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Objectives: To suggest the presence of a hyperimmune state in patients, and indicate that immune system attack on glycosylphosphatidylinositol (+) (GPI + ) cells while escaping GPI - cell immunity., Methods: We retrospective the immune cell subtypes in peripheral blood from 25 patients visiting Tianjin Medical University General Hospital, Tianjin, China, with classical paroxysmal nocturnal hemoglobinuria (PNH) and 50 healthy controls., Results: The total CD3 + and CD3 + CD8 + cell levels were higher in patients with PNH. The CD3 + cells are positively, correlated with lactate dehydrogenase (LDH; r=0.5453, p =0.0040), indirect bilirubin (r=0.4260, p =0.0379) and Flear - cells in monocytes (r=0.4099, p =0.0303). However, a negative correlation was observed between CD3 + cells and hemoglobin (r= -0.4530, p =0.0105). The total CD19 + cells decreased in patients, and CD19 + cells were negatively correlated with LDH (r= -0.5640, p =0.0077) and Flear - cells in monocytes (r= -0.4432, p =0.0341). Patients showed an increased proportion of total dendritic cells (DCs), with a higher proportion of myeloid DCs (mDCs) within the DC population. Moreover, the proportion of mDC/DC was positively correlated with CD59 - cells (II + III types) in red cells (r=0.7941, p =0.0004), Flear - cells in granulocytes (r=0.5357, p =0.0396), and monocytes (r=0.6445, p =0.0095)., Conclusion: Our results demonstrated that immune abnormalities are associated with PNH development., (Copyright: © Saudi Medical Journal.)

Neospora caninum causes abortion in ruminants, leading to important economic losses and no efficient treatment or vaccine against neosporosis is available. Considering the complexity of the strategies developed by intracellular apicomplexan parasites to escape immune system, future vaccine formulations should associate the largest panel of antigens and adjuvants able to better stimulate immune responses than natural infection. A mucosal vaccine, constituted of di-palmitoyl phosphatidyl glycerol-loaded nanoparticles (DGNP) and total extract (TE) of soluble antigens of Toxoplasma gondii, has demonstrated its efficacy, decreasing drastically the parasite burden. Here, DGNP were loaded with N. caninum TE and glycosylphosphatidylinositol (GPI) of N. caninum as Toll-like receptor (TLR) adjuvant able to induce specific cellular and humoral immune responses. Activation of TLR2 and TLR4 signalling pathway in HEK reporter cells induced by GPI was abrogated after its incorporation into DGNP. However, in murine bone marrow-derived dendritic cells, an adjuvant effect of GPI was observed with higher levels of interleukin (IL)-1β, reduced levels of IL-6, IL-12p40 and IL-10, and decreased expression of major histocompatibility complex (MHC) molecules. GPI also modulated the responses of bovine peripheral blood mononuclear cells, by increasing the production of IFN-γ and by decreasing the expression of MHC molecules. Altogether, these results suggest that GPI delivered by the DGNP might modulate cell responses through the activation of an intracellular pathway of signalisation in a TLR-independent manner. In vivo experiments are needed to confirm the potent adjuvant properties of N. caninum GPI in a vaccine strategy against neosporosis., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Glycosylphosphatidylinositols (GPIs) are glycolipids described as toxins of protozoan parasites due to their inflammatory properties in mammalian hosts characterized by the production of interleukin (IL)-1, IL-12 and tumor necrosis factor (TNF)-α. In the present work, we studied the cytokines produced by antigen presenting cells in response to ten different GPI species extracted from Babesia divergens, responsible for babesiosis. Interestingly, B. divergens GPIs induced the production of anti-inflammatory cytokines (IL-2, IL-5) and of the regulatory cytokine IL-10 by macrophages and dendritic cells. In contrast to all protozoan GPIs studied until now, GPIs from B. divergens did not stimulate the production of TNF-α and IL-12, leading to a unique Th1/Th2 profile. Analysis of the carbohydrate composition of the B. divergens GPIs indicated that the di-mannose structure was different from the evolutionary conserved tri-mannose structure, which might explain the particular cytokine profile they induce. Expression of major histocompatibility complex (MHC) molecules on dendritic cells and apoptosis of mouse peritoneal cells were also analysed. B. divergens GPIs did not change expression of MHC class I, but decreased expression of MHC class II at the cell surface, while GPIs slightly increased the percentages of apoptotic cells. During pathogenesis of babesiosis, the inflammation-coagulation auto-amplification loop can lead to thrombosis and the effect of GPIs on coagulation parameters was investigated. Incubation of B. divergens GPIs with rat plasma ex vivo led to increase of fibrinogen levels and to prolonged activated partial thromboplastin time, suggesting a direct modulation of the extrinsic coagulation pathway by GPIs., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Staphylococcus aureus is frequently isolated from patients with community-acquired pneumonia and acute respiratory distress syndrome (ARDS). ARDS is associated with staphylococcal phosphatidylinositol-specific phospholipase C (PI-PLC); however, the role of PI-PLC in the pathogenesis and progression of ARDS remains unknown. Here, we showed that recombinant staphylococcal PI-PLC possesses enzyme activity that causes shedding of glycosylphosphatidylinositol-anchored CD55 and CD59 from human umbilical vein endothelial cell surfaces and triggers cell lysis via complement activity. Intranasal infection with PI-PLC-positive S. aureus resulted in greater neutrophil infiltration and increased pulmonary oedema compared with a plc-isogenic mutant. Although indistinguishable proinflammatory genes were induced, the wild-type strain activated higher levels of C5a in lung tissue accompanied by elevated albumin instillation and increased lactate dehydrogenase release in bronchoalveolar lavage fluid compared with the plc - mutant. Following treatment with cobra venom factor to deplete complement, the wild-type strain with PI-PLC showed a reduced ability to trigger pulmonary permeability and tissue damage. PI-PLC-positive S. aureus induced the formation of membrane attack complex, mainly on type II pneumocytes, and reduced the level of CD55/CD59, indicating the importance of complement regulation in pulmonary injury. In conclusion, S. aureus PI-PLC sensitised tissue to complement activation leading to more severe tissue damage, increased pulmonary oedema, and ARDS progression., (© 2019 John Wiley & Sons Ltd.)

Toxoplasmosis, while often an asymptomatic parasitic disease in healthy individuals, can cause severe complications in immunocompromised persons and during pregnancy. The most common method to diagnose Toxoplasma gondii infections is the serological determination of antibodies directed against parasite protein antigens. Here we report the use of a bead-based multiplex assay containing a synthetic phosphoglycan portion of the Toxoplasma gondii glycosylphosphatidylinositol (GPI 1 ) for the detection of GPI 1 -specific antibodies in human sera. The glycan was conjugated to beads at the lipid site to retain its natural orientation and its immunogenic groups. We compared the response against GPI 1 with that against the protein antigen SAG1, a common component of commercial serological assays, via the detection of parasite-specific human IgG and IgM antibodies, respectively. The GPI 1 -based test is in excellent agreement with the results for the commercial ELISA, as the ROC analysis of the GPI 1 test shows 97% specificity and 98% sensitivity for the assay. GPI 1 was a more reliable predictor for a parasite-specific IgM response compared to SAG1, indicating that a bead-based multiplex assay using GPI 1 in combination with SAG1 may strengthen Toxoplasma gondii serology, in particular in seroepidemiological studies.

A long-standing challenge in malaria is the limited understanding of B cell immunity, previously hampered by lack of tools to phenotype rare antigen-specific cells. Our aim was to develop a method for identifying carbohydrate-specific B cells within lymphocyte populations and to determine whether a candidate vaccine generated functional memory B cells (MBCs) that reactivated upon challenge with Plasmodium (pRBCs). To this end, a new flow cytometric probe was validated and used to determine the kinetics of B cell activation against the candidate vaccine glycosylphosphatidylinositol conjugated to Keyhole Limpet Haemocyanin (GPI-KLH). Additionally, immunized C57BL/6 mice were rested (10 weeks) and challenged with pRBCs or GPI-KLH to assess memory B cell recall against foreign antigen. We found that GPI-specific B cells were detectable in GPI-KLH vaccinated mice, but not in Plasmodium -infected mice. Additionally, in previously vaccinated mice GPI-specific IgG1 MBCs were reactivated against both pRBCs and synthetic GPI-KLH, which resulted in increased serum levels of anti-GPI IgG in both challenge approaches. Collectively our findings contribute to the understanding of B cell immunity in malaria and have important clinical implications for inclusion of carbohydrate conjugates in malaria vaccines.

Malaria remains a significant worldwide public health problem. To address biological questions, researchers rely on the experimental murine model. For decades, chloroquine (CQ) and pyrimethamine (Pyr) have been used to clear Plasmodium infections in experimental animals using standardised accepted protocols and, because of this, drug-treated controls are rarely included. However, there is limited data available on the modulation of anti-malarial immunity, including generation of memory B cells, when these drugs are administered days after malaria infection. We investigated B cell responses to an important malaria glycolipid, glycosylphosphatidylinositol (GPI), and the hapten nitrophenol (NP), with or without standard CQ and Pyr treatment using the murine model. At day 14, CQ/Pyr treatment significantly suppressed the frequency of NP + IgG1 + memory B cells in NP-KLH-immunised mice. Furthermore, CQ/Pyr-treated NP-KLH-immunised mice did not have significantly higher cellular counts of NP + B cells, germinal centre B cells, nor NP + IgG1 + memory B cells than naïve mice (CQ/Pyr treated and untreated). CQ/Pyr-treated GPI-KLH-immunised mice did not have significantly higher cellular counts of GPI + B cells than naïve untreated mice. By day 28, this effect appeared to resolve since all immunised mice, whether treated or untreated, had significantly higher B cell proliferative responses than naïve mice (CQ/Pyr treated and untreated) for the majority of B cell phenotypes. The current study emphasises the potential for drug modulation of antigenic B cell responses when using standardised malaria treatment protocols in the experimental murine model. It is recommended that drug-treated controls are included when using experimental malaria infections to address biological questions.

Background: Tropical theileriosis is widely distributed from North Africa to East Asia. It is a tick-borne disease caused by Theileria annulata, an obligate two-host intracellular protozoan parasite of cattle. Theileria annulata use leukocytes and red blood cells for completion of the life-cycle in mammalian hosts. The stage of Theileria annulata in monocytes and B lymphocytes of cattle is an important step in pathogenicity and diagnosis of the disease. Glycosylphosphatidylinositols (GPIs) are a distinct class of glycolipid structures found in eukaryotic cells and are implicated in several biological functions. GPIs are particularly abundant in protozoan parasites, where they are found as free glycolipids or attached to proteins in the plasma membrane., Results: In this study we first isolated and purified schizonts of Theileria annulata from infected leukocytes in Theileria annulata vaccine cell line (S15) by aerolysin-percoll technique. Then, the free GPIs of schizont stage and isolated GPI from cell membrane glycoproteins were purified by high performance liquid chromatography (HPLC) and confirmed by gas chromatography-mass spectrometry (GC-MS). Furthermore, enzyme linked immunosorbent assay (ELISA) on the serum samples obtained from naturally infected, as well as Theileria annulata-vaccinated animals, confirmed a significant (P < 0.01) high level of anti-GPI antibody in their serum., Conclusions: The results presented in this study show, to our knowledge for the first time, the isolation of GPI from the schizont stage of Theileria annulata and demonstrate the presence of anti-GPI antibody in the serum of naturally infected as well as vaccinated animals. This finding is likely to be valuable in studies aimed at the evaluation of chemically structures of GPIs in the schizont stage of Theileria annulata and also for pathogenicity and immunogenicity studies with the aim to develop GPI-based therapies or vaccines.

Malaria, one of the most common vector borne human diseases, is a major world health issue. In 2015 alone, more than 200 million people were infected with malaria, out of which, 429 000 died. Even though artemisinin-based combination therapies (ACT) are highly effective at treating malaria infections, novel efforts toward development of vaccines to prevent transmission are still needed. Pfs25, a postfertilization stage parasite surface antigen, is a leading transmission-blocking vaccine (TBV) candidate. It is postulated that Pfs25 anchors to the cell membrane using a glycosylphosphatidylinositol (GPI) linker, which itself possesses pro-inflammatory properties. In this study, Escherichia coli derived extract (XtractCF +TM ) was used in cell free protein synthesis [CFPS] to successfully express >200 mg/L of recombinant Pfs25 with a C-terminal non-natural amino acid (nnAA), namely, p-azidomethyl phenylalanine (pAMF), which possesses a reactive azide group. Thereafter, a unique conjugate vaccine (CV), namely, Pfs25-GPI was generated with dibenzocyclooctyne (DBCO) derivatized glycan core of malaria GPI using a simple but highly efficient copper free click chemistry reaction. In mice immunized with Pfs25 or Pfs25-GPI, the Pfs25-GPI group showed significantly higher titers compared to the Pfs25 group. Moreover, only purified IgGs from Pfs25-GPI group were able to significantly block transmission of parasites to mosquitoes, as judged by a standard membrane feeding assay [SMFA]. To our knowledge, this is the first report of the generation of a CV using Pfs25 and malaria specific GPI where the GPI is shown to enhance the ability of Pfs25 to elicit transmission blocking antibodies.

Background: Further reduction in malaria prevalence and its eventual elimination would be greatly facilitated by the development of biomarkers of exposure and/or acquired immunity to malaria, as well as the deployment of effective vaccines against Plasmodium falciparum and Plasmodium vivax. A better understanding of the acquisition of immunity in naturally-exposed populations is essential for the identification of antigens useful as biomarkers, as well as to inform rational vaccine development., Methods: ELISA was used to measure total IgG to a synthetic form of glycosylphosphatidylinositol from P. falciparum (PfGPI) in a cohort of 1-3 years old Papua New Guinea children with well-characterized individual differences in exposure to P. falciparum and P. vivax blood-stage infections. The relationship between IgG levels to PfGPI and measures of recent and past exposure to P. falciparum and P. vivax infections was investigated, as well as the association between antibody levels and prospective risk of clinical malaria over 16 months of follow-up., Results: Total IgG levels to PfGPI were low in the young children tested. Antibody levels were higher in the presence of P. falciparum or P. vivax infections, but short-lived. High IgG levels were associated with higher risk of P. falciparum malaria (IRR 1.33-1.66, P = 0.008-0.027), suggesting that they are biomarkers of increased exposure to P. falciparum infections. Given the cross-reactive nature of antibodies to PfGPI, high IgG levels were also associated with reduced risk of P. vivax malaria (IRR 0.65-0.67, P = 0.039-0.044), indicating that these antibodies are also markers of acquired immunity to P. vivax., Conclusions: This study highlights that in young children, IgG to PfGPI might be a useful marker of immune-status to both P. falciparum and P. vivax infections, and potentially useful to help malaria control programs to identify populations at-risk. Further functional studies are necessary to confirm the potential of PfGPI as a target for vaccine development.

Interleukin 17 (IL-17) cytokines play a crucial role in host defense and inflammatory diseases. Of the six mammalian IL-17 members five are described in the chicken (gg) genome. A novel method that attached cytokines to the surface of cells via a GPI linker was established to generate two chicken IL-17A and one chicken IL-17F specific mab. Recombinant gg IL-17A and gg IL-17F that showed dimerization in Western blot were used to verify the antibodies specificity. The mab could detect gg IL-17 by intracellular cytokine staining as demonstrated on cells expressing recombinant IL-17. Furthermore IL-17A and lower amounts of IL-17F were detectable in CD4 positive T cells of stimulated splenocytes. In conclusion, we have generated novel tools to analyze chicken IL-17 in more detail and demonstrated that the surface expression of cytokines is a reliable method to generate specific mab applicable for intracellular cytokine staining., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Vaccination is an efficient means of combating infectious disease burden globally. However, routine vaccines for the world's major human parasitic diseases do not yet exist. Vaccines based on carbohydrate antigens are a viable option for parasite vaccine development, given the proven success of carbohydrate vaccines to combat bacterial infections. We will review the key components of carbohydrate vaccines that have remained largely consistent since their inception, and the success of bacterial carbohydrate vaccines. We will then explore the latest developments for both traditional and non-traditional carbohydrate vaccine approaches for three of the world's major protozoan parasitic diseases-malaria, toxoplasmosis, and leishmaniasis. The traditional prophylactic carbohydrate vaccine strategy is being explored for malaria. However, given that parasite disease biology is complex and often arises from host immune responses to parasite antigens, carbohydrate vaccines against deleterious immune responses in host-parasite interactions are also being explored. In particular, the highly abundant glycosylphosphatidylinositol molecules specific for Plasmodium, Toxoplasma , and Leishmania spp. are considered exploitable antigens for this non-traditional vaccine approach. Discussion will revolve around the application of these protozoan carbohydrate antigens for vaccines currently in preclinical development.

Glycosylphosphatidylinositol (GPI) anchor of Plasmodium falciparum origin is considered an important toxin leading to severe malaria pathology through stimulation of pro-inflammatory responses from innate immune cells. Even though the GPI-induced immune response is widely described to be mediated by pattern recognition receptors such as TLR2 and TLR4, previous studies have revealed that these two receptors are dispensable for the development of severe malaria pathology. Therefore, this study aimed at the identification of potential alternative Plasmodium GPI receptors. Herein, we have identified the host protein moesin as an interaction partner of Plasmodium GPI in vitro . Given previous reports indicating the relevance of moesin especially in the LPS-mediated induction of pro-inflammatory responses, we have conducted a series of in vitro and in vivo experiments to address the physiological relevance of the moesin- Plasmodium GPI interaction in the context of malaria pathology. We report here that although moesin and Plasmodium GPI interact in vitro , moesin is not critically involved in processes leading to Plasmodium -induced pro-inflammatory immune responses or malaria-associated cerebral pathology.

A large number of proteins in malaria parasites are anchored using glycophosphatidylinositols (GPIs) with lipid tails. These GPIs are structurally distinct from human GPIs. Plasmodium falciparum GPIs have been considered as potential vaccine candidates because these molecules are involved in inducing inflammatory responses in human hosts, and natural anti-GPI antibody responses have been shown to be associated with protection against severe disease. GPIs can also be considered as targets for rapid diagnostic tests. Because isolation of native GPIs in large quantities is challenging, development of synthetic GPI molecules can facilitate further exploration of GPI molecules for diagnostics. Here, we report synthesis and immunological characterization of a panel of malaria-specific GPI analogues. A total of three GPI analogues were chemically synthesized and conjugated to a carrier protein to immunize and generate antibodies in rabbits. The rabbit immune sera showed reactivity with synthetic GPIs and native GPIs extracted from P. falciparum parasite, as determined by Luminex and ELISA methods.

Glycoscience research has been significantly impeded by the complex compositions of the glycans present in biological molecules and the lack of convenient tools suitable for studying the glycosylation process and its function. Polysaccharides and glycoconjugates are not encoded directly by genes; instead, their biosynthesis relies on the differential expression of carbohydrate enzymes, resulting in heterogeneous mixtures of glycoforms, each with a distinct physiological activity. Access to well-defined structures is required for functional study, and this has been provided by chemical and enzymatic synthesis and by the engineering of glycosylation pathways. This review covers general methods for preparing glycans commonly found in mammalian systems and applying them to the synthesis of therapeutically significant glycoconjugates (glycosaminoglycans, glycoproteins, glycolipids, glycosylphosphatidylinositol-anchored proteins) and the development of carbohydrate-based vaccines.

In this study, we hypothesized that the inhibition of epithelial to mesenchymal transition (EMT) program by knockdown of Zinc-finger E-box binding homeobox 1 (ZEB1) or administration of miR200c agomir would strengthen the B16F10 cells transfected with GPI-anchored IL-21 (B16F10/GPI-IL-21) vaccine efficacy in inhibiting the melanoma metastasis. Our findings from the current study indicated that, when compared with the mice immunized with the B16F10/GPI-IL-21 vaccine alone, the mice immunized with B16F10/GPI-IL-21 vaccine combined with injection of shZEB1 plasmid or miR200c agomir not only meaningfully inhibited EMT of melanoma, reduced the EMT characteristic molecular expression in tumor tissues, but also significantly decreased the Treg cells and TGF-β1, enhanced the cytotoxicities of NK cells and cytotoxic T lymphocytes and the IFN-γ level. Furthermore, the immunotherapeutic combination resulted in inhibiting the melanoma growth and lung metastasis. Our study demonstrated that using the B16F10/GPI-IL-21 vaccine in combination with the down-regulated ZEB1 or miR200c administration effectively elicited anti-tumor immunity and reduced melanoma metastasis by inhibiting the EMT program in the B16F10 melanoma-bearing mice., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

Vaccination against the ubiquitous parasite Toxoplasma gondii would provide the most efficient prevention against toxoplasmosis-related congenital, brain and eye diseases in humans. We investigated the immune response elicited by pathogen-specific glycosylphosphatidylinositol (GPI) glycoconjugates using carbohydrate microarrays in a BALB/c mouse model. We further examined the protective properties of the glycoconjugates in a lethal challenge model using the virulent T. gondii RH strain. Upon immunization, mice raised antibodies that bind to the respective GPIs on carbohydrate microarrays, but were mainly directed against an unspecific GPI epitope including the linker. The observed immune response, though robust, was unable to provide protection in mice when challenged with a lethal dose of viable tachyzoites. We demonstrate that anti-GPI antibodies raised against the here described semi-synthetic glycoconjugates do not confer protective immunity against T. gondii in BALB/c mice., (© The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Recombinant virus-like nanoparticles (VLPs) are a promising nanoparticle platform to develop safe vaccines for many viruses. Herein, we describe a novel and rapid protein transfer process to enhance the potency of enveloped VLPs by decorating influenza VLPs with exogenously added glycosylphosphatidylinositol-anchored immunostimulatory molecules (GPI-ISMs). With protein transfer, the level of GPI-ISM incorporation onto VLPs is controllable by varying incubation time and concentration of GPI-ISMs added. ISM incorporation was dependent upon the presence of a GPI-anchor and incorporated proteins were stable and functional for at least 4weeks when stored at 4°C. Vaccinating mice with GPI-granulocyte macrophage colony-stimulating factor (GM-CSF)-incorporated-VLPs induced stronger antibody responses and better protection against a heterologous influenza virus challenge than unmodified VLPs. Thus, VLPs can be enriched with ISMs by protein transfer to increase the potency and breadth of the immune response, which has implications in developing effective nanoparticle-based vaccines against a broad spectrum of enveloped viruses., From the Clinical Editor: The inherent problem with current influenza vaccines is that they do not generate effective cross-protection against heterologous viral strains. In this article, the authors described the development of virus-like nanoparticles (VLPs) as influenza vaccines with enhanced efficacy for cross-protection, due to an easy protein transfer modification process., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background/aim: In previous work, we found that prostate stem cell antigen (PSCA) gene, encoding a glycosylphosphatidylinositol-anchored protein, is a presumable tumor suppressor in gastric cancer and gallbladder cancer (GBC). The introduction of PSCA cDNA into GBC cell lines significantly suppressed tumorigenecity of cells in mice. The PSCA protein is thought to be involved in some form of intracellular signaling that remains to be elucidated., Materials and Methods: Using microarrays, we conducted gene-expression profiling on tumors generated by a GBC cell line TGBC-1TKB, with and without expression of PSCA, which was implanted into mice. Genes whose expression was down-regulated by PSCA were selected, and their down-regulation was confirmed by real-time PCR., Results: We identified several immune-related genes down-regulated by PSCA, including interleukin 8 (IL8), IL1 receptor antagonist (IL1RN) and S100 calcium-binding proteins A8 (S100A8) and A9 (S100A9)., Conclusion: PSCA signaling may suppress tumor growth in vivo by modulating immunological characteristics of GBC cells., (Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Several questions regarding the production and functioning of autoantibodies (AAb) during malaria infection remain open. Here we provide an overview of studies conducted in our laboratory that shed some light on the questions of whether antiphospholipid antibodies (aPL) and other AAb associated with autoimmune diseases (AID) can recognize Plasmodia antigens and exert anti-parasite activity; and whether anti-parasite phospholipid antibodies, produced in response to malaria, can inhibit phospholipid-induced inflammatory responses and protect against the pathogenesis of severe malaria. Our work showed that sera from patients with AID containing AAb against dsDNA, ssDNA, nuclear antigens (ANA), actin, cardiolipin (aCL) and erythrocyte membrane antigens recognize plasmodial antigens and can, similarly to monoclonal AAb of several specificities including phospholipid, inhibit the growth of P. falciparum in vitro. However, we did not detect a relationship between the presence of anti-glycosylphosphatidylinositol (GPI) antibodies in the serum and asymptomatic malaria infection, although we did register a relationship between these antibodies and parasitemia levels in infected individuals. Taken together, these results indicate that autoimmune responses mediated by AAb of different specificities, including phospholipid, may have anti-plasmodial activity and protect against malaria, although it is not clear whether anti-parasite phospholipid antibodies can mediate the same effect. The potential effect of anti-parasite phospholipid antibodies in malarious patients that are prone to the development of systemic lupus erythematosus or antiphospholipid syndrome, as well as the (possibly protective?) role of the (pathogenic) aPL on the malaria symptomatology and severity in these individuals, remain open questions., (© The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.)

Purpose: VLPs displaying tumor targeting single-chain variable fragments (VLP-rscFvs) which targets tumor-associated glycoprotein-72 (TAG-72) marker protein have a potential for immunotherapy against colon carcinoma tumors. In this study, scFvs anchored on VLPs using glycosylphosphatidylinositol (GPI) were prepared to target colon carcinoma spheroids in vitro., Methods: VLPs-rscFvs were produced by co-injecting two types of Bombyx mori nucleopolyhedrovirus (BmNPV) bacmids, encoding RSV-gag and rscFvs cDNA into silkworm larvae. Large unilamellar vesicles (LUVs) of 100 nm in diameter were made using 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and packaged with Sulforhodamine B (SRB). LUV-SRB was used to associate with VLP-rscFvs assisted by GP64 present on VLP-rscFvs to produce VLP-rscFv associated SRB (VLP-rscFvs-SRB) at pH 7.5., Results: The antigenicity of the purified VLPs-rScFvs was confirmed by enzyme-linked immunosorbent assay (ELISA) using TAG-72 as antigen. LUV-SRB made of DOPC was used to associate with 100 μg of VLP-rscFvs to produce VLP-rscFv-SRB. Specific delivery and penetration of SRB up to 100 μm into the spheroids shows the potential of the new model., Conclusions: The current study demonstrated the display, expression and purification of VLP-rscFvs efficiently. As a test model VLP-rscFv-SRB were prepared which can be used for immunotherapy. rscFvs provide the specificity needed to target tumors and VLPs serve as carrier transporting the dye to target.

Background: Genetically modified cells have been shown to be one of the most effective tumor vaccine strategies. However, in many cases, such as in melanoma, induction of a potent immune responses against the disease still remains a major challenge. Thus, novel strategies to reinforce tumor vaccine efficacy are needed. Using microRNA (miR) and Zinc-finger E-box binding homeobox (ZEB) have received much attention for potentially regulating tumor progression. To elicit a potent antitumor efficacy against melanoma, we used tumor vaccine in combination with miR200c overexpression or ZEB1 knockdown to assess the efficacy of treatment of murine melanoma., Methods: B16F10 cell vaccine expressing interleukin 21 (IL-21) in the glycosylpho- sphatidylinositol (GPI)-anchored form (B16F10/GPI-IL-21) were developed. The vaccine was immunized into mice challenged by B16F10 cells or B16F10 cells stably transduced with lentiviral-miR200c (B16F10/miR200c) or transfected with the ZEB1-shRNA recombinant (B16F10/shZEB1) or the B16F10/GPI-IL-21 vaccine. The immune responses, tumorigenicity and lung metastasis in mice were evaluated, respectively., Results: The vaccination with B16F10/GPI-IL-21 markedly increased the serum cytokine levels of IFN-γ, TNF-α, IL-4 and decreased TGF-β level as well as augmented the cytotoxicity of splenocytes in immunized mice compared with control mice. In addition, the tumor vaccine B16F10/GPI-IL-21 significantly inhibited the tumor growth and reduced counts of lung metastases in mice challenged by B16F10/GPI-IL-21, B16F10/shZEB1 and B16F10/miR200c respectively compared with the control mice challenged by B16F10 cells. The efficacy mechanisms may involve in reinforcing immune responses, increasing expression of miR200c, E-cadherin and SMAD-7 and decreasing expression of TGF-β, ZEB1, Vimentin and N-cadherin in tumor tissues from the immunized mice., Conclusions: These results indicate that the tumor vaccine B16F10/GPI-IL-21 in combination with miR200c overexpression or ZEB1 knockdown effectively inhibited melanoma growth and metastasis a murine model. Such a strategy may, therefore, be used for the clinical trials.

Anti-glycosylphosphatidylinositol (GPI) antibodies (Abs) may reflect and mediate, at least partially, anti-disease immunity in malaria by neutralising the toxic effect of parasitic GPI. Thus, we assessed the anti-GPI Ab response in asymptomatic individuals living in an area of the Brazilian Amazon that has a high level of malaria transmission. For comparative purposes, we also investigated the Ab response to a crude extract prepared from Plasmodium falciparum, the merozoite surface protein (MSP)3 antigen of P. falciparum and the MSP 1 antigen of Plasmodium vivax (PvMSP1-19) in these individuals and in Angolan patients with acute malaria. Our data suggest that the Ab response against P. falciparum GPI is not associated with P. falciparum asymptomatic infection in individuals who have been chronically exposed to malaria in the Brazilian Amazon. However, this Ab response could be related to ongoing parasitaemia (as was previously shown) in the Angolan patients. In addition, our data show that PvMSP1-19may be a good marker antigen to reflect previous exposure to Plasmodium in areas that have a high transmission rate of P. vivax.

During malaria infection, elevated levels of pro-inflammatory mediators and nitric oxide production have been associated with pathogenesis and disease severity. Previous in vitro and in vivo studies have proposed that both Plasmodium falciparum hemozoin and glycosylphosphatidylinositols are able to modulate blood mononuclear cells, contributing to stimulation of signal transduction and downstream regulation of the NF-κB signaling pathway, and subsequently leading to the production of pro-inflammatory cytokines, chemokines, and nitric oxide. The present review summarizes the published in vitro and in vivo studies that have investigated the mechanism of intracellular signal transduction and activation of the NF-κB signaling pathway in blood mononuclear cells after being inducted by Plasmodium falciparum malaria components. Particular attention is paid to hemozoin and glycosylphosphatidylinositols which reflect the important mechanism of signaling pathways involved in immune response.

Glycosylphosphatidylinositols (GPI) are complex glycolipids that are covalently linked to the C terminus of proteins as a post-translational modification and tether proteins to the plasma membrane. One of the most striking features of GPI-anchored proteins (APs) is their enrichment in lipid rafts. The biosynthesis of GPI and its attachment to proteins occur in the endoplasmic reticulum. In the Golgi, GPI-APs are subjected to fatty acid remodeling, which replaces an unsaturated fatty acid at the sn-2 position of the phosphatidylinositol moiety with a saturated fatty acid. We previously reported that fatty acid remodeling is critical for the enrichment of GPI-APs in lipid rafts. To investigate the biological significance of GPI-AP enrichment in lipid rafts, we generated a PGAP3 knock-out mouse (PGAP3(-/-)) in which fatty acid remodeling of GPI-APs does not occur. We report here that a significant number of aged PGAP3(-/-) mice developed autoimmune-like symptoms, such as increased anti-DNA antibodies, spontaneous germinal center formation, and enlarged renal glomeruli with deposition of immune complexes and matrix expansion. A possible cause for this was the impaired engulfment of apoptotic cells by resident peritoneal macrophages in PGAP3(-/-) mice. Mice with conditional targeting of PGAP3 in either B or T cells did not develop such autoimmune-like symptoms. In addition, PGAP3(-/-) mice exhibited the tendency of Th2 polarization. These data demonstrate that PGAP3-dependent fatty acid remodeling of GPI-APs has a significant role in the control of autoimmunity, possibly by the regulation of apoptotic cell clearance and Th1/Th2 balance.

Background: C4.4A is a glycolipid-anchored membrane protein expressed in several human malignancies. We examined clinical relevance of C4.4A expression in 111 esophageal squamous cell carcinoma (ESCC) tissue samples., Methods: Anti-human C4.4A antibody that recognizes the glycosylphosphatidyl inositol (GPI) anchor signaling sequence (C4.4A-GPI Ab) and anti-human C4.4A-119 polyclonal antibody (C4.4A-119 Ab) were used for immunohistochemistry and Western blot testing., Results: Both antibodies detected the C4.4A protein expression at the parabasal layer of normal epithelium of the esophagus. In tumor tissues, the C4.4A protein was detected in 66 (59.5 %) and 95 (85.6 %) of 111 ESCCs by the C4.4A-GPI Ab and the C4.4A-119 Ab, respectively. The C4.4A-GPI Ab mainly detected membranous C4.4A expression (83.3 %, 55 of 66 positive cases), while the C4.4A-119 Ab exclusively detected cytoplasmic C4.4A expression (100 %, 73 cytoplasm alone and 22 cytoplasm plus membrane in 95 positive cases). Western blot analysis indicated that normal epithelium expressed the band of C4.4A at 70 kDa, whereas the tumor tissues displayed the band at the lower molecular weight. Survival analysis indicated that the C4.4A-positive ESCCs had significantly worse 5-year overall survival than the C4.4A-negative ESCC samples (P = 0.021) when using the C4.4A-GPI Ab, but not when using the C4.4A-119 Ab. This difference was most evident with membranous expression of C4.4A (P = 0.005)., Conclusions: C4.4A expression was associated with a poor prognosis of ESCC when the GPI-related antibody was used. On the other hand, the C4.4A-119 Ab may be a useful diagnostic tool for ESCC because of its high detection rate.

Infection with the intracellular protozoan parasite Leishmania mexicana causes chronic disease in C57BL/6 mice, in which cutaneous lesions persist for many months with high parasite burdens (10(7)-10(8) parasites). This chronic disease process requires host IL-10 and FcγRIII. When Leishmania amastigotes are released from cells, surface-bound IgG can induce IL-10 and suppress IL-12 production from macrophages. These changes decrease IFN-γ from T cells and nitric oxide production in infected cells, which are both required for Leishmania control. However, antibodies targets and the kinetics of antibody production are unknown. Several groups have been unsuccessful in identifying amastigote surface proteins that bind IgG. We now show that glycoinositol phospholipids (GIPLs) of L. mexicana are recognized by mouse IgG1 by 6 weeks of infection, with a rapid increase between 12 and 16 weeks, consistent with the timing of chronic disease in C57BL/6 mice vs. healing in FcγRIII-deficient mice. A single prominent spot on TLC is recognized by IgG, and the glycolipid is a glycosyl phosphatidylinositol containing a branched mannose structure. We show that the lipid structure of the GIPL (the sn-2 fatty acid) is required for antibody recognition. This GIPL is abundant in L. mexicana amastigotes, rare in stationary-phase promastigotes, and absent in L. major, consistent with a role for antibodies to GIPLs in chronic disease. A mouse monoclonal anti-GIPL IgG recognizes GIPLs on the parasite surface, and induces IL-10 from macrophages. The current work also extends this mouse analysis to humans, finding that L. mexicana-infected humans with localized and diffuse cutaneous leishmaniasis have antibodies that recognize GIPLs, can bind to the surface of amastigotes, and can induce IL-10 from human monocytes. Further characterization of the target glycolipids will have important implications for drug and vaccine development and will elucidate the poorly understood role of glycolipids in the immunology of infections.

Glycosylphosphatidylinositols (GPIs) are complex glycolipids that are covalently linked to the C-terminus of proteins as a posttranslational modification. They anchor the attached protein to the cell membrane and are essential for normal functioning of eukaryotic cells. GPI-anchored proteins are structurally and functionally diverse. Many GPIs have been structurally characterized but comprehension of their biological functions, beyond the simple physical anchoring, remains largely speculative. Work on functional elucidation at a molecular level is still limited. This Review focuses on the roles of GPI unraveled by using synthetic molecules and summarizes the structural diversity of GPIs, as well as their biological and chemical syntheses., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

B cell tolerance to self-antigen is critical to preventing antibody-mediated autoimmunity. Previous work using B cell antigen receptor transgenic animals suggested that self-antigen-specific B cells are either deleted from the repertoire, enter a state of diminished function termed anergy, or are ignorant to the presence of self-antigen. These mechanisms have not been assessed in a normal polyclonal repertoire because of an inability to detect rare antigen-specific B cells. Using a novel detection and enrichment strategy to assess polyclonal self-antigen-specific B cells, we find no evidence of deletion or anergy of cells specific for antigen not bound to membrane, and tolerance to these types of antigens appears to be largely maintained by the absence of T cell help. In contrast, a combination of deleting cells expressing receptors with high affinity for antigen with anergy of the undeleted lower affinity cells maintains tolerance to ubiquitous membrane-bound self-antigens.

Studies have shown that glycosylphosphatidylinositols (GPIs) of Plasmodium falciparum activate macrophages mainly through Toll-like receptor 2 (TLR2)-mediated signalling and to certain extent through TLR4-mediated signalling to induce proinflammatory cytokine production. However, the ability of parasite GPIs to activate dendritic cells (DCs) has not been reported. Here, we show that parasite GPIs efficiently activate DCs through TLR2-mediated signalling mechanism and induce the production of TNF-α and IL-12. We also studied the role of scavenger receptor CD36 in P. falciparum GPI- and merozoite-induced cytokine responses by DCs. The results indicate that CD36 modulates the cytokine-inducing activity of the parasite GPIs by collaborating with TLR2 in DCs. Furthermore, our data reveal that CD36 modulates the activity of P. falciparum merozoites, likely by the contribution of phagocytosis-coupled CD36-mediated signalling to the signalling induced by merozoites. Altogether, these results contribute towards understanding of signalling mechanisms in malaria parasite-induced activation of the innate immune system., (© 2012 Blackwell Publishing Ltd.)

Paroxysmal Nocturnal Haemoglobinuria (PNH) is due to pathological expansion of a stem progenitor bearing a somatic mutation of PIG-A gene involved in the biosynthesis of the glycosyl-phosphatidyl-inositol (GPI) anchor. Numerous data suggest a role for immune-mediated mechanisms in the selection/expansion of GPI-defective clone. Haemolytic anaemia in PNH is dependent on the effect of complement against GPI-defective red cells. Eculizumab, an anti-C5 monoclonal antibody, is dramatically effective in controlling haemolysis and thrombosis, in reducing fatigue and in improving quality of life of patients. However, this therapy presents new challenges that need to be properly faced. Here, we report the decrease in B, Natural Killer (NK) and regulatory T cells (Treg), an altered cytokine profile of invariant-NKT cells (NKTi) and the increasing of C-X-C chemokine receptor type 4 (CXCR4) receptor in PNH patients before the Eculizumab therapy. Treatment significantly affects some of these alterations: after Eculizumab, the number of B lymphocytes, the cytokine secretion of NKTi and CXCR4 expression on CD8 T cells became similar to healthy donors. No effects were observed on NK and Treg. The amplitude of the GPI-defective compartment remained unchanged., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

This is a review of ten previously published studies of the human sperm proteome. Proteins expressed on the sperm cell surface were identified and characterized by a combination of vectorial labelling with radioiodine and biotin, PI-PLC treatment, two-dimensional gel electrophoresis, immuno and lectin blotting procedures, affinity overlay assays with radioactive nucleotide triphosphates and 45Ca, and mass spectrometry analysis. Examination of capacitation-induced modifications of the human sperm proteome led to the cloning and characterisation of two new phospho-regulated cancer-testis antigens, which we named Fibrous Sheath Protein 95 (FSP95) and CABYR (calcium-binding tyrosine phosphorylation regulated). A protein kinase A RII binding domain is present between amino acids 124 and 141 identifying FSP95 (now commonly known as AKAP3) as a member of the A kinase anchoring protein-family which provides spatial and temporal specificity to the cAMP-PKA pathway. In addition to scaffolding PKA, PDE and protein phosphatases, AKAPs also bind to a group of four proteins that share homology to the RII dimerization/docking (R2D2) domain of PKA' regulatory subunit. CABYR, which is one of these four proteins, also interacts with a diverse array of signal tranducers via its SH3-, R2D2-, and proline-rich extension-like domains. AKAP3 and CABYR appear to associate in high molecular weight multi-protein complexes, which regulate the flagella' energy supply and movements. Diagonal gel electrophoresis experiments suggest that the high molecular weight signal-integrating scaffold partly is established by homo- and hetero-oligomerization of lower molecular weight splice variants of CABYR. The putative role of CABYR in lung cancer cells is finally discussed.

Glycosylphosphatidylinositol (GPI) is a complex glycolipid that serves as a membrane anchor for many cell-surface proteins, such as Thy-1 and CD48. GPI-anchored proteins (GPI-APs) play important roles in many biological processes, such as signal transduction and cell-cell interaction, through their association with lipid rafts. Fatty acid remodeling of GPI-APs in the Golgi apparatus is required for their efficient association with lipid rafts, i.e., the unsaturated fatty acid at the sn-2 position of the PI moiety is exchanged for the saturated fatty acid by PGAP2 and PGAP3. To investigate the immunological role of the fatty acid remodeling of GPI-APs, we generated a Pgap3 knockout mouse. In this mouse, GPI-APs are expressed on the cell surface without fatty acid remodeling, and fail to associate with lipid rafts. Male Pgap3 knockout mice were born alive at a ratio lower than expected from Mendel's law, whereas the number of female mice followed Mendel's law. All mice exhibited growth retardation and abnormal reflexes such as limb grasping. We focused T cell function in these mice and found that T cell development in the absence of Pgap3 was normal. However, the response of T cells was enhanced in Pgap3 knockout mice in both in vitro and in vivo studies, including alloreactive response, antigen-specific immune response, and experimental autoimmune encephalomyelitis. Cross-linking of Thy-1 in wild-type cells inhibited the signal transduced by the T cell receptor (TCR), whereas cross-linking of Thy-1 in Pgap3 knockout cells enhanced the TCR signal. These results suggest that GPI-APs localized in lipid rafts may modulate signaling through the TCR., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The flatworm Schistosoma mansoni is a blood fluke parasite that causes schistosomiasis, a debilitating disease that occurs throughout the developing world. Current schistosomiasis control strategies are mainly based on chemotherapy, but many researchers believe that the best long-term strategy to control schistosomiasis is through immunization with an antischistosomiasis vaccine combined with drug treatment. In the search for potential vaccine candidates, numerous tegument antigens have been assessed. As the major interface between parasite and mammalian host, the tegument plays crucial roles in the establishment and further course of schistosomiasis. Herein, we evaluated the potential of a GPI fraction, containing representative molecules located on the outer surface of adult worms, as vaccine candidate. Immunization of mice with GPI-anchored proteins induced a mixed Th1/Th2 type of immune response with production of IFN-γ and TNF-α, and low levels of IL-5 into the supernatant of splenocyte cultures. The protection engendered by this vaccination protocol was confirmed by 42% reduction in worm burden, 45% reduction in eggs per gram of hepatic tissue, 29% reduction in the number of granulomas per area, and 53% reduction in the granuloma fibrosis. Taken together, the data herein support the potential of surface-exposed GPI-anchored antigens from the S. mansoni tegument as vaccine candidate.

Toxoplasma gondii glycosylphosphatidylinositols (GPIs) bind to galectin-3 to induce TNF-α production in macrophages via Toll-like receptors 2 and 4. Here we show that T. gondii GPIs stimulate human macrophages to synthesize matrix metalloproteinase-9 in a TNF-α-dependent pathway and degrade extracellular galectin-3., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

A key event and potential therapeutic target in allergic and asthmatic diseases is signaling by the IgE receptor FcεRI, which depends on its interactions with Src family kinases (SFK). Here we tested the hypothesis that glycosylphosphatidylinositiol-anchored proteins (GPI-AP) are involved in FcεRI signaling, based on previous observations that GPI-AP colocalize with and mediate activation of SFK. We generated mice with a hematopoietic cell-specific GPI-AP deficiency by targeted disruption of the GPI biosynthesis gene PigA. In these mice, IgE-mediated passive cutaneous anaphylaxis was largely abolished. PigA-deficient mast cells cultured from these mice showed impaired degranulation in response to stimulation with IgE and antigen in vitro, despite normal IgE binding and antigen-induced FcεRI aggregation. On stimulation of these cells with IgE and antigen, coprecipitation of the FcεRI α-chain with the γ-chain and β-chain was markedly reduced. As a result, IgE/antigen-induced FcεRI-Lyn association and γ-chain tyrosine phosphorylation were both impaired in PigA-deficient cells. These data provide genetic evidence for an unanticipated key role of GPI-AP in FcεRI interchain interactions and early FcεRI signaling events, necessary for antigen-induced mast cell degranulation.

Here we discuss CD160 an essential NK cell activating receptor that remains poorly understood. CD160 receptor exhibits a number of unique structural and functional characteristics that are not common to other killer immunoglobulin-like receptors that recognize major histocompatibility complex (MHC) class I molecules: (1) In addition to humans and mice, the cd160 gene is conserved in several other mammal species; (2) cd160 is located outside the NK gene complex and the Leukocyte Receptor Complex in humans; (3) CD160 expression is associated to the CD56(dim) CD16+ cytotoxic NK cell phenotype; (4) both human and mouse CD160 recognize MHC class Ia and Ib molecules; (5) unlike the other MHC class I-dependent activating NK receptors, CD160 is a glycosylphosphatidylinositol-anchored molecule with a single immunoglobulin-like domain, and does not bear immunoreceptor tyrosine-based activation motifs. Consequently, CD160 cannot signal by itself, requiring the recruitment of adaptor proteins. CD160 recruits phosphoinositide-3 kinase to trigger cytotoxicity and cytokine secretion; (6) specific engagement of NK CD160 receptor expressed by circulating NK cells produces proinflammatory cytokines IFN-γ, TNF-α, and, most notably, IL-6 and IL-8 as well as MIP1-β chemokine. The level of CD160-mediated IFN-γ production is always higher than the one observed after engagement of the CD16 receptor., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The glycosylphosphatidylinositols (GPIs) of Plasmodium falciparum have been shown to activate macrophages and produce inflammatory responses. The activation of macrophages by malarial GPIs involves engagement of Toll like receptor 2 (TLR2) resulting in the intracellular signaling and production of cytokines. In the present study, we investigated the requirement of TLR1 and TLR6 for the TLR2 mediated cell signaling and proinflammatory cytokine production by macrophages. The data demonstrate that malarial GPIs, which contain three fatty acid substituents, preferentially engage TLR2-TLR1 dimeric pair than TLR2-TLR6, whereas their derivatives, sn-2 lyso GPIs, that contain two fatty acid substituents recognize TLR2-TLR6 with slightly higher selectivity as compared to TLR2-TLR1 heteromeric pair. These results are analogous to the recognition of triacylated bacterial and diacylated mycoplasmal lipoproteins, respectively, by TLR2-TLR1 and TLR2-TLR6 dimers, suggesting that the lipid portions of the microbial GPI ligands play essential role in determining their TLR recognition specificity., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The glycosylphosphatidylinositol (GPI)-anchored neutrophil-specific receptor NB1 (CD177) presents the autoantigen proteinase 3 (PR3) on the membrane of a neutrophil subset. PR3-ANCA-activated neutrophils participate in small-vessel vasculitis. Since NB1 lacks an intracellular domain, we characterized components of the NB1 signaling complex that are pivotal for neutrophil activation. PR3-ANCA resulted in degranulation and superoxide production in the mNB1(pos)/PR3(high) neutrophils, but not in the mNB1(neg)/PR3(low) subset, whereas MPO-ANCA and fMLP caused similar responses. The NB1 signaling complex that was precipitated from plasma membranes contained the transmembrane receptor Mac-1 (CD11b/CD18) as shown by MS/MS analysis and immunoblotting. NB1 co-precipitation was less for CD11a and not detectable for CD11c. NB1 showed direct protein-protein interactions with both CD11b and CD11a by surface plasmon resonance analysis (SPR). However, when these integrins were presented as heterodimeric transmembrane proteins on transfected cells, only CD11b/CD18 (Mac-1)-transfected cells adhered to immobilized NB1 protein. This adhesion was inhibited by mAb against NB1, CD11b, and CD18. NB1, PR3, and Mac-1 were located within lipid rafts. In addition, confocal microscopy showed the strongest NB1 co-localization with CD11b and CD18 on the neutrophil. Stimulation with NB1-activating mAb triggered degranulation and superoxide production in mNB1(pos)/mPR3(high) neutrophils, and this effect was reduced using blocking antibodies to CD11b. CD11b blockade also inhibited PR3-ANCA-induced neutrophil activation, even when β2-integrin ligand-dependent signals were omitted. We establish the pivotal role of the NB1-Mac-1 receptor interaction for PR3-ANCA-mediated neutrophil activation.

Invariant natural killer T (iNKT) cells are a small subset of lymphocytes that recognize glycolipid antigens in the context of CD1d and consequently produce large quantities of pro-inflammatory and/or anti-inflammatory cytokines. Several transmembrane glycoproteins have been implicated in the co-stimulation of iNKT cell responses. However, whether glycosylphosphatidylinositol (GPI)-anchored proteins can function in this capacity is not known. Here, we demonstrate that antibody-mediated cross-linking of the prototype mouse GPI-anchored protein Thy-1 (CD90) on the surface of a double-negative (CD4⁻CD8⁻) iNKT cell line leads to cytokine production at both the mRNA and protein levels. In addition, Thy-1 triggering enhanced cytokine secretion by iNKT cells that were concomitantly stimulated with α-galactosylceramide (αGC), consistent with a co-stimulatory role for Thy-1 in iNKT cell activation. This was also evident when a CD4+ mouse iNKT cell line or primary hepatic NKT cells were stimulated with αGC and/or anti-Thy-1 antibody. Cross-linking Ly-6A/E, another GPI-anchored protein, could also boost cytokine secretion by αGC-stimulated iNKT cells, suggesting that the observed effects reflect a general property of GPI-anchored proteins. To extend these results from mouse to human cells, we focused on CD55, a GPI-anchored protein that, unlike Thy-1, is expressed on human iNKT cells. Cross-linking CD55 augmented αGC-induced iNKT cell responses as judged by more vigorous proliferation and higher CD69 expression. Collectively, these findings demonstrate for the first time that GPI-anchored proteins are able to co-stimulate CD1d-restricted, glycolipid-reactive iNKT cells in both mice and humans., (© 2010 The Authors. Immunology © 2010 Blackwell Publishing Ltd.)

The experimental studies of Brucei group trypanosomes presented here demonstrate that the balance of host and parasite factors, especially IFN-γ GPI-sVSG respectively, and the timing of cellular exposure to them, dictate the predominant MP and DC activation profiles present at any given time during infection and within specific tissues. The timing of changes in innate immune cell functions following infection consistently support the conclusion that the key events controlling host resistance occur within a short time following initial exposure to the parasite GPI substituents. Once the changes in MP and DC activities are initiated, there appears little that the host can do to reverse these changes and alter the final outcome of these regulatory events. Instead, despite the availability of multiple innate and adaptive immune mechanisms that can control parasites, there is an inability to control trypanosome numbers sufficiently to prevent the emergence and establishment of virulent trypanosomes that eventually kill the host. Overall it appears that trypanosomes have carefully orchestrated the host innate and adaptive immune response so that parasite survival and transmission, and alterations of host immunity, are to its ultimate benefit.

Background: Identification of broad neutralization epitopes in HIV-1 envelope spikes is paramount for HIV-1 vaccine development. A few broad neutralization epitopes identified so far are present on the surface of native HIV-1 envelope spikes whose recognition by antibodies does not depend on conformational changes of the envelope spikes. However, HIV-1 envelope spikes also contain transiently-exposed neutralization epitopes, which are more difficult to identify., Results: In this study, we constructed single chain Fvs (scFvs) derived from seven human monoclonal antibodies and genetically linked them with or without a glycosyl-phosphatidylinositol (GPI) attachment signal. We show that with a GPI attachment signal the scFvs are targeted to lipid rafts of plasma membranes. In addition, we demonstrate that four of the GPI-anchored scFvs, but not their secreted counterparts, neutralize HIV-1 with various degrees of breadth and potency. Among them, GPI-anchored scFv (X5) exhibits extremely potent and broad neutralization activity against multiple clades of HIV-1 strains tested. Moreover, we show that GPI-anchored scFv (4E10) also exhibited more potent neutralization activity than its secretory counterpart. Finally, we demonstrate that expression of GPI-anchored scFv (X5) in the lipid raft of plasma membrane of human CD4+ T cells confers long-term resistance to HIV-1 infection, HIV-1 envelope-mediated cell-cell fusion, and the infection of HIV-1 captured and transferred by human DCs., Conclusions: Thus GPI-anchored scFv could be used as a general and effective way to identify antibodies that react with transiently-exposed neutralization epitopes in envelope proteins of HIV-1 and other enveloped viruses. The GPI-anchored scFv (X5), because of its breadth and potency, should have a great potential to be developed into anti-viral agent for HIV-1 prevention and therapy.

Background: Glycosyl-phosphatidylinositol (GPI)-negative blood cells are diagnostic for Paroxysmal Nocturnal Hemoglobinuria (PNH). Marrow failure states are often associated with GPI-negative cell populations. Quantification of small clonal populations of GPI-negative cells influences clinical decisions to administer immunosuppressive therapy in marrow failure states (aplastic anemia or myelodysplastic syndrome) and to monitor minimal residual disease after allogeneic blood or marrow transplantation (BMT). We studied the reliability of high-resolution flow cytometry markers operating at the limits of detection., Methods: We performed serial quantification of the PNH clone size in 38 samples using multiparameter flow cytometry. Granulocytes, monocytes, and RBCs were gated using forward and side scatter as well as lineage-specific markers. The GPI-linked markers fluorescent aerolysin (FLAER), CD55, and CD59 were comparatively evaluated. We also evaluated CD16 on granulocytes and CD14 on monocytes. The sensitivity of detection by each marker was further defined by serial dilution experiments on a flow-sorted sample. Two patients had quantification of their GPI-negative clones before and after allogeneic BMT., Results: FLAER was the most discriminant marker and allowed identification of 0.1% of GPI-negative cells despite other markers having superior signal-to-noise characteristics. CD14 and CD16 were inferior to CD55 at lower concentrations and in clinical application., Conclusions: Multiparameter flow cytometry permits quantification of small GPI-negative clones with a sensitivity limit of about 0.1%. The single most reliable marker to monitor small granulocyte or monocyte PNH clones is FLAER, especially in conditions such as myelodysplastic syndromes or BMT, when traditional GPI-linked surface marker expression can be significantly altered. © 2010 International Clinical Cytometry Society., (Copyright © 2010 International Clinical Cytometry Society.)

Parasite-derived glycosylphosphatidylinositol (GPI) is believed to be a major inducer of the pathways leading to pathology and morbidity during Plasmodium falciparum infection and has been termed a malaria "toxin." The generation of neutralizing anti-GPI ("antitoxic") antibodies has therefore been hypothesized to be an important step in the acquisition of antidisease immunity to malaria; however, to date the GPI-neutralizing capacity of antibodies induced during natural Plasmodium falciparum infection has not been evaluated. Here we describe the development of an in vitro macrophage-based assay to assess the neutralizing capacity of malarial GPI-specific IgG. We demonstrate that IgG from Plasmodium falciparum-exposed individuals can significantly inhibit the GPI-induced activation of macrophages in vitro, as shown by reduced levels of tumor necrosis factor production and attenuation of CD40 expression. The GPI-neutralizing capacity of individual IgG samples was directly correlated with the anti-GPI antibody titer. IgG from malaria-exposed individuals also neutralized the macrophage-activating effects of P. falciparum schizont extract (PfSE), but there was only a poor correlation between PfSE-neutralizing activity and the anti-GPI antibody titer, suggesting that PfSE contains other macrophage-activating moieties, in addition to GPI. In conclusion, we have established an in vitro assay to test the toxin-neutralizing activities of antimalarial antibodies and have shown that anti-GPI antibodies from malaria-immune individuals are able to neutralize GPI-induced macrophage activation; however, the clinical relevance of anti-GPI antibodies remains to be proven, given that malarial schizonts contain other proinflammatory moieties, in addition to GPI.

Induction of sterilizing immunity by vaccination is extremely difficult because of the evasion mechanisms developed by parasites, and identification of new targets for therapy is therefore important. Glycosylphosphatidylinositols (GPIs) of parasites are glycolipids that participate in pathogenicity of parasitic diseases. Studies of Plasmodium falciparum and Trypanosoma brucei indicate that GPIs are good candidates for developing vaccines against malaria and sleeping sickness, respectively. By contrast, fatty acids isolated from P. falciparum and Toxoplasma gondii can inhibit the production of inflammatory cytokines induced by the GPIs in macrophages. GPIs are considered to be toxins that, if present in large amounts, induce irreversible damages to the host, and treatment with fatty acids could reduce this effect., (Copyright 2010 Elsevier Ltd. All rights reserved.)