CD317 mediates immunocytolysis resistance by RICH2/cytoskeleton-dependent membrane protection.

Immune evasion is a common hallmark of cancers. Immunotherapies that aim at restoring or increasing the immune response against cancers have revolutionized outcomes for patients, but the mechanisms of resistance remain poorly defined. Here, we report that CD317, a surface molecule with a unique topology that is double anchored into the membrane, protects tumor cells from immunocytolysis. CD317 knockdown in tumor cells renders more severe death in response to NK or chimeric antigen receptor-modified NK cells challenge. Such effects of CD317 silencing might be the results of increasing sensitivity of tumor cells to immune killing rather than strengthening immune response, since neither effector-target cell contact nor the activation of effector cells was affected, and the enhanced cytolysis was also not counteracted by the addition of recombinant CD317 proteins. Mechanistically, CD317 might endow tumor cells with more flexibility to modulate cytoskeleton through its association with RICH2, thereby protects membrane integrity against perforin and consequently promotes survival in response to immunocytolysis. These results reveal a new mechanism of immunocytolysis resistance and suggest CD317 as an attractive target which can be exploited for improving the efficacy of cancer immunotherapies.

Bacterial membrane vesicles as promising vaccine candidates.

Both Gram-positive and Gram-negative bacteria can release nano-sized lipid bilayered structures, known as membrane vesicles (MVs). These MVs play an important role in bacterial survival by orchestrating interactions between bacteria and between bacteria and host. The major constituents of MVs are proteins, lipids and nucleic acids. Due to the immunogenicity of the membrane lipids and/or proteins of the MVs, in combination with adjuvant danger signals and the repeating patterns on the nanosized surface, MVs can effectively stimulate the innate and adaptive immune system. Since they are non-replicating, they are safer than attenuated vaccines. In addition, by genetic engineering of the donor cells, further improvements to their safety profile, immunogenicity and yield can be achieved. To date, one MV-based vaccine against Neisseria meningitidis (N. meningitidis) serogroup B was approved. Other (engineered) MVs in the pipeline study are mostly in the preclinical phase.

Roles of secreted phospholipase A2 group IIA in inflammation and host defense.

Among all members of the secreted phospholipase A2 (sPLA2) family, group IIA sPLA2 (sPLA2-IIA) is possibly the most studied enzyme. Since its discovery, many names have been associated with sPLA2-IIA, such as "non-pancreatic", "synovial", "platelet-type", "inflammatory", and "bactericidal" sPLA2. Whereas the different designations indicate comprehensive functions or sources proposed for this enzyme, the identification of the precise roles of sPLA2-IIA has remained a challenge. This can be attributed to: the expression of the enzyme by various cells of different lineages, its limited activity towards the membranes of immune cells despite its expression following common inflammatory stimuli, its ability to interact with certain proteins independently of its catalytic activity, and its absence from multiple commonly used mouse models. Nevertheless, elevated levels of the enzyme during inflammatory processes and associated consistent release of arachidonic acid from the membrane of extracellular vesicles suggest that sPLA2-IIA may contribute to inflammation by using endogenous substrates in the extracellular milieu. Moreover, the remarkable potency of sPLA2-IIA towards bacterial membranes and its induced expression during the course of infections point to a role for this enzyme in the defense of the host against invading pathogens. In this review, we present current knowledge related to mammalian sPLA2-IIA and its roles in sterile inflammation and host defense.

Immunization with lipopolysaccharide-free outer membrane complexes protects against Acinetobacter baumannii infection.

Outer membrane complex (OMC) vaccines, which contain antigens from the bacterial outer membrane, have been developed for multiple Gram-negative bacteria. However, OMC vaccines demonstrate high endotoxin activity due to the presence of lipopolysaccharide in the bacterial outer membrane, thus precluding their use in humans. We isolated OMCs from an LPS-deficient strain of A. baumannii (IB010) which completely lacks LPS due to a mutation in the lpxD gene. OMCs from IB010 demonstrated a more than 10,000-fold reduction in endotoxin activity compared to OMCs from wild type A. baumannii. Vaccination with IB010 OMCs produced similar levels of antigen-specific IgG and IgM after two administrations compared to wild type OMCs, and resulted in a similar reduction in post-infection spleen bacterial loads and serum pro-inflammatory cytokine levels. Vaccination with IB010 OMCs provided significant protection against infection compared to control mice, indicating the LPS-free OMCs could contribute to vaccine strategies for preventing infection by A. baumannii.

The Trypanosoma cruzi Surface, a Nanoscale Patchwork Quilt.

The Trypanosoma cruzi trypomastigote membrane provides a major protective role against mammalian host-derived defense mechanisms while allowing the parasite to interact with different cell types and trigger pathogenesis. This surface has been historically appreciated as a rather unstructured 'coat', mainly consisting of a continuous layer of glycolipids and heavily O-glycosylated mucins, occasionally intercalated with different developmentally regulated molecules displaying adhesive and/or enzymatic properties. Recent findings, however, indicate that the trypomastigote membrane is made up of multiple, densely packed and discrete 10-150nm lipid-driven domains bearing different protein composition; hence resembling a highly organized 'patchwork quilt' design. Here, we discuss different aspects underlying the biogenesis, assembly, and dynamics of this cutting-edge fashion outfit, as well as its functional implications.

Potency of Both Human Th1 and NK Helper Cell Activation is Determined by IL-12p70-Producing PAMP-Matured DCs.

Besides T helper (Th) cells, natural killer (NK) cells have also been described to participate in the shaping of dendritic cell (DC)-mediated adaptive immune responses. At present, it remains unclear to what extent the induction of these NK helper cell immune mechanisms is coupled with Th responses and whether both helper immune responses are induced by the same DC upon specific pathogen recognition receptor (PRR) stimulation. In this study, we demonstrate that maturation of DCs with a cocktail containing FMKp (membrane fragments of Klebsiella pneumoniae) mounts both Th cell and NK cell helper responses in a PRR-triggered dose-dependent manner as determined by the capacity of the helper cells to produce IFN-γ. Furthermore, by triggering an additional PRR pathway [FMKp in combination with poly(I:C) lyovec], we reveal that both approaches modulate the amount of DC-derived IL-12p70 and that this cytokine is the key determinant of the DC-induced Th1 and NK cell helper responses. Moreover, all PRR triggers able to induce IL-12-producing mature DCs are sufficient to induce these helper responses. We propose the existence of a single program used by DCs to induce potent cellular immune responses by stimulating both T helper and NK cell helper processes. This knowledge can help to select the proper PRR triggers in preventive and therapeutic vaccine design.

Membrane fractions from Strongyloides venezuelensis in the immunodiagnosis of human strongyloidiasis.

Strongyloides venezuelensis is a parasitic nematode of rodents frequently used to obtain heterologous antigens for the immunological diagnosis of human strongyloidiasis. The aim of this study was to evaluate membrane fractions from S. venezuelensis for human strongyloidiasis immunodiagnosis. Soluble and membrane fractions were obtained in phosphate saline (SS and SM) and Tris-HCl (TS and TM) from filariform larvae of S. venezuelensis. Ninety-two serum samples (n = 92) were obtained from 20 strongyloidiasis patients (Group I), 32 from patients with other parasitic diseases (Group II), and 40 from healthy individuals (Group III), and were analyzed by enzyme-linked immunosorbent assay (ELISA). Soluble fractions (SS and TS) showed 90.0% sensitivity and 88.9% specificity, whereas the membrane fractions (SM and TM) showed 95.0% sensitivity and 94.4% specificity. The present results suggest the possible use of membrane fractions of S. venezuelensis as an alternative antigen for human strongyloidiasis immunodiagnosis.

Membrane fractions from Strongyloides venezuelensis in the immunodiagnosis of human strongyloidiasis / Frações de membrana de Strongyloides venezuelensis para o imunodiagnóstico da estrongiloidíase humana

Strongyloides venezuelensis is a parasitic nematode of rodents frequently used to obtain heterologous antigens for the immunological diagnosis of human strongyloidiasis. The aim of this study was to evaluate membrane fractions from S. venezuelensis for human strongyloidiasis immunodiagnosis. Soluble and membrane fractions were obtained in phosphate saline (SS and SM) and Tris-HCl (TS and TM) from filariform larvae of S. venezuelensis. Ninety-two serum samples (n = 92) were obtained from 20 strongyloidiasis patients (Group I), 32 from patients with other parasitic diseases (Group II), and 40 from healthy individuals (Group III), and were analyzed by enzyme-linked immunosorbent assay (ELISA). Soluble fractions (SS and TS) showed 90.0% sensitivity and 88.9% specificity, whereas the membrane fractions (SM and TM) showed 95.0% sensitivity and 94.4% specificity. The present results suggest the possible use of membrane fractions of S. venezuelensis as an alternative antigen for human strongyloidiasis immunodiagnosis.

Strongyloides venezuelensis é um nematódeo parasita de roedores, frequentemente usado como antígeno heterólogo para o diagnóstico imunológico da estrongiloidíase humana. O objetivo deste estudo foi avaliar frações de membrana de S. venezuelensis para o imunodiagnóstico da estrongiloidíase humana. Para tanto, frações solúveis e de membrana foram obtidas em solução salina fosfato (SS e MS) e Tris-HCl (ST e MT) de larvas filarioides de S. venezuelensis. Amostras de soro de 92 indivíduos, sendo 20 com estrongiloidíase (Grupo I); 32 com outras parasitoses (Grupo II), e 40 indivíduos saudáveis (Grupo III), foram analisadas pelo teste Imunoenzimático (ELISA). As frações solúveis (SS e ST) apresentaram 90,0% e 88,9%, enquanto que as frações de membrana (MS e MT) demonstraram 95,0% e 94,4%, de sensibilidade e especificidade, respectivamente. Os resultados obtidos permitem indicar as frações de membranas como antígeno alternativo para o diagnóstico da estrongiloidíase humana.

Structure and partial protein profiles of the peritrophic membrane (PM) from the gut of the shrimp Litopenaeus vannamei.

Peritrophic membrane (PM) is a non-cellular structure surrounding the food bolus in invertebrate's midgut. In this study, the shrimp Litopenaeus vannamei was found continuously secreting a tube-like PM enclosing the fecal pellets. The PM structure was membranous in this penaeid shrimp which was similar to that in Sicyonia ingentis studied and was primarily composed of chitin and proteins. Chitin was detected along the whole PM. By using the approach of gel electrophoresis and mass spectrometry of tryptic peptides, the most extracted proteins from the shrimp PMs were identified mainly including digestion-related, immune-related, antioxidant proteins and proteins related to PM structure. This suggests that PM may participate in modulating its permeability and immobilizating the digestive enzymes, actively protect the gut from pathogen contact, and play an important role in the gut immune system.

Mycobacteria release active membrane vesicles that modulate immune responses in a TLR2-dependent manner in mice.

Bacteria naturally release membrane vesicles (MVs) under a variety of growth environments. Their production is associated with virulence due to their capacity to concentrate toxins and immunomodulatory molecules. In this report, we show that the 2 medically important species of mycobacteria, Mycobacterium tuberculosis and Mycobacterium bovis bacille Calmette-Guérin, release MVs when growing in both liquid culture and within murine phagocytic cells in vitro and in vivo. We documented MV production in a variety of virulent and nonvirulent mycobacterial species, indicating that release of MVs is a property conserved among mycobacterial species. Extensive proteomic analysis revealed that only MVs from the virulent strains contained TLR2 lipoprotein agonists. The interaction of MVs with macrophages isolated from mice stimulated the release of cytokines and chemokines in a TLR2-dependent fashion, and infusion of MVs into mouse lungs elicited a florid inflammatory response in WT but not TLR2-deficient mice. When MVs were administered to mice before M. tuberculosis pulmonary infection, an accelerated local inflammatory response with increased bacterial replication was seen in the lungs and spleens. Our results provide strong evidence that actively released mycobacterial vesicles are a delivery mechanism for immunologically active molecules that contribute to mycobacterial virulence. These findings may open up new horizons for understanding the pathogenesis of tuberculosis and developing vaccines.

The 2008 ESPEN Sir David Cuthbertson Lecture: Fatty acids and inflammation--from the membrane to the nucleus and from the laboratory bench to the clinic.

Many chronic conditions involve excessive inflammation that is damaging to host tissues. Excessive or inappropriate inflammation and immunosuppression are components of the response to surgery, trauma, injury and infection in some individuals and these can lead, progressively, to sepsis and septic shock. Hyperinflammation is characterised by the production of inflammatory cytokines, eicosanoids and other inflammatory mediators, while the immunosuppression is characterised by impairment of antigen presentation and of certain T cell responses. N-6 fatty acids may contribute to the hyperinflamed and immunosuppressed states. N-3 fatty acids from fish oil decrease the production of inflammatory cytokines and eicosanoids. They act both directly (by replacing arachidonic acid as an eicosanoid precursor) and indirectly (by altering the expression of inflammatory genes through effects on transcription factor activation). Thus, these fatty acids are potentially useful anti-inflammatory agents and may be of benefit in patients with chronic inflammatory diseases or at risk of hyperinflammation and sepsis. An emerging application of n-3 fatty acids is in surgical or critically ill patients where they may be added to parenteral or enteral formulas. Studies to date are suggestive of clinical benefits from these approaches, although more robust data are needed especially in critically ill patients.

Glycosidase-induced fusion of isoprenoid gentiobiosyl lipid membranes at acidic pH.

A difficulty in explaining the mechanism whereby archaeal lipid membrane vesicles (archaeosomes) deliver entrapped protein antigens to the MHC class I cytosolic pathway from phagolysosomes of antigen-presenting cells has been the observation that they tend not to fuse. Here, we determine that archaeosomes, composed of archaeal isoprenoid mixtures of glyco and phospholipids, can be highly fusogenic when exposed to the pH and enzymes found in late phagolysosomes. Fusions were strictly dependent on acidic pH and the presence of alpha- or beta-glucosidase. Resonance energy transfer (RET) assays demonstrated that fusion conditions induced lipid mixing of archaeosome lipids with self-unlabeled archaeosomes. Because PC/PG/cholesterol liposomes by themselves did not fuse, it was possible to unequivocally show a fusion of rhodamine-labeled liposomes with archaeosomes by fluorescence microscopy and to demonstrate lipid mixing between labeled liposomes and archaeosomes by the RET assay. Radiotracer and (1)H NMR studies revealed that glycolipids in fused archaeosomes were not degraded significantly by glucosidase treatment during fusion. Rather, the glucosidases dramatically induced small archaeosomes to rapidly and visually aggregate at pH 4.8, but not 6.8, thus bringing membranes together appropriately as a first step in the fusion process. (1)H NMR was used to demonstrate that conditions causing aggregation correlated with binding of glucosidase to the archaeosomes. Binding at acidic pH occurred by the electrostatic interaction of positively charged glucosidase with the anionic phospholipids, although the interaction also occurred with the gentiobiosyl lipids. The data indicate a mechanism of membrane-membrane fusion for archaeal glycolipid membranes induced by glycosidase and illustrate the importance for inclusion of glycolipids in compositions of vesicles designed to deliver protein antigens to the cytosol for MHC class I presentation.

Toxoplasma gondii Hsp20 is a stripe-arranged chaperone-like protein associated with the outer leaflet of the inner membrane complex.

BACKGROUND INFORMATION: Toxoplasma gondii is among the most successful parasites, with nearly half of the human population chronically infected. T. gondii has five sHsps [small Hsps (heat-shock proteins)] located in different subcellular compartments. Among them, Hsp20 showed to be localized at the periphery of the parasite body. sHsps are widespread, constituting the most poorly conserved family of molecular chaperones. The presence of sHsps in membrane structures is unusual. RESULTS: The localization of Hsp20 was further analysed using high-resolution fluorescent light microscopy as well as electron microscopy, which revealed that Hsp20 is associated with the outer surface of the IMC (inner membrane complex), in a set of discontinuous stripes following the same spiralling trajectories as the subpellicular microtubules. The detergent extraction profile of Hsp20 was similar to that of GAP45 [45 kDa GAP (gliding-associated protein)], a glideosome protein associated with the IMC, but was different from that of IMC1 protein. Although we were unable to detect interacting protein partners of Hsp20 either in normal or stressed tachyzoites, an interaction of Hsp20 with phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate phospholipids could be observed. CONCLUSIONS: Hsp20 was shown to be associated with a specialized membranous structure of the parasite, the IMC. This discontinuous striped-arrangement is unique in T. gondii, indicating that the topology of the outer leaflet of the IMC is not homogeneous.

Autoantibodies to novel membrane and cytosolic antigens of the lachrymal gland in primary Sjögren's syndrome.

Sjögren's syndrome (SS) is a prototypical systemic autoimmune disease, where autoimmune processes lead to the dysfunction of the exocrine glands. The key feature of the disease is autoimmune exocrinopathy, causing reduced tear secretion and subsequent keratoconjunctivitis sicca (KCS). The aim of this study was to investigate the connection between the presence of autoantibodies to lachrymal gland antigens and the reduced tear production in patients with primary SS. Ninety-nine patients, 90 women and 9 men, were investigated in the study. Twenty healthy young women served as controls. Enzyme-linked immunosorbent assay (ELISA) and Western blotting were applied to detect autoantibodies to antigen fractions prepared from the human lachrymal gland membrane and cytosolic fractions. Autoantibodies of the IgG, IgA and IgM isotypes to the lachrymal membrane and cytosolic fractions were detected in about one third (27%) of the patients with primary SS. IgA antobodies to the membrane and cytosolic fractions occurred most frequently in SS patients. A significant difference was found in the presence of IgA antibodies to the membrane lachrymal fraction between patients and controls given in ELISA indices (1.23 +/- 0.3 vs 1 +/- 0.19, p < 0.001). IgG, IgA, and IgM isotypes of autoantibodies directed to the membrane lachrymal fraction of 200-180, 120-116, 80-70, 58, 50, 48.5, 40 and 28.8 kDa were also identified in patients. Membrane IgG antibody levels showed a positive correlation (R = 0.998; p = 0.045) with the clinical loss of secretory function (Schirmer's test values). Positive correlation was found between membrane IgM and anti-SS-A levels (R = 0.962; p = 0.038) and also between cytosolic IgM antibodies and anti-SS-A levels (R = 0.982; p = 0.018). IgG, IgA and IgM types of autoantibodies may play a role in the development of the impaired lachrymal secretion and therefore may be involved in the pathogenesis of KCS.

Autoantibody response to microsomal epoxide hydrolase in hepatitis C and A.

Autoimmune responses were observed in a large proportion of hepatitis C cases and are suspected to be part of viral pathogenesis. The AN6520 antigen (AN-Ag) is a normal cellular protein mainly expressed in liver that was found associated with non-A, non-B hepatitis. To elucidate its pathogenic role in hepatitis C, we developed an IgM capture assay using purified AN-Ag and confirmed that the antibody response to AN-Ag is associated almost exclusively with hepatitis C cases (29%). Screening of a human liver expression library revealed that AN-Ag is mainly the microsomal epoxide hydrolase (mEH), a drug-metabolizing enzyme that plays an important role in the metabolism of some mutagenic and carcinogenic epoxides. Using the purified recombinant human mEH as an antigen, we now found that antibodies against this protein are associated with nearly 82% of hepatitis C virus infections and surprisingly with 46% of patients with hepatitis A. The appearance of AN-Ag/mEH in the incubation period of hepatitis C as previously reported and the antibody responses shown here indicate that this enzyme may be a marker for or even a cause of some of the pathology associated with hepatitis C and A.

Stationary phase culture supernatant containing membrane vesicles induced immunity to rainbow trout Oncorhynchus mykiss fry syndrome.

Flavobacterium psychrophilum is the causative agent of bacterial cold water disease (BCWD) and rainbow trout (Oncorhynchus mykiss) fry syndrome (RTFS). Logarithmic phase formalin-killed cells (FKC) of F. psychrophilum induced immunity to BCWD in ayu (Plecoglossus altivelis) by using an oral administration. In this study, we investigated the effective antigens of logarithmic phase cells in rainbow trout. Rainbow trout fry immunized with logarithmic phase FKC resulted in near complete protection, but the vaccine effect was low in fry immunized with stationary phase FKC. Scanning electron microscopy showed that logarithmic phase cells had many membrane vesicles (MVs) on the surface of F. psychrophilum cells. The MVs were released into medium at the stationary phase. MVs rich supernatant was collected from the stationary phase culture supernatant by using an ammonium precipitation method. Immunization with MVs rich supernatant combined with stationary phase FKC resulted in a relative percentage survival (RPS) of 94-100%, but immunization with MVs rich supernatant only resulted in no protection against F. psychrophilum infection. These data show that MVs have an adjuvant efficacy and suggest that combination of MVs and cells is necessary to obtain efficient protection.

The membranes' role in the HIV-1 neutralizing monoclonal antibody 2F5 mode of action needs re-evaluation.

2F5, a monoclonal antibody that neutralizes HIV-1 primary isolates, recognizes an epitope in the membrane proximal region of the glycoprotein gp41 ectodomain. It is believed that binding to the viral membrane is a step in the antibody mode of action, as usual in ligand membrane receptor interactions. We investigated the interaction of 2F5 with membrane model systems, namely large unilamellar vesicles, by means of fluorescence techniques. There were no significant interactions of 2F5 with model viral membranes or with model target cell membranes. Thus, the usual three-step 'membrane catalysis' method is not followed by 2F5 in its mode of action.

Protection studies in sheep using affinity-purified and recombinant cysteine proteinases of adult Haemonchus contortus.

Vaccination with a membrane-bound thiol Sepharose-binding fraction (TSBP) of adult Haemonchus contortus has been shown to confer significant levels of protection against homologous challenge in sheep. This fraction is greatly enriched for cysteine proteinase activity. Following fractionation of TSBP by anion-exchange chromatography on MonoQ, protection was found to partition with those fractions further enriched for cysteine proteinase activity. In this study, the cysteine proteinases of adult H. contortus TSBP were specifically purified by affinity chromatography using recombinant H. contortus cystatin, a potent cysteine proteinase inhibitor. Although only 1-1.5% of total TSBP bound to cystatin-Sepharose, this fraction contained 100% of the cysteine proteinase activity, as determined by gelatin substrate gel analysis. When used to immunise sheep, less than 3microg per dose of this cysteine proteinase fraction was found to confer a substantial and repeatable level of protection against homologous challenge infection, reducing faecal egg counts by 48 and 28% and worm burdens by 44 and 46% over two trials. Host serum immunoglobulin levels and abomasal mast cell and eosinophil numbers were evaluated, although no correlation with protection was observed. Three cathepsin B-like cysteine proteinases present in TSBP (hmcp1, 4 and 6) have been identified previously by cDNA library immunoscreening. The predicted mature forms of these three cysteine proteinases were expressed in bacteria as insoluble, GST-fusion proteins. Following solubilisation in urea/DTT, the protective capacity of a cocktail of recombinant proteins was evaluated in sheep. Although no reduction in faecal egg counts was observed, sheep vaccinated with recombinant cysteine proteinases showed a highly significant 38% reduction (P <0.01) in worm burdens.

Mouse-heart grafts expressing an incompatible carbohydrate antigen. II. Transition from accommodation to tolerance.

BACKGROUND: Immune response to incompatible ABO antigens on allografts may result in rejection, accommodation, or immune tolerance. Our objective has been to develop a model for studying these three types of immune response to incompatible carbohydrate antigen in alpha1,3-galactosyltransferase knockout (KO) mice. KO mice lack the alpha-gal epitope and can produce the anti-Gal antibody against it after immunization with pig kidney membranes (PKM) that express this epitope. METHODS: KO mice were transplanted with syngeneic wild-type (WT) heart expressing alpha-gal epitopes. Subsequently, the mice were lethally irradiated and received lymphocytes including memory anti-Gal B cells from PKM immunized KO mice. Immune response to incompatible alpha-gal epitopes on the graft was determined by transplanted-heart function and by production of anti-Gal after PKM immunizations. RESULTS: Anti-Gal B cells exposed for 1 to 2 weeks to alpha-gal epitopes of WT hearts differentiate into cells producing noncytolytic accommodating antibodies. Exposure for longer periods (2-4 weeks) induces a transition from accommodation into tolerance, indicated by the inability of mice to produce anti-Gal antibodies despite repeated PKM immunizations. WT hearts in accommodating and in tolerized mice continue to function for months. CONCLUSIONS: In the absence of T-cell help, anticarbohydrate B cells exposed to incompatible carbohydrate antigens of transplanted organs differentiate first into cells capable of producing accommodating antibodies, but, after prolonged exposure, these B cells gradually become tolerized. These findings suggest that prolonged T-cell suppression in recipients of ABO-incompatible allografts may result in a similar induction of tolerance to incompatible blood-group antigens.