Hypersialylated type-I lactosamine-containing N-glycans found in Artiodactyla sera are potential xenoantigens.

There is increasing interest in biologics, i.e. human-originated biological pharmaceutics. Most of the protein drugs developed so far, such as immunoglobulins and erythropoietin, are secreted glycoproteins; as a result, any non-human-type glycans, such as αGal and NeuGc, derived from animal cells and sera must be removed to circumvent undesirable immunogenic reactions. In this study, we made an extensive search for potential xenoantigenic glycans among a panel of mammalian sera. As a result, sera belonging to the order Artiodactyla, i.e. bovine, lamb and goat sera, were found to contain substantial amounts of hypersialylated biantennary glycans closely associated with a type-I lactosamine structure containing a unique tetrasaccharide, Siaα2-3Galß1-3(Siaα2-6)GlcNAc. In all three Artiodactyla sera, the most abundant structure was Siaα2-3Galß1-3(Siaα2-6)GlcNAcß1-2Manα1-3[Siaα2-6Galß1-4GlcNAcß1-2Manα1-6]Manß1-4GlcNAcß1-4GlcNAc. A dually hypersialylated biantennary structure, Siaα2-3Galß1-3(Siaα2-6)GlcNAcß1-2Manα1-3[Siaα2-3Galß1-3(Siaα2-6)GlcNAcß1-2Manα1-6]Manß1-4GlcNAcß1-4GlcNAc, was also abundant (10%) in bovine serum. The amount of hypersialylated glycans among total sialylated glycans was 46, 26 and 23% in bovine, lamb and goat sera, respectively. On the other hand, such structures could not be detected in the sera of other animals including human. The biological functions and the immunogenicity of the hypersialylated glycans in these animals remain to be elucidated; however, it is worth noting that glycoproteins biosynthesized from Artiodactyla cells and those contaminated with bovine serum might enhance undesirable antigenicity in human patients.

IgA detection in human neurocysticercosis using different preparations of heterologous antigen.

Neurocysticercosis (NC) is the most important neurological disease of parasitic origin in humans. IgA and IgG detection in serum from neurocysticercosis patients was tested using some antigenic preparations of total saline extract from Taenia saginata: detergent (D) and aqueous (A) phases extracted with Triton X-114 and the jacalin bound (JBF) and unbound fractions (JUF) obtained by affinity chromatography using jacalin column. Samples were obtained from 45 patients with definitive NC, who were subdivided into active-NC group and inactive-NC group; 35 patients with other parasitoses; and 30 apparently healthy individuals. Sensitivity and specificity were calculated. Specificity to detect IgA and IgG for D phase, respectively, were 89.8% and 86.9% and for IgG detection 91.3% and 76.8% when using D phase and JUF, respectively. D phase and JBF proved to be specific and efficient and could be efficiently utilized as an alternative antigen for IgA detection in NC, with comparable results with IgG.

IgG1, IgG4, and IgE antibody responses in human strongyloidiasis by ELISA using Strongyloides ratti saline extract as heterologous antigen.

The aim of this study was to evaluate total IgG, IgG1, IgG4, and IgE antibody responses in human strongyloidiasis by enzyme-linked immunosorbent assay (ELISA) using Strongyloides ratti saline extract as heterologous antigen for a possible clinical utility of the assay. A total of 40 serum samples of patients who were shedding Strongyloides stercoralis larvae in feces (group I), 30 sera from patients with other intestinal parasites (group II), and 30 sera from subjects with negative results in three parasitological assays (group III) were analyzed to detect total IgG, IgG1, IgG4, and IgE to Strongyloides spp. by ELISA and expressed in ELISA index. Levels of total IgG anti-Strongyloides spp. were significantly higher in patients of group I than in groups II (p=0.0005) and III (p<0.0001). Levels of specific IgG1, IgG4, and IgE of group I were also significantly higher than in groups II and III, respectively. There was a significant positive correlation between specific IgE and IgG4 (r=0.6524; p=0.0084) and IgG1 and IgG4 (r=0.5398; p=0.0171). It can be concluded that the detection of specific IgE, IgG1, and IgG4 subclasses rather than total IgG antibodies to Strongyloides spp. using the S. ratti antigen showed to be an additional tool for improving the serodiagnosis of human strongyloidiasis.

Presence and elimination of the xenoantigen gal (alpha1, 3) gal in tissue-engineered heart valves.

Tissue engineering of heart valves promises to create functional autologous tissue with the potential for regeneration and growth without the limitations of current heart valve prostheses. The appropriate valve matrix is essential. Porcine heart valves are attractive because of their anatomical similarity. Decellularization is used for antigen reduction. The efficacy of published protocols varies, however. The absence of a specific immunological or unspecific inflammatory reaction is mandatory. The porcine cell-specific alpha-Gal epitope is known to be responsible for hyperacute rejection in xenotransplantation. In tissue engineering residual alpha-Gal epitope may induce severe inflammation in humans and may lead to graft failure. In this study porcine pulmonary conduits were decellularized with Triton X-100, sodium deoxycholate, Igepal CA-630, and ribonuclease treatment and were compared with specimens of the commercially available porcine decellularized SynerGraft regarding cell removal and elimination of the alpha-Gal epitope. In addition, samples of a porcine bioprosthesis were examined for the presence of the alpha-Gal epitope. In conclusion, we describe for the first time the presence of the alpha-Gal epitope in clinically used porcine bioprostheses and the first generation of a commercial tissue-engineered heart valve. In contrast, complete cell and alpha-Gal removal was achieved by a decellularization procedure developed by our group.

Decellularization of bovine pericardium for tissue-engineering by targeted removal of xenoantigens.

BACKGROUND AND AIM OF STUDY: Assessment of decellularization of xenogeneic biological scaffolds for tissue engineering has relied primarily on histological cellularity, though this may not ensure the removal of known xenogeneic antigens such as galactose-alpha1,3-galactose (alpha-gal) and MHC I. METHODS: Bovine pericardium (BP) underwent standard (Std) decellularization consisting of hypotonic lysis and treatment with DNAse/RNAse. In addition to Std decellularization, tissues were treated for 24 h with either 0.5% Triton X-100, 0.5% sodium deoxycholate (SD), 0.1% sodium dodecyl sulfate (SDS), alpha-galactosidase (5 U/ml) or phospholipase (PL) A2 (150 U/ml). Tissues underwent a 96-h washout under gentle agitation at 27 degrees C, and then evaluated by light microscopy for % cellularity, and by immunohistochemistry and Western blot for alpha-gal, bovine MHC I and smooth muscle alpha-actin. RESULTS: Standard treatment of BP resulted in only partial removal histological cellularity and persistence of alpha-gal, MHC I and alpha-actin. Adding SD treatment resulted in apparent acellularity, but persistence of xenogeneic antigens. Only the addition of SDS resulted in complete histological acellularity and removal of xenogeneic antigens. Treatment with alpha-galactosidase selectively removed alpha-gal from BP. CONCLUSION: Histological cellularity is not an adequate end-point for assuring removal of antigenicity from xenogeneic biological scaffolds. However, known xenogeneic antigens can be targeted for removal by novel decellularization treatments such as alpha-galactosidase.

Bacillus subtilis as a tool for vaccine development: from antigen factories to delivery vectors.

Bacillus subtilis and some of its close relatives have a long history of industrial and biotechnological applications. Search for antigen expression systems based on recombinant B. subtilis strains sounds attractive both by the extensive genetic knowledge and the lack of an outer membrane, which simplify the secretion and purification of heterologous proteins. More recently, genetically modified B. subtilis spores have been described as indestructible delivery vehicles for vaccine antigens. Nonetheless both production and delivery of antigens by B. subtilis strains face some inherent obstacles, as unstable gene expression and reduced immunogenicity that, otherwise, can be overcome by already available gene technology approaches. In the present review we present the status of B. subtilis-based vaccine research, either as protein factories or delivery vectors, and discuss some alternatives for a better use of genetically modified strains.

Peach profilin: cloning, heterologous expression and cross-reactivity with Bet v 2.

BACKGROUND: Peach is among the main foods causing allergic reactions in the Mediterranean adult population. Only a single peach allergen, named Pru p 3, has been characterized. However, a potential role of profilin has also been suggested in grass pollen-associated allergy to peach. METHODS: Complementary DNA clones for two different peach profilin isoforms were obtained by reverse transcriptase polymerase chain reaction using non-degenerated primers. Expression of recombinant peach profilin was performed in Escherichia coli, and confirmed using rabbit polyclonal antibodies to sunflower pollen profilin. Twenty-nine individual sera from patients with peach allergy proved by double-blind, placebo-controlled food challenges (DBPCFC), either with (n = 15) or without (n = 14) specific IgE to Bet v 2, were used in immunodetection assays to test recombinant peach profilin reactivity. RESULTS: Each peach profilin cDNA included an open reading frame coding for a 131 amino acid protein. The peach profilin isoforms, designated Pru p 4.01 and Pru p 4.02, showed 80% of amino acid sequence identity, and were very similar (>70% identity) to allergenic profilins from plant foods and pollens. Recombinant Pru p 4.01 was expressed in E. coli as a nonfusion protein, displaying the expected molecular size and reacting with anti-profilin antibodies. rPru p 4.01 was recognized by all sera (15 of 15) with specific IgE to Bet v 2, whereas no sera (zero of 14) without IgE to this birch allergen reacted with rPru p 4.01. CONCLUSIONS: Peach profilin Pru p 4 is very closed to other allergenic profilins from plant foods and pollens. A complete correlation between reactivity to rPru p 4 and rBet v 2 has been found in sera from peach allergic patients.

Heterologous antigen extract in ELISA for the detection of human IgE anti-Strongyloides stercoralis.

Strongyloides ratti larval extract was used for the standardization of ELISA to detect genus-specific IgE in human strongyloidiasis. Forty serum samples from monoinfected patients shedding S. stercoralis larvae (Group I), 40 from patients with other intestinal parasites (Group II), and 40 from copronegative healthy subjects (Group III) were analyzed. Genus-specific IgE levels (ELISA Index: EI) were significantly higher in the group I (EI = 1.43) than groups II (EI = 0.70) and III (EI = 0.71), showing positivity rates of 55%, 2.5% and 0%, respectively. Similarly, sera from copropositive patients had significantly higher levels of total IgE (866 IU/mL) as compared to those from group II (302 IU/mL) and III (143 IU/mL). A significant positive correlation was found between levels of Strongyloides specific-IgE and total IgE in sera from patients with strongyloidiasis. In conclusion, S. ratti heterologous extract showed to be a useful tool for detecting genus-specific IgE by ELISA, contributing for a better characterization of the immune response profile in human strongyloidiasis.

[A comparative study of the nonhistone chromosomal proteins (pp23) from rat and bull kidneys]. / Sravnitel'noe izuchenie khromosomnykh negistonovykh belkov (pp23) iz pochek krysy i byka.

The bovine nonhistone protein pp23 differs from that of rat origin both immunologically (precipitation, specific interaction with monoclonal antibodies to rat pp23) and functionally (capability to stimulate expression of hetero-organic antigens on the membrane of intact hepatocytes cultured in suspension). These differences, however, are not recognized in the experiments on stimulation of DNA synthesis in Swiss 3T3 cell resting culture, when a more general cell function (proliferation) is dealt with, rather than a narrow specific one, such as synthesis of tumor-associated hetero-organic antigen of kidney origin.

Comparison of purified 12 kDa and recombinant 15 kDa Fasciola hepatica antigens related to a Schistosoma mansoni fatty acid binding protein

Vaccines in schistosomiasis using homologous antigens have been studied extensively in experimentally infected mammalian hosts. Vaccines using heterologous antigens have received comparatively less attention. This review summarizes recent work on a heterologous 12 kDa Fasciola hepatica antigenic polypeptide which cross reacts with Schistosoma mansoni. A cDNA has been cloned and sequenced, and the predicted amino acid sequence of the recombinant protein has been shown to have significant (44) identity with a 14 kDa S. mansoni fatty acid binding protein. Thus in the parasitic trematodes fatty acid binding proteins may be potential vaccine candidates. The F. hepatica recombinant protein has been overexpressed and purified and denoted rFh15. Preliminary rFh15 migrates more slowly (i.e. may be slightly larger) than nFh12 on SDS-PAGE and has a predicted pI of 6.01 vs. observed pI of 5.45. Mice infected with F. hepatica develop antibodies to nFh12 by 2 weeks of infection vs. 6 weeks of infection to rFh15; on the other hand, mice with schistosomiasis mansoni develop antibodies to both nFh12 and rFh15 by 6 weeks of infection. Both the F. hepatica and S. mansoni cross-reactive antigens may be cross-protective antigens with the protection inducing capability against both species.

Comparison of purified 12 kDa and recombinant 15 kDa Fasciola hepatica antigens related to a Schistosoma mansoni fatty acid binding protein.

Vaccines in schistosomiasis using homologous antigens have been studied extensively in experimentally infected mammalian hosts. Vaccines using heterologous antigens have received comparatively less attention. This review summarizes recent work on a heterologous 12 kDa Fasciola hepatica antigenic polypeptide which cross reacts with Schistosoma mansoni. A cDNA has been cloned and sequenced, and the predicted amino acid sequence of the recombinant protein has been shown to have significant (44%) identity with a 14 kDa S. mansoni fatty acid binding protein. Thus in the parasitic trematodes fatty acid binding proteins may be potential vaccine candidates. The F. hepatica recombinant protein has been overexpressed and purified and denoted rFh15. Preliminary rFh15 migrates more slowly (i.e. may be slightly larger) than nFh12 on SDS-PAGE and has a predicted pI of 6.01 vs. observed pI of 5.45. Mice infected with F. hepatica develop antibodies to nFh12 by 2 weeks of infection vs. 6 weeks of infection to rFh15; on the other hand, mice with schistosomiasis mansoni develop antibodies to both nFh12 and rFh15 by 6 weeks of infection. Both the F. hepatica and S. mansoni cross-reactive antigens may be cross-protective antigens with the protection inducing capability against both species.

[The isolation and characteristics of a protein from human and bovine kidneys close in molecular and biochemical properties to non-histone chromosomal antigen PP23]. / Vydelenie i kharakteristika belka iz pochek cheloveka i byka, blizkogo po molekuliarno-biokhimicheskim svoistvam NGB-antigenu PP23.

By ion-exchange gradient chromatography, autophosphorylated protein fractions were separated from nonhistone chromosomal protein (NHCP) samples isolated from bovine and human kidney and liver. Fractions, eluted from the phosphocellulose column with 0.4-0.5 M NaCl, could occur only in the case of kidney rather than liver NHCP-fractionation. The molecular weight of the major phosphorylated protein component, eluted in this zone, is about 20-26 kDa, which is close to the molecular weight of protein pp23, previously observed during similar fractionation of NHCP from rat kidney rather than from liver.

Carbohydrate antigens of pig tissues reacting with human natural antibodies as potential targets for hyperacute vascular rejection in pig-to-man organ xenotransplantation.

Pig tissues were screened by immunofluorescence with lectins, mAb, and human natural antibodies for the presence of carbohydrate antigens, which may be potential targets for hyperacute vascular rejection in pig to man xenotransplantation. The unfucosylated monomorph linear B-antigen was found at the surface of all porcine vascular endothelial cells. This pig linear-B antigen reacts strongly with the anti-alpha Gal isolectin B4 from Griffonia simplicifolia 1 and with human natural anti-alpha Gal antibodies specifically purified by affinity chromatography on synthetic oligosaccharides containing the terminal nonreducing alpha Gal1-->3 beta Gal-R disaccharide. This antigenic activity is destroyed by treatment of pig tissues with alpha-galactosidase. The localization of this linear-B epitope on vascular endothelium and its reactivity with natural human anti-alpha Gal antibodies suggest that it may play a major role in the hyperacute vascular rejection of pig to man organ xenografts. The lectin from Maackia amurensis reacting with alpha NeuAc2-->3 beta Gal1-->4GlcNAc/Glc was also positive on pig vascular endothelium, but we do not know yet whether there are human natural antibodies reacting with the carbohydrate recognized by this lectin. Epithelial cells of pig renal proximal convoluted tubules, respiratory epithelium, pancreatic ducts, and epidermis express the linear-B antigen, but they are less likely to trigger a hyperacute vascular rejection because they are not directly exposed to the blood. The genetically defined pig A+/A- system controls the expression of A and H antigens in pig epithelial cells from renal distal and collecting tubules, biliary ducts, pancreatic ducts, large bronchi, and digestive mucosa. The pig A antigen may trigger an immune response in human O or B recipients if they are transplanted with organs from A+ pigs, but the pig A antigen is probably not involved in the hyperacute vascular rejection of a xenograft because it is not expressed on vascular endothelium.

[Heterophile infectious mononucleosis antigen used in the latex diagnostic test. I. A method of antigen isolation and its serologic activity]. / Heterofilny antygen mononukleozowy w zastosowaniu do lateksowego testu diagnostycznego. I. Metodyka uzyskiwania antygenu i jego serologiczna aktywnosc.

The aim of this study was to elaborate a method of heterophile mononucleosis antigen preparation useful for latex coating. This antigen was isolated from bovine red blood cells stroma by the technique of Schwarzweiss and Tomcsik with author's own modification, in which introductory extraction of erythrocytes stroma ++ was performed by means of trichloracetic acid, aqueous extraction and elution of active substance with 80% ethanol. Besides of heterophile antigen preparation obtained by the method of Schwerzweiss and Tomcsik (preparation S-T) two serologically++ active preparations were obtained (fraction I and IV), which ability to inhibit PBD agglutinating reaction and bovine red blood cells haemolysis was 16 and 8 times lower, respectively, than S-T preparation. The preparation of heterophile mononucleosis antigen obtained differed in latex coating efficacy. In order to prepare latex reagent MZ-I (from fraction I) a solution of preparation of 125 micrograms/ml concentration was used, for MZ-II (from fraction IV)--50 micrograms and for MZ-III (from preparation S-T)--15 micrograms/ml. The reagent MZ-I showed, the highest activity in agglutinating test with human serum containing heterophile mononucleosis antibodies while two others reacted with 2-4 times lover serum dilutions. Similar differentiated reactivity with these reagents was found in latex test with 15 sera from patients suspected of having infectious mononucleosis.

Heterogeneity of the Ro/SSA antigen. Different molecular forms in lymphocytes and red blood cells.

Ro(SSA) is an intracellular ribonucleoprotein against which autoantibodies are found in a portion of patients with Sjögren's syndrome and systemic lupus erythematosus. A form of Ro(SSA) is described in red blood cells that shares a line of identity with purified Ro(SSA) from bovine spleen and human lymphocytes in counterimmunoelectrophoresis, but has different molecular properties. Ro(SSA) from red blood cells exists in association with only two small RNAs as opposed to four in other cell types, as determined by RNA extraction of protein A-assisted immunoprecipitates. In addition to the common 60-kD Ro(SSA) protein, Western blot analysis revealed an additional 52-kD protein in lymphocytes and a 54-kD protein in red blood cells. The 60-kD form of Ro(SSA) in red cells was found to be antigenically distinct from that in the lymphocyte, because sera were identified that bound each exclusively. Finally, a rabbit antibovine Ro(SSA) serum distinguished red cell from lymphocyte Ro(SSA). These results suggest two distinctive populations of Ro(SSA) proteins and distributions of Ro(SSA) RNAs in the lymphocyte and red blood cell.