Understanding the Chemistry and Biology of Glycosylation with Glycan Synthesis.

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.

Neospora caninum glycosylphosphatidylinositols used as adjuvants modulate cellular immune responses induced in vitro by a nanoparticle-based vaccine.

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)-1ß, 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.

Staphylococcal phosphatidylinositol-specific phospholipase C potentiates lung injury via complement sensitisation.

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.

Detection of Anti-Toxoplasma gondii Antibodies in Human Sera Using Synthetic Glycosylphosphatidylinositol Glycans on a Bead-Based Multiplex Assay.

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 (GPI1) for the detection of GPI1-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 GPI1 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 GPI1-based test is in excellent agreement with the results for the commercial ELISA, as the ROC analysis of the GPI1 test shows 97% specificity and 98% sensitivity for the assay. GPI1 was a more reliable predictor for a parasite-specific IgM response compared to SAG1, indicating that a bead-based multiplex assay using GPI1 in combination with SAG1 may strengthen Toxoplasma gondii serology, in particular in seroepidemiological studies.

Early suppression of B cell immune responses by low doses of chloroquine and pyrimethamine: implications for studying immunity in malaria.

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.

Malaria Derived Glycosylphosphatidylinositol Anchor Enhances Anti-Pfs25 Functional Antibodies That Block Malaria Transmission.

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.

IgG antibodies to synthetic GPI are biomarkers of immune-status to both Plasmodium falciparum and Plasmodium vivax malaria in young children.

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.

Toward the Development of the Next Generation of a Rapid Diagnostic Test: Synthesis of Glycophosphatidylinositol (GPI) Analogues of Plasmodium falciparum and Immunological Characterization.

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.

Investigation of the protective properties of glycosylphosphatidylinositol-based vaccine candidates in a Toxoplasma gondii mouse challenge model.

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.

Protein transfer-mediated surface engineering to adjuvantate virus-like nanoparticles for enhanced anti-viral immune responses.

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.

Significance of glycosylphosphatidylinositol-anchored protein enrichment in lipid rafts for the control of autoimmunity.

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.

Regulation gene expression of miR200c and ZEB1 positively enhances effect of tumor vaccine B16F10/GPI-IL-21 on inhibition of melanoma growth and metastasis.

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-ß 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-ß, 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.

Autoimmunity, phospholipid-reacting antibodies and malaria immunity.

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.

A model for targeting colon carcinoma cells using single-chain variable fragments anchored on virus-like particles via glycosyl phosphatidylinositol anchor.

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.

The immunologic abnormalities in patients with paroxysmal nocturnal hemoglobinuria are associated with disease progression.

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.

C4.4A expression is associated with a poor prognosis of esophageal squamous cell carcinoma.

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.

Impaired FcεRI stability, signaling, and effector functions in murine mast cells lacking glycosylphosphatidylinositol-anchored proteins.

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 ß-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.

Asymptomatic infection in individuals from the municipality of Barcelos (Brazilian Amazon) is not associated with the anti-Plasmodium falciparum glycosylphosphatidylinositol antibody response.

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.