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Biomedical subjects

A L Rothman

Publications and source records attributed to A L Rothman.

At least 19 recordsLinked to original sources
PubMed2001

Induction of T lymphocyte responses to dengue virus by a candidate tetravalent live attenuated dengue virus vaccine.

Development of a safe and immunogenic tetravalent dengue virus (DV) vaccine has been designated as a priority by the World Health Organization. We characterized the T cell response to DV induced by a candidate live attenuated tetravalent DV vaccine as part of a phase I study. Proliferation and cytotoxic T lymphocyte (CTL) responses to multiple DV serotypes were detected in six of six and four of four subjects studied, respectively. Proliferation responses were higher to DV serotypes 1 and 3 than to serotypes 2 and 4. CTL responses were higher to DV serotypes 2 and 3 than to serotype 1, and included serotype cross-reactive responses. Production of interferon-gamma, but not IL-4, was observed in response to DV stimulation. This candidate vaccine is immunogenic for both CD4+ and CD8+ T lymphocytes. However, T cell responses to the four DV serotypes were not equivalent, suggesting that the vaccine could be further optimized.

Antibodies, Viral↗
PubMed2001

MIP-1 alpha and MIP-1 beta induction by dengue virus.

Dengue virus (DV) infection can result in either a mild febrile illness known as dengue fever (DF) or a life-threatening disease called dengue hemorrhagic fever (DHF). DHF is more prevalent in patients undergoing secondary DV infection. This observation has led to the hypothesis that DHF may be the result of immune reactions to the secondary DV infection; an event termed immunopathology. Two cellular factors, MIP-1 alpha and MIP-1 beta, have been found to be induced by infection with DV. MIP-1 induction by DV infection was observed in a myelomonocytic cell line, as well as in peripheral blood mononuclear cells isolated from a dengue naive donor. MIP-1 induction was not due to factors secreted by infected cells. In fact, replication-competent virus was required to induce MIP-1. Evidence is also provided that MIP-1 genes are expressed in patients with dengue disease. It is hypothesized that these chemokines may have roles in the immunopathology of dengue infections and may contribute to fever and bone marrow suppression observed in patients with DV infections.

Cells, Cultured↗
PubMed2001

Analysis of plasma viral RNA levels during acute dengue virus infection using quantitative competitor reverse transcription-polymerase chain reaction.

There is increasing recognition of the potential importance of viral burden in the pathogenesis of dengue hemorrhagic fever (DHF). There is little data available, however, describing the kinetics of viral replication in humans with natural dengue virus (DV) infection. Standard procedures for measuring titers of infectious virus in clinical specimens are either laborious or insensitive. We developed a method for measurement of DV RNA in plasma samples based on reverse transcription-polymerase chain reaction (RT-PCR) using a mutant RNA target as a competitor. This technique was reproducible and accurate for samples containing any of the four DV serotypes, and could be applied to samples containing as few as 250 copies of RNA per reaction. We examined plasma viral RNA levels in 80 children with acute DV infection; sequential plasma samples were tested in 34 of these children. Plasma viral RNA levels ranged as high as 10(9) RNA copies/ml, and correlated with titers of infectious virus measured in mosquitoes (r= 0.69). Plasma viral RNA levels fell rapidly during the last several days of the febrile period. We did not find a significant difference in maximal plasma viral RNA levels between children with DHF and children with dengue fever, but peak viral RNA levels were identified in only 16 subjects. We conclude that this quantitative RT-PCR method will be valuable for further studies of natural DV infections.

Adolescent↗
PubMed2000

Transient decreases in human T cell proliferative responses following vaccinia immunization.

To further study the immunosuppression associated with virus infections, we analyzed the proliferative responses of serial PBMC samples obtained following vaccinia virus immunization. In four of five volunteers, responses to PHA, anti-CD3, vaccinia virus, and recall antigens were markedly decreased at at least one time point between days 5 and 29 after vaccination. Responses to PHA were restored by the addition of IL-2 or irradiated autologous healthy PBMC in the two volunteers tested, suggesting that the proliferation defect is attributable to accessory cell dysfunction. In one donor, immobilized anti-CD3 failed to induce proliferation, but addition of immobilized anti-CD28 partially restored proliferation. These results indicate that vaccinia virus infection can transiently suppress proliferative responses of PBMC, in part by causing accessory cell dysfunction. Our findings extend the list of viral infections associated with systemic immunologic effects and demonstrate that suppression of proliferation can occur with localized virus infections.

Antibodies↗
PubMed2000

Dengue viremia titer, antibody response pattern, and virus serotype correlate with disease severity.

Viremia titers in serial plasma samples from 168 children with acute dengue virus infection who were enrolled in a prospective study at 2 hospitals in Thailand were examined to determine the role of virus load in the pathogenesis of dengue hemorrhagic fever (DHF). The infecting virus serotype was identified for 165 patients (DEN-1, 46 patients; DEN-2, 47 patients; DEN-3, 47 patients, DEN-4, 25 patients). Patients with DEN-2 infections experienced more severe disease than those infected with other serotypes. Eighty-one percent of patients experienced a secondary dengue virus infection that was associated with more severe disease. Viremia titers were determined for 41 DEN-1 and 46 DEN-2 patients. Higher peak titers were associated with increased disease severity for the 31 patients with a peak titer identified (mean titer of 107.6 for those with dengue fever vs. 108.5 for patients with DHF, P=.01). Increased dengue disease severity correlated with high viremia titer, secondary dengue virus infection, and DEN-2 virus type.

Adolescent↗
PubMed1999

Partial agonist effect influences the CTL response to a heterologous dengue virus serotype.

Activation of dengue serotype-cross-reactive memory CTL during secondary dengue virus (DV) infection is thought to be important in the pathogenesis of dengue hemorrhagic fever. To model this effect, we studied the CTL responses to DV types 2 (D2V) and 3 (D3V) in PBMC from an individual previously infected with D3V. DV-specific CD8+ CTL from this donor recognized two HLA-B62-restricted epitopes on the NS3 protein, aa 71-79 (SVKKDLISY) and 235-243 (AMKGLPIRY). Both D3V-specific and D2V/D3V-cross-reactive CTL clones were detected for each epitope; all D2V-reactive CTL clones could lyse D2V-infected autologous cells. CTL responses to both epitopes were detected in bulk cultures stimulated with D3V, but PBMC stimulated with D2V recognized only the 235-243 epitope. IFN-gamma enzyme-linked immunospot assay showed that the D2V (71-79) peptide (DVKKDLISY) did not efficiently activate T cells. Analysis of a CTL clone suggests that the D2V (71-79) peptide acts as a partial agonist, able to sensitize target cells for lysis and inducing only minimal proliferation at high concentrations. These results suggest that variant peptide sequences present in the heterologous DV serotype can influence the CTL response in vivo during secondary DV infection.

Adult↗
PubMed1999

Impaired T cell proliferation in acute dengue infection.

Decreased proliferative responses to mitogens and recall Ags have been observed in PBMC obtained during several acute human viral infections. To determine whether cell-mediated responses are altered during acute dengue infection, we examined the proliferative responses of PBMC from children enrolled in a prospective study of dengue infections in Thailand. All responses of PBMC during acute illness were compared with the same patients' PBMC obtained at least 6 mo after their infection. Proliferative responses to PHA, anti-CD3, tetanus toxoid, and dengue Ags were decreased significantly in PBMC obtained during the acute infection. The proliferative responses to PHA were restored by the addition of gamma-irradiated autologous convalescent or allogeneic PBMC. Cell contact with the irradiated PBMC was necessary to restore proliferation. Non-T cells from the acute PBMC of dengue patients did not support proliferation of T cells from control donors in response to PHA, but T cells from the PBMC of patients with acute dengue proliferated if accessory cells from a control donor were present. Addition of anti-CD28 Abs restored anti-CD3-induced proliferation of the PBMC of some patients. The percentage of monocytes was reduced in the acute sample of PBMC of the dengue patients. Addition of IL-2 or IL-7, but not IL-4 or IL-12, also restored proliferation of acute PBMC stimulated with anti-CD3. The results demonstrate that both quantitative and qualitative defects in the accessory cell population during acute dengue illness result in a depression of in vitro T cell proliferation.

Acute Disease↗
PubMed1999

High levels of cytokine-producing cells in the lung tissues of patients with fatal hantavirus pulmonary syndrome.

Hantavirus pulmonary syndrome (HPS) is characterized by the rapid onset of pulmonary edema and a high case-fatality rate. Hantavirus antigens have been demonstrated in pulmonary capillary endothelial cells, but the mechanisms causing capillary leakage remain unclear. Immunohistochemical staining was used to enumerate cytokine-producing cells (monokines: interleukin [IL]-1alpha, IL-1beta, IL-6, and tumor necrosis factor [TNF]-alpha; lymphokines: interferon-gamma, IL-2, IL-4, and TNF-beta) in tissues obtained at autopsy from subjects with HPS. High numbers of cytokine-producing cells were seen in the lung and spleen tissues of HPS patients, but only low numbers in the livers and kidneys. A modest increase in the numbers of cytokine-producing cells was detected in the lungs of patients who died with non-HPS acute respiratory distress syndrome (ARDS), and very few (or no) cytokine-producing cells were detected in the lungs of patients who died of causes other than ARDS. These results suggest that local cytokine production may play an important role in the pathogenesis of HPS.

Adult↗
PubMed1999

Early immune activation in acute dengue illness is related to development of plasma leakage and disease severity.

T lymphocyte activation and increased cytokine levels have been described in retrospective studies of children presenting with dengue hemorrhagic fever (DHF). Serial plasma samples obtained in a prospective study of Thai children presenting with <72 h of fever were studied. Plasma levels of 80-kDa soluble tumor necrosis factor receptors (sTNFRs) were higher in children who developed DHF than in those with dengue fever (DF) or other nondengue febrile illnesses (OFIs) and were correlated with the degree of subsequent plasma leakage. Soluble CD8 and soluble interleukin-2 receptor levels were also elevated in children with DHF compared with those with DF. Interferon-gamma and sTNFR 60-kDa levels were higher in children with dengue than in those with OFIs. TNF-alpha was detectable more often in DHF than in DF or OFIs (P<.05). These results support the hypothesis that immune activation contributes to the pathogenesis of DHF. Further studies evaluating the predictive value of sTNFR80 for DHF are warranted.

Acute Disease↗
PubMed1999

Early CD69 expression on peripheral blood lymphocytes from children with dengue hemorrhagic fever.

Recent reports have demonstrated immune activation in dengue hemorrhagic fever (DHF) by cytokine and soluble receptor detection in blood. The goal of this study was to determine which cell types are activated and likely to be responsible for cytokine production. Whole blood specimens from 51 Thai children presenting within 72 h of fever onset and with detectable plasma dengue viral RNA were studied by flow cytometry. Absolute CD4 T cell, CD8 T cell, NK cell, and gammadelta T cell counts were decreased in children with DHF compared with those with dengue fever (DF) early in the course of illness. The percent of cells expressing CD69 was increased on CD8 T cells and NK cells in children who developed DHF more than in those with DF. These data directly demonstrate that cellular immune activation is present early in acute dengue and is related to disease severity.

Antigens, CD↗
PubMed1999

Analysis of murine CD8(+) T-cell clones specific for the Dengue virus NS3 protein: flavivirus cross-reactivity and influence of infecting serotype.

Serotype-cross-reactive dengue virus-specific cytotoxic T lymphocytes (CTL) induced during a primary dengue virus infection are thought to play a role in the immunopathogenesis of dengue hemorrhagic fever (DHF) during a secondary dengue virus infection. Although there is no animal model of DHF, we previously reported that murine dengue virus-specific CTL responses are qualitatively similar to human dengue virus-specific CTL responses. We used BALB/c mice to study the specificity of the CTL response to an immunodominant epitope on the dengue virus NS3 protein. We mapped the minimal H-2Kd-restricted CTL epitope to residues 298 to 306 of the dengue type 2 virus NS3 protein. In short-term T-cell lines and clones, the predominant CD8(+) CTL to this epitope in mice immunized with dengue type 2 virus or vaccinia virus expressing the dengue type 4 virus NS3 protein were cross-reactive with dengue type 2 or type 4 virus, while broadly serotype-cross-reactive CTL were a minority population. In dengue type 3 virus-immunized mice, the predominant CTL response to this epitope was broadly serotype cross-reactive. All of the dengue virus-specific CTL clones studied also recognized the homologous NS3 sequences of one or more closely related flaviviruses, such as Kunjin virus. The critical contact residues for the CTL clones with different specificities were mapped with peptides having single amino acid substitutions. These data demonstrate that primary dengue virus infection induces a complex population of flavivirus-cross-reactive NS3-specific CTL clones in mice and suggest that CTL responses are influenced by the viral serotype. These findings suggest an additional mechanism by which the order of sequential flavivirus infections may influence disease manifestations.

Amino Acid Sequence↗
PubMed1999

Bystander target cell lysis and cytokine production by dengue virus-specific human CD4(+) cytotoxic T-lymphocyte clones.

Dengue hemorrhagic fever, the severe form of dengue virus infection, is believed to be an immunopathological response to a secondary infection with a heterologous serotype of dengue virus. Dengue virus capsid protein-specific CD4(+) cytotoxic T-lymphocyte (CTL) clones were shown to be capable of mediating bystander lysis of non-antigen-presenting target cells. After activation by anti-CD3 or in the presence of unlabeled antigen-presenting target cells, these clones could lyse both Jurkat cells and HepG2 cells as bystander targets. Lysis of HepG2 cells suggests a potential role for CD4(+) CTL in the liver involvement observed during dengue virus infection. Three CD4(+) CTL clones were demonstrated to lyse cognate, antigen-presenting target cells by a mechanism that primarily involves perforin, while bystander lysis occurred through Fas/Fas ligand interactions. In contrast, one clone used a Fas/Fas ligand mechanism to lyse both cognate and bystander targets. Cytokine production by the CTL clones was also examined. In response to stimulation with D2 antigen, CD4(+) T-cell clones produced gamma interferon, tumor necrosis factor alpha (TNF-alpha) and TNF-beta. The data suggest that CD4(+) CTL clones may contribute to the immunopathology observed upon secondary dengue virus infections through direct cytolysis and/or cytokine production.

Antigen Presentation↗
PubMed1998

Inefficient recognition of autologous viral sequences by intrahepatic hepatitis C virus-specific cytotoxic T lymphocytes in chronically infected subjects.

Cytotoxic T lymphocytes (CTL) capable of recognizing prototype hepatitis C virus (HCV) sequences have been shown to localize to the liver in chronically infected individuals, where they are thought to influence hepatic inflammation and viral replication. We isolated three intrahepatic CD8(+) CTL clones from two individuals with chronic HCV infection and compared the recognition of prototype and autologous HCV sequences. These CTL recognized epitopes within the NS2 (amino acids 957-964) or NS3 (amino acids 1402-1410 and 1406-1415) proteins in the context of HLA B37, B8, or A2.1, respectively. The corresponding predominant autologous HCV sequences (SDWAANGL, ELAAKLVGL, and ALRGMGVNAV, respectively) differed from the HCV-1 sequences used for screening (RDWAHNGL, ELAAKLVAL, and KLVALGINAV, respectively) at one to five residues. For each CTL clone, recognition of the autologous HCV sequence required significantly higher peptide concentrations than did recognition of the HCV-1 sequence; for two of the clones, recognition was minimal or absent at peptide concentrations as high as 25 microM. These data show that intrahepatic HCV-specific CD8(+) CTL clones can be relatively inefficient at recognizing autologous viral epitopes. Inefficient recognition of autologous HCV sequences should influence the interpretation of data generated using prototype HCV sequences and might have implications in vivo.

Amino Acid Sequence↗
PubMed1998

Microplate-reverse hybridization method to determine dengue virus serotype.

A reverse transcriptase-polymerase chain reaction (RT-PCR) and microplate-reverse hybridization method were developed to detect and type dengue viruses in patients plasma specimens. A silica method was used to isolate RNA; and 3'-noncoding region universal primers were used to amplify dengue virus RNA. Using RT-PCR and ethidium bromide staining we could detect dengue virus in serum spiked with serially diluted dengue virus with a level of sensitivity similar to that of a quantitative fluorescent focus assay of dengue viruses in cell culture, i.e. 1.4 fluorescent focus units per reaction. Applying this assay to 14 dengue-positive plasma samples and 13 dengue-negative samples, dengue viremia was detectable by RT-PCR with a sensitivity comparable to mosquito inoculation. To determine the serotypes, digoxigenin-labeled PCR products from plasma samples and six laboratory adapted dengue viruses were hybridized in stringent conditions to serotype-specific DNA probes immobilized on microplates, and the hybridized product was detected with a colorimetric assay. Serotypes of dengue viruses, in cell culture and in patient plasma specimens, were identified using this method.

Dengue↗
PubMed1998

Predominance of HLA-restricted cytotoxic T-lymphocyte responses to serotype-cross-reactive epitopes on nonstructural proteins following natural secondary dengue virus infection.

We examined the memory cytotoxic T-lymphocytic (CTL) responses of peripheral blood mononuclear cells (PBMC) obtained from patients in Thailand 12 months after natural symptomatic secondary dengue virus infection. In all four patients analyzed, CTLs were detected in bulk culture PBMC against nonstructural dengue virus proteins. Numerous CD4+ and CD8+ CTL lines were generated from the bulk cultures of two patients, KPP94-037 and KPP94-024, which were specific for NS1.2a (NS1 and NS2a collectively) and NS3 proteins, respectively. All CTL lines derived from both patients were cross-reactive with other serotypes of dengue virus. The CD8+ NS1.2a-specific lines from patient KPP94-037 were HLA B57 restricted, and the CD8+ NS3-specific lines from patient KPP94-024 were HLA B7 restricted. The CD4+ CTL lines from patient KPP94-037 were HLA DR7 restricted. A majority of the CD8+ CTLs isolated from patient KPP94-024 were found to recognize amino acids 221 to 232 on NS3. These results demonstrate that in Thai patients after symptomatic secondary natural dengue infections, CTLs are mainly directed against nonstructural proteins and are broadly cross-reactive.

CD4-Positive T-Lymphocytes↗