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.

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.

Nonrandom inclusion of H-2K and H-2D antigens in Friend virus particles from mice of various strains.

Friend murine leukemia virus (FV), isolated from infectious serum of several mouse strains, has been examined for the presence of H-2 antigens. Following banding of the virus on a discontinuous sucrose gradient, pelleting, and disruption with Nonidet P-40 detergent, virus preparations were tested for their capacity to inhibit the lysis of target cells mediated by various anti-H-2K or anti-H-2D antisera. Virus from mice homozygous for the H-2b, H-2d, H-2g H-2k, and H-2ol haplotypes or heterozygous for the H-2g/H-2k, H-2b/H-2d, and H-2b/H-2k haplotypes was used. Of the six H-2D or H-2K alleles examined, the products of only two, H-2Db and H-2Kk were detected. Virus preparations contained no, one, or both antigens, depending on the genotype of the host animal. Control preparations from normal mouse serum and preparations in which the virus had not been disrupted demonstrated no H-2 activity. Furthermore, attempts to neutralize FV spleen focus forming activity with anti-H-2Db or anti-H-2Kk antisera yielded negative results.

Differential expression of MHC class I antigens on the placenta of the rat. A mechanism for the survival of the fetal allograft.

In some mating combinations in rats, there is a maternal antibody response to the maternal antigenic components of the placenta without any previous immunization of the mother. The highest response occurs in the WF (u) female mated to the DA (a) male, and it is against a unique MHC-encoded class I antigen, the Pa antigen, and not against the major allele-specific transplantation antigen of the DA strain, RT1.Aa. The development of mAbs to the Pa and Aa antigens allowed us to localize these antigens on the placenta and to explore the reason for the differential antibody response to them using immunohistochemical and biochemical techniques. Both antibodies reacted with the WF X DA placenta and stained the endovascular and interstitial trophoblast of the decidua, the basal trophoblast, Reichert's membrane, and the yolk sac epithelium, but they did not stain the labyrinthine trophoblast. Blocking studies showed that each antibody reacted with a separate molecule in the placenta. Anti-class II mAbs reactive with the a or u haplotype did not stain the WF X DA, DA X DA, or WF X WF placenta; hence, there are no class II antigens in the placenta. Electron microscopic studies of the semiallogeneic WF X DA placenta using the immunogold technique with both single- and double-labeling showed that only the Pa antigen was expressed on the surface of the basal trophoblast, but that both the Pa and Aa antigens were in the cytoplasm of these cells; neither antigen was found in the labyrinthine trophoblast. By contrast, the placenta from the syngeneic DA X DA mating expressed both the Pa and Aa antigens on the surface of the basal trophoblast as well as in the cytoplasm; neither antigen was found in the labyrinthine trophoblast. These observations were quantified morphometrically using electron photomicrographs of single-labeled tissues. Both the Pa and Aa antigens isolated from the plasma membrane of lymphocytes have heavy chains of 46 kD, but those antigens isolated from the plasma membrane of basal trophoblast cells have heavy chains of 43 kD. Based on densitometric measurements of autoradiographs, the Pa/Aa ratio in the basal trophoblast membrane is 23.5, whereas it is 0.46 in lymphocyte membranes. These studies show that there is differential regulation of the expression of class I antigens on basal trophoblast cells in semiallogeneic pregnancies, but not in syngeneic pregnancies, such that the major allele-specific transplantation antigen is scarcely expressed on the surface of the basal trophoblast.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

Characterization of rat liver subcellular membranes. Demonstration of membrane-specific autoantigens.

The induction of acute hepatocellular necrosis in rats resulted in the production of complement fixing, IgM autoantibodies directed toward inner and outer mitochondrial membranes, microsomal membrane, lysosomal membrane, nuclear membrane, cytosol, but not to plasma membrane. Utilizing selective absorption procedures it was demonstrated that each subcellular membrane fraction possessed unique autoantigenic activity with little or no cross-reactivity between the various membrane fractions. It is proposed that the development of membrane-specific autoantibodies may provide an immunological marker useful in the differential characterization of various subcellular membranes.

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.

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.

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.

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.

Vaccination with Legionella pneumophila membranes induces cell-mediated and protective immunity in a guinea pig model of Legionnaires' disease. Protective immunity independent of the major secretory protein of Legionella pneumophila.

We have examined the capacity of Legionella pneumophila membranes to induce cell-mediated immune responses and protective immunity in a guinea pig model of Legionnaires' disease. Guinea pigs immunized by aerosol with L. pneumophila membranes developed strong cell-mediated immune responses to L. pneumophila membranes as demonstrated by cutaneous delayed-type hypersensitivity and in vitro splenic lymphocyte proliferation. Guinea pigs immunized by aerosol or by subcutaneous inoculation with L. pneumophila membranes developed strong protective immunity against lethal aerosol challenge with L. pneumophila. Overall, in six independent experiments, 39 of 49 (80%) guinea pigs immunized with L. pneumophila membranes survived challenge compared with 2 of 40 (5%) sham-immunized controls (P = 2 x 10(-13). In contrast, guinea pigs immunized by aerosol with formalin-killed L. pneumophila did not develop either a strong cell-mediated immune response to L. pneumophila antigens or protective immunity to lethal aerosol challenge. The capacity of L. pneumophila membranes to induce protective immunity was independent of the major secretory protein of L. pneumophila, which we previously demonstrated is an immunoprotective molecule. Purified L. pneumophila membranes did not contain detectable major secretory protein (MSP) on immunoblots; immunization of guinea pigs with L. pneumophila membranes did not induce anti-MSP antibody; and guinea pigs developed comparable protective immunity after immunization with membranes from either an L. pneumophila strain that secretes the major secretory protein or an isogenic mutant that does not. This study demonstrates that (a) immunization with L. pneumophila membranes but not formalin-killed L. pneumophila induces strong cell-mediated immune responses and protective immunity, (b) L. pneumophila membranes contain immunoprotective molecules distinct from the major secretory protein of L. pneumophila, and (c) L. pneumophila membranes have potential as a vaccine against Legionnaires' disease.

Consumption of classical complement components by heart subcellular membranes in vitro and in patients after acute myocardial infarction.

Experiments were conducted to characterize the antibody-independent activation of complement in human serum by isolated human heart mitochondrial membranes in vitro and to determine whether similar patterns of complement consumption occurred in patients after acute myocardial infarction. Direct evidence for the interaction of C1 and heart mitochondrial membranes was obtained by mitochondria-C1 binding and elution experiments. Exposure of normal human sera to isolated human heart mitochondria at 37 degrees C resulted in the consumption of C1, C4, C2, and C3 without significant consumption of the terminal components of the complement system (C6 through C9). The consumption occurred in the absence of detectable anti-heart mitochondria autoantibody, was demonstrated to be calcium dependent, and was inhibited by either 0.01 M EDTA or ethylene glycol bis(bets-aminoethyl ether) N,N,N',N',-tetraacetic acid (EDTA). Although specific absorption of C1q from human sera inhibited the mitochondria-dependent activation of C4, C3 donsumption was not affected. These data indicate that the consumption of C4 and C2 likely occurred due to the mitochondrial membrane-mediated activation of C1, but that the consumption of the C3 did not necessarily involve either the classical or alternative complement pathways. After the in vitro characterization of the mitochondria-dependent activation of the complement system, additional studies were performed to determine whether similar consumption occurred in patients after acute myocaridal infarction. During a 72-h period after hospital admission significant decreases in C1, C4, and C3 occurred in six patients with recent chest pain but no evidence of acute myocardial infarction. These studies suggest that myocardial cell necrosis results in the release of subcellular membrane constituents capable of activating the complement system in the absence of detectable anti-heart autoantibodies; such activation may be responsible in part for the development of acute inflammation and evolution of the infarct size following coronary artery occulusion.

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.

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.

In vitro induction of tumor necrosis factor alpha, tumor cytolysis, and blast transformation by Spiroplasma membranes.

Membranes of Spiroplasma sp. strain MQ-1 (hereafter referred to as MQ-1) were potent inducers of tumor necrosis factor alpha (TNF alpha) secretion and of blast transformation. Specific anti-recombinant murine TNF alpha antibodies markedly inhibited macrophage-mediated tumor cytolysis of A9 fibrosarcoma target cells following activation by MQ-1 membranes. Thus, TNF alpha plays a major role in mediation of tumor cytolysis induced by MQ-1 membranes, which is similar to its role in lipopolysaccharide (LPS)-induced tumor cytolysis. Two findings, however, suggested that the mechanism of macrophage activation by MQ-1 membranes differs from that by LPS: (a) macrophages, taken from C3H/HeJ mice showing a low responsiveness to LPS, were activated by MQ-1 membranes to enhanced TNF alpha secretion, resulting in a high-level tumor cytolysis compared with the negligible tumor cytolysis induced by LPS; and (b) MQ-1 membranes and LPS synergized to highly augment TNF alpha secretion by macrophages of C57BL/6 mice. MQ-1 membranes were capable of inducing blast transformation of murine lymphocytes as well. In addition, they activated human monocytes to secrete high levels of TNF alpha. Further studies need to be carried out using in vivo models to evaluate the therapeutic potential of MQ-1 membranes in the treatment of malignant diseases.

Immunization of mice against murine mammary tumor virus infection and mammary tumor development.

Formalin-inactivated whole murine mammary tumor virus (MuMTV), VuMTV membranes, the acid-soluble component of MuMTV, and purified MuMTV glycoprotein with a molecular weight of 55,000 (gp55; also designated as gp52) were used as vaccines in an attempt to identify the MuMTV antigen(s) that can protect mice from exogenous MuMTV infection and subsequent tumor development. Formalin-inactivated whole MuMTV, MuMTV membranes, and purified MuMTV gp55 were effective immunogens, whereas the acid-soluble component of MuMTV (which consists mainly of MuMTV gp55) failed to protect mice from challenge with live virus. These results suggest that (a) MuMTV gp55 is the major immunizing antigen and (b) its native conformation must be maintained for it to be an effective vaccine.

Association of antigen expression and DNA ploidy in human colorectal tumors.

Fifty colorectal tumors were screened by indirect immunofluorescence and flow cytometry for antigen expression using a panel of monoclonal antibodies that recognize determinants preferentially expressed on tumor cells (carcinoembryonic antigen, Y haptenic blood group, 791T/36 defined antigen 791T-P72). Fifty % of the tumors expressed all three antigens, 41%, two, and 9%, one. Over a third reacted strongly with at least one monoclonal antibody, although the majority of tumors stained with a moderate intensity. Extranuclear membranes from tumors showed similar antigen expression to disaggregated tumor cells and were particularly useful for providing the relative tumor:normal tissue binding ratios. The carcinoembryonic antigen specific monoclonal antibody showed the strongest tumor selectivity with a tumor:normal tissue ratio of 24 +/- 7:1. Lack of correlation between expression of the three antigens suggested that the monoclonal antibodies recognizing them may have potential as a "cocktail." One-third of the tumors contained cells with an aneuploid DNA content and expressed elevated levels of carcinoembryonic antigen and Y haptenic blood group antigen when compared to tumors with diploid DNA content. Aneuploid cells within a tumor were also preferentially stained with all of the monoclonal antibodies.