Cellular Origin of Human Lymphotoxin and its Purification

The ability of various subsets of human mononuclear cells to produce human lymphotoxin (LT) was examined. Peripheral blood mononuclear cells were seperated into OKT4+. OKT8+ and Leu-lla+ subpopulations by flow cytometry

Lymphostatin, a virulence factor of attaching and effacing Escherichia coli, inhibits proliferation and cytokine responses of human T cells in a manner associated with cell cycle arrest but not apoptosis or necrosis

Lymphostatin is a virulence factor of enteropathogenic E. coli (EPEC) and non-O157 serogroup enterohaemorrhagic E. coli. Previous studies using whole-cell lysates of EPEC showed that lymphostatin inhibits the mitogen-activated proliferation of bulk human peripheral blood mononuclear cells (PBMCs) and the production of cytokines IL-2, IL-4, IL-5, and IFN-γ. Here, we used highly purified lymphostatin and PBMC-derived T cells to show that lymphostatin inhibits anti-CD3/anti-CD28-activated proliferation of human CD4+ and CD8+ T cells and blocks the synthesis of IL-2, IL-4, IL-10 and IFN-γ without affecting cell viability and in a manner dependent on an N-terminal DTD glycosyltransferase motif. Such inhibition was not observed with T cells activated by phorbol 12-myristate 13-acetate and ionomycin, implying that lymphostatin targets T cell receptor signaling. Analysis of the expression of CD69 indicated that lymphostatin suppresses T cell activation at an early stage and no impacts on apoptosis or necrosis were observed. Flow cytometric analysis of the DNA content of lymphostatin-treated CD4+ and CD8+ T cells showed a concentration- and DTD-dependent accumulation of the cells in the G0/G1 phase of the cell cycle, and corresponding reduction of the percentage of cells in S phase. Consistent with this, we found a marked reduction in the abundance of cyclins D3, E and A and loss of phosphorylated Rb over time in activated T cells from 8 donors treated with lymphostatin. Moreover, the cyclin-dependent kinase (cdk) inhibitor p27kip1, which inhibits progression of the cell cycle at G1 by acting on cyclin E-cdk2 or cyclin D-cdk4 complexes, was found to be accumulated in lymphostatin-treated T cells. Analysis of the abundance of phosphorylated kinases involved in signal transduction found that 30 of 39 were reduced in abundance following lymphostatin treatment of T cells from 5 donors, albeit not significantly so. Our data provide novel insights into the mode of action of lymphostatin on human T lymphocytes

Long-Lasting Protective Immune Response to the 19-Kilodalton Carboxy-Terminal Fragment of Plasmodium yoelii Merozoite Surface Protein 1 in Mice

Merozoite surface protein 1 (MSP1) is the major protein on the surface of the plasmodial merozoite, and its carboxy terminus, the 19-kDa fragment (MSP1(19)), is highly conserved and effective in induction of a protective immune response against malaria parasite infection in mice and monkeys. However, the duration of the immune response has not been elucidated. As such, we immunized BALB/c mice with a standard four-dose injection of recombinant Plasmodium yoelii MSP1(19) formulated with Montanide ISA51 and CpG oligodeoxynucleotide (ODN) and monitored the MSP1(19)-specific antibody levels for up to 12 months. The antibody titers persisted constantly over the period of time without significant waning, in contrast to the antibody levels induced by immunization with Freund's adjuvant, where the antibody levels gradually declined to significantly lower levels 12 months after immunization. Investigation of immunoglobulin G (IgG) subclass longevity revealed that only the IgG1 antibody level (Th2 type-driven response) decreased significantly by 6 months, while the IgG2a antibody level (Th1 type-driven response) did not change over the 12 months after immunization, but the boosting effect was seen in the IgG1 antibody responses but not in the IgG2a antibody responses. After challenge infection, all immunized mice survived with negligibly patent parasitemia. These findings suggest that protective immune responses to MSP1(19) following immunization using oil-based Montanide ISA51 and CpG ODN as an adjuvant are very long-lasting and encourage clinical trials for malaria vaccine development

Immunostimulatory CpG Oligodeoxynucleotide Confers Protection in a Murine Model of Infection with Burkholderia pseudomallei

Although CpG oligodeoxynucleotides (CpG ODNs) are known to enhance resistance against infection in a number of animal models, little is known about the CpG-induced protection against acute fatal sepsis such as that associated with the highly virulent bacterium Burkholderia pseudomallei. We previously demonstrated in an in vitro study that immunostimulatory CpG ODN 1826 enhances phagocytosis of B. pseudomallei and induces nitric oxide synthase and nitric oxide production by mouse macrophages. In the present study, CpG ODN 1826 given intramuscularly to BALB/c mice 2 to 10 days prior to B. pseudomallei challenge conferred better than 90% protection. CpG ODN 1826 given 2 days before the bacterial challenge rapidly enhanced the innate immunity of these animals, judging from the elevated serum levels of interleukin-12 (IL-12)p70 and gamma interferon (IFN-γ) over the baseline values. No bacteremia was detected on day 2 in 85 to 90% of the CpG-treated animals, whereas more than 80% of the untreated animals exhibited heavy bacterial loads. Although marked elevation of IFN-γ was found consistently in the infected animals 2 days after the bacterial challenge, it was ameliorated by the CpG ODN 1826 pretreatment (P = 0.0002). Taken together, the kinetics of bacteremia and cytokine profiles presented are compatible with the possibility that protection by CpG ODN 1826 against acute fatal septicemic melioidosis in this animal model is associated with a reduction of bacterial load and interference with the potential detrimental effect of the robust production of proinflammatory cytokines associated with B. pseudomallei multiplication

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

A blunted blood plasmacytoid dendritic cell response to an acute systemic viral infection is associated with increased disease severity

At least two distinct human dendritic cell (DC) subsets are produced in the bone marrow and circulate in the peripheral blood-precursor myeloid DCs (pre-mDCs) and plasmacytoid DCs (PDCs). Both lineages of DCs are instrumental in antiviral innate immunity and shaping Th1 adaptive immune responses. PDCs are the most potent IFN-alpha-producing cells to viral pathogens. Dengue, an acute flavivirus disease, provides a model to study DC responses to a self-limited human viral infection. We analyzed circulating DC subsets in a prospective study of children with dengue across a broad range of illness severities: healthy controls; mild, nondengue, presumed viral infections; moderately ill dengue fever; and, the most severe form of illness, dengue hemorrhagic fever. We also examined PDC responses in monkeys with asymptomatic dengue viremia and to dengue virus exposure in vitro. The absolute number and frequency of circulating pre-mDCs early in acute viral illness decreased as illness severity increased. Depressed pre-mDC blood levels appeared to be part of the typical innate immune response to acute viral infection. The frequency of circulating PDCs trended upward and the absolute number of circulating PDCs remained stable early in moderately ill children with dengue fever, mild other, nondengue, febrile illness, and monkeys with asymptomatic dengue viremia. However, there was an early decrease in circulating PDC levels in children who subsequently developed dengue hemorrhagic fever. A blunted blood PDC response to dengue virus infection was associated with higher viremia levels, and was part of an altered innate immune response and pathogenetic cascade leading to severe disease

Superior protection in a relapsing Plasmodium cynomolgi rhesus macaque model by a chemoprophylaxis with sporozoite immunization regimen with atovaquone-proguanil followed by primaquine

Abstract Background To gain a deeper understanding of protective immunity against relapsing malaria, this study examined sporozoite-specific T cell responses induced by a chemoprophylaxis with sporozoite (CPS) immunization in a relapsing Plasmodium cynomolgi rhesus macaque model. Methods The animals received three CPS immunizations with P. cynomolgi sporozoites, administered by mosquito bite, while under two anti-malarial drug regimens. Group 1 (n = 6) received artesunate/chloroquine (AS/CQ) followed by a radical cure with CQ plus primaquine (PQ). Group 2 (n = 6) received atovaquone-proguanil (AP) followed by PQ. After the final immunization, the animals were challenged with intravenous injection of 104 P. cynomolgi sporozoites, the dose that induced reliable infection and relapse rate. These animals, along with control animals (n = 6), were monitored for primary infection and subsequent relapses. Immunogenicity blood draws were done after each of the three CPS session, before and after the challenge, with liver, spleen and bone marrow sampling and analysis done after the challenge. Results Group 2 animals demonstrated superior protection, with two achieving protection and two experiencing partial protection, while only one animal in group 1 had partial protection. These animals displayed high sporozoite-specific IFN-γ T cell responses in the liver, spleen, and bone marrow after the challenge with one protected animal having the highest frequency of IFN-γ+ CD8+, IFN-γ+ CD4+, and IFN-γ+ γδ T cells in the liver. Partially protected animals also demonstrated a relatively high frequency of IFN-γ+ CD8+, IFN-γ+ CD4+, and IFN-γ+ γδ T cells in the liver. It is important to highlight that the second animal in group 2, which experienced protection, exhibited deficient sporozoite-specific T cell responses in the liver while displaying average to high T cell responses in the spleen and bone marrow. Conclusions This research supports the notion that local liver T cell immunity plays a crucial role in defending against liver-stage infection. Nevertheless, there is an instance where protection occurs independently of T cell responses in the liver, suggesting the involvement of the liver's innate immunity. The relapsing P. cynomolgi rhesus macaque model holds promise for informing the development of vaccines against relapsing P. vivax