28], and, on defervescence, circulating levels of T lymphocyte–activation markers (interferon–γ, soluble IL-2 receptor, and soluble CD8) are higher in subjects with DHF than in those with DF [32]. Elimination of DV likely prompts the apoptosis of terminally differentiated effector T lymphoctyes, resulting in modulation of the adaptive immune response [10]

A previous study of children with dengue reported that 5%–20% of all circulating CD8+ T lymphocytes around the time of defervescence were apoptotic; TUNEL staining was performed on cryopreserved PBMCs from a small number of subjects (n= 8), and the study did not compare DF and DHF [22]. In contrast, we examined apoptosis in directly frozen and fixed PBMCs from a larger number of children and were able to compare CD8+ T lymphoctye apoptosis in children with DF and DHF. We found that, around the time of defervescence, a much smaller percentage of the total circulating CD8+ T lymphocytes were apoptotic in children with DF and DHF (0.05%–0.1%, assuming that CD8+ T lymphoctyes make up 30% of the circulating lymphocytes in DV-infected children [28]). Differences in cell preservation and TUNEL-staining techniques between the 2 studies may be responsible for the 100-fold difference seen in circulating apoptotic CD8+ T lymphoctyes, but additional investigation is necessary. At the peak percentage of apoptotic cells in the circulation, CD8+ T lymphoctyes accounted for nearly half of the lymphocytes and mononuclear cells undergoing apoptosis. Children with DHF had a greater proportion of PBMCs undergoing apoptosis than those with DF, but CD8+ T lymphoctyes made up the same percentage of apoptotic cells in both groups. Our data highlight that the differences in CD8+ T lymphoctye apoptosis between DHF and DF are primarily quantitative rather than qualitative

CD95/CD95L-mediated apoptosis is an important pathway for antigen-dependent, activation-induced apoptosis in effector T lymphocytes [10], including DV NS3–specific CD8+ T lymphoctyes [22]. The elevated plasma sCD95 levels in subjects with DHF, compared with that in subjects with DF, likely reflect greater T lymphoctye activation and subsequent CD95-mediated apoptosis of T lymphoctyes in subjects with DHF. Increased levels of sCD95 have been reported in the serum of HIV-positive patients and correlate with surface CD95 expression in apoptotic T lymphocytes [38]. Our data suggest that CD95-mediated apoptosis plays a role in CD8+ T lymphoctye regulation during acute DV infections but does not exclude involvement of TNF-α/TNF receptor or other mechanisms of apoptosis [10]. Plasma levels of sCD95 continued to increase after peak levels of PBMC apoptosis were achieved in subjects with DF and DHF. The difference in kinetics of plasma sCD95 levels and PBMC apoptosis levels may be due to the longer circulatory half-life of sCD95, compared with that of apoptotic cells, which are rapidly cleared by the reticuloendothelial system. Alternatively, sCD95 production and release may be delayed in acute viral infections to act as a counterregulatory mechanism for CD95/CD95L–mediated apoptosis. The addition of sCD95 to cells in vitro has an antiapoptotic effect [39]

The effect that DV infection has on target cells is less clear. Infection of neuroblastoma, Kupffer, hepatocyte, and endothelial cells in vitro can induce apoptosis [16–19], but apoptosis of myeloid dendritic cells is delayed after DV infection [20]. Cytotoxic T lymphocytes can also drive the apoptosis of cells containing viral antigens [40], and this may be an important mechanism for controlling dengue viremia. We were unable to perform dual staining for apoptosis and DV, because apoptotic PBMCs exhibited nonspecific staining with the anti-DV hyperimmune mouse ascitic fluid. Additional studies are needed to identify apoptotic DV-containing cells and their phenotypes and to address the potential roles that they might play in viral clearance and immunomodulation

In DV infections, the viral burden and degree of immune activation are positively associated with disease severity and the development of DHF. Similarly, measures of apoptosis in PBMCs are positively associated with disease severity and DHF. Apoptosis likely plays several roles in modulating the viral burden and evolution of the immune response to DV infection. Deactivation of the CD8+ T lymphocyte response appears to be a key function of apoptosis in DV infections. Future studies of cellular apoptosis in dengue and other viral hemorrhagic fevers will expand our knowledge of the steps involved in the pathogenesis of these potentially deadly, emerging viral diseases

Previous SectionNext Section

Acknowledgments

We thank Nawarat Charoensri, for performance of the soluble CD95 assay and assistance with cell counting on the cytospin slides; Panor Srisongkram and Somkiat Changnak, for serologic testing; Nonglak Ongsakorn, Naowayubol Nutkumhang, and Somsak Imlarp, for virus isolation and identification; Tipawan Kungvanrattana and Chitchai Hemachudha, for data processing; the research nurses of the Department of Virology, Armed Forces Research Institute of Medical Sciences, for collection of clinical specimens; and Mammen P. Mammen, Jr., for thoughtful review of the manuscript

Previous SectionNext Section

Footnotes

  • ↵Potential conflicts of interest: none reported

    Financial support: National Institutes of Health (grant NIH-P01AI34533); US Army Medical Research and Materiel Command

    The contents of this article are solely the responsibility of the authors and do not necessarily represent the official views of the National Institutes of Health or the Department of Defense

  • ↵Present affiliations: Division of Communicable Diseases and Immunology, Walter Reed Army Institute of Research, Silver Spring, Maryland (T.P.E.); Military Infectious Diseases Research Program, US Army Medical Research and Materiel Command, Fort Detrick, Maryland (D.W.V.)

  • Received January 11, 2006.
  • Accepted April 24, 2006.
Previous Section
 

References

  1. ↵
    The dengue viruses. Clin Microbiol Rev 1990;3:376-96.
    Abstract/FREE Full Text
  2. ↵
    1. Monath TP
    . Dengue: the risk to developed and developing countries. Proc Natl Acad Sci USA 1994;91:2395-400.
    Abstract/FREE Full Text
  3. ↵
    1. Pinheiro FP,
    2. Corber SJ
    . Global situation of dengue and dengue haemorrhagic fever, and its emergence in the Americas. World Health Stat Q 1997;50:161-9.
    Medline
  4. ↵
    1. Burke DS,
    2. Nisalak A,
    3. Johnson D,
    4. Scott RM
    . A prospective study of dengue infections in Bangkok. Am J Trop Med Hyg 1988;38:172-80.
    Abstract/FREE Full Text
  5. ↵
    1. Guzman MG,
    2. Kouri GP,
    3. Bravo J,
    4. Soler M,
    5. Vazquez S
    . Dengue hemorrhagic fever in Cuba. Am J Trop Med Hyg 1990;42:179-84.
    Abstract/FREE Full Text
  6. ↵
    1. Halstead SB,
    2. Nimmannitya S,
    3. Cohen SN
    . Observations related to pathogenesis of dengue hemorrhagic fever. IV: Relation of disease severity to antibody response and virus recovered. Yale J Biol Med 1970;42:311-28.
    MedlineWeb of Science
  7. ↵
    1. Halstead SB
    . Antibody, macrophages, dengue virus infection, shock, and hemorrhage: a pathogenetic cascade. Rev Infect Dis 1989;11:S830-9.
    CrossRefMedlineWeb of Science
  8. ↵
    1. Rothman AL,
    2. Ennis FA
    . Immunopathogenesis of dengue hemorrhagic fever. Virology 1999;257:1-6.
    CrossRefMedlineWeb of Science
  9. ↵
    1. Everett H,
    2. McFadden G
    . Apoptosis: an innate immune response to virus infection. Trends Microbiol 1999;7:160-5.
    CrossRefMedlineWeb of Science
  10. ↵
    1. Lenardo M,
    2. Chan FKM,
    3. Hornung F,
    4. et al
    . Mature T lymphocyte apoptosis-immune regulation in a dynamic and unpredictable antigenic environment. Ann Rev Immunol 1999;17:221-53.
    CrossRefMedlineWeb of Science
  11. ↵
    1. Itoh N,
    2. Yonehara S,
    3. Ishii A,
    4. et al
    . The polypeptide encoded by the cDNA for human cell surface antigen Fas can mediate apoptosis. Cell 1991;66:233-43.
    CrossRefMedlineWeb of Science
  12. ↵
    1. Jones LA,
    2. Chin LT,
    3. Longo DL,
    4. Kruisbeek AM
    . Peripheral clonal elimination of functional T cells. Science 1990;250:1726-9.
    Abstract/FREE Full Text
  13. ↵
    1. Oyaizu N,
    2. Pahwa S
    . Role of apoptosis in HIV disease pathogenesis. J Clin Immunol 1995;15:217-31.
    CrossRefMedlineWeb of Science
  14. ↵
    1. Pignata C,
    2. Fiore M,
    3. De Filippo S,
    4. Cavalcanti M,
    5. Gaetaniello L
    . Apoptosis as a mechanism of peripheral blood mononuclear cell death after measles and varicella-zoster virus infections in children. Pediatr Res 1998;43:77-83.
    MedlineWeb of Science
  15. ↵
    1. Suda KT,
    2. Fukuyama H,
    3. Adachi M,
    4. Nagata S
    . Essential roles of the Fas ligand in the development of hepatitis. Nat Med 1997;3:409-13.
    CrossRefMedlineWeb of Science
  16. ↵
    1. Despres P,
    2. Flamand M,
    3. Ceccaldi P-E,
    4. Deubel V
    . Human isolates of dengue type 1 virus induce apoptosis in mouse neuroblastoma cells. J Virol 1996;70:4090-96.
    Abstract/FREE Full Text
  17. ↵
    1. Marianneau P,
    2. Steffan AM,
    3. Royer C,
    4. et al
    . Infection of primary cultures of human Kupffer cells by dengue virus: no viral progeny synthesis, but cytokine production is evident. J Virol 1999;73:5201-6.
    Abstract/FREE Full Text
  18. ↵
    1. Marianneau P,
    2. Cardona A,
    3. Edelman L,
    4. Deubel V,
    5. Despres P
    . Dengue virus replication in human hepatoma cells activates NF-κB which in turn induces apoptotic cell death. J Virol 1997;71:3244-9.
    Abstract/FREE Full Text
  19. ↵
    1. Avirutnan P,
    2. Malasit P,
    3. Seliger B,
    4. Bhakdi S,
    5. Husmann M
    . Dengue virus infection of human endothelial cells leads to chemokine production, complement activation, and apoptosis. J Immunol 1998;161:6338-46.
    Abstract/FREE Full Text
  20. ↵
    1. Lo LJ,
    2. Wang JJ,
    3. Shaio MF,
    4. et al
    . Infection of human dendritic cells by dengue virus causes cell maturation and cytokine production. J Immunol 2001;166:1499-506.
    Abstract/FREE Full Text
  21. ↵
    1. Huerre MR,
    2. Lan NT,
    3. Marianneau P,
    4. et al
    . Liver histopathology and biological correlates in five cases of fatal dengue fever in Vietnamese children. Virchows Archiv 2001;438:107-15.
    CrossRefMedlineWeb of Science
  22. ↵
    1. Mongkolsapaya J,
    2. Dejnirattisai W,
    3. Xu XN,
    4. et al
    . Original antigenic sin and apoptosis in the pathogenesis of dengue hemorrhagic fever. Nat Med 2003;9:921-7.
    CrossRefMedlineWeb of Science
  23. ↵
    1. Vaughn DW,
    2. Green S,
    3. Kalayanarooj S,
    4. et al
    . Dengue in the early febrile phase: viremia and antibody responses. J Infect Dis 1997;176:322-30.
    Abstract/FREE Full Text
  24. ↵
    1. Innis BL,
    2. Nisalak A,
    3. Nimmannitya S,
    4. et al
    . An enzyme-linked immunosorbent assay to characterize dengue infections where dengue and Japanese encephalitis co-circulate. Am J Trop Med Hyg 1989;40:418-27.
    Abstract/FREE Full Text
  25. ↵
    1. Clarke DH,
    2. Casals J
    . Techniques for hemagglutination and hemagglutination-inhibition with arthropod borne viruses. Am J Trop Med Hyg 1958;7:561-73.
    Abstract/FREE Full Text
  26. ↵
    1. World Health Organization
    . Dengue haemorrhagic fever: diagnosis, treatment, prevention and control. 2nd ed. Geneva: World Health Organization; 1997.
  27. ↵
    1. Lanciotti RS,
    2. Calisher CH,
    3. Gubler DJ,
    4. Chang GJ,
    5. Vorndam AV
    . Rapid detection and typing of dengue viruses from clinical samples by using reverse transcriptase-polymerase chain reaction. J Clin Microbiol 1992;30:545-51.
    Abstract/FREE Full Text
  28. ↵
    1. Green S,
    2. Pichyangkul S,
    3. Vaughn DW,
    4. et al
    . Early CD69 expression on peripheral blood lymphocytes from children with dengue hemorrhagic fever. J Infect Dis 1999;180:1429-35.
    Abstract/FREE Full Text
  29. ↵
    1. Krammer PH
    . CD95’s deadly mission in the immune system. Nature 2000;407:789-95.
    CrossRefMedline
  30. ↵
    1. Vaughn DW,
    2. Green S,
    3. Kalayanarooj S,
    4. et al
    . Dengue viremia titer, antibody response pattern, and virus serotype correlate with disease severity. J Infect Dis 2000;181:2-9.
    Abstract/FREE Full Text
  31. ↵
    1. Libraty DH,
    2. Endy TP,
    3. Houng HS,
    4. et al
    . Differing influences of virus burden and immune activation on disease severity in secondary dengue-3 virus infections. J Infect Dis 2002;185:1213-21.
    Abstract/FREE Full Text
  32. ↵
    1. Green S,
    2. Vaughn DW,
    3. Kalayanarooj S,
    4. et al
    . Early immune activation in acute dengue illness is related to development of plasma leakage and disease severity. J Infect Dis 1999;179:755-62.
    Abstract/FREE Full Text
  33. ↵
    1. Murgue B,
    2. Roche C,
    3. Chungue E,
    4. Deparis X
    . Prospective study of the duration and magnitude of viraemia in children hospitalised during the 1996–97 Dengue-2 outbreak in French Polynesia. J Med Virol 2000;60:432-438.
    CrossRefMedlineWeb of Science
  34. ↵
    1. Green S,
    2. Vaughn DW,
    3. Kalayanarooj S,
    4. et al
    . Elevated plasma interleukin-10 levels in acute dengue correlate with disease severity. J Med Virol 1999;59:329-34.
    CrossRefMedlineWeb of Science
  35. ↵
    1. Taga K,
    2. Chretien J,
    3. Cherney B,
    4. Diaz L,
    5. Brown M
    . Interleukin-10 inhibits apoptotic cell death in infectious mononucleosis T cells. J Clin Invest 1994;94:251-60.
    MedlineWeb of Science
  36. ↵
    1. Pantaleo G,
    2. Demarest JF,
    3. Soudeyns H,
    4. et al
    . Major expansion of CD8+ T cells with a predominant V beta usage during the primary immune response to HIV. Nature 1994;370:463-7.
    CrossRefMedline
  37. ↵
    1. Callan MF,
    2. Steven N,
    3. Krausa P,
    4. et al
    . Large clonal expansions of CD8+ T cells in acute infectious mononucleosis. Nat Med 1996;2:906-11.
    CrossRefMedlineWeb of Science
  38. ↵
    1. Hosaka N,
    2. Oyaizu N,
    3. Kaplan MH,
    4. Yagita H,
    5. Pahwa S
    . Membrane and soluble forms of Fas (CD95) and Fas ligand in peripheral blood mononuclear cells and in plasma from HIV infected persons. J Infect Dis 1998;178:1030-9.
    Abstract/FREE Full Text
  39. ↵
    1. Papoff G,
    2. Cascino I,
    3. Eramo A,
    4. Starace G,
    5. Lynch DH
    . A N-terminal domain shared by Fas/Apo-1 (CD95) soluble variants prevents cell death in vitro. J Immunol 1996;156:4622-30.
    Abstract
  40. ↵
    1. Razvi ES,
    2. Welsh RM
    . Apoptosis in viral infections. Adv Virus Res 1995;45:1-60.
    MedlineWeb of Science
| Table of Contents

This Article

  1. J Infect Dis. (2006) 194 (5): 600-607. doi: 10.1086/506451
  1. AbstractFree
  2. » Full Text (HTML)Free
  3. Full Text (PDF)Free

Classifications

Share

  1. Email this article

Navigate This Article

Current Issue

  1. March 1, 2012 205 (5)
  1. Journal of Infectious Diseases
  1. Alert me to new issues

Most