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

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.

Tissue Distribution of Memory T and B Cells in Rhesus Monkeys following Influenza A Infection.

Studies of influenza-specific immune responses in humans have largely assessed systemic responses involving serum Ab and peripheral blood T cell responses. However, recent evidence indicates that tissue-resident memory T (TRM) cells play an important role in local murine intrapulmonary immunity. Rhesus monkeys were pulmonary exposed to 2009 pandemic H1N1 virus at days 0 and 28 and immune responses in different tissue compartments were measured. All animals were asymptomatic postinfection. Although only minimal memory immune responses were detected in peripheral blood, a high frequency of influenza nucleoprotein-specific memory T cells was detected in the lung at the "contraction phase," 49-58 d after second virus inoculation. A substantial proportion of lung nucleoprotein-specific memory CD8(+) T cells expressed CD103 and CD69, phenotypic markers of TRM cells. Lung CD103(+) and CD103(-) memory CD8(+) T cells expressed similar levels of IFN-γ and IL-2. Unlike memory T cells, spontaneous Ab secreting cells and memory B cells specific to influenza hemagglutinin were primarily observed in the mediastinal lymph nodes. Little difference in systemic and local immune responses against influenza was observed between young adult (6-8 y) and old animals (18-28 y). Using a nonhuman primate model, we revealed substantial induction of local T and B cell responses following 2009 pandemic H1N1 infection. Our study identified a subset of influenza-specific lung memory T cells characterized as TRM cells in rhesus monkeys. The rhesus monkey model may be useful to explore the role of TRM cells in local tissue protective immunity after rechallenge and vaccination.

Controlling the cytokine storm in severe bacterial diarrhoea with an oral Toll-like receptor 4 antagonist.

Shigella dysenteriae causes the most severe of all infectious diarrhoeas and colitis. We infected rhesus macaques orally and also treated them orally with a small and non-absorbable polypropyletherimine dendrimer glucosamine that is a Toll-like receptor-4 (TLR4) antagonist. Antibiotics were not given for this life-threatening infection. Six days later, the clinical score for diarrhoea, mucus and blood was 54% lower, colon interleukin-8 and interleukin-6 were both 77% lower, and colon neutrophil infiltration was 75% less. Strikingly, vasculitis did not occur and tissue fibrin thrombi were reduced by 67%. There was no clinical toxicity or adverse effect of dendrimer glucosamine on systemic immunity. This is the first report in non-human primates of the therapeutic efficacy of a small and orally bioavailable TLR antagonist in severe infection. Our results show that an oral TLR4 antagonist can enable controlled resolution of the infection-related-inflammatory response and can also prevent neutrophil-mediated gut wall necrosis in severe infectious diarrhoeas.

Evaluation of the safety and immunogenicity in rhesus monkeys of a recombinant malaria vaccine for Plasmodium vivax with a synthetic Toll-like receptor 4 agonist formulated in an emulsion.

Plasmodium vivax is the major cause of malaria outside sub-Saharan Africa and inflicts debilitating morbidity and consequent economic impacts in developing countries. In order to produce a P. vivax vaccine for global use, we have previously reported the development of a novel chimeric recombinant protein, VMP001, based on the circumsporozoite protein (CSP) of P. vivax. Very few adjuvant formulations are currently available for human use. Our interest is to evaluate second-generation vaccine formulations to identify novel combinations of adjuvants capable of inducing strong, long-lasting immune responses. In this study rhesus monkeys were immunized intramuscularly three times with VMP001 in combination with a stable emulsion (SE) or a synthetic Toll-like receptor 4 (TLR4) agonist (glucopyranosyl lipid A [GLA]) in SE (GLA-SE). Sera and peripheral blood mononuclear cells (PBMCs) were tested for the presence of antigen-specific humoral and cellular responses, respectively. All groups of monkeys generated high titers of anti-P. vivax IgG antibodies, as detected by enzyme-linked immunosorbent assays (ELISAs) and immunofluorescence assays. In addition, all groups generated a cellular immune response characterized by antigen-specific CD4(+) T cells secreting predominantly interleukin-2 (IL-2) and lesser amounts of tumor necrosis factor (TNF). We conclude that the combination of VMP001 and GLA-SE is safe and immunogenic in monkeys and may serve as a potential second-generation vaccine candidate against P. vivax malaria.

Antiviral immune responses in H5N1-infected human lung tissue and possible mechanisms underlying the hyperproduction of interferon-inducible protein IP-10.

Information on the immune response against H5N1 within the lung is lacking. Here we describe the sustained antiviral immune responses, as indicated by the expression of MxA protein and IFN-alpha mRNA, in autopsy lung tissue from an H5N1-infected patient. H5N1 infection of primary bronchial/tracheal epithelial cells and lung microvascular endothelial cells induced IP-10, and also up-regulated the retinoic acid-inducible gene-I (RIG-I). Down-regulation of RIG-I gene expression decreased IP-10 response. Co-culturing of H5N1-infected pulmonary cells with TNF-alpha led to synergistically enhanced production of IP-10. In the absence of viral infection, TNF-alpha and IFN-alpha also synergistically enhanced IP-10 response. Methylprednisolone showed only a partial inhibitory effect on this chemokine response. Our findings strongly suggest that both the H5N1 virus and the locally produced antiviral cytokines; IFN-alpha and TNF-alpha may have an important role in inducing IP-10 hyperresponse, leading to inflammatory damage in infected lung.

Expression of toll-like receptors on antigen-presenting cells in patients with falciparum malaria.

The continuous release of blood-stage malaria parasites and their products can activate components of the innate immune system and induce the production of proinflammatory cytokines. Toll-like receptors (TLRs) have emerged as pattern-recognition receptors, residing on/in innate immune cells whose function is recognizing specific conserved components on different microbes. The aim of this study was to determine the expression of TLR2, TLR4 and TLR9 on antigen-presenting cells (APCs) in patients with mild and severe forms of falciparum malaria. Healthy individuals were used as controls. Peripheral blood mononuclear cells (PBMCs) were stained with specific monoclonal antibodies (mAbs) to investigate the percentage and the level of TLR expression by flow cytometry. Patients with severe and mild malaria showed increased surface expression of TLR2 and TLR4 on CD14(+)monocytes and myeloid dendritic cells (MDCs) and decreased intracellular expression of TLR9 on plasmacytoid dendritic cells (PDCs), compared to those of healthy controls. A significant decrease in the percentage of circulating CD14(+)monocytes and MDCs expressing TLR2 was found in both severe and mild malaria patients. These findings suggested that TLRs might play role in innate immune recognition in which the differential expression of TLRs on APCs could be regulated by the P. falciparum parasite.

Expression and function of Toll-like receptors on dendritic cells and other antigen presenting cells from non-human primates.

Antigen presenting cells (APCs), especially dendritic cells (DCs), play a crucial role in immune responses against infections by sensing microbial invasion through Toll-like receptors (TLRs). In this regard, TLR ligands are attractive candidates for use in humans and animal models as vaccine adjuvants. So far, no studies have been performed on TLR expression in non-human primates such as rhesus macaques. Therefore, we studied the TLR expression patterns in different subsets of APC in rhesus macaques and compared them to similar APC subsets in human. Also, expression was compared with corresponding DC subsets from different organs from mice. Here we show by semi-quantitative RT-PCR, that blood DC subsets of rhesus macaque expressed the same sets of TLRs as those of human but substantially differed from mouse DC subsets. Macaque myeloid DCs (MDCs) expressed TLR3, 4, 7 and 8 whereas macaque plasmacytoid DCs (PDCs) expressed only TLR7 and 9. Additionally, TLR expression patterns in macaque monocyte-derived dendritic cells (mo-DCs) (i.e., TLR3, 4, 8 and 9), monocytes (i.e., TLR4, 7, and 8) and B cells (i.e., TLR4, 7, 8, and 9) were also similar to their human counterparts. However, the responsiveness of macaque APCs to certain TLR ligands partially differed from that of human in terms of phenotype differentiation and cytokine production. Strikingly, in contrast to human mo-DCs, no IL-12p70 production was observed when macaque mo-DCs were stimulated with TLR ligands. In addition, CD40 and CD86 phenotypic responses to TLR8 ligand (poly U) in mo-DCs of macaque were higher than that of human. Despite these functional differences, our results provide important information for a rational design of animal models in evaluating TLR ligands as adjuvant in vivo.

Chemoprophylaxis with sporozoite immunization in P. knowlesi rhesus monkeys confers protection and elicits sporozoite-specific memory T cells in the liver.

Whole malaria sporozoite vaccine regimens are promising new strategies, and some candidates have demonstrated high rates of durable clinical protection associated with memory T cell responses. Little is known about the anatomical distribution of memory T cells following whole sporozoite vaccines, and immunization of nonhuman primates can be used as a relevant model for humans. We conducted a chemoprophylaxis with sporozoite (CPS) immunization in P. knowlesi rhesus monkeys and challenged via mosquito bites. Half of CPS immunized animals developed complete protection, with a marked delay in parasitemia demonstrated in the other half. Antibody responses to whole sporozoites, CSP, and AMA1, but not CelTOS were detected. Peripheral blood T cell responses to whole sporozoites, but not CSP and AMA1 peptides were observed. Unlike peripheral blood, there was a high frequency of sporozoite-specific memory T cells observed in the liver and bone marrow. Interestingly, sporozoite-specific CD4+ and CD8+ memory T cells in the liver highly expressed chemokine receptors CCR5 and CXCR6, both of which are known for liver sinusoid homing. The majority of liver sporozoite-specific memory T cells expressed CD69, a phenotypic marker of tissue-resident memory (TRM) cells, which are well positioned to rapidly control liver-stage infection. Vaccine strategies that aim to elicit large number of liver TRM cells may efficiently increase the efficacy and durability of response against pre-erythrocytic parasites.

Randomized, controlled, double-blind trial of daily oral azithromycin in adults for the prophylaxis of Plasmodium vivax malaria in Western Thailand.

We assessed the prophylactic efficacy of azithromycin (250 mg/day) against malaria in 276 adults in western Thailand in a randomized, double-blind, placebo-controlled trial. After antimalarial suppressive treatment, volunteers were randomized in a 2:1 ratio to either the azithromycin or placebo, respectively. Study medication was given for an average of 74 days. The azithromycin group (n = 179) had five endpoint parasitemias (1 Plasmodium vivax and 4 P. falciparum), and the placebo group (n = 97) had 28 endpoint parasitemias (21 P. vivax, 5 P. falciparum, and 2 mixed infections). Adverse events and compliance and withdrawal rates were similar in both groups. The protective efficacy (PE) of azithromycin was 98% for P. vivax (95% confidence interval [CI] = 88-100%). There were too few cases to reliably estimate the efficacy of azithromycin for P. falciparum (PE =71%, 95% C =-14-94%). We conclude that daily azithromycin was safe, well-tolerated, and had a high efficacy for the prevention of P. vivax malaria.

Robust in vitro replication of Plasmodium falciparum in glycosyl-phosphatidylinositol-anchored membrane glycoprotein-deficient red blood cells.

Red blood cells (RBCs) infected with Plasmodium falciparum are protected from complement-mediated lysis by surface membrane glycosyl-phosphatidylinositol (GPI)-anchored proteins, which include decay accelerating factor (DAF or CD55) and CD59. To determine if P. falciparum avoids or replicates less efficiently in GPI protein-deficient cells at a higher risk for complement-mediated lysis, we compared P. falciparum infectivity among control RBCs with those from subjects with paroxysmal nocturnal hemoglobinuria (PNH), a condition in which RBCs express variable levels of DAF (negative and positive) and CD59 (negative [-], intermediate [I], and high [H]). Co-cultures of 19 matched samples of control and PNH RBCs were infected with P. falciparum to directly compare parasitic invasion. Each PNH RBC sample was then assessed for P. falciparum infectivity across the spectrum of GPI protein deficiency. Identification methods included biotin-streptavidin for RBC populations, fluorescein isothiocyanate-labeled antibodies to DAF and CD59, hydroethidine for parasite DNA, and flow cytometry. The mean +/- SD parasitemias in co-cultured PNH and control RBCs were 24.7 +/- 6.9% versus 21.0 +/- 5.9% (P = 0.12). For individual PNH samples, parasitemias were significantly higher in DAF (-) cells versus DAF (+) cells (25.0 +/- 8.9% versus 19.1 +/- 8.7%; P < 0.001) and in CD59 (-) cells versus I/H cells (22.5 +/- 6.4% versus 17.6 +/- 4.2%; P < 0.0003). Across the CD59 spectrum, mean parasitemias were highest in CD59 (-) cells (24.5 +/- 6.4%), followed by CD59-H cells (19.5 +/- 5.4%), and CD59-I cells (16.4 +/- 4.8%). Expression of DAF in 12 (63%) of 19 infected PNH samples was reduced. Thus, P. falciparum does not selectively avoid RBCs with fewer GPI proteins and parasite replication in PNH cells is at least as robust as in normal RBCs.

Pre-existing cross-reactive antibodies to avian influenza H5N1 and 2009 pandemic H1N1 in US military personnel.

We studied cross-reactive antibodies against avian influenza H5N1 and 2009 pandemic (p) H1N1 in 200 serum samples from US military personnel collected before the H1N1 pandemic. Assays used to measure antibodies against viral proteins involved in protection included a hemagglutination inhibition (HI) assay and a neuraminidase inhibition (NI) assay. Viral neutralization by antibodies against avian influenza H5N1 and 2009 pH1N1 was assessed by influenza (H5) pseudotyped lentiviral particle-based and H1N1 microneutralization assays. Some US military personnel had cross-neutralizing antibodies against H5N1 (14%) and 2009 pH1N1 (16.5%). The odds of having cross-neutralizing antibodies against 2009 pH1N1 were 4.4 times higher in subjects receiving more than five inactivated whole influenza virus vaccinations than those subjects with no record of vaccination. Although unclear if the result of prior vaccination or disease exposure, these pre-existing antibodies may prevent or reduce disease severity.

Comparison of the immune responses induced by soluble and particulate Plasmodium vivax circumsporozoite vaccine candidates formulated in AS01 in rhesus macaques.

We have designed a pre-erythrocytic vaccine candidate based on the Plasmodium vivax circumsporozoite (CSV) protein, which includes its N- and C-terminal parts and a truncated region containing repeat sequences from both the VK210 and the VK247 P. vivax subtypes. Two versions of this vaccine candidate were made: a soluble recombinant protein expressed in Escherichia coli, designated VMP001 and a particulate antigen expressed in Saccharomyces cerevisiae, designated CSV-S,S. The latter is composed of CSV-S, a fusion protein between VMP001 and hepatitis B surface antigen (HBsAg), and free HBsAg co-expressed in yeast and self-assembling into mixed particles. Both antigen versions, adjuvanted with AS01, were shown to be immunogenic in rhesus monkeys. CSV-S,S/AS01 induced higher levels of VMP001-specific antibodies than did VMP001/AS01. Antibody responses against the N- and C-terminal regions of CSV and the VK210 repeat motif were of a similar magnitude following immunization with either the soluble or the particulate antigen. However, antibodies against the AGDR region, a potentially protective B cell epitope, were only detected after immunization with CSV-S,S. Analysis of the induced CD4(+) T cells highlighted different cytokine profiles depending on the antigen form. These results warrant further clinical evaluation of these two vaccine candidates to assess the added value of a particulate versus soluble form of CSV, in terms of both immunogenicity and protective efficacy.

Evaluation of in vitro cross-reactivity to avian H5N1 and pandemic H1N1 2009 influenza following prime boost regimens of seasonal influenza vaccination in healthy human subjects: a randomised trial.

INTRODUCTION: Recent studies have demonstrated that inactivated seasonal influenza vaccines (IIV) may elicit production of heterosubtypic antibodies, which can neutralize avian H5N1 virus in a small proportion of subjects. We hypothesized that prime boost regimens of live and inactivated trivalent seasonal influenza vaccines (LAIV and IIV) would enhance production of heterosubtypic immunity and provide evidence of cross-protection against other influenza viruses. METHODS: In an open-label study, 26 adult volunteers were randomized to receive one of four vaccine regimens containing two doses of 2009-10 seasonal influenza vaccines administered 8 (±1) weeks apart: 2 doses of LAIV; 2 doses of IIV; LAIV then IIV; IIV then LAIV. Humoral immunity assays for avian H5N1, 2009 pandemic H1N1 (pH1N1), and seasonal vaccine strains were performed on blood collected pre-vaccine and 2 and 4 weeks later. The percentage of cytokine-producing T-cells was compared with baseline 14 days after each dose. RESULTS: Subjects receiving IIV had prompt serological responses to vaccine strains. Two subjects receiving heterologous prime boost regimens had enhanced haemagglutination inhibition (HI) and neutralization (NT) titres against pH1N1, and one subject against avian H5N1; all three had pre-existing cross-reactive antibodies detected at baseline. Significantly elevated titres to H5N1 and pH1N1 by neuraminidase inhibition (NI) assay were observed following LAIV-IIV administration. Both vaccines elicited cross-reactive CD4+ T-cell responses to nucleoprotein of avian H5N1 and pH1N1. All regimens were safe and well tolerated. CONCLUSION: Neither homologous nor heterologous prime boost immunization enhanced serum HI and NT titres to 2009 pH1N1 or avian H5N1 compared to single dose vaccine. However heterologous prime-boost vaccination did lead to in vitro evidence of cross-reactivity by NI; the significance of this finding is unclear. These data support the strategy of administering single dose trivalent seasonal influenza vaccine at the outset of an influenza pandemic while a specific vaccine is being developed. TRIAL REGISTRATION: ClinicalTrials.gov NCT01044095.

High susceptibility of human dendritic cells to avian influenza H5N1 virus infection and protection by IFN-alpha and TLR ligands.

There is worldwide concern that the avian influenza H5N1 virus, with a mortality rate of >50%, might cause the next influenza pandemic. Unlike most other influenza infections, H5N1 infection causes a systemic disease. The underlying mechanisms for this effect are still unclear. In this study, we investigate the interplay between avian influenza H5N1 and human dendritic cells (DC). We showed that H5N1 virus can infect and replicate in monocyte-derived and blood myeloid DC, leading to cell death. These results suggest that H5N1 escapes viral-specific immunity, and could disseminate via DC. In contrast, blood pDC were resistant to infection and produced high amounts of IFN-alpha. Addition of this cytokine to monocyte-derived DC or pretreatment with TLR ligands protected against infection and the cytopathic effects of H5N1 virus.

Characterization of circulating monocytes expressing HLA-DR or CD71 and related soluble factors for 2 weeks after severe, non-thermal injury.

BACKGROUND: Severe injury is associated with changes in monocytes that may contribute to poor outcomes. Longitudinal characterization of monocyte response patterns after trauma may provide added insight into these immunological alterations. METHODS: Venous blood obtained seven times during post-injury days 1 through 13 from 61 patients with an injury severity score >20 was assessed by flow cytometry for monocytes (CD14+) expressing HLA-DR or CD71 (transferrin receptor) and for circulating levels of interleukin (IL) 1alpha, IL-1beta, IL-6, soluble CD14 (sCD14), tumor necrosis factor-alpha (TNF-alpha), prostaglandin E(2) (PGE(2)), thromboxane B(2) (TXB(2)), and endotoxin. Urine neopterin was measured by high-pressure liquid chromatography, expressed as a neopterin-creatinine ratio. RESULTS: Trauma patients had leucocytosis days 1 through 13, monocytosis days 5 through 13, reduced proportions of CD14+HLA-DR+ cells days 2 through 5, and elevated proportions of CD14+CD71+ cells days 1 through 13. Neopterin was elevated all days, peaking on day 10. sCD14 was elevated days 2 through 13, and there were sporadic elevations of IL-1alpha, IL-1beta, IL-6, TNF-alpha, PGE(2), TXB(2), and endotoxin. Sepsis syndrome patients (n = 6) had larger and more prolonged reductions in CD14+HLA-DR+ cells and higher neopterin values, in comparison with uneventful patient outcomes. CONCLUSIONS: Altered proportions of monocytes expressing HLA-DR and CD71 and elevated sCD14 and urine neopterin levels, for up to 2 weeks after severe injury, underscores an extended period of profound immunological effects. Additional studies to more fully assess temporal monocyte response patterns after severe injury, including activation, may be warranted.

Generation of gingival T cell lines/clones specific with Porphyromonas gingivalis pulsed dendritic cells from periodontitis patients.

OBJECTIVES AND BACKGROUND: It is well documented that in periodontitis lesions, most infiltrated gingival T cells are antigen-specific memory T cells. These cells play an important role as regulators and effector cells in the pathogenesis of periodontitis. In this study, we used dendritic cells (DCs) as antigen-presenting cells to generate human gingival T cell lines and clones specific for Porphyromonas gingivalis from periodontitis patients. METHODS: Autologous DCs were derived from the patients' adherent monocytes using granulocyte-macrophage colony-stimulating factor and interleukin (IL)-4. Lymphocytes were isolated from gingival biopsies using collagenase enzyme digestion and the number was increased by subsequent culturing in IL-2-containing medium. T cells were then negatively sorted using flow cytometry, cocultured with P. gingivalis-pulsed DCs and subsequently expanded in the culture medium containing IL-2. T cells were kept viable and active by periodic exposure to antigen-pulsed DCs. The specificity of the T cell lines was tested against four plaque bacteria: P. gingivalis, Actinobacillus actinomycetemcomitans, Prevotella intermedia and Actinomyces viscosus. The established T cell lines were then cloned. Three P. gingivalis-specific T cell lines and 12 gingival T cell clones were generated. They all showed good specificity against P. gingivalis but not to other plaque bacteria. RESULTS: All T cell clones were positive for CD4 and the majority of them produced interferon gamma, but a minimal or negligible amount of IL-5. CONCLUSIONS: The data obtained clearly showed that monocyte-derived DCs could be used as powerful antigen-presenting cells to generate antigen-specific T cells from periodontitis tissues.

Upregulation of co-stimulatory molecule expression and dendritic cell marker (CD83) on B cells in periodontal disease.

T cells and their cytokines are well known for their important role in the pathogenesis of periodontitis. To date, the role of antigen presenting cells (APCs), which are known to be critical in the regulation of T cell response, has been poorly investigated in periodontitis. In this study, we analyzed the expression of co-stimulatory molecules (CD80 and CD86) and CD83, which is a marker of mature dendritic cells, on gingival cells that were isolated from severe periodontitis tissues, with the use of flow cytometry. Significant upregulation of CD86 and CD83 expression was detected in periodontitis lesions, and most of this occurred on B cells. In vitro peripheral blood mononuclear cell cultures showed that stimulation with different periodontopathic bacteria, that included Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, Prevotella intermedia, and Actinomyces viscosus, upregulated both CD86 and CD83 expression on B cells. Therefore, the presence of plaque bacteria may be responsible for the enhanced expression seen in vivo on gingival B cells. APC function by bacterial-activated B cells was further investigated using allogeneic mixed leukocyte reactions. After 24 h culture with either A. actinomycetemcomitans or P. gingivalis, these activated B cells performed as potent APCs in mixed leukocyte reactions, and they stimulated T cells to produce high levels of gamma interferon and minimal interleukin-5. In conclusion, periodontopathic bacterial-induced B cell activation with upregulation of CD86 and CD83 may be associated with enhanced APC function. The results of this study suggest, therefore, that infiltrated gingival B cells have a possible role as APCs in the regulation and maintenance of local T cell response in periodontitis.

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.

Malaria blood stage parasites activate human plasmacytoid dendritic cells and murine dendritic cells through a Toll-like receptor 9-dependent pathway.

A common feature of severe Plasmodium falciparum infection is the increased systemic release of proinflammatory cytokines that contributes to the pathogenesis of malaria. Using human blood, we found that blood stage schizonts or soluble schizont extracts activated plasmacytoid dendritic cells (PDCs) to up-regulate CD86 expression and produce IFN-alpha. IFN-alpha production was also detected in malaria-infected patients, but the levels of circulating PDCs were markedly reduced, possibly because of schizont-stimulated up-regulation of CCR7, which is critical for PDC migration. The schizont-stimulated PDCs elicited a poor T cell response, but promoted gamma delta T cell proliferation and IFN-gamma production. The schizont immune stimulatory effects could be reproduced using murine DCs and required the Toll-like receptor 9 (TLR9)-MyD88 signaling pathway. Although the only known TLR9 ligand is CpG motifs in pathogen DNA, the activity of the soluble schizont extract was far greater than that of schizont DNA, and it was heat labile and precipitable with ammonium sulfate, unlike the activity of bacterial DNA. These results demonstrate that schizont extracts contain a novel and previously unknown ligand for TLR9 and suggest that the stimulatory effects of this ligand on PDCs may play a key role in immunoregulation and immunopathogenesis of human falciparum malaria.