A comparative study of the immunity region of lambdoid phages including Shiga-toxin-converting phages: molecular basis for cross immunity

Comparison of eight lambdoid phages, including three Shiga-toxin converting phages, has been carried out with respect to the immunity region, especially the recognition helices of their repressor and CRO proteins on the one hand, and operator sequences on the other. Some as yet unassigned components of the regulatory circuits have been inferred by computer search. The cross immunity phenomenon shown by phages VT2-Sa and lambda is explained on the basis of similarity in their sequences. In addition, the similarity of 933W and HK022 in the sequences of their recognition helices of repressor and CRO, on the one hand, and operators, on the other, has led us to predict that they will have identical or similar immunity specificity. This homology has enabled us also to locate the OL (and consequently PL) of phage 933W that has been thought to be non-existent.     

The versatility of the αβ T‐cell antigen receptor

The T‐cell antigen receptor is a heterodimeric αβ protein (TCR) expressed on the surface of T‐lymphocytes, with each chain of the TCR comprising three complementarity‐determining regions (CDRs) that collectively form the antigen‐binding site. Unlike antibodies, which are closely related proteins that recognize intact protein antigens, TCRs classically bind, via their CDR loops, to peptides (p) that are presented by molecules of the major histocompatibility complex (MHC). This TCR‐pMHC interaction is crucially important in cell‐mediated immunity, with the specificity in the cellular immune response being attributable to MHC polymorphism, an extensive TCR repertoire and a variable peptide cargo. The ensuing structural and biophysical studies within the TCR‐pMHC axis have been highly informative in understanding the fundamental events that underpin protective immunity and dysfunctional T‐cell responses that occur during autoimmunity. In addition, TCRs can recognize the CD1 family, a family of MHC‐related molecules that instead of presenting peptides are ideally suited to bind lipid‐based antigens. Structural studies within the CD1‐lipid antigen system are beginning to inform us how lipid antigens are specifically presented by CD1, and how such CD1‐lipid antigen complexes are recognized by the TCR. Moreover, it has recently been shown that certain TCRs can bind to vitamin B based metabolites that are bound to an MHC‐like molecule termed MR1. Thus, TCRs can recognize peptides, lipids, and small molecule metabolites, and here we review the basic principles underpinning this versatile and fascinating receptor recognition system that is vital to a host's survival.     

Effects of colchicine- and cytochalasin B-treatment on the intracellular distribution of yolk droplets, lipid bodies, and Golgi apparatus of the chick neuroepithelial cells

Treatment with colchicine (antimicrotubular agent) and cytochalasin B (antimicrofilamentous agent) has been used to investigate the possible role played by the cytoskeleton in the maintenance of intracellular distribution of yolk droplets, lipid bodies, and Golgi apparatus of the chick neuroepithelial cells. On the one hand, embryos treated with colchicine showed modifications in their distribution patterns of yolk droplets and lipid bodies, which suggests the involvement of the microtubular integrity of neuroepithelial cells in the maintenance of normal distribution patterns. On the other hand, the close relationships between vitelline and lipid inclusions and Golgi apparatus observed in untreated embryos seems to be kept in the embryos treated with colchicine and cytochalasin B. Moreover, from the effects of colchicine on Golgi apparatus position a possible functional role for the microtubular system in the maintenance of Golgi apparatus polarity in the chick neuroepithelial cells can be proposed. The results provided here constitute new information about the cellular mechanisms involved in chick neurulation.     

Lipid droplet hijacking by intracellular pathogens

Lipid droplets were long considered to be simple storage structures, but they have recently been shown to be dynamic organelles involved in diverse biological processes, including emerging roles in innate immunity. Various intracellular pathogens, including viruses, bacteria, and parasites, specifically target host lipid droplets during their life cycle. Viruses such as hepatitis C, dengue, and rotaviruses use lipid droplets as platforms for assembly. Bacteria, such as mycobacteria and Chlamydia, and parasites, such as trypanosomes, use host lipid droplets for nutritional purposes. The possible use of lipid droplets by intracellular pathogens, as part of an anti‐immunity strategy, is an intriguing question meriting further investigation in the near future.     

Cytochrome P-450 involvement in the NADPH-dependent lipid peroxidation in human placental mitochondria

The NADPH-dependent lipid peroxidation in human placental mitochondria has been found to be inhibited strongly by amphenone B, aminoglutethimide and carbon monoxide, inhibitors of cytochrome P-450-mediated reactions, but was hardly affected by respiratory chain inhibitors. Cytochrome c, an exogenous electron acceptor which is known to compete with cytochrome P-450 for the reducing equivalents, showed an inhibitory effect on NADPH-dependent lipid peroxidation. The observed NADPH-dependent superoxide generation was also strongly inhibited by amphenone B and aminoglutethimide. Moreover, the lipid peroxidation in placental mitochondria was demonstrated to be stimulated by xanthine/xanthine oxidase added as superoxide generating system. This peroxidation was not affected by amphenone B and aminoglutethimide. On the other hand, the superoxide dismutase was found to inhibit both the xanthine oxidase- and NADPH-dependent lipid peroxidation. These data provide evidence that cytochrome P-450 is involved in NADPH-dependent mitochondrial lipid peroxidation. It is suggested that superoxide liberated from cytochrome P-450, in combination with iron, may be responsible for initiation of NADPH-dependent lipid peroxidation in human placental mitochondria.     

Partial characterization of the inhibitory effect of lipid peroxidation on the ouabain-insensitive Na-ATPase of rat kidney cortex plasma membranes

The present work evaluates the effect of lipid peroxidation on the ouabain-insensitive Na-ATPase of basolateral plasma membranes from rat kidney proximal tubular cells as an indirect way to study the lipid dependence of this enzyme. An inverse relationship between lipid peroxidation and Na-ATPase activity was found. This effect was due neither to a change in the optimalK _m of the system for Na⁺ nor for the substrate Mg : ATP, nor the optimal pH value of the medium. The optimal temperature value, however, was shifted toward a higher value. There was also an increase of the apparent energy of activation in the region of temperatures above the transition point (20°C) with increase in lipid peroxidation. Peroxidized membranes incubated with phosphatidylcholine from soybean restored their Na-ATPase activity. On the other hand, the Na-ATPase activity was sensitive to oleoly lysophosphatidylcholine. These results suggest that lipid peroxidation might be affecting the Na-ATPase activity through either an increase of peroxidized phospholipids, which might change the membrane fluidity of the lipid microenvironment of the ATPase molecules, or through a direct effect of lysophospholipids released during the lipid peroxidation.     

Combinatorial Contextualization of Peptidic Epitopes for Enhanced Cellular Immunity

Invocation of cellular immunity by epitopic peptides remains largely dependent on empirically developed protocols, such as interfusion of aluminum salts or emulsification using terpenoids and surfactants. To explore novel vaccine formulation, epitopic peptide motifs were co-programmed with structural motifs to produce artificial antigens using our “motif-programming” approach. As a proof of concept, we used an ovalbumin (OVA) system and prepared an artificial protein library by combinatorially polymerizing MHC class I and II sequences from OVA along with a sequence that tends to form secondary structures. The purified endotoxin-free proteins were then examined for their ability to activate OVA-specific T-cell hybridoma cells after being processed within dendritic cells. One clone, F37A (containing three MHC I and two MHC II OVA epitopes), possessed a greater ability to evoke cellular immunity than the native OVA or the other artificial antigens. The sensitivity profiles of drugs that interfered with the F37A uptake differed from those of the other artificial proteins and OVA, suggesting that alteration of the cross-presentation pathway is responsible for the enhanced immunogenicity. Moreover, F37A, but not an epitopic peptide, invoked cellular immunity when injected together with monophosphoryl lipid A (MPL), and retarded tumor growth in mice. Thus, an artificially synthesized protein antigen induced cellular immunity in vivo in the absence of incomplete Freund''s adjuvant or aluminum salts. The method described here could be potentially used for developing vaccines for such intractable ailments as AIDS, malaria and cancer, ailments in which cellular immunity likely play a crucial role in prevention and treatment.     

Parameters and techniques to determine intestinal health of poultry as constituted by immunity,integrity, and functionality

The intestinal microflora lives in intimate contact with its surrounding intestinal wall and the bacteria can exert beneficial or deleterious effects on the host, depending on whether they are classified as probiotics or as pathogens. The interaction is determined on one hand by characteristics of the microorganisms, and on the other hand by characteristics of the intestinal wall. Together they determine the health status of the intestine. This review describes parameters and techniques (with advantages and disadvantages) available for poultry to identify the characteristics of the intestinal health, as constituted by three components: immunity, integrity, and functionality. To investigate intestinal immunity, in situ detection of various cell populations of the immune system with specific monoclonal antibodies using immunocytochemical staining is a reliable, semi-quantitative method. In vitro assays to measure functional aspects of T lymphocytes, B lymphocytes, plasma cells, natural killer cells, macrophages, and phagocytes are applicable to intestinal wall tissue. For investigation of intestinal integrity, in situ detection of villous height and crypt depth and their ratio, and villus arrangement is still an easy, routine histological method. In addition, expression levels of specific molecules, such as E-cadherin, different growth factors, and trefoil factor, seem promising parameters. To investigate functionality of the intestine, the permeability can be measured as the rate of transport of tracer molecules across the epithelial surface. Furthermore, determination of the level of mucus secretion and its composition are a valuable tool. These parameters for immunity, integrity and functionality, or a combination thereof, are indispensable to investigate the influence of intestinal microorganisms on intestinal health.     

Interrelationships between serum and secretory antibodies in immunization with live influenza vaccine]

A study was made of the accumulation of antibodies in the blood serum and the secretions of the respiratory tracts of persons immunized with the living influenza vaccine. The duration of inductive phase and the dynamics of the antibody accumulation in the secretions occurred irrespective of their initial level in the blood sera, this pointing to the autonomic character of the local immunity system. On the other hand the functional condition of the system of local immunity influenced the intensity of the antibody formation in the system of the general immunity. If before the immunization the antibody titre in the secretions were 1:4 and greater, the antibody accumulation in the blood sera took place less intensively. An analogous phenomenon was also observed when the antibodies were absent in the secretions before the immunization, but their formation took place as soon as the first week after it. The mechanism of this peculiar "competition for the antigen" of the systems of local and general immunity consisted in the neutralization of the influenza virus in the area of the porta of infection.     

A rapid, small-scale procedure for the structural characterization of lipid A applied to Citrobacter and Bordetella strains: discovery of a new structural element

Endotoxins [lipopolysaccharides (LPSs)] are part of the outer cell membrane of Gram-negative bacteria. Their biological activities are associated mainly with the lipid component (lipid A) and even more specifically with discrete aspects of their fine structure. The need for a rapid and small-scale analysis of lipid A motivated us to develop a procedure that combines direct isolation of lipids A from bacterial cells with sequential release of their ester-linked fatty acids by a mild alkali treatment followed by MALDI-MS analysis. This method avoids the multiple-step LPS extraction procedure and lipid A isolation. The whole process can be performed in a working day and applied to lyophilized bacterial samples as small as 1 mg. We illustrate the method by applying it to the analysis of lipids A of three species of Citrobacter that were found to be identical. On the other hand, when applied to two batches of Bordetella bronchiseptica strain 4650, it highlighted the presence, in one of them, of hitherto unreported hexosamine residues substituting the lipid A phosphate groups, possibly a new camouflage opportunity to escape a host defense system.     

Trypanosoma cruzi adjuvants potentiate T cell-mediated immunity induced by a NY-ESO-1 based antitumor vaccine

Immunological adjuvants that induce T cell-mediate immunity (TCMI) with the least side effects are needed for the development of human vaccines. Glycoinositolphospholipids (GIPL) and CpGs oligodeoxynucleotides (CpG ODNs) derived from the protozoa parasite Trypanosoma cruzi induce potent pro-inflammatory reaction through activation of Toll-Like Receptor (TLR)4 and TLR9, respectively. Here, using mouse models, we tested the T. cruzi derived TLR agonists as immunological adjuvants in an antitumor vaccine. For comparison, we used well-established TLR agonists, such as the bacterial derived monophosphoryl lipid A (MPL), lipopeptide (Pam3Cys), and CpG ODN. All tested TLR agonists were comparable to induce antibody responses, whereas significant differences were noticed in their ability to elicit CD4(+) T and CD8(+) T cell responses. In particular, both GIPLs (GTH, and GY) and CpG ODNs (B344, B297 and B128) derived from T. cruzi elicited interferon-gamma (IFN-γ) production by CD4(+) T cells. On the other hand, the parasite derived CpG ODNs, but not GIPLs, elicited a potent IFN-γ response by CD8(+) T lymphocytes. The side effects were also evaluated by local pain (hypernociception). The intensity of hypernociception induced by vaccination was alleviated by administration of an analgesic drug without affecting protective immunity. Finally, the level of protective immunity against the NY-ESO-1 expressing melanoma was associated with the magnitude of both CD4(+) T and CD8(+) T cell responses elicited by a specific immunological adjuvant.     

Membrane protein insertase YidC in bacteria and archaea

The insertion of proteins into the prokaryotic plasma membrane is catalyzed by translocases and insertases. On one hand, the Sec translocase operates as a transmembrane channel that can open laterally to first bind and then release the hydrophobic segments of a substrate protein into the lipid bilayer. On the other hand, YidC insertases interact with their substrates in a groove‐like structure at an amphiphilic protein–lipid interface thus allowing the transmembrane segments of the substrate to slide into the lipid bilayer. The recently published high‐resolution structures of YidC provide new mechanistic insights of how transmembrane proteins achieve the transition from an aqueous environment in the cytoplasm to the hydrophobic lipid bilayer environment of the membrane.     

A new cloning system based on the OprI lipoprotein for the production of recombinant bacterial cell wall-derived immunogenic formulations

The conjugation of antigens with ligands of pattern recognition receptors (PRR) is emerging as a promising strategy for the modulation of specific immunity. Here, we describe a new Escherichia coli system for the cloning and expression of heterologous antigens in fusion with the OprI lipoprotein, a TLR ligand from the Pseudomonas aeruginosa outer membrane (OM). Analysis of the OprI expressed by this system reveals a triacylated lipid moiety mainly composed by palmitic acid residues. By offering a tight regulation of expression and allowing for antigen purification by metal affinity chromatography, the new system circumvents the major drawbacks of former versions. In addition, the anchoring of OprI to the OM of the host cell is further explored for the production of novel recombinant bacterial cell wall-derived formulations (OM fragments and OM vesicles) with distinct potential for PRR activation. As an example, the African swine fever virus ORF A104R was cloned and the recombinant antigen was obtained in the three formulations. Overall, our results validate a new system suitable for the production of immunogenic formulations that can be used for the development of experimental vaccines and for studies on the modulation of acquired immunity.     

Muscovite (mica) allows the characterisation of supported bilayers by ellipsometry and confocal fluorescence correlation spectroscopy

We demonstrate for the first time that ellipsometry and confocal fluorescence correlation spectroscopy (FCS) are complementary methods for the characterisation of supported planar phospholipid bilayers (SPBs) formed on mica, a mineral used in atomic force microscopy investigations of SPBs. Addition of small unilamellar vesicles containing 20% dioleoyl-phosphatidylserine (DOPS) and 80% dioleoyl-phosphatidylcholine (DOPC) to an oxidised borosilicate surface, on the other hand, results in a planar lipid system characterised by lateral diffusion coefficients which are three time smaller than those obtained for SPBs. Moreover, seven labelled phospholipids were tested for their suitability in the FCS characterisation of vesicles as well as of SPBs.     

The First Structure of a Lantibiotic Immunity Protein,SpaI from Bacillus subtilis,Reveals a Novel Fold

Lantibiotics are peptide-derived antibiotics that inhibit the growth of Gram-positive bacteria via interactions with lipid II and lipid II-dependent pore formation in the bacterial membrane. Due to their general mode of action the Gram-positive producer strains need to express immunity proteins (LanI proteins) for protection against their own lantibiotics. Little is known about the immunity mechanism protecting the producer strain against its own lantibiotic on the molecular level. So far, no structures have been reported for any LanI protein. We solved the structure of SpaI, a LanI protein from the subtilin producing strain Bacillus subtilis ATCC 6633. SpaI is a 16.8-kDa lipoprotein that is attached to the outside of the cytoplasmic membrane via a covalent diacylglycerol anchor. SpaI together with the ABC transporter SpaFEG protects the B. subtilis membrane from subtilin insertion. The solution-NMR structure of a 15-kDa biologically active C-terminal fragment reveals a novel fold. We also demonstrate that the first 20 N-terminal amino acids not present in this C-terminal fragment are unstructured in solution and are required for interactions with lipid membranes. Additionally, growth tests reveal that these 20 N-terminal residues are important for the immunity mediated by SpaI but most likely are not part of a possible subtilin binding site. Our findings are the first step on the way of understanding the immunity mechanism of B. subtilis in particular and of other lantibiotic producing strains in general.     

Solubilization of lipids from hamster bile-canalicular and contiguous membranes and from human erythrocyte membranes by conjugated bile salts.

We have demonstrated in vitro the efficacy of the taurine-conjugated dihydroxy bile salts deoxycholate and chenodeoxycholate in solubilizing both cholesterol and phospholipid from hamster liver bile-canalicular and contiguous membranes and from human erythrocyte membrane. On the other hand, the dihydroxy bile salt ursodeoxycholate and the trihydroxy bile salt cholate solubilize much less lipid. The lipid solubilization by the four bile salts correlated well with their hydrophobicity: glycochenodeoxycolate, which is more hydrophobic than the tauro derivative, also solubilized more lipid. All the dihydroxy bile salts have a threshold concentration above which lipid solubilization increases rapidly; this correlates approximately with the critical micellar concentration. The non-micelle-forming bile salt dehydrocholate solubilized no lipid at all up to 32 mM. All the dihydroxy bile acids are much more efficient at solubilizing phospholipid than cholesterol. Cholate does not show such a pronounced discrimination. Lipid solubilization by chenodeoxycholate was essentially complete within 1 min, whereas that by cholate was linear up to 5 min. Maximal lipid solubilization with chenodeoxycholate occurred at 8-12 mM; solubilization by cholate was linear up to 32 mM. Ursodeoxycholate was the only dihydroxy bile salt which was able to solubilize phospholipid (although not cholesterol) below the critical micellar concentration. This similarity between cholate and ursodeoxycholate may reflect their ability to form a more extensive liquid-crystal system. Membrane specificity was demonstrated only inasmuch as the lower the cholesterol/phospholipid ratio in the membrane, the greater the fractional solubilization of cholesterol by bile salts, i.e. the total amount of cholesterol solubilized depended only on the bile-salt concentration. On the other hand, the total amount of phospholipid solubilized decreased with increasing cholesterol/phospholipid ratio in the membrane.     

Interferon-gamma increases monocyte HLA-DR expression without effects on glucose and fat metabolism in postoperative patients.

Tissue injury is associated with decreased cellular immunity and enhanced metabolism. Immunodepression is thought to be counteracted by interferon (IFN)-gamma, which increases human leukocyte antigen (HLA)-DR expression. Hypermetabolism could be enhanced by IFN-gamma because cytokines induce a hypermetabolic response to stress. In healthy humans, IFN-gamma enhanced HLA-DR expression without effects on glucose and fat metabolism. In the present study, we evaluated whether IFN-gamma lacks potential harmful side effects on metabolic and endocrine pathways while maintaining its beneficial effects on the immune system under conditions in which the inflammatory response system is activated. In 13 patients scheduled for major surgery, we studied HLA-DR expression on peripheral blood monocytes before surgery and postoperatively randomized the patients into an intervention and a placebo group. Subsequently, we evaluated the effects of a single dose of IFN-gamma vs. saline on short-term monocyte activation, glucose and lipid metabolism, and glucose and lipid regulatory hormones. HLA-DR expression on monocytes was restored from postoperative levels of 54% (42-60%; median and interquartiles) to 92% (91-96%) 24 h after IFN-gamma administration but stayed low in the placebo-treated patients. IFN-gamma did not affect glucose metabolism (plasma glucose, rate of appearance and disappearance of glucose) and lipid metabolism (plasma glycerol, plasma free fatty acids, and rates of appearance and disappearance of glycerol). IFN-gamma had no effect on plasma cortisol, adrenocorticotropic hormone, growth hormone, insulin, C-peptide, glucagon, epinephrine, and norepinephrine concentrations. We conclude that IFN-gamma exerts a favorable effect on cell-mediated immunity in patients after major surgery without effects on glucose and lipid metabolism.     

Immune cell functions, lipids and host natural resistance

Nutritional status may exert a profound effect on immune system functions. Hence, several parameters of immune system are modified by dietary lipid administration, as lymphocyte proliferation, cytokine production, natural killer activity, antigen presentation, etc. Thus, numerous studies have indicated the key role of lipids as immune response modulators. These properties have been applied in the treatment of autoimmune and inflammatory diseases. As a result, the reduction or suppression of immune status due to lipid incorporation promotes an impairment in the ability of host natural response to eliminate infectious microorganisms as bacteria or parasites. In the present review, we analyze the current status about the relationship among dietary lipids, reduction of immune parameters and reduction of host natural response against infectious diseases. Many discrepancies are discussed, although several studies indicate a close association between dietary lipid manipulation and impairment in the elimination of bacteria, viruses or parasites. On the other hand, other studies point out a beneficial effect of dietary lipid manipulation on the host natural response. Future investigations will determine the events involved in the regulation of immune response by fatty acids and their role in the elimination of pathogenic agents.     

Malondialdehyde epitopes as mediators of sterile inflammation

Enhanced lipid peroxidation occurs during oxidative stress and results in the generation of lipid peroxidation end products such as malondialdehyde (MDA), which can attach to autologous biomolecules, thereby generating neo-self epitopes capable of inducing potentially undesired biological responses. Therefore, the immune system has developed mechanisms to protect from MDA epitopes by binding and neutralizing them through both cellular and soluble effectors. Here, we briefly discuss innate immune responses targeting MDA epitopes and their pro-inflammatory properties, followed by a review of physiological carriers of MDA epitopes that are relevant in homeostasis and disease. Then we discuss in detail the evidence for cellular responses towards MDA epitopes mainly in lung, liver and the circulation as well as signal transduction mechanisms and receptors implicated in the response to MDA epitopes. Last, we hypothesize on the role of MDA epitopes as mediators of inflammation in diseases and speculate on their contribution to disease pathogenesis. This article is part of a Special Issue entitled: Lipid modification and lipid peroxidation products in innate immunity and inflammation edited by Christoph J. Binder.     

An Immunogenic Process Leading to Cancer in the Context of Immunodeficiency

Colorectal MSI tumors are associated with an increased immune response. On the other hand, immunodeficient patients are more prone to lymphomas with the MSI phenotype. These facts are of wide interest in understanding the complex link between immunity and cancer, particularly of the MSI phenotype. Specific attention to the host immune system should thus be considered in the treatment of patients with MSI tumors.