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Objective: To assess the enzymatic activity and biochemical status of dipeptidyl peptidase IV (DPP IV), an enzyme that participates in the degradation of proinflammatory molecules, in sera from a group of patients with rheumatoid arthritis (RA; n = 15) treated with a human anti-tumor necrosis factor-a (anti-TNF-alpha) antibody (adalimumab) for 32 weeks. IgG antibody titers against chaperone Bip (GRP78), phosphoglucose isomerase (PGI), lactate dehydrogenase (LDH), fibronectin (FN), and actin were also studied., Methods: DPP IV activity was measured in sera using Gly-Pro-p-nitroanilide as substrate. The biochemical profile of circulating DPP IV glycoforms was assessed by isoelectric focusing gel electrophoresis. All IgG autoantibody titers and their sialylation levels were determined by ELISA., Results: Patients showed significant increases in serum DPP IV enzymatic activity from basal values (3.554 +/- 1.096) with respect to those obtained at 32 weeks (4.787 +/- 0.953; p < 0.05). Changes in the biochemical profile of circulating DPP IV from acidic to more neutral isoelectric point glycoforms were also seen during treatment. The elevated titers of anti-GRP78 and anti-PGI IgG observed at the beginning of treatment decreased significantly during therapy, whereas those of anti-LDH, anti-FN, and anti-actin IgG remained unchanged. At the end of treatment, sialylation levels of anti-GRP78 and anti-PGI IgG antibodies increased to nearly normal levels. The DPP IV biochemical changes were accompanied by a significant improvement of the Disease Activity Score (DAS28)., Conclusion: The reduced activity of DPP IV along with increased titers of circulating antibodies to GRP78 and PGI may play a role in the pathogenesis of RA and can be successfully modified by administration of adalimumab.

Objective: To explore the important role of the terminal residues containing sialic acid (SA) in Campylobacter jejuni (CJ) lipopolysaccharide (LPS) as the critical antigen to induce nerve damage, and also to identify immunopathological evidence for the hypothesis of molecular mimicry and cross-immunity between CJ LPS and gangliosides., Methods: A mutant of Pen O:19 CJ with neuB1 gene inactivated and LPS outer core terminal residues losing SA was to be constructed. PCR and RT-PCR were used to confirm the mutant. Capability of CJ LPS binding to cholera toxin B subunit (CTB) was tested. Guinea pigs were systematically immunized with LPS of the wild and the mutant strains, respectively. Titers of anti-LPS and anti-ganglioside GM(1) IgG antibodies in sera of immunized guinea pigs were detected by ELISA. Pathological study for sciatic nerves of both Guinea pigs either immunized systematically or perineural injection with their immunized serum was finished., Results: (1) The mutant of CJ O:19 strain with inactivated neuB1 gene was successfully constructed and lost transcriptional activity of neuB1 gene in the mutant strain was confirmed by PCR and RT-PCR. SA was well demonstrated by both acidic ninhydrin reaction and periodate-resorcinol reaction in the LPS of wild strain but not in the mutant LPS; (2) Compared with the titers before immunization, the titers of anti-GM(1) IgG antibody increased in sera of guinea pigs immunized with LPS of the wild strain. However there were no detectable anti-GM(1) IgG antibody in sera of the animals immunized with mutant LPS and PBS. (3) The incidence of pathological fibers of sciatic nerves in wild CJ LPS group (17.3%) was significantly higher than the mutant CJ LPS group (chi(2) = 125, P < 0.01); the difference between the mutant CJ LPS group and control group was not statistically significant (chi(2) = 1.633, P > 0.05). (4) After perineural injection with immunized serum, the incidence of pathological fibers of sciatic nerves in wild strain group (67.8%) was also significantly higher than the incidence of mutant group (P < 0.01)., Conclusion: A mutant of CJ O:19 strain neuB1 gene inactivated and SA component of terminal structure of LPS lost was successfully constructed. And it no longer expressed SA component which is the normal terminal structure of LPS in wild strain. The capability of the wild strain to induce increased titers of anti-GM(1) antibody and immune-mediated nerve damage was simultaneously lost for the mutant strain. It could be a strong immunopathologic evidence to identify the molecular mimicry hypothesis between CJ LPS and ganglioside epitope in nerve on the pathogenesis of CJ related GBS. The terminal residues containing SA should be as the basic GM1-like structure in CJ LPS.

Serum-circulating antibody can be linked to poor outcomes in some cancer patients. To investigate the role of human antibodies in regulating tumor cell growth, we constructed a recombinant cDNA expression library of human IgG Fab from a patient with breast cancer. Clones were screened from the library with breast tumor cell lysate. Sequence analysis of the clones showed somatic hypermutations when compared to their closest VH/VL germ-line genes. Initial characterizations focused on five clones. All tested clones displayed stronger binding to antigen derived from primary breast cancers and established breast cancer cell lines than to normal breast tissues. In vitro functional studies showed that four out of five tested clones could stimulate the growth of MDA-MB-231 breast cancer cell lines, and one out of five was able to promote MCF-7 cell growth as well. Involvement of ERK2 pathway was observed. By 1H-NMR spectra and Western blot analysis, it was evident that two tested antibody Fabs are capable of interacting with sialic acid. Our study suggests a possible role for human antibody in promoting tumor cell growth by direct binding of IgG Fab to breast tumor antigen. Such studies prompt speculation regarding the role of serum antibodies in mediating tumor growth as well as their contribution to disease progression.

Molecular mimicry of Campylobacter jejuni lipo-oligosaccharides (LOS) with gangliosides in nervous tissue is considered to induce cross-reactive antibodies that lead to Guillain-Barre syndrome (GBS), an acute polyneuropathy. To determine whether specific bacterial genes are crucial for the biosynthesis of ganglioside-like structures and the induction of anti-ganglioside antibodies, we characterized the C. jejuni LOS biosynthesis gene locus in GBS-associated and control strains. We demonstrated that specific types of the LOS biosynthesis gene locus are associated with GBS and with the expression of ganglioside-mimicking structures. Campylobacter knockout mutants of 2 potential GBS marker genes, both involved in LOS sialylation, expressed truncated LOS structures without sialic acid, showed reduced reactivity with GBS patient serum, and failed to induce an anti-ganglioside antibody response in mice. We demonstrate, for the first time, to our knowledge, that specific bacterial genes are crucial for the induction of anti-ganglioside antibodies.

Introduction: Polyneuropathies associated with IgM monoclonal gammopathy were recently recognized. Antibodies can react with glycoproteins such as myelin associated glycoprotein (MAG), or gangliosides containing one sialosyl epitope such as GM1 or several sialosyl epitopes (polysialyted gangliosides) including GD2, GD3, GT1b, GT1a, GQ1b., Methods: We report on three patients presenting oculomotor dysfunction, chronic sensitive ataxic polyneuropathy, high sedimentation rate, IgM monoclonal paraprotein of unknown signification and antidisialosyl IgM antibodies and for two of them cold agglutinins. Such features have been previously described under the acronym "CANOMAD" (chronic ataxic neuropathy with ophthalmoplegia, M protein, agglutination and disialosyl antibodies)., Results: One of the patients presents extramembranous glomerulopathy and severe motor disability associated with this syndrome. The pathophysiology of the glomerulopathy seems to be linked with the polyneuropathy. Patients were treated either by intravenous immunoglobulin, corticosteroids or cyclophosphamid. Response to treatment differs in the three cases and there is currently no consensus., Conclusion: Our study demonstrates that spectrum of polyneuropathy associated with monoclonal polyneuropathy may be larger than originally described.

Background: FB21 is reactive with glomerular endothelial cells and distal tubules of the human kidney and is bound to a sialic-acid-dependent cell-surface antigen. We evaluated FB21 staining in fetal kidneys and kidneys of children and adults with normal kidneys and glomerulonephritis and investigated whether FB21 can be used as a marker for endothelial cell injury., Methods: This study was performed on 6 children, 10 adults, and 12 fetuses with normal kidneys and 113 patients diagnosed with primary and secondary glomerulonephritis. We evaluated renal staining for FB21 in children with normal kidneys and glomerulonephritis and measured serum E-selectin concentrations in patients with hemolytic uremic syndrome (HUS) and Henoch-Schönlein purpura nephritis (HSPN)., Results: (1) FB21 was reactive with endothelial cells of normal kidneys and detected on the surface of endothelial cells by immunoelectron microscopy. (2) FB21 was reactive with endothelial cells in kidneys of fetuses older than 32 weeks. (3) Endothelial cell FB21 staining scores in the first renal biopsy specimens of patients with HUS and HSPN were lower than those in normal kidneys of children and correlated negatively with serum E-selectin concentrations. (4) Endothelial cell FB21 staining of crescentic and sclerotic glomerular lesions in patients with immunoglobulin A nephropathy, membranoproliferative glomerulonephritis, and focal glomerulosclerosis was weaker than that in normal kidneys., Conclusion: These results suggest that FB21 can be used as a marker for glomerular endothelial cell injury in various types of glomerulonephritis.

Erythrocyte clearance is reviewed in the context of what is known in 2003 on clearance of apoptotic cells in vitro and in vivo. Thus, emphasis is put on the role of the innate immune system comprised of naturally occurring autoantibodies (NAbs) and complement. Oxidative damage, cellular senescence and diffusion-controlled exoplasmic cross-linking appear to generate oligomers of band 3 (anion transport protein) that are a prerequisite for anti-band 3 NAb binding to human red blood cells (RBC). Similar processes seem to be responsible for premature RBC clearance in hemoglobinopathies and membrane protein deficiencies. The review discusses why NAb binding alone is insufficient and how bound NAbs may enhance complement deposition. Clearance of RBC is not only the result of cell-bound opsonins, but is enhanced by the loss of RBC membrane constituents, such as CD47 and sialic acids. As long as these constituents are present on RBC in normal numbers and topologic arrangement, they bind to their respective receptors on macrophages, elicit a negative signal that appears to prevent the macrophage from engulfing bound RBC. Exposure of phosphatidylserine is not a primary signal for RBC removal and where exposed it initiates binding of CRP or of beta-2-glycoprotein I and NAbs.

A few years ago a hypothesis was proposed suggesting that elements of the innate immune system, such as acute phase reactants, contribute to the development of Type 2 diabetes mellitus. Acute phase reactants such as C-reactive protein and sialic acid may thus predict risk of developing Type 2 diabetes mellitus, as well as being markers of diabetes microvascular and macrovascular complications. This article discusses these issues.

Background and Objectives: In cold agglutinin disease, monoclonal red blood cell autoantibodies, termed cold agglutinins, induce haemolysis in patients exposed to the cold. Commonly, these autoantibodies are directed against the developmentally regulated I/i blood groups. A second blood group system, the Pr system (located on glycophorins), is involved less frequently. Anti-Pr cold agglutinins recognize either alpha 2,3- or alpha 2,6-linked N-acetylneuraminic acid as the immunodominant group. Cold agglutinins of anti-I/i specificity show a remarkable restriction in their genomic repertoire of the immunoglobulin heavy and light-chain immunoglobulin-variable domain (i.e. exclusive use of VH4-34 in heavy chains). For anti-Pr cold agglutinins, preliminary data on the repertoire of the light-chain variable domain indicate a preference for the subgroup Vkappa IV. To elucidate restrictions in the light-chain variable-domain subgroup repertoire of anti-Pr cold agglutinins systematically, and to discuss these results in the context of their anti-Pr(1-3) subclassification and immunodominant sialic acid, light chains in 13 anti-Pr cold agglutinins were investigated., Materials and Methods: The anti-Pr light chains were isolated using temperature-dependent absorption/elution techniques. Subsequently, they were subjected to N-terminal Edman degradation, and the light chain Vkappa subgroup was affiliated using the Kabat database., Results: Five of 13 (38%) light chains belonged to Vkappa IV, five of 13 (38%) to Vkappa I and three of 13 (23%) to Vkappa III. Anti-Pr with Vkappa IV subgroup light chains exclusively recognized alpha 2,3-linked N-acetylneuraminic acid., Conclusions: Including data from the literature, the repertoire of the light-chain variable domain in pathological anti-Pr cold agglutinins exhibits a clear bias towards the use of the single germline gene-derived subgroup, Vkappa IV (eight of 17 or 47%). The association of Vkappa IV subgroup light chain-containing anti-Pr cold agglutinins with binding to alpha 2,3-, but not alpha 2,6-linked N-acetyneuraminic acid raises speculations about a possible role of subgroup-derived determinants in anti-Pr binding.

The specificity of the MY.1E12 mAb that was generated by immunizing mice with human milk fat globule (HMFG) was investigated. Fluorescein isothiocyanate (FITC)-conjugated peptides corresponding to a portion of the MUC1 tandem repeat were enzymatically glycosylated with N-acetylgalactosamine, galactose, and then sialic acid. The MY.1E12 mAb was examined for its affinity to the resulting glycopeptides by fluorescence polarization. Its affinity for the peptide whose Thr within the VTS sequence bears a Neu5Ac alpha 2-3Gal beta 1-3GalNAc trisaccharide (K(d)=1.4 x 10(-7) M) was significantly higher than for the same peptide whose Thr bears an unsialylated disaccharide (K(d)=3.9 x 10(-6) M). The MY.1E12 mAb also bound strongly to a purified recombinant MUC1 fusion protein with six tandem repeats that was expressed by transfected MCF-7 breast cancer cells. The removal of sialic acids from the fusion protein significantly decreased MY.1E12 mAb reactivity, much more so than the MUC1-specific 115D8 antibody, whose epitope is known to be destroyed by desialylation. Thus, the attachment of the sialyl alpha 2-3Gal beta 1-3 beta 1-3GalNAc trisaccharide onto the Thr within the VTS motif significantly increases the binding of the MY.1E12 antibody to the MUC1 repeat sequence.

Recent studies of the major cell types involved in the initiation and progression of allergic inflammation have revealed that they express an unexpectedly large number of surface receptors that inhibit the release of proinflammatory mediators from mast cells and basophils in vitro. Moreover, analyses of animals deficient in some of these receptors, for example, Fc(gamma)RIIB, gp49B1 and paired Ig-like receptor (PIR)-B, have shown that the molecules indeed suppress allergic responses driven by the adaptive immune response in vivo. These findings support the emerging concept that allergic diseases are caused not only by excessive activation of cells but also from deficiencies in receptors that suppress these activation responses.

Purpose: To examine the role of GM3 monosialoganglioside and sialic acid in the antitumor activity of a vaccine based on GM3, hydrophobically conjugated with the outer-membrane-protein complex from Neisseria meningitidis (GM3/VSSP)., Methods: In order to evaluate the relationship between antitumor activity and the presence of GM3 on the surface of tumor cells, we used two murine tumor cell lines with different ganglioside expression. Syngeneic mice were immunized with four i.m. doses of GM3/VSSP (120 micro g) at 14-day intervals and challenged subcutaneously with tumor cells., Results: B16 melanoma cells showed GM3 on cell surface and GM3-dependent in vitro growth. As expected, preimmunization with the vaccine significantly inhibited tumor formation and prolonged survival in mice challenged with B16 cells. In contrast, no antitumor effect was observed in mice challenged with GM3-negative F3II mammary carcinoma cells. The reactivity of sera from immunized mice against B16 cells was confirmed by flow cytometry and immunoperoxidase staining. Depletion of sialic acid residues from the cell surface completely abolished antibody response against melanoma cells., Conclusions: These results indicate that the antitumor activity of GM3/VSSP is associated with GM3 expression on tumor cell surface and demonstrate a major role of sialic acid in the humoral response of vaccinated mice.

The immune reactivity implicated in the pathogenesis of Guillain-Barré syndrome (GBS) and related diseases, which occur following infection with specific strains of Campylobacter jejuni bearing sialylated lipopolysaccharide structures that cross-react with specific gangliosides, is consistent with provocation of inflammation via molecular mimicry. In this review, we have focused upon microbial characteristics and structures, the fine structure of the essential carbohydrate determinants, and the application of our proposed criteria, modified from those of Koch for causation of infectious and of Witebsky for autoimmune diseases, to the circumstance of infectious induction of autoimmune disorder.

Several microbial infections, including Mycoplasma pneumoniae respiratory infection, are capable, in man, of transiently inducing the expression of anti-red blood cell autoantibody called cold agglutinins (CA). To analyze the mechanisms by which immune tolerance is broken following a mycoplasma infection, we used transgenic mice expressing a pathogenic human CA, designated CA-GAS, specific for sialylated carbohydrates. In these mice peripheral deletion of autoreactive B lymphocytes and receptor editing, prevent the development of autoimmune hemolytic anemia. Experimental infections of transgenic mice with Mycoplasma pulmonis resulted in a high anti-mycoplasma antibody response (despite a severe B cell depletion at the onset of infection), and an important induction of serum CA concentrations, reaching in some mice pathological titers. Whereas in naïve mice, only a small percentage of CA-expressing cells could be detected, in infected mice, a majority of circulating B lymphocytes were large B220(-) cells, which expressed the transgenic immunoglobulin. Immunization of the transgenic mice with keyhole limpet hemocyanin and Freund's adjuvant, to nonspecifically stimulate the expression of the passenger transgenes, only moderately increased the CA titers. These results indicate that M. pulmonis infection is capable of breaking immune tolerance in the CA-transgenic mice, in part through specific activation of CA-expressing B lymphocytes. This experimental infection mimics the induction of CA in humans and provide an animal model for studying the genesis of the autoimmune hemolytic anemia.

We generated the 1E10 gamma-type anti-idiotype mAb (Ab2) specific to an Ab1 mAb able to react specifically with N-glycolyl-containing gangliosides and with Ags expressed on human melanoma and breast carcinoma cells. This Ab2 mAb induced an Ab response in animal models sharing immunochemically defined idiotopes with the Ab1. The treatment of tumor-bearing mice with 1E10 mAb induced a strong antitumor activity. A clinical trial was conducted in 20 patients with advanced malignant melanoma. Patients were treated with six intradermal injections of aluminum hydroxide-precipitated 1E10 anti-Id mAb given at 2-wk intervals. Sixteen of the 17 patients who received at least four doses of the anti-Id vaccine develop Ab3 Abs capable of inhibiting Ab2 binding to Ab1 (Ab3Id+). In contrast to the incapacity of 1E10 mAb to generate Ab3 Abs with the same antigenic specificity as the Ab1 mAb in mice, a very specific and strong Ab3 response against N-glycolyl-containing gangliosides was induced in 16 patients (Ab3Ag+). No evidence of serious or unexpected adverse effects has been observed in this clinical trial. 1E10 anti-Id vaccine was safe, well tolerated, and immunologically effective, with most patients being able to generate a specific immune response against 1E10 and Neu-glycolyl-GM(3) ganglioside.

Salivary agglutinin is a Streptococcus mutans binding protein and a member of the scavenger receptor cysteine-rich superfamily. It is identical to lung gp-340 and brain DMBT1, which possibly play a role in innate immunity and tumor suppression, respectively. The goal of this study was to localize salivary agglutinin in human salivary glands. Two monoclonal antibodies, directed against gp-340, were characterized. mAb 213-1 reacted with sialic acid epitopes and cross-reacted with MUC7. The reaction with mAb 213-6 disappeared after reduction, suggesting that a protein epitope was recognized. In the parotid gland, immunohistochemical labeling with mAb 213-6 was found in the duct cells. In the submandibular gland and labial gland, both serous acini and demilune cells were labeled. In the labial gland, labeling was found at the luminal side of the duct cells. Salivary agglutinin was distinctly localized in salivary glands, but in distinct glandular secretions, no differences in electrophoretic behavior were observed.

We have studied the interaction of the Gal-GalNAc-reactive champedak lectin-C with neuraminidase-treated and untreated IgA1 from IgA nephropathy patients. The binding ability of the lectin to untreated IgA1 from IgA nephropathy patients was significantly lower as compared to the untreated IgA1 from normal controls. This differential lectin-binding capacity was abrogated when the experiment was performed on neuraminidase-treated sera. Treatment of the serum IgA1 with neuraminidase also abrogated the differential charge distribution between the alpha-heavy chains of IgA nephropathy patients and normal controls., (Copyright 2001 S. Karger AG, Basel)

An antibody against a transition state analog (TSA) may share some common features with an enzyme that produces such a transition state. SIC172 antibody binds specifically to Neu2en5Ac, a TSA of Neu5Ac in the sialidase reaction, but has no catalytic activity. To understand how the antibody recognizes Neu2en5Ac and to find out if it is possible to convert it to a catalytic antibody, we made and sequenced the SIC172 ScFv, and constructed a 3-D model of it. The VH-CDR3 contains a unique sequence with Cys at H95. The 3-D model showed that Cys-H95 is exposed inside the antigen-binding cavity. After affinity docking, 4 types emerged. In type I, the carboxyl group of Neu2en5Ac is located near the Cys-H95 and neighboring positively charged residues. The change of Cys-H95 to Asp by site-directed mutation decreased the binding activity, while a change to Arg did not. These and other mutation experiments, and further modeling of mutant Fv, support the 3-D model and docking type I. A comparison with sialidase indicates that SIC172 antibody appears to have some groups of residues that are conserved at the active site of the enzyme. The possibility of Neu2en5Ac-binding antibody being converted to a catalytic antibody is discussed., (Copyright 2001 Wiley-Liss, Inc.)

Initial studies have revealed an enhanced surface expression of O-acetylated sialoglycoconjugates (O-AcSGs) on lymphoblasts concomitant with high titres of IgG in childhood Acute Lymphoblastic Leukaemia (ALL) (Mandal C, Chatterjee M, Sinha D, Br J Haematol 110, 801-12, 2000). In our efforts to identify disease specific markers for ALL, we have affinity-purified IgM directed against O-AcSGs that reacts with three disease specific O-AcSGs present on membrane proteins derived from peripheral blood mononuclear cells (PBMC) of ALL patients. Antibody specificity towards O-AcSGs was confirmed by selective binding to erythrocytes bearing surface O-AcSGs, decreased binding with de-O-acetylated BSM and following pretreatment with O-acetyl esterase. Competitive inhibition ELISA demonstrated a higher avidity of IgM for O-AcSG than IgG. Flow cytometry demonstrated the diagnostic potential of purified O-AcSA IgM as binding was specific with ALL patients and minimal with other haematological disorders and normal individuals. It therefore may be adopted as a non-invasive approach for detection of childhood ALL. Taken together, the data indicates that carbohydrate epitopes having terminal O-AcSA alpha2 --> 6 GalNAc determinants induce disease specific IgG and IgM, potentially useful molecular markers for childhood ALL.

The CD60 antigen is expressed on a majority of T cells in autoimmune lesions, and anti-CD60 can activate T lymphocytes. CD60 has been defined as the GD3 ganglioside, and subsequently as the 9-O-acetylated form of GD3. However, other evidence suggests that anti-CD60 recognizes a glycoprotein or family of glycoproteins expressed by T lymphocytes. The current studies were undertaken to better define the identity of the CD60 antigen on both T cells and non-T cells. Treatment of intact cells with neuraminidases of various specificities confirmed that detection of the CD60 epitope depends on expression of an alpha2, 8-disialic acid carbohydrate linkage, as is found in GD3 and related gangliosides. However, the sialicacid polymer colominic acid inhibited anti-GD2 and anti-GD3, but not anti-CD60 from binding to cell surfaces. Expression of CD60 did not correlate with expression of GD3 on a variety of cell lines and T cell populations. Expression of CD60 and 9-O-acetyl-GD3 was roughly parallel on some non-T cell lines such as melanoma cells, but on T cells expression of CD60 was consistently greater. Antibodies to GD2, GD3 and 9-O-acetyl-GD3 were ineffective at inhibiting binding of anti-CD60 to CD60+ cells. Activation responses of T cells to anti-CD60 were inducible in either the presence or absence of a response to anti-GD3. A novel inhibitor of glucosyl ceramide synthesis, D-threo-1-phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol (D-t-P4) reduced expression of GD3 much more than CD60 on activated T lymphocytes. Following biotinylation of HUT78 T cells, anti-CD60 immunoprecipitated a 70 kDa antigen. Taken together, the present data and previous findings suggest that anti-CD60 can recognize both a modified form of the GD3 ganglioside and a carbohydrate-dependent complex epitope present on one or more glycoproteins. This glycoprotein epitope may be the more abundant and functionally significant CD60 antigen on T lymphocytes, while 9-O-acetyl-GD3 is likely to be the principal structure recognized by anti-CD60 on melanoma cells. These findings emphasize the complexity of understanding the functional roles of carbohydrate epitopes in cell activation.

Pulmonary infection with Pseudomonas aeruginosa in patients with cystic fibrosis (CF) causes a chronic destructive bronchitis. A xenograft model was used to study the susceptibility of the CF respiratory epithelium to P. aeruginosa strain PAK and the virulence of certain mutants. Despite an early trend toward increased susceptibility, colonization of CF xenografts (ID(95), 62 colony-forming units [cfu]) was not statistically different (P=.5) than in xenografts with normal respiratory cells (ID(95), 1.2x10(3) cfu). Infection severity in 12 CF xenografts (mean polymorphonuclear leukocyte [PMNL] density, 1.88x10(6)+/-1.75x10(6)/xenograft) was similar to that in 16 non-CF xenografts (3.19x10(6)+/-2.45x10(6) PMNL/xenograft; P=.38), despite slightly greater bacterial density in the CF xenografts (mean, 1.57+/-2.73x10(6) cfu/xenograft) versus xenografts with normal epithelium (mean, 1.03+/-1.3x10(6) cfu/xenograft). P. aeruginosa mutants pilA and fliF, but not rpoN, colonized normal respiratory xenografts, indicating that colonization and infection in this model depend on an uncharacterized RpoN-controlled gene. This model appears to be suitable for genetic study of P. aeruginosa virulence but not of the CF respiratory tract's unique susceptibility.

Foreign particles and damaged host cells can activate the complement system leading to their destruction by the host defense system. Factor H (fH) plays a vital role in restricting complement activation on host cells through interactions with polyanions such as heparin, while allowing activation to proceed on foreign surfaces. Complement activation by damaged host cells is also down regulated by fH, which is localized to injured areas through interactions with C-reactive protein (CRP). A number of pathogens have developed mechanisms by which they can also bind fH and thus exploit its protective properties. One such organism is Group A Streptococcus (GAS) which mediates fH binding via its surface expressed M-protein. fH consists of 20 conserved short consensus repeat (SCR) units and mutagenesis studies indicate that the seventh repeat is responsible for interactions with heparin, CRP and M-protein. We recently performed molecular modelling of fH SCR 7 and identified a cluster of positively charged residues on one face of the domain. By alanine replacement mutagenesis, we demonstrated that these residues are involved in heparin, CRP and M protein binding, which indicates that there is a common site within fH SCR 7 responsible for multiple ligand recognition.

Background: Sialic acid is a component of many tumor-associated oligosaccharide antigens. The repertoire of sialic acids presented by cells can be expanded to include unnatural variants by intercepting the sialic acid biosynthetic pathway with unnatural precursors. We explored whether unnatural cell surface sialosides produced by metabolism can act as neo-antigens and modulate the immunogenicity of cells., Results: Immunization of rabbits with synthetic conjugates of an unnatural sialic acid bound to keyhole limpet hemocyanin produced significant titers of antibodies that were specific for the structurally altered sialic acid. The antibodies recognized cells that were fed the unnatural biosynthetic precursor, and were capable of directing complement-mediated lysis., Conclusions: Structural alteration of sialic acids replaces a tolerized self-antigen with an antigenic determinant. Incorporation of unnatural sialosides into cell surface glycoconjugates through biosynthetic means can alter the immunoreactivity of cells, providing new possibilities for tumor immunotherapy.

A study on the binding interaction of lectins from Artocarpus heterophyllus (jacalin), Glycine max and Sambucus nigra with standardised quantity of IgA from the IgA nephropathy patients and normal controls was performed. The Glycine max lectin demonstrated higher affinity towards the serum IgA of IgAN patients as compared to normal controls. However, the affinity binding was lower in cases ofjacalin and the Sambucus nigra lectin. When serum samples were treated with neuraminidase, the differential jacalin affinity binding between IgA1 of patients and normal controls was abrogated. Our data are in support of the view that the O-linked oligosaccharide moieties of the patients IgA1 were generally lacking in galactose and sialic acid residues.

Iscador activated (in vivo and in vitro) splenocytes were found to inhibit metastatic tumour growth in C57BL/6 mice. In order to check whether NK cells are involved in the antimetastatic activity of Iscador activated splenocytes ,animals were depleted of NK cells using antiasialo GMI antibodies. When spleen cells activated in vivo with Iscador were injected into animals pretreated with Antiasialo GM I antibodies, there was an average of 44.6 tumour nodules on 21st day indicating that stimulation of NK cell activity produced by the Iscador compensate for the NK cell depletion by Antiasialo GM I antibody. Animals treated with Iscador activated splenocytes showed an average survival period of 68 days whereas that of control tumour bearing animals treated with Ab the average survival was 19.3 days. The lung collagen hydroxyproline content, serum sialic acid levels, markers of metastasis were also significantly (P<0.001) lowered in these animals compared to those of the untreated tumour bearing animals. gamma-glutamyl transpeptidase a marker of neoplastic growth, was also significantly reduced (P<0.001) in animals treated with activated splenocytes.

An autoimmune hypothesis for the etiology of Meniere's disease has been proposed. In this study, we focused on gangliosides as potential antigens for autoantibodies in Meniere's disease patients. In an attempt to investigate ganglioside antigens which respond to the serum of patients with Meniere's disease, we analyzed gangliosides of human acoustic neurinomas, and used them as antigens to broadly explore gangliosides that react to serum. All the acoustic neurinoma samples used in the present study showed a similar ganglioside profile on TLC (thin-layer chromatography). For the microscale ganglioside analysis, a newly developed TLC blotting/secondary ion mass spectrometry (SIMS) system together with TLC immunostaining method was employed. Most of the ganglioside bands could be analyzed, and they were identified as GM3, GM2, SPG, GM1a, GD3, S-i (sialyl-i ganglioside) and GD1a. GD1a was the predominant ganglioside and many neolactoseries gangliosides were recognized by immunological analysis. Next, the immune reactivity of serum samples, from patients with Meniere's disease, with the acoustic neurinoma gangliosides was studied by TLC immunostaining. The result showed that five of 11 patients with Meniere's disease and one of eight normal subjects reacted with a specific band, which was identified as S-i by the TLC blotting/SIMS system. The findings of the present study indicate that S-i ganglioside is an autoantigen and possibly involved in the pathogenesis of Meniere's disease.

Sialic acids typically present as terminal sugars of oligo-saccharides are reported to be modified by O-acetylation at the C-9 position on lymphoblasts of childhood acute lymphoblastic leukemia (ALL) patients (Sinha et al., 1999a, Leukaemia, 13, 119-125). We now report high titers of IgG antibodies directed against O-acetylated derivatives of sialic acids (O-AcSA) in serum of ALL patients. These antibodies were purified using bovine submaxillary mucin (BSM) and the IgG distribution was confined to IgG(1)and IgG(2)subclasses; their binding was totally abolished with de-O-acetylation confirming their specificity towards O-AcSA determinants. Flow cytometry demonstrated binding of these antibody fractions to peripheral blood mononuclear cells (PBMC) of both T- and B-ALL patients having increased cell surface 9-O-AcSA determinants. Western blotting of membranes derived from PBMC of ALL patients confirmed binding of the antibody to O-acetylated sialoglycoconjugates corresponding to 144, 135, 120, 90, and 36 kDa whereas binding to PBMC from normal individuals corresponded to 144 and 36 kDa. Specificity of the antibody fraction towards 9-O-AcSA was substantiated by hemagglutination and hemagglutination-inhibition assays. The antibody purified from ALL serum selectively mediates complement dependent cytolysis of lymphoblasts expressing O-AcSAs and thereby possibly confers passive protection. The enhanced anti O-AcSA antibody levels allowed for development of a serodiagnostic assay (BSM-ELISA) specific for ALL. Minimal crossreactivity was observed with other hematological disorders like acute myeloid leukemia (n = 16), chronic myeloid leukemia (n = 6), chronic lymphocytic leukemia (n = 7) and non-Hodgkin's lymphoma (n = 3) as well as normal healthy individuals (n = 28). The BSM-ELISA therefore provides a simple, noninvasive alternative diagnostic approach for ALL and merits clinical consideration.

Objective: To determine whether clinical outcome during pregnancy in rheumatoid arthritis (RA) is associated with changes in the levels of exposed immunoglobulin G (IgG) terminal sugars., Methods: Serum IgG glycosylation from 23 pregnant patients with RA was analyzed during the prenatal, antenatal, and post-partum periods. Patients were randomly selected on the basis of whether they achieved spontaneous remission (n = 11) or did not remit (n = 12); of the latter group 6 patients experienced a relapse in disease activity. Levels of exposed terminal IgG sugars, galactose (Gal), N-acetylglucosamine (GlcNAc), and sialic acid (SA), were estimated in a lectin binding assay using Ricinis (communis, Bandeiraea simplicifolia II, and Sambucus nigra, respectively., Results: Exposed Gal levels increased (p<0.02) and GlcNAc levels decreased (p<0.05) in the antenatal period, and returned to preconception levels during post-partum. GlcNAc rebound was instantaneous (p<0.005), whereas Gal remained high for a further 10 weeks. SA did not undergo any major changes. Remission was associated with an earlier and significantly greater antenatal reduction in GlcNAc (2nd and 3rd trimester; p<0.02) in comparison to the groups that did not experience a decrease in disease activity. Analysis of individual IgG samples during the first trimester revealed a significant negative correlation between Gal and GlcNAc in the remission group (r = -0.80; p<0.05), which was opposite to that found in the relapse group (r = +0.87; p<0.03). There was no significant difference between the groups with regard to the timing and/or incidence of a post-partum flare of disease., Conclusion: Temporal changes in the levels of IgG terminal sugars, in particular exposed GlcNAc, are integrally associated with the clinical manifestation of RA in pregnancy. Generation of IgG sugar micro-heterogeneity is complex and understanding it may help unravel pathogenic features associated with RA.

Many tumor-associated epitopes possess carbohydrate as a key component, and thus changes in the activity of glycosyltransferases could play a role in generating these epitopes. In this report we describe the stable transfection of a human pancreatic adenocarcinoma cell line, Panc1-MUC1, with the cDNA for mucin core 2 GlcNAc-transferase (C2GnT), which creates the core 2 beta-1,6 branch in mucin-type glycans. These cells lack endogenous C2GnT activity but express a recombinant human MUC1 cDNA. C2GnT-transfected clones expressing different levels of C2GnT were characterized using monoclonal antibodies CC49, CSLEX-1, and SM-3, which recognize tumor-associated epitopes. Increased C2GnT expression led to greatly diminished expression of the CC49 epitope, which we identified as NeuAcalpha2,6(Galbeta1,3)GalNAcalpha-Ser/Thr in the Panc1-MUC1 cells. This was accompanied by the emergence of the CSLEX-1 epitope, sialyl Lewis x (NeuAcalpha2,3Galbeta1,4(Fucalpha1,3)GlcNAc-R), an important selectin ligand. Despite this, however, the C2GnT transfectants could not bind to selectins. Increased C2GnT expression also led to masking of the SM-3 peptide epitope, which persisted after the removal of sialic acid, further suggesting greater complexity of the core 2-associated O-glycans on MUC1. The results of this study suggest that C2GnT could play a regulatory role in the expression of certain tumor-associated epitopes.

We have described new monoclonal antibodies (FUN-1, -2, FK61, FB1, FB21) which recognize B cells in the peripheral blood and lymphoid tissues. In preliminary reports FB1 and FUN-1 were described previously as anti-CD20 and -CD86 antibodies, respectively. FB1 and FB21 recognize an intracytoplasmic epitope (35, 38 kD) and a sialic acid-dependent carbohydrate epitope, respectively. FB1 reacts with pan B cells and FB21 with a subpopulation of B cells. In addition, FB21 shows relatively specific reaction with papillary or follicular carcinoma of the thyroid gland, but not with normal thyroid follicules and most benign thyroid gland tumors. Since FB1 and FB21 can be used with formalin-fixed paraffin-embedded tissue sections, they are useful for diagnosis of B cell lymphoma or thyroid carcinoma. FUN-1 recognizes surface antigen (CD86) on activated B cells, monocytes in peripheral blood and germinal center B cells in lymphoid tissues. CD86 has an important role in T cell activation and the antigen-specific T-cell dependent immune response.

The highly polysialylated neural cell adhesion molecule (PSA-NCAM) is one of the most promising molecules that contributes to plasticity in the central nervous system. We evaluated PSA-NCAM immunoreactivity in the hippocampal formation of Alzheimer's disease (AD) patients. We found significant increases over control levels in the optical density ratios of PSA-NCAM immunoreactivity in the outer molecular layer/granule cell layer (ODoml/grl) and in the inner molecular layer/granule cell layer (ODiml/grl) in the dentate gyrus of AD patients. The optical density of the granule cell layer in the dentate gyrus did not differ significantly between AD patients and control subjects. However, the number of PSA-NCAM-immunopositive infragranule cells was higher in the AD group compared with control subjects. The major finding in the CA1, subiculum and entorhinal cortex of AD patients was the disorganization of PSA-NCAM-immunoreactive fibres. These results indicate that neuronal remodelling occurs, especially in the dentate gyrus of patients with AD.

The reactivity of monoclonal antibody FB21, which recognizes a sialic acid-dependent carbohydrate epitope, was tested with 94 non-neoplastic and neoplastic thyroid tissue specimens using immunohistochemical methods on formalin-fixed, paraffin-embedded tissue sections. These specimens included 11 cases of adenomatous goiter, three cases of Basedow's disease, 30 cases of follicular adenoma, 20 cases of papillary carcinoma, 15 cases of follicular carcinoma, six cases of medullary carcinoma, and nine cases of anaplastic carcinoma. FB21 reacted with 14 of 15 cases of follicular carcinoma that showed a microfollicular or trabecular pattern, and with nine of 20 cases of papillary carcinoma. A positive reaction was found on the cell surface membranes or apical parts of neoplastic follicles. FB21 also reacted with five of 30 cases of follicular adenoma. These cases showed a follicular pattern and positive staining pattern similar to that in follicular carcinoma. Adenomatous goiters, Basedow's disease, medullary carcinomas, and anaplastic carcinomas were negative for FB21 reactivity. Although the different reactivities of FB21 with papillary carcinoma and follicular carcinoma remain to be investigated, the high frequency of reactivity with FB21 suggests that it may be useful as a complement to morphological diagnosis in follicular carcinoma.

It has been known for over a decade that sialidase (neuraminidase) treatment could substantially enhance the capacity of resting B cells to stimulate the proliferation of allogeneic and antigen specific, syngeneic T cells. Thus, cell-surface sialic acid was implicated as a potential modulator of immune cell interaction. However, little progress has been made in either identifying explicit roles for sialic acid in this system or in hypothesizing mechanisms to explain the "neuraminidase effect." Here we show for the first time that cell surface sialic acid on medium incubated B cells blocks access to costimulatory molecules on the B cell surface, and that this is the most likely explanation for the neuraminidase effect. Further, we show that it is likely to be upregulation of ICAM-1 and its subsequent engagement of LFA-1 rather than loss of cell surface sialic acid that in part regulates access to CD86 and other costimulatory molecules. However, we cannot exclude a role for CD86-bound sialic acid on the B cell in modulating binding to T cell CD28. Because sialidase treatment of resting B cells but not resting T cells enables T cell activation, we suggest that sialidase treatment may still be an analogue for an authentic step in B cell activation, and show that for highly activated B cells (activated with polyclonal anti-IgM plus INF-gamma) there is specific loss 2, 6-linked sialic acid. Potential roles for sialic acid in modulating B cell/T cell collaboration are discussed.

Neu2en5Ac is a minor component of body fluids and is abundant in sialuria, but no antibody to detect it has been reported. 5-Acetamido-2,6-anhydro-9-glutaramido-3,5,9-trideoxy-D-glycero-D- galacto-non-2-enonic acid has been synthesized and conjugated with keyhole limpet hemocyanin (KLH) for immunization. A hybridoma named SIC172 was obtained that produces a monoclonal antibody (MAb) to Neu2en5Ac. SIC172 MAb in culture supernatant bound strongly to the hapten conjugated to BSA in ELISA, but slightly to fetuin, a glycoprotein which is rich in Neu5Ac. SIC172 MAb (IgG3(kappa)), purified with a protein A/G affinity column, bound strongly to fetuin. Neu2en5Ac competed with the MAb in binding in amounts as low as 3 microM, while the competition of Neu5Ac appeared at amounts of more than 300 microM. SIC172 MAb is a unique MAb specific to Neu2en5Ac and might be useful for detecting Neu2en5Ac, which occurs naturally and in sialuria.

Meningococcal meningitis is a severe childhood disease which often results in significant disability or death. Two major etiological agents of meningitis are the group B meningococci and capsular type K1 E. coli. The virulence of these organisms is attributable to structural mimicry between their common alpha(2-8)-polysialic acid capsular polysaccharide and human tissue antigens, which allows the bacteria to evade immune surveillance. There is currently no effective vaccine to protect against this infection. It has been demonstrated that the capsular polysaccharide of the bacteria can adopt a unique 'antigenic conformation'. This antigenic conformation has formed the basis for the development of an N-propionylated polysialic acid vaccine. Immunization trials in mice with this vaccine show the production of two groups of antibodies, of which only N-propionylated polysialic acid-specific were protective. Knowledge of the structure of the antigen-binding site which recognizes the protective epitope is essential to determining the antigenic conformation of the polysaccharides, and is a critical aspect in understanding and improving the action of potential vaccines. The antigen-binding fragments (Fab) of one protective (13D9) and one non-protective (6B9) monoclonal antibody specific for the capsular polysaccharides of group B meningococci have been crystallized and have undergone preliminary X-ray diffraction analysis. Both crystals are observed to scatter X-rays to approximately 1.7 A resolution at the A1 station at the Cornell High-Energy Synchrotron Source. 13D9 has an orthorhombic unit cell with a = 41.8, b = 102.3, c = 134.7 A, with space group P212121. Fab 6B9 has an orthorhombic unit cell with a = 89.6, b = 132.0 and c = 36.9 A, with space group P21212.

By using flow cytometric analysis of cells in whole blood expressing high levels of CD14, we found a subpopulation of monocytes (8% of total) with higher scatter parameters, high capacity to produce reactive oxygen species (ROS), stronger expression of Lewis-X (CD15), sialyl-Lewis-X, CD11b and CD18 antigens, as well as an increased polymerized actin content. The size of this subpopulation increased after stimulation with lipopolysaccharide at the expense of the remaining monocytes, suggesting that its features were inducible. The membrane increase in Lewis-X and sialyl-Lewis-X expression observed during this conversion was largely due to the translocation of these carbohydrate structures from intracellular pools. Moreover, this subpopulation behaved as a primed monocyte subpopulation producing large amounts of H2O2 in response to N-formyl-methionyl-leucyl-phenylalanine. Increased H2O2 production was inhibited not only by anti-CD14 but also by anti-CD15 and anti-sialyl-Lewis-X monoclonal antibodies when added before lipopolysaccharide. These results show that lipopolysaccharide priming is regulated, at least in part, by Lewis-X and sialyl-Lewis-X structures expressed on the monocyte membrane. All together, this highly reactive and inducible subpopulation of monocytes, which share phenotypic and functional characteristics with neutrophils, might play an important role in host defenses and inflammatory responses.

Fusobacterium nucleatum has been identified as significantly associated with sites with active periodontal disease and, as a group, the oral fusobacteria coaggregate with members of all oral bacteria genera tested. Monoclonal antibodies were prepared and used in conjunction with other potential inhibitors, such as simple sugars and amino acids, to characterize coaggregation interactions, of F. nucleatum PK1594. Four unique monoclonal antibodies, 5H11, 14C7, 19F2 and 29C12, were obtained by their ability to inhibit coaggregation of F. nucleatum PK1594 with Actinomyces israelii PK16. They were also capable of inhibiting other coaggregations including Streptococcus oralis H1, S. oralis J22, Capnocytophaga ochracea ATCC33596, Prevotella denticola PK1277 and Prevotella intermedia PK1511. All of these interactions were completely inhibited by N-acetylneuraminic acid. Neither N-acetylneuraminic acid nor monoclonal antibody 5H11 had any inhibitory effect on other F. nucleatum PK1594 interactions, including all galactose-inhibitable coaggregations. The results indicate that F. nucleatum PK1594 expresses upon its surface a distinct type of adhesin that mediates coaggregation interactions that are inhibited by N-acetylneuraminic acid.

Background and Objectives: Pr1,2,3, PrM, Sa and Sia-l1, -b1, -lb1 are sialic acid (NeuNAc)-dependent antigens recognized by human cold agglutinins. Pr and Sa antigens are the O-glycans of glycophorins containing alpha 2,3NeuNAc (to galactose) and/or alpha 2,6NeuNAc (to galactosamine). The antigens of the Sia-l1, -b1, -lb1 complex are gangliosides that may carry alpha 2,3NeuNAc (to galactose) and/or alpha 2,8NeuNAc (to NeuNAc). We studied the NeuNAc groups involved in the antigens., Materials and Methods: From 74 sera with cold agglutinins against NeuNAc-dependent antigens, anti-T-free preparations were made and tested against human red cells, treated with an alpha 2,3-specific recombinant sialidase., Results: Most (51/62) Pr antigens use alpha 2,3NeuNAc, some (8/62) use alpha 2,6NeuNAc and a few (3/62) use both sialyl groups as immunodominant components on glycophorins. The immunodominant component of Sa and Sia-l1, -b1, -lb1 determinants was alpha 2,3NeuNAc in all cases., Conclusion: The red cell target structures for cold agglutinins against NeuNAc-dependent antigens have been identified. We propose a Pr nomenclature to reflect this. The binding of anti-Pr to gangliosides may be the basis for anti-Pr-induced peripheral neuropathy.

Serogroup B meningococci express sialic acids on their surfaces as a modification of the lipooligosaccharide (LOS) and as capsular material consisting of alpha2,8-linked sialic acid homopolymers. The aim of this study was to elucidate the impact of each sialic acid component on the deposition of complement factor C3 and serum resistance. For this purpose, we used isogenic mutants deficient in capsule expression (a polysialyltransferase mutant) or sialylation of the LOS (a galE mutant) or both (a mutant with a deletion of the cps gene locus). Bactericidal assays using 40% normal human serum (NHS) demonstrated that both the capsule and LOS sialic acid are indispensable for serum resistance. By immunoblotting with monoclonal antibody MAb755 that is specific for the C3 alpha-chain, we were able to demonstrate that C3 from 40% NHS was covalently linked to the surface structures of meningococci as C3b and iC3b, irrespective of the surface sialic acid compounds. However, C3b linkage was more pronounced and occurred on a larger number of target molecules in galE mutants with nonsialylated LOS than in meningococci with wild-type LOS, irrespective of the capsule phenotype. C3b deposition was caused by both the classical pathway (CP) and the alternative pathway of complement activation. Use of 10% NHS revealed that at low serum concentrations, C3 deposition occurred via the CP and was detected primarily on nonsialylated-LOS galE mutants, irrespective of the capsular phenotype. Accordingly, immunoglobulin M (IgM) binding to meningococci from heat-inactivated NHS was demonstrated only in both encapsulated and unencapsulated galE mutants. In contrast, inhibition of IgA binding required both encapsulation and LOS sialylation. We conclude that serum resistance in wild-type serogroup B meningococci can only be partly explained by an alteration of the C3b linkage pattern, which seems to depend primarily on the presence of wild-type LOS, since a serum-resistant phenotype also requires capsule expression.

In previous studies we have examined the roles of meningococcal surface structures (capsule, lipopolysaccharides, pili and opacity proteins: Opa and Opc) in bacterial interactions with human epithelial, endothelial and mononuclear phagocytic cells. In the current investigations, using defined derivatives of a serogroup A strain C751 and a serogroup B strain MC58, we studied the roles of these structures with human polymorphonuclear phagocytes (PMN). In addition, we examined the potential influence of the pilus-associated protein, PilC, previously known to affect epithelial cell interactions. The data show, that, as with monocytes, opacity proteins affect bacterial interactions with PMN and require surface sialic acids (on capsule and LPS) to be down-modulated in order to function. Also, in contrast to their role in human epithelial and endothelial adherence, neither pili nor PilC expression had any effect on phagocytic cell interactions with respect to induction of chemiluminescence as well as phagocytic killing. Examination of the relative influence of Opa and Opc indicated that Opa proteins are more effective than Opc in PMN interactions whereas the reverse was the case with monocytes. These results suggest that Opa and Opc mediate interactions with phagocytic cells via distinct mechanisms. Observations presented here and reported previously collectively show that the structural requirements of meningococci for interacting with phagocytes, in the absence of opsonins, are present in the phenotype which is often isolated from the nasopharynx (asialylated, piliated, Opa/Opc+) whereas the phenotype prevalent in the blood (sialyted, piliated, Opa/Opc+) retains the ability to adhere to endothelial cells (via pili) but appears to be refractory to interactions with phagocytic cells.

An analysis was made of the neutralizing antibody repertoire, for influenza virus hemagglutinin (HA) of transgenic mice expressing a human immunoglobulin mu (IgH) minigene, by monoclonal antibody (MAb) selection and sequencing of the HA genes of X31 (H3N2 subtype) laboratory variants. Whereas previously reported laboratory variants, selected in ovo with high-affinity murine MAbs of the IgG class, differed from wild-type virus by a single amino acid residue change in one of the major antigenic sites, neutralizing MAbs from transgenic donors selected novel variant viruses with altered receptor-binding specificity and contained residue changes in both the receptor-binding pocket (HA1 225 or HA1 226) and an antigenic site (HA1 135, HA1 145, or HA1 158). Changes in receptor-binding specificities of the variant viruses were confirmed by their resistance to inhibition by horse serum glycoproteins and altered binding to neoglycoproteins. The residue changes in variant virus V-21.2 (HA1 135 G-->R, 225 G-->D) abrogated neutralization by each of the MAbs; nevertheless V-21.2 was recognized by its own selecting MAb in enzyme-linked immunosorbent assay and therefore qualified as an adsorptive mutant rather than an antigenic variant. We consider that a low-affinity neutralizing antibody response may preferentially select for receptor-binding variants of influenza virus HA.

Activation of T lymphocytes by pokeweed mitogen (PWM) is strictly monocyte (Mo)-dependent and results in T-cell mitogenesis and interleukin-2 (IL-2) secretion, coupled with an inability to utilize IL-2 due to an impaired expression of functional IL-2 receptor (IL-2R). Such IL-2R impairment could arise in PWM-activated T cells themselves or, alternatively, be the result of Mo-derived influences, as it is known that PWM binds Mo strongly and does not or poorly binds lymphocytes, and Mo becomes rapidly destroyed in PWM-stimulated cultures of blood mononuclear cells or T cells plus Mo. The present study investigated these possibilities. The results show for the first time that desialylation of T lymphocytes strongly increases their PWM-binding capacity and, in addition, overcomes the Mo requirement for PWM to induce T-cell mitogenesis and IL-2 secretion. Such secreted IL-2 levels were even higher that those found in cultures of Mo-dependent PWM-activated T lymphocytes but similarly to the latter, PWM-activated desialylated purified T lymphocytes exhibited negligible high-affinity IL-2 binding capacity and an inability to utilize the IL-2 they produced. These effects were not due to desialylation itself, as indicated by data obtained with peanut agglutinin, a lectin that becomes strongly reactive with desialylated T lymphocytes. The data clearly indicate the existence of PWM-related events capable of impairing the expression of functional IL-2R without affecting IL-2 secretion, and indicate that such events are due to mechanisms arising at the level of PWM-activated T cells themselves.

The Sia-lb1 epitope, recognized by anti-Sia-lb1 cold agglutinins, is unique since it is represented by the alpha-N-acetylneuraminic acid (alpha NeuNAc) monosaccharide. Chemical modifications of the chain at C-5 of alpha NeuNAc have shown that the natural 2-carbon and the artificial 3-carbon chains are optimal for anti-Sia-lb1 binding. Sia-lb1 antigenicity of alpha NeuNAc could be tenfold enhanced by replacement of the carbonyl oxygen by sulphur. The structural requirements of the Sia-lb1 epitope for optimal antibody binding were identified.

Group B Streptococcus (GBS) is an important perinatal pathogen. Because transplacentally acquired maternal antibodies to the GBS capsular polysaccharides (CPS) confer protection, prevention of infant disease may be possible after immunization of women. Unfortunately, the purified CPS of GBS are only variably immunogenic in adults; therefore to enhance immunogenicity we have designed and developed a CPS-protein conjugate vaccine. The lability of a conformationally dependent epitope on the III CPS containing a critical sialic acid residue was important to consider in vaccine design. 100 women were randomized to receive GBS type III CPS-tetanus toxoid conjugate (III-TT) vaccine at one of three doses; unconjugated GBS type III CPS; or saline. Serum samples were obtained before immunization and 2, 4, 8, and 26 wk thereafter, and specific antibody to type III CPS was measured. Vaccines were well tolerated. In sera from recipients of the highest dose of III-TT, CPS-specific IgG levels rose from a geometric mean of 0.09 microg/ml before immunization to 4.53 microg/ml 8 wk later, whereas levels in recipients of unconjugated type III CPS rose from 0.21 microg/ml to 1.41 microg/ml. Lower doses resulted in lower antibody levels. A > or = 4-fold rise in antibody concentration was achieved in 90% of recipients of III-TT compared with 50% of those that received III CPS (P = 0.0015). Antibodies evoked by the conjugate vaccine recognized a conformationally dependent epitope of the III-CPS, promoted opsonophagocytosis and killing of GBS, and, after maternal immunization, protected neonatal mice from lethal challenge with type III GBS. We conclude that directed coupling of type III GBS polysaccharide to a carrier protein yielded a conjugate vaccine with preserved expression of a highly labile conformational epitope involving sialic acid and enhanced immunogenicity compared with uncoupled CPS.

Two monoclonal antibodies (MAbs) to a 36-kDa extracellular metalloprotease (PSCP) from Burkholderia (Pseudomonas) cepacia were found to react with thermolysin, Pseudomonas aeruginosa elastase, alkaline protease (Apr) and LasA, Serratia marcescens protease (SMP), Aeromonas hydrophila protease (AhP), and both the lethal factor (LF) and protective antigen (PA) of Bacillus anthracis on immunoblots. The MAbs were capable of neutralising the proteolytic activity of thermolysin, P. aeruginosa elastase and PSCP but not that of Apr, SMP, and AhP. These results suggest that these MAbs may be able to differentiate between the thermolysin and serralysin family of metalloproteases on the basis of their neutralisation capability and could, therefore, be useful tools in the characterisation of new bacterial proteases.