Expression of the plasminogen system in the physiological mouse ovary and in the pathological polycystic ovary syndrome (PCOS) state.

BACKGROUND: The fibrinolytic system and its inhibitors play a number of roles, apart from their function in blood haemostasis and thrombosis, namely in ovarian folliculogenesis and in ovulation. Plasminogen is converted to active plasmin at the time of follicular rupture through a decrease in plasminogen activator inhibitor-1 (PAI-1) and an increase in plasminogen activators. Oligo-/anovulation and follicle arrest are key characteristics of PCOS, but studies evaluating fibrinolytic/proteolytic markers within human or animal PCOS ovaries are lacking. We aimed to investigate and compare the expression and distribution of the plasminogen system markers in PCOS and control ovaries. METHODS: A hyperandrogenised PCOS mouse model was used that mimics the ovarian, endocrine and metabolic features of the human condition. Immunohistochemistry and digital image analysis were used to investigate and compare fibrinolytic/proteolytic markers plasminogen, plasminogen/plasmin, tissue plasminogen activator, urokinase plasminogen activator and inhibitor PAI-1 in PCOS and control ovaries. Student's t-test was used to compare data sets for normally distributed data and Wilcoxon-Mann Whitney test for non-normally distributed data. RESULTS: We noted differences in the ovarian distribution of PAI-1 that was expressed throughout the PCOS ovary, unlike the peripheral distribution observed in control ovaries. Plasminogen was present in small follicles only in PCOS ovaries but not in small follicles of control ovaries. When we assessed and compared PAI-1 expression within follicles of different developmental stages we also noted significant differences for both the PCOS and control ovaries. While we noted differences in distribution and expression within specific ovarian structures, no differences were noted in the overall ovarian expression of markers assessed between acyclical PCOS mice and control mice at the diestrus stage of the estrous cycle. CONCLUSIONS: Our novel study, that comprehensively assessed the fibrinolytic/proteolytic system in the mouse ovary, showed the expression, differential localisation and a potential role for the plasminogen system in the physiological mouse ovary and in PCOS. Androgens may be involved in regulating expression of the ovarian plasminogen system. Further studies evaluating these markers at different time-points of ovulation may help to further clarify both physiological and potential pathological actions these markers play in ovulatory processes distorted in PCOS.

Siah2-deficient mice show impaired skin wound repair.

Hypoxia is associated with the dermal wound healing process and hypoxia signaling is presumed to be crucial for normal wound repair. The Siah2 ubiquitin ligase controls the abundance of hypoxia-inducible factor-1 alpha, and loss of Siah2 results in destabilization of hypoxia-inducible factor-1 alpha under hypoxia. Utilizing Siah2(-/-) mice we demonstrate that cutaneous wound healing is impaired in these mice. Wounds in Siah2(-/-) mice heal slower and are associated with delayed induction of myofibroblast infiltration and reduced collagen deposition. This coincides with delayed angiogenesis and reduced macrophage infiltration into the wounds of Siah2(-/-) mice. We furthermore demonstrate that primary Siah2(-/-) dermal fibroblasts have reduced migratory capacities and produce less collagen than wild-type fibroblasts. Additionally, Siah2(-/-) fibroblasts showed conserved responses to transforming growth factor-ß at the receptor level (pSmad 2C activation) but reduced responses downstream. Together, our data show, for the first time, that Siah2 is involved as a positive regulator in the wound healing response. Understanding the role of hypoxia signaling in tissue repair and fibrosis and interference with the hypoxia signaling pathway via regulation of Siah2 may provide new targets for clinical regulation of fibrosis and scarring.

Characterization of blood monocyte phenotype in patients with endometrial cancer.

BACKGROUND: Endometrial tumors induce various tumor escape mechanisms that result in immunosuppression in patients and, ultimately, tumor progression. Blood monocytes are able to exhibit potent cytotoxic action against tumor cells where novel immunotherapeutics targeting antigen-presenting cells including dendritic cells, and blood monocytes are being used as a means of delivering immunogens to stimulate an antitumor and, ultimately, therapeutic response. This study shows that peripheral blood monocytes from patients with endometrial cancer show functional deficiencies, and these deficiencies can be characterized by phenotypic changes as well as altered cytokine secretion. METHODS: This study assessed the phenotypic changes of peripheral blood monocytes by flow cytometry as well as the functional status via cytokine production measured by enzyme-linked immunosorbent assay in patients with endometrial cancer versus controls. RESULTS: Altered blood monocyte phenotype incorporating a decrease in costimulatory and adhesion factor expression and increased expression of vascular endothelial growth factor receptor 1 in patients with endometrial cancer versus controls. Increased interleukin 12 and decreased interleukin 10 secretion by blood monocytes in patients with endometrial cancer were also observed. CONCLUSIONS: These findings showed that peripheral blood monocytes from patients with endometrial cancer show an altered phenotype and cytokine secretion when compared with controls. Limitations to this study include the small sample size, the need to investigate the effect of phenotype and cytokine changes in functional assays, as well as future studies investigating the effect on tumor-associated macrophages from endometrial tissue from cancer versus control patients. Nevertheless, these findings suggest that peripheral blood monocyte induced immunosuppression in endometrial cancer and implications in the design of future immunotargeting therapies remain to be elucidated.

Cell-penetrating peptides: application in vaccine delivery.

Recent years have seen a surge in interest in cell-penetrating peptides (CPP) as an efficient means for delivering therapeutic targets into cellular compartments. The cell membrane is impermeable to hydrophilic substances yet linking to CPP can facilitate delivery into cells. Thus the unique translocatory property of CPP ensures they remain an attractive carrier, with the capacity to deliver cargoes in an efficient manner having applications in drug delivery, gene transfer and DNA vaccination. Fundamental for an effective vaccine is the delivery of antigen epitopes to antigen-presenting cells, ensuing processing and presentation and induction of an immune response. Vaccination with proteins or synthetic peptides incorporating CTL epitopes have proven limited due to the failure for exogenous antigens to be presented efficiently to T cells. Linking of antigens to CPP overcomes such obstacles by facilitating cellular uptake, processing and presentation of exogenous antigen for the induction of potent immune responses. This review will encompass the various strategies for the delivery of whole proteins, T cell epitopes and preclinical studies utilizing CPP for cancer vaccines.

A membrane penetrating multiple antigen peptide (MAP) incorporating ovalbumin CD8 epitope induces potent immune responses in mice.

Cell penetrating peptides (CPP) represent a novel approach to facilitate cytoplasmic delivery of macromolecules. The DNA binding domain of Drosophila Antennapedia contains 60 amino acids and consists of 3 α-helices, with internalizing activity mapped to a 16-amino acid peptide penetratin (Antp) within the third α-helix. Here, we report on the use of penetratin to deliver a multiple antigen peptide (MAP) incorporating the immunodominant CD8 epitope of ovalbumin, SIINFEKL (MAPOVACD8). We demonstrate that penetratin linked to the MAPOVACD8 construct either by a disulfide (SS) or thioether (SC) linkage promotes the uptake, cross presentation and subsequent in vivo proliferation and generation of OVACD8 (SIINFEKL)-specific T cells. The MAPOVACD8 construct without penetratin is not presented by MHC class I molecules nor does it generate an in vivo IFN-γ response in C57BL/6 mice. Moreover, we clearly define the uptake and intracellular processing pathways of AntpMAPOVACD8 SS and SC revealing the majority of AntpMAPOVACD8 is taken up by DC via an endocytic, proteasome and tapasin independent mechanism. We also show that the uptake mechanism of AntpMAPOVACD8 is dose dependent and uptake or intracellular processing is not altered by the type of chemical linkage.

Whole protein and defined CD8(+) and CD4(+) peptides linked to penetratin targets both MHC class I and II antigen presentation pathways.

Cytoplasmic delivery and cross-presentation of proteins and peptides is necessary for processing and presentation of antigens for the generation of cytotoxic T cells. We previously described the use of the 16 amino acid peptide penetratin from the Drosophila Antennapedia homeodomain (penetratin, Antp) to transport cytotoxic T lymphocyte epitopes derived from ovalbumin (OVA) or the Mucin-1 tumor-associated antigen into cells. We have now shown that penetratin covalently conjugated to OVA protein and linked in tandem to CD4(+) and/or CD8(+) T-cell epitopes from OVA-stimulated T cells in vitro (B3Z T-cell hybridoma and OT-I and OT-II T cells). The induction of these responses was directly mediated by the penetratin peptide as linking a nonspecific 16-mer peptide to OVA or mixing did not induce CD8(+) or CD4(+) T-cell responses in vitro. Furthermore, interferon (IFN)-γ-secreting CD4(+) and CD8(+) T cells were induced which suppressed B16.OVA tumor growth in C57BL/6 mice. Tumor protection was mediated by a CD8(+) T-cell-dependent mechanism and did not require CD4(+) help to protect mice 7 days after a boost immunization. Alternatively, 40 days after a boost immunization, the presence of CD4(+) help enhanced antigen-specific IFN-γ-secreting CD8(+) T cells and tumor protection in mice challenged with B16.OVA. Long-term CD8 responses were equally enhanced by antigen-specific and universal CD4 help. In addition, immunization with AntpOVA significantly delayed growth of B16.OVA tumors in mice in a tumor therapy model.

The adjuvanticity of a mannosylated antigen reveals TLR4 functionality essential for subset specialization and functional maturation of mouse dendritic cells.

The evidence that dendritic cell (DC) subsets produce differential cytokines in response to specific TLR stimulation is robust. However, the role of TLR stimulation in Ag presentation and phenotypic maturation among DC subsets is not clear. Through the adjuvanticity of a novel mannosylated Ag, mannosylated dendrimer OVA (MDO), as a pathogen-associated molecular pattern Ag, we characterized the functionality of GM-CSF/IL-4-cultured bone marrow DC and Flt3 ligand (Flt3-L) DC subsets by Ag presentation and maturation assays. It was demonstrated that both bone marrow DCs and Flt3-L DCs bound, processed, and presented MDO effectively. However, while Flt3-L CD24(high) (conventional CD8(+) equivalent) and CD11b(high) (CD8(-) equivalent) DCs were adept at MDO processing by MHC class I and II pathways, respectively, CD45RA(+) plasmacytoid DCs presented MDO poorly to T cells. Successful MDO presentation was largely dependent on competent TLR4 for Ag localization and morphological/phenotypic maturation of DC subsets, despite the indirect interaction of MDO with TLR4. Furthermore, Toll/IL-1 receptor-domain-containing adaptor-inducing IFN-beta, but not MyD88, as a TLR4 signaling modulator was indispensable for MDO-induced DC maturation and Ag presentation. Taken together, our findings suggest that DC subsets differentially respond to a pathogen-associated molecular pattern-associated Ag depending on the intrinsic programming and TLRs expressed. Optimal functionality of DC subsets in Ag presentation necessitates concomitant TLR signaling critical for efficient Ag localization and processing.

Immunomodulation in endometrial cancer.

Endometrial cancer is the most frequent gynecological cancer and the fourth most common cancer in women in the developed world. Over the last decade, immunotherapy has been the focus of intense investigation as a form of cancer treatment whereby the treatment initiates a host immune response ultimately eradicating the tumor. It has been suggested that in endometrial cancer and many other forms of cancer, immunosuppression poses a significant obstacle at inducing antitumor immunity by immunotherapy. This review will look at the different studies that have identified immunomodulation of T cells, cytokines and macrophages, and regulation of apoptotic and angiogenic factors in endometrial cancer patients that may contribute to the inefficiency of immunotherapy.

Pilot phase III immunotherapy study in early-stage breast cancer patients using oxidized mannan-MUC1 [ISRCTN71711835].

INTRODUCTION: Mucin 1 (MUC1) is a high molecular weight glycoprotein overexpressed on adenocarcinoma cells and is a target for immunotherapy protocols. To date, clinical trials against MUC1 have included advanced cancer patients. Herein, we report a trial using early stage breast cancer patients and injection of oxidized mannan-MUC1. METHOD: In a randomized, double-blind study, 31 patients with stage II breast cancer and with no evidence of disease received subcutaneous injections of either placebo or oxidized mannan-MUC1, to immunize against MUC1 and prevent cancer reoccurrence/metastases. Twenty-eight patients received the full course of injections of either oxidized mannan-MUC1 or placebo. Survival and immunological assays were assessed. RESULTS: After more than 5.5 years had elapsed since the last patient began treatment (8.5 years from the start of treatment of the first patient), the recurrence rate in patients receiving the placebo was 27% (4/15; the expected rate of recurrence in stage II breast cancer); those receiving immunotherapy had no recurrences (0/16), and this finding was statistically significant (P = 0.0292). Of the patients receiving oxidized mannan-MUC1, nine out of 13 had measurable antibodies to MUC1 and four out of 10 had MUC1-specific T cell responses; none of the placebo-treated patients exhibited an immune response to MUC1. CONCLUSION: The results suggest that, in early breast cancer, MUC1 immunotherapy is beneficial, and that a larger phase III study should be undertaken.

Quantitative proteomics of bronchoalveolar lavage fluid in lung adenocarcinoma.

BACKGROUND: The most commonly reported primary lung cancer subtype is adenocarcinoma, which is associated with a poor prognosis and short survival. Proteomic studies on human body fluids such as bronchoalveolar lavage fluid (BALF) have become essential methods for biomarker discovery, examination of tumor pathways and investigation of potential treatments. AIM: This study used quantitative proteomics to investigate the up-regulation of novel proteins in BALF from patients with primary lung adenocarcinoma in order to identify potential biomarkers. MATERIALS AND METHODS: BALF samples from individuals with and without primary lung adenocarcinoma were analyzed using liquid chromatography-mass spectrometry. RESULTS: One thousand and one hundred proteins were identified, 33 of which were found to be consistently overexpressed in all lung adenocarcinoma samples compared to non-cancer controls. A number of overexpressed proteins have been previously shown to be related to lung cancer progression including S100-A8, annexin A1, annexin A2, thymidine phosphorylase and transglutaminase 2. CONCLUSION: The overexpression of a number of specific proteins in BALF from patients with primary lung adenocarcinoma may be used as a potential biomarker for lung adenocarcinoma.

Alveolar Macrophage Polarisation in Lung Cancer.

The role of alveolar macrophages in lung cancer is multifaceted and conflicting. Alveolar macrophage secretion of proinflammatory cytokines has been found to enhance antitumour functions, cytostasis (inhibition of tumour growth), and cytotoxicity (macrophage-mediated killing). In contrast, protumour functions of alveolar macrophages in lung cancer have also been indicated. Inhibition of antitumour function via secretion of the anti-inflammatory cytokine IL-10 as well as reduced secretion of proinflammatory cytokines and reduction of mannose receptor expression on alveolar macrophages may contribute to lung cancer progression and metastasis. Alveolar macrophages have also been found to contribute to angiogenesis and tumour growth via the secretion of IL-8 and VEGF. This paper reviews the evidence for a dual role of alveolar macrophages in lung cancer progression.

Blood classical monocytes phenotype is not altered in primary non-small cell lung cancer.

AIM: To evaluate the M1 and M2 monocyte phenotype in patients with non-small cell lung cancer (NSCLC) compared to controls. Also, to examine the expression of Th1 and Th2 cytokines in plasma of NSCLC vs controls. METHODS: Freshly prepared peripheral blood mononuclear cells samples were obtained from patients with NSCLC (lung adenocarcinoma and squamous cell lung carcinoma) and from non-cancer controls. Flow cytometry was performed to investigate M1 and M2 phenotypes in peripheral monocytes (classical monocytes CD14+, CD45+ and CD16-) using conventional surface markers. Th1 and Th2 cytokine production was also analysed in the plasma using cytometric bead array technique. RESULTS: There were no significant difference in expression of M1 (HLA-DR) and/or M2 markers (CD163 and CD36) markers on classical monocytes in patients with NSCLC compared to non-cancer controls. Expression of CD11b, CD11c, CD71 and CD44 was also shown to be similar in patients with NSCLC compared to non-cancer controls. Th1 and Th2 cytokines [interleukin (IL)-1ß, IL-2, IL-4, IL-5, IL-8, IL-10, IL-12 (p70), tumor necrosis factor (TNF)-α, TNF-ß, and interferon-γ] analysis revealed no significant difference between patients with NSCLC and non-cancer controls. CONCLUSION: This study shows no alteration in peripheral monocyte phenotype in circulating classical monocytes in patients with NSCLC compared to non-cancer controls. No difference in Th1 and Th2 cytokine levels were noted in the plasma of these patients.

Delivery of antigen using a novel mannosylated dendrimer potentiates immunogenicity in vitro and in vivo.

Antigen mannosylation has been shown to be an effective approach to potentiate antigen immunogenicity, due to the enhanced antigen uptake and presentation by APC. To overcome disadvantages associated with conventional methods used to mannosylate antigens, we have developed a novel mannose-based antigen delivery system that utilizes a polyamidoamine (PAMAM) dendrimer. It is demonstrated that mannosylated dendrimer ovalbumin (MDO) is a potent immune inducer. With a strong binding avidity to DC, MDO potently induced OVA-specific T cell response in vitro. It was found that the immunogenicity of MDO was due not only to enhanced antigen presentation, but also to induction of DC maturation. Mice immunized with MDO generated strong OVA-specific CD4(+)/CD8(+) T cell and antibody responses. MDO also targeted lymph node DC to cross-present OVA, leading to OTI CD8(+) T cell proliferation. Moreover, upon challenge with B16-OVA tumor cells, tumors in mice pre-immunized with MDO either did not grow or displayed a much more delayed onset, and had slower kinetics of growth than those of OVA-immunized mice. This mannose-based antigen delivery system was applied here for the first time to the immunization study. With several advantages and exceptional adjuvanticity, we propose mannosylated dendrimer as a potential vaccine carrier.

Mannan-mediated gene delivery for cancer immunotherapy.

Recent years have seen a resurgence in interest in the development of efficient non-viral delivery systems for DNA vaccines and gene therapy. We have previously used oxidized and reduced mannan as carriers for protein delivery to antigen-presenting cells by targeting the receptors that bind mannose, resulting in efficient induction of cellular responses. In the present study, oxidized mannan and reduced mannan were used as receptor-mediated gene transfer ligands for cancer immunotherapy. In vivo studies in C57BL/6 mice showed that injection of DNA encoding ovalbumin (OVA) complexed to oxidized or reduced mannan-poly-L-lysine induced CD8 and CD4 T-cell responses as well as antibody responses leading to protection of mice from OVA+ tumours. Both oxidized and reduced mannan delivery was superior to DNA alone or DNA-poly-L-lysine. These studies demonstrate the potential of oxidized and reduced mannan for efficient receptor-mediated gene delivery in vivo, particularly as DNA vaccines for cancer immunotherapy.

Penetratin tandemly linked to a CTL peptide induces anti-tumour T-cell responses via a cross-presentation pathway.

Recently there has been increasing evidence to suggest that membrane translocating peptides enter cells by a receptor-dependent pathway. There have been some studies on the mechanism of major histocompatibility complex (MHC) class I presentation of membrane translocating peptides incorporating cytotoxic T lymphocyte epitopes. However, these have been on different cell lines and only a limited number of inhibitors of the antigen presentation pathway were used. Herein, we demonstrate a comprehensive study utilizing a full spectrum of inhibitors to various pathways of MHC class I to elucidate the mechanism of the membrane translocating peptide, penetratin from Antennapedia (Int). It is clear that Int, RQIKIWFQNRRMKWKK when tandemly linked to a cytotoxic T lymphocyte peptide of ovalbumin, SIINFEKL (IntSIIN) is endocytosed via phagocytosis or macropinocytosis by dendritic cells in an ATP-dependent manner and is processed by a proteasome- and tapasin-independent pathway for presentation and loading to MHC class I molecules. In addition, the majority of antigen is taken up by negatively charged receptors. IntSIIN activates T cells in vitro and in vivo and protects mice against challenge with an ovalbumin-expressing tumour.

Mannan derivatives induce phenotypic and functional maturation of mouse dendritic cells.

Mannan, a polysaccharide isolated from yeast binds to C-type lectins of the mannose receptor family, expressed by antigen-presenting cells (APCs) including dendritic cells (DCs) and macrophages. As these receptors mediate endocytosis, they have been targeted with ligands to deliver antigens into APCs to initiate immune responses. Immunization with tumour antigen MUC1 conjugated to oxidized mannan (OM) or reduced mannan (RM) induced differential immune responses in mice, and only mice immunized with OM-MUC1 elicited strong MUC1-specific cytotoxic T lymphocyte responses and protected mice from a MUC1 tumour challenge. In this study, the adjuvant effect of mannan and its derivatives including OM and RM, in comparison to lipopolysaccharide, on DCs were investigated. Mannan, OM and RM were capable of stimulating mouse bone marrow-derived DC in vitro, eliciting enhanced allogeneic T-cell proliferation and enhancing OTI/OTII peptide-specific T-cell responses. Injection of mice with mannan, OM and RM induced a mature phenotype of lymph node and splenic DCs. Analysis by reverse transcription-polymerase chain reaction indicated that Manna, OM and RM also stimulated up-regulation of inflammatory cytokines including interleukin-1beta and tumour necrosis factor-alpha, and differential T helper 1 (Th1)/Th2 cytokines. Subsequent experiments demonstrated that activation of DCs was Toll-like receptor-4-dependent. The data presented here, together with evidence reported previously on OM and RM in induction of immune responses in vivo, suggest that OM and RM exert a dual capacity to target antigen to APCs as well as mature DCs.

Delivery of tumor associated antigens to antigen presenting cells using penetratin induces potent immune responses.

Cytoplasmic delivery of proteins or CTL epitopes is crucial for the presentation of antigen for the generation of CTL. We previously described the use of the 16-amino acid peptide penetratin from the Drosophila Antennapedia domain (Int) to transport CTL epitopes into cells. Here we show that, Int, incorporating MUC1 CTL epitopes in tandem is able to facilitate their rapid uptake by macrophages and dendritic cells (DC) in an energy-dependent endocytic pathway. We also demonstrate for the first time that Int conjugated proteins are also able to be efficiently taken up by DC. Furthermore, C57BL/6 and HLA-A2 transgenic mice immunized with the Int-peptides or Int-proteins induce strong IFN-gamma secreting T cells and weak IgG1 antibodies. Immunized C57BL/6 mice were protected against the growth of a MUC1(+) tumor cell line.

Selectively impaired CD8+ but not CD4+ T cell cycle arrest during priming as a consequence of dendritic cell interaction with plasmodium-infected red cells.

Individuals living in malaria-endemic areas show generally low T cell responses to malaria Ags. In this study, we show murine dendritic cell (DC) interaction with parasitized erythrocytes (pRBC) arrested their maturation, resulting in impaired ability to stimulate naive, but not recall T cell responses in vitro and in vivo. Moreover, within the naive T cell population, pRBC-treated DC were selectively deficient in priming CD8(+) but not CD4(+) T cells. Indeed, DC that had taken up pRBC were shown for the first time to efficiently prime CD4(+) T cell responses to a known protective merozoite Ag, MSP4/5. In contrast, impaired priming resulted in decreases in both proliferation and cytokine production by CD8(+) T cells. Deficient priming was observed to both a model and a Plasmodium berghei-specific CD8(+) T cell epitope. The mechanisms underlying the inability of parasite-treated DC to prime CD8(+) T cells were explored. pRBC treatment of DC from wild-type C57BL/6, but not from IL-10 knockout animals, suppressed DC-mediated T cell priming across a Transwell, suggesting active IL-10-dependent suppression. CD8(+) T cells were arrested at the G(0) stage of the cell cycle after two cell divisions post-Ag stimulation. The proliferation arrest was partially reversible by the addition of IL-2 or IL-7 to responder cultures. These results suggest that in malaria-endemic areas, priming of CD8(+) T cell responses may be more difficult to induce via vaccination than the priming of CD4(+) T cells. Moreover, pathogens may selectively target the CD8(+) T cell arm of protective immunity for immune evasion.

Dendritic cells induce immunity and long-lasting protection against blood-stage malaria despite an in vitro parasite-induced maturation defect.

Dendritic cells (DC) suffer a maturation defect following interaction with erythrocytes infected with malaria parasites and become unable to induce protective malaria liver-stage immunity. Here we show that, by contrast, maturation-arrested DC in vitro are capable of the successful induction of antigen-specific gamma interferon (IFN-gamma) and interleukin 4 (IL-4) T-cell responses, antibody responses, and potent protection against lethal blood-stage malaria challenge in vivo. Similar results were found with DC pulsed with intact parasitized Plasmodium yoelii or Plasmodium chabaudi erythrocytes. Cross-strain protection was also induced. High levels of protection (80 to 100%) against lethal challenge were evident from 10 days after a single immunization and maintained up to 120 days. Interestingly, correlation studies versus blood-stage protection at different time points suggest that the immune effector mechanisms associated with protection could change over time. Antibody-independent, T-cell- and IL-12-associated protection was observed early after immunization, followed by antibody and IL-4-associated, IFN-gamma-independent protection in long-term studies. These results indicate that DC, even when clearly susceptible to parasite-induced maturation defect effects in vitro, can be central to the induction of protection against blood-stage malaria in vivo.