Class II MHC molecules are present in macrophage lysosomes and phagolysosomes that function in the phagocytic processing of Listeria monocytogenes for presentation to T cells.

Phagocytic processing of heat-killed Listeria monocytogenes by peritoneal macrophages resulted in degradation of these bacteria in phagolysosomal compartments and processing of bacterial antigens for presentation to T cells by class II MHC molecules. Within 20 min of uptake by macrophages, Listeria peptide antigens were expressed on surface class II MHC molecules, capable of stimulating Listeria-specific T cells. Within this period, degradation of labeled bacteria to acid-soluble low molecular weight catabolites also commenced. Immunoelectron microscopy was used to evaluate the compartments involved in this processing. Upon uptake of the bacteria, phagosomes containing Listeria fused rapidly with both lysosomes and endosomes. Class II MHC molecules were present in a tubulo-vesicular lysosome compartment, which appeared to fuse with phagosomes, as well as in the resulting phagolysosomes containing internalized Listeria; these compartments were all positive for Lamp 1 and cathepsin D and lacked 46-kD mannose-6-phosphate receptors. In addition, class II MHC and Lamp 1 were co-localized in vesicles of the trans Golgi reticulum, where they were segregated from 46-kD mannose-6-phosphate receptors. Vesicles containing both Listeria-derived components and class II MHC molecules were also observed; some of these may represent vesicles recycling from phagolysosomes, potentially bearing processed immunogenic peptides complexed with class II MHC. These results support a central role for lysosomes and phagolysosomes in the processing of bacterial antigens for presentation to T cells. Tubulo-vesicular lysosomes appear to represent an important convergence of endocytic, phagocytic and biosynthetic pathways, where antigens may be processed to allow binding to class II MHC molecules and recycling to the cell surface.     

Pathways for lipid antigen presentation by CD1 molecules: nowhere for intracellular pathogens to hide

A crucial feature of peptide antigen presentation by major histocompatibilty complex (MHC) class I and II molecules is their differential ability to sample cytosolic and extracellular antigens. Intracellular viral infections and bacteria that are taken up in phagosomes, but then escape from the endocytic compartment efficiently, enter the class I pathway via the cytosol. In contrast, phagosome-resident bacteria yield protein antigens that are sampled deep in the endocytic compartment and presented in a vacuolar acidification-dependent pathway mediated by MHC class II molecules. Despite this potential for antigen sampling, microbes have evolved a variety of evasive mechanisms that affect peptide transport in the MHC class I pathway or blockade of endosomal acidification and inhibition of phagosome–lysosome fusion that may compromise the MHC class II pathway of antigen presentation. Thus, besides MHC class I and II, a third lineage of antigen-presenting molecules that bind lipid and glycolipid antigens rather than peptides exists and is mediated by the family of CD1 proteins. CD1 isoforms (CD1a, b, c, and d) differentially sample both recycling endosomes of the early endocytic system and late endosomes and lysosomes to which lipid antigens are differentially delivered. These CD1 pathways include vacuolar acidification-independent pathways for lipid antigen presentation. These features of presenting lipid antigens, independently monitoring various antigen-containing intracellular compartments and avoiding certain evasive techniques employed by microbes, enable CD1 molecules to provide distinct opportunities to function in host defense against the microbial world.     

Presentation of Phagocytosed Antigens by MHC Class I and II

Phagocytosis provides innate immune cells with a mechanism to take up and destroy pathogenic bacteria, apoptotic cells and other large particles. In some cases, however, peptide antigens from these particles are preserved for presentation in association with major histocompatibility complex (MHC) class I or class II molecules in order to stimulate antigen‐specific T cells. Processing and presentation of antigens from phagosomes presents a number of distinct challenges relative to antigens internalized by other means; while bacterial antigens were among the first discovered to be presented to T cells, analyses of the cellular mechanisms by which peptides from phagocytosed antigens assemble with MHC molecules and by which these complexes are then expressed at the plasma membrane have lagged behind those of conventional model soluble antigens. In this review, we cover recent advances in our understanding of these processes, including the unique cross‐presentation of phagocytosed antigens by MHC class I molecules, and in their control by signaling modalities in phagocytic cells.     

The role of host immune cells and <Emphasis Type="Italic">Borrelia burgdorferi</Emphasis> antigens in the etiology of Lyme disease

Lyme disease is a zoonosis caused by infection with bacteria belonging to the Borrelia burgdorferi species after the bite of an infected tick. Even though an infection by this bacterium can be effectively treated with antibiotics, when the infection stays unnoticed B. burgdorferi can persist and chronic post-treatment Lyme disease syndrome is able to develop. Although a cellular and humoral response is observed after an infection with the Borrelia bacteria, these pathogens are still capable to stay alive. Several immune evasive mechanisms have been revealed and explained and much work has been put into the understanding of the contribution of the innate and adaptive immune response. This review provides an overview with the latest findings regarding the cells of the innate and adaptive immune systems, how they recognize contribute and mediate in the killing of the B. burgdorferi spirochete. Moreover, this review also elaborates on the antigens that are expressed by on the spirochete. Since antigens drive the adaptive and, indirectly, the innate response, this review will discuss briefly the most important antigens that are described to date. Finally, there will be a brief elaboration on the escape mechanisms of B. burgdorferi with a focus on tick salivary proteins and spirochete antigens.     

Live, attenuated strains of Listeria and Salmonella as vaccine vectors in cancer treatment

Live, attenuated strains of many bacteria that synthesize and secrete foreign antigens are being developed as vaccines for a number of infectious diseases and cancer. Bacterial-based vaccines provide a number of advantages over other antigen delivery strategies including low cost of production, the absence of animal products, genetic stability and safety. In addition, bacterial vaccines delivering a tumor-associated antigen (TAA) stimulate innate immunity and also activate both arms of the adaptive immune system by which they exert efficacious anti-tumor effects. Listeria monocytogenes and several strains of Salmonella have been most extensively studied for this purpose. A number of attenuated strains have been generated and used to deliver antigens associated with infectious diseases and cancer. Although both bacteria are intracellular, the immune responses invoked by Listeria and Salmonella are different due to their sub-cellular locations. Upon entering antigen-presenting cells by phagocytosis, Listeria is capable of escaping from the phagosomal compartment and thus has direct access to the cell cytosol. Proteins delivered by this vector behave as endogenous antigens, are presented on the cell surface in the context of MHC class I molecules, and generate strong cell-mediated immune responses. In contrast, proteins delivered by Salmonella, which lacks a phagosomal escape mechanism, are treated as exogenous antigens and presented by MHC class II molecules resulting predominantly in Th2 type immune responses. This fundamental disparity between the life cycles of the two vectors accounts for their differential application as antigen delivery vehicles. The present paper includes a review of the most recent advances in the development of these two bacterial vectors for treatment of cancer. Similarities and differences between the two vectors are discussed.     

Identification of major histocompatibility complex class II-restricted antigens and epitopes of the Epstein-Barr virus by a novel bacterial expression cloning approach

Epstein-Barr virus (EBV)-specific T cells have been successfully used to treat or prevent EBV-positive lymphoproliferative disease in hematopoietic stem cell transplant recipients, but the antigens recognized by the infused CD4+ T cells have remained unknown. Here, we describe a simple procedure that permits the identification of viral T-helper (TH)-cell antigens and epitopes. This direct antigen identification method is based on the random expression of viral polypeptides fused to chloramphenicol acetyltransferase (CAT) in bacteria, which are subsequently fed to major histocompatibility complex class II+ antigen-presenting cells and probed with antigen-specific T cells. The fusion of antigenic fragments to CAT offers several advantages. First, chloramphenicol treatment allows the selection of bacteria expressing antigen-CAT fusion proteins in frame, which greatly reduces the number of colonies to be screened. Second, antigenic fragments fused to CAT are expressed at high levels, even when derived from proteins that are toxic to bacteria. Third, the uniformly high expression level of antigen-CAT fusion proteins permits the establishment of large and representative pool sizes. Finally, antigen identification does not require knowledge of the restriction element and often leads directly to the identification of the T-cell epitope. Using this approach, the BALF4 and BNRF1 proteins were identified as targets of the EBV-specific T-helper-cell response, demonstrating that lytic cycle antigens are a relevant component of the EBV-specific TH-cell response.     

Immune surveillance of intracellular pathogens via autophagy

MHC class II molecules are thought to present peptides derived from extracellular proteins to CD4+ T cells, which are important mediators of adaptive immunity to infections. In contrast, autophagy delivers constitutively cytosolic material for lysosomal degradation and has so far been recognized as an efficient mechanism of innate immunity against bacteria and viruses. Recent studies, however, link these two pathways and suggest that intracellular cytosolic and nuclear antigens are processed for MHC class II presentation after autophagy.     

Serological Specificity of the Lipopolysaccharides, the Major Antigens of Pseudomonas syringae

The nature of major antigens of Pseudomonas syringae was studied on one strain of four pathovars (pvs aptata, mors-prunorum, phaseolicola and tabaci) belonging to four separate serogroups. Bacterial antigens were prepared by 4 procedures: extraction by phenol-water (PW), by citrate-NaCl (CN), by trichloracetic acid (TCA), and precipitation of a glycoproteic extracellular complex (GP). 3-Deoxy-2-octulosonic acid (KDO) revelation in all the extracts showed that the four procedures led to antigens containing similar amounts of lipopolysaccharide (LPS). Twenty polyclonal antisera were raised in rabbits against whole bacteria and the different extracts. Serological reactions were tested by gel double diffusion (DD) and indirect immunofluorescent staining (IF). The anti-whole cell sera were shown to contain mostly anti-LPS antibodies. For each pathovar, whole bacteria used as antigens in DD gave precipitation bands identical to the bands given by the LPS extracts (PW, CN or TCA), identical to the heated bacteria (HB), and identical to LPS sidechain preparations. The GP extract itself was shown to be rich in LPS. To serotype P. syringae, it is advised to raise antisera against either whole bacteria or GP extracts; whereas the reacting antigens for DD would be heated bacteria.     

Delivery of protein antigens and DNA by virulence-attenuated strains of Salmonella typhimurium and Listeria monocytogenes

Two different plasmid-vector systems were developed which allow the efficient production and presentation of protein antigens in antigen-presenting cells (APC) by means of virulence-attenuated bacteria. The first antigen-delivery system is based on the secretion machinery of the Escherichia coli hemolysin (HlyA-type I secretion system), which transports proteins, possessing the specific HlyA secretion signal (HlyA(s)) at the C-terminus, across both membranes of gram-negative bacteria. This system functions in all gram-negative bacteria that possess the TolC-analogous protein in the outer membrane. This outer membrane protein is necessary for the stable anchoring of the type I secretion apparatus in the cell envelope. Suitable HlyA(s)-fused antigens are secreted with high efficiency by E. coli and by virulence-attenuated strains of Salmonella, Shigella, Vibrio cholerae and Yersinia enterocolitica. The other vector system expresses the heterologous antigen under the control of an eukaryotic promoter in a similar fashion as in plasmids commonly used for vaccination with naked DNA. This plasmid DNA is introduced into APCs with the help of virulence-attenuated self-destructing Listeria monocytogenes mutants. After synthesis of the heterologous protein, epitopes of the antigen are presented by the APC together with MHC class I molecules. This system functions in macrophages and dendritic cells in vitro and can also be used in a modified form in animal models.     

Vaccine strategies against latent tuberculosis infection

The leading tuberculosis (TB) vaccines currently in clinical trials are all designed as prophylactic vaccines. Although these vaccines are highly active, they will most probably not result in sterilizing immunity and, therefore, will not solve the global problem of latent TB. An attractive strategy is to target the remaining dormant bacteria with vaccines based upon antigens induced as the bacteria change from active multiplication to non-replicating dormancy (latency antigens) or during reactivation as dormant bacteria resume active metabolism (resuscitation antigens). These late-stage antigens might have potential as post-exposure vaccines or could form the basis for a multi-stage vaccine strategy, in which they are combined with prophylactic vaccines based on early antigens from replicating bacteria.     

The presence and possible significance of cross-reactive antigens inRhizobium — legume associations

Soluble antigens of threeRhizobium species were compared by the agar-gel double-diffusion technique with those of eight legumes representing compatible and non-compatible hosts. Cross-reactive antigens were found between all the legume hosts and the three rhizobia. These common antigens among hosts and bacteria were not related to the specificity of compatibleRhizobium-legume associations. The cross-reactive antigens were absent between rhizobia and eight non-legume plants tested, but present between five out of eleven gramnegative phytopathogenic bacteria and legumes. The potential significance of cross-reactive antigens betweenRhizobium and legume hosts in nodule development is discussed.     

Serological relationship between three varieties of Bacillus popilliae

The serological relationship between three varieties of Bacillus popilliae was investigated by double diffusion and immunoelectrophoresis, using antigens prepared from homogenized vegetative cells in the logarithmic growth phase. The results showed the existence of a close serological relationship among the three varieties in agreement with a recently proposed taxonomic scheme for the milky disease bacteria (Wyss, 1971).     

Analysis of bacterial DNA in synovial tissue of Tunisian patients with reactive and undifferentiated arthritis by broad-range PCR, cloning and sequencing

Introduction

Bacteria and/or their antigens have been implicated in the pathogenesis of reactive arthritis (ReA). Several studies have reported the presence of bacterial antigens and nucleic acids of bacteria other than those specified by diagnostic criteria for ReA in joint specimens from patients with ReA and various arthritides. The present study was conducted to detect any bacterial DNA and identify bacterial species that are present in the synovial tissue of Tunisian patients with reactive arthritis and undifferentiated arthritis (UA) using PCR, cloning and sequencing.

Methods

We examined synovial tissue samples from 28 patients: six patients with ReA and nine with UA, and a control group consisting of seven patients with rheumatoid arthritis and six with osteoarthritis (OA). Using broad-range bacterial PCR producing a 1,400-base-pair fragment from the 16S rRNA gene, at least 24 clones were sequenced for each synovial tissue sample. To identify the corresponding bacteria, DNA sequences were compared with sequences from the EMBL (European Molecular Biology Laboratory) database.

Results

Bacterial DNA was detected in 75% of the 28 synovial tissue samples. DNA from 68 various bacterial species were found in ReA and UA samples, whereas DNA from 12 bacteria were detected in control group samples. Most of the bacterial DNAs detected were from skin or intestinal bacteria. DNA from bacteria known to trigger ReA, such as Shigella flexneri and Shigella sonnei, were detected in ReA and UA samples of synovial tissue and not in control samples. DNA from various bacterial species detected in this study have not previously been found in synovial samples.

Conclusion

This study is the first to use broad-range PCR targeting the full 16S rRNA gene for detection of bacterial DNA in synovial tissue. We detected DNA from a wide spectrum of bacterial species, including those known to be involved in ReA and others not previously associated with ReA or related arthritis. The pathogenic significance of some of these intrasynovial bacterial DNAs remains unclear.     

Salmonella infection of bone marrow-derived macrophages and dendritic cells: influence on antigen presentation and initiating an immune response

Traditionally macrophages (MPhi) have been considered to be the key type of antigen presenting cells (APC) to combat bacterial infections by phagocytosing and destroying bacteria and presenting bacteria-derived antigens to T cells. However, data in recent years have demonstrated that dendritic cells (DC), at their immature stage of differentiation, are capable of phagocytosing particulate antigens including bacteria. Thus, DC may also be important APC for initiating an immune response to bacterial infections. Our studies focus on studying how DC and MPhi process antigens derived from bacteria with no known mechanism of phagosomal escape (i.e. Salmonella typhimurium) for T cell stimulation as well as what role these APC types have in Salmonella infection in vivo. Using an in vitro antigen processing and presentation assay with bone marrow-derived (BM) APC showed that, in addition to peritoneal elicited MPhi and BMMPhi, BMDC can phagocytose and process Escherichia coli and S. typhimurium for peptide presentation on major histocompatibility complex (MHC) class I (MHC-I) and class II MHC-II. These studies showed that both elicited peritoneal MPhi and BMMPhi use an alternate MHC-I presentation pathway that does not require the transporter associated with antigen processing (TAP) or the proteasome and involves peptide loading onto a preformed pool of post-Golgi MHC-I molecules. In contrast, DC process E. coli and S. typhimurium for peptide presentation on MHC-I using the cytosolic MHC-I presentation pathway that requires TAP, the proteasome and uses newly synthesized MHC-I molecules. We further investigated the interaction of Salmonella with BMDC and BMMPhi by analyzing surface molecule expression and cytokine secretion following S. typhimurium infection of BMDC and BMMPhi. These data reveal that Salmonella co-incubation with BMDC as well as BMMPhi results in upregulation of MHC-I and MHC-II as well as several co-stimulatory molecules including CD80 and CD86. Salmonella infection of BMDC or BMMPhi also results in secretion of cytokines including IL-6 and IL-12. Finally, injecting mice with BMDC that have been loaded in vitro with S. typhimurium primes na?ve CD4(+) and CD8(+) T cells to Salmonella-encoded antigens. Taken together, our data suggest that DC may be an important type of APC that contributes to the immune response to Salmonella.     

Lysis of cells infected with typhus group rickettsiae by a human cytotoxic T cell clone

Cytolytic human T cell clones generated in response to the intracellular bacterium Rickettsia typhi were characterized. Growing clones were tested for their ability to proliferate specifically in response to antigens derived from typhus group rickettsiae or to lyse targets infected with R. typhi or Rickettsia prowazekii. Two clones were able to lyse targets infected with typhus group rickettsiae. One of these clones was more fully characterized because of its rapid growth characteristics. This cytolytic clone was capable of lysing an autologous infected target as well as a target matched for class I and II histocompatibility leukocyte antigens (HLA). It was not capable, however, of lysing either a target mismatched for both class I and II HLA or a target partially matched for class I HLA. In addition, the clone exhibited specificity in that it was able to lyse an autologous target infected with typhus group rickettsiae, but did not lyse an autologous target infected with an antigenically distinct rickettsial species, Rickettsia tsutsugamushi. These results demonstrate, for the first time, that cells infected with intracellular bacteria can be lysed by human cytotoxic T lymphocytes.     

Immunoelectron microscopy investigation of the cell surface of <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> strains

The genus-specific surface protein antigens of Azospirillum brasilense strains were visualized immunochemically. The procedure used for cell sample preparation was optimized to ensure that the surface protein structures were detected on cells in situ. Gold and gold-silver nanoparticles were conjugated to antibodies raised against the flagellin of A. brasilense type strain Sp7, against the lipopolysaccharide of A. brasilense Sp245, and against the genus-specific protein determinants of A. brasilense Sp7. Electron microscopic analysis using nanoparticle-labeled antibodies revealed antigenic determinants of the polar flagellum on the A. brasilense Sp245 cell surface, which in these bacteria are normally screened from the surroundings by a lipopolysaccharide sheath. Pili-like structures were detected on the Sp245 wild-type strain and on its Fla^– Swa^– Omegon-Km mutant SK048, which are presumably involved in microcolonial spreading in these bacteria.     

Biochemical evidence that equine leucocyte antigens W13, W22 and W23 are present on horse major histocompatibility complex class II molecules

A number of horse alloantisera were characterized biochemically as being directed against MHC class I or class II antigens by immunoprecipitation of the corresponding antigens from lysates of biosynthetically radioactively labelled lymphocytes and determination of their molecular weights by SDS-PAGE and fluorography. Sera recognizing A2 and A3 specificities precipitated antigens of 44,000 Daltons molecular weight (class I heavy chain), whereas sera with specificities W13, W22 and W23 precipitated antigens corresponding to class II dimers (30,000 and 32,000 Daltons). Comparison with antigens precipitated from horse lymphocyte lysates using (cross-reacting) antibodies to human class I and class II MHC molecules confirmed the results obtained.     

Selection of cell wall antigens for the rapid detection of bacteria by immunological methods.

Some proteins extracted from the cell wall of Gram-positive and Gram-negative bacteria that affect the time for which meat can be stored are antigens produced by the strains from our laboratory collection or from field samples. Moreover, the rapid detection of bacteria (within 8 h for Gram-positive or 5 h for Gram-negative) that influence the quality of meat is made possible by immunochemical techniques such as ELISA or by introducing simultaneous detection of these antigens, as no cross-reactions have been observed.     

Serospecific antigens of Legionella pneumophila.

Serospecific antigens isolated by EDTA extraction from four serogroups of Legionella pneumophila were analyzed for their chemical composition, molecular heterogeneity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and immunological properties. The antigens were shown to be lipopolysaccharides and to differ from the lipopolysaccharides of other gram-negative bacteria. The serospecific antigens contained rhamnose, mannose, glucosamine, and two unidentified sugars together with 2-keto-3-deoxyoctonate, phosphate, and fatty acids. The fatty acid composition was predominantly branched-chain acids with smaller amounts of 3-hydroxymyristic acid. The antigens contain periodate-sensitive groups; mannosyl residues were completely cleaved by periodate oxidation. Hydrolysis of the total lipopolysaccharide by acetic acid resulted in the separation of a lipid A-like material that cross-reacted with the antiserum to lipid A from Salmonella minnesota but did not comigrate with it on sodium dodecyl sulfate gels. None of the four antigens contained heptose. All of the antigen preparations showed endotoxicity when tested by the Limulus amebocyte lysate assay. The results of this study indicate that the serogroup-specific antigens of L. pneumophila are lipopolysaccharides containing an unusual lipid A and core structure and different from those of other gram-negative bacteria.     

Modulation of the surface antigens of Salmonella-phagocytized U937 cells]

In this study, modulation of the surface antigens of Salmonella enteritidis-phagocytized U937 cells and morphology of the bacteria in these cells were analyzed by the indirect immunofluorescence technique. The results are as follows: (1) Morphological studies revealed that the bacteria phagocytized by the U937 cells were transformed to a small coccoid form. (2) The expression of CD14 antigen was observed 24 to 48 h after phagocytosis. (3) The levels of CD11b and CD23 antigens were clearly enhanced 48 h after phagocytosis. (4) No modulation of HLA-class II (DR, DQ and DP) antigens was observed after phagocytosis.