Association of histochemical expression of Maackia amurensis leukoagglutinin-positive glycoconjugates with behaviour of human gastric cancer.

AIMS: To investigate the relationship between sialylation of glycoconjugates and clinicopathological characteristics of gastric cancer. METHODS AND RESULTS: Sialylation of glycoconjugates was examined histochemically in 71 gastric cancers using Maackia amurensis leukoagglutinin (MAL), a lectin that recognizes the trisaccharide sequence NeuAc alpha 2,3Gal beta 1,4GlcNAc/Glc. Positive staining with MAL was observed in the tumour region of all of the samples, but the populations of MAL-positive tumour cells in the tumour region varied among the samples. In the corresponding non-cancerous regions, however, no positive staining was observed. Calculating the percentage of MAL-positive tumour cells as part of the total tumour cells with respect to the MAL-staining index (MI) allowed the gastric cancer to be classified into two distinct groups: high and low levels of MI, with a cut-off level of 40% of MI. Furthermore, statistical analyses using the MI level and clinicopathological characteristics of the tumour indicated that a high MI level in gastric tumour tissues is related to a poorer prognosis. CONCLUSIONS: The appearance of MAL-positive glycoconjugates in gastric tumour cells is associated with the behaviour of gastric cancer.

Macrophages are involved in DNA degradation of apoptotic cells in murine thymus after administration of hydrocortisone.

In the present study, we undertook kinetic analyses of DNA degradation and acid DNase activity in murine thymus after administration of hydrocortisone. Hydrocortisone induced apoptosis in thymocytes, and a large number of cortical thymocytes became TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end labelling)-positive (TUNEL+). F4/80+ macrophages infiltrated through the cortico-medullay junction into the cortical region, and thereafter engulfed apoptotic cells in the cortex of thymus. The distribution of acid DNase-active cells appeared to be similar to that of F4/80+ macrophages. Eighteen hours after the injection, although the foci of apoptotic cells were situated within massively distended F4/80+ macrophages, oligonucleosomal DNA fragments on an agarose gel were undetectable. Our results showed that macrophages were involved in the disappearance of oligonucleosomal DNA fragments in apoptotic thymocytes. Taken together, macrophages play a role in the hydrolysis of DNA in apoptotic cells upon their phagocytosis of the dead cells.

Isotype-specific selection of high affinity memory B cells in nasal-associated lymphoid tissue.

Mucosal immunoglobulin (Ig)A dominance has been proposed to be associated with preferential class switch recombination (CSR) to the IgA heavy chain constant region, Calpha. Here, we report that B cell activation in nasal-associated lymphoid tissue (NALT) upon stimulation with the hapten (4-hydroxy-3-nitrophenyl)acetyl (NP) coupled to chicken gamma globulin caused an anti-NP memory response dominated by high affinity IgA antibodies. In the response, however, NP-specific IgG(+) B cells expanded and sustained their number as a major population in germinal centers (GCs), supporting the view that CSR to IgG heavy chain constant region, Cgamma, operated efficiently in NALT. Both IgG(+) and IgA(+) GC B cells accumulated somatic mutations, indicative of affinity maturation to a similar extent, suggesting that both types of cell were equally selected by antigen. Despite the selection in GCs, high affinity NP-specific B cells were barely detected in the IgG memory compartment, whereas such cells dominated the IgA memory compartment. Taken together with the analysis of the V(H) gene clonotype in GC and memory B cells, we propose that NALT is equipped with a unique machinery providing IgA-specific enrichment of high affinity cells into the memory compartment, facilitating immunity with high affinity and noninflammatory secretory antibodies.

Abnormal IgG galactosylation and arthritis in MRL-Fas(lpr) or MRL-FasL(gld) mice are under the control of the MRL genetic background.

MRL mice bearing the lpr (Fas) or gld (Fas ligand) mutation, MRL-Fas(lpr) or MRL-FasL(gld), respectively, develop arthritis similar to rheumatoid arthritis, but C3H and C57BL/6 mice bearing such mutations do not. In MRL-Fas(lpr) mice, agalactosylated oligosaccharides in serum IgG increase significantly in comparison to MRL-+/+ mice without arthritis. In this study, an increased level of agalactosylation in IgG, as compared to MRL-+/+, was found in both MRL-Fas(lpr) and MRL-FasL(gld) mice. In contrast, the incidence of IgG without galactose was comparable among C3H-Fas(lpr), C3H-FasL(gld), and C3H-+/+ mice as well as between C57BL/6-Fas(lpr) and C57BL/6-+/+ mice. These results suggest that the increase in agalactosylated IgG and the development of arthritis in MRL-Fas(lpr) and MRL-FasL(gld) mice are controlled by the MRL genetic background.

An ancient lectin-dependent complement system in an ascidian: novel lectin isolated from the plasma of the solitary ascidian, Halocynthia roretzi.

Mannose-binding lectin (MBL) is a C-type lectin involved in the first line of host defense against pathogens and it requires MBL-associated serine protease (MASP) for activation of the complement lectin pathway. To elucidate the origin and evolution of MBL, MBL-like lectin was isolated from the plasma of a urochordate, the solitary ascidian Halocynthia roretzi, using affinity chromatography on a yeast mannan-Sepharose. SDS-PAGE of the eluted proteins revealed a major band of approximately 36 kDa (p36). p36 cDNA was cloned from an ascidian hepatopancreas cDNA library. Sequence analysis revealed that the carboxy-terminal half of the ascidian lectin contains a carbohydrate recognition domain (CRD) that is homologous to C-type lectin, but it lacks a collagen-like domain that is present in mammalian MBLs. Purified p36 binds specifically to glucose but not to mannose or N-acetylglucosamine, and it was designated glucose-binding lectin (GBL). The two ascidian MASPs associated with GBL activate ascidian C3, which had been reported to act as an opsonin. The removal of GBL-MASPs complex from ascidian plasma using Ab against GBL inhibits C3-dependent phagocytosis. These observations strongly suggest that GBL acts as a recognition molecule and that the primitive complement system, consisting of the lectin-proteases complex and C3, played a major role in innate immunity before the evolution of an adaptive immune system in vertebrates.

Requirement of ceramide for adhesion of Helicobacter pylori to glycosphingolipids.

Direct adhesion of Helicobacter pylori to immobilized glycosphingolipids (GSLs) was compared to that of their corresponding oligosaccharide-conjugated neoglycoconjugates in order to clarify the roles of the carbohydrate and lipid portions of GSLs in H. pylori adhesion. These bacteria were found to adhere to sulfatide, GM3, GalCer and LacCer, but not to ceramide, sphingomyelin, or polyacrylamides conjugated with beta-galactose, lactose, 3'-sialyllactose or 3'-sulfo-beta-galactose. Furthermore, neoglycolipids or bovine serum albumin derivatives with corresponding oligosaccharides were unable to serve as the ligands. H. pylori adhesion to GalCer with alpha-hydroxyl fatty acid was much stronger than GalCer with the non-hydroxyl fatty acid. These results suggest that H. pylori recognize the conformation of GSL with alpha-hydroxyl fatty acid on solid phase.

Rapid and simple preparation of N-linked oligosaccharides by cellulose-column chromatography.

As a means of preparing N-linked oligosaccharides from hydrazinolysates of glycoproteins in a rapid and simple manner, a method has been developed using cellulose-column chromatography. Hydrazinolysates of human IgG, containing a series of biantennary complex type oligosaccharides, were applied to a cellulose column equilibrated with (4:1:1, v/v) 1-butanol-ethanol-water. The N-linked oligosaccharides were eluted with (1:1, v/v) ethanol-water, and analyzed by HPLC in combination with sequential glycosidase digestion. The oligosaccharides, with or without sialic acid, were quantitatively recovered in the fraction eluted with (1:1, v/v) ethanol-water without UV-detectable contamination by impurities derived from protein or the cellulose. Other types of N-linked oligosaccharides of alpha1-acid glycoprotein (tetraantennary complex-type), ovalbumin (hybrid-type), and ribonuclease B (high mannose-type) were also quantitatively prepared from the hydrazinolysates by elution of the cellulose column with (1:1, v/v) ethanol-water and these had as high a quality as those prepared by conventional paper chromatography.

Role of galactosylation in the renal pathogenicity of murine immunoglobulin G3 monoclonal cryoglobulins.

Cryoglobulin activity associated with murine immunoglobulin G3 (IgG3) has been shown to play a significant role in the development of murine lupuslike glomerulonephritis. A fraction, but not all, IgG3 monoclonal antibodies are capable of inducing a severe acute lupuslike glomerulonephritis as a result of direct localization of IgG3 cryoglobulins, suggesting the importance of qualitative features of cryoglobulins in their nephritogenic activities. Here a remarkable difference is shown in the renal pathogenicity of 2 murine IgG3 monoclonal cryoglobulins, identical in the amino acid sequences of their heavy and light chains but different in galactosylation patterns of oligosaccharide side chains because of their synthesis in different myeloma cells. The antibody lacking the capacity to induce severe glomerulonephritis displayed an increased proportion of galactosylated heavy chains. Changes in conformation, as revealed by gel filtration analysis, reduced cryoglobulin activity, and accelerated clearance could account for the lack of the renal pathogenicity of the more galactosylated variant. This observation provides a direct demonstration for the role of IgG galactosylation in the pathogenic potential of cryoglobulins. (Blood. 2001;97:3537-3543)

Simultaneous quantification of components of neoglycolipid-coated liposomes using high-performance liquid chromatography with evaporative light scattering detection.

Liposomes composed of dipalmitoylphosphatidylcholine (DPPC), cholesterol and a neoglycolipid, mannopentaose-conjugated dipalmitoylphosphatidylethanolamine (Man5-DPPE), have been shown to have a strong adjuvant effect in inducing the antigen-specific cellular immunity. In this study, a rapid and simple analytical method using a HPLC system with an evaporative light scattering detector was developed for simultaneous quantification of the liposome components Man5-DPPE, cholesterol and DPPC. The chromatographic separation of these components was performed using a trimethylsilane column with an isocratic mobile phase of chloroform-methanol-water (1:33:6, v/v) after disrupting the liposomes with chloroform-methanol-water (10:10:3, v/v). This HPLC method provided sufficient reproducibility and linearity of calibration curves for the quantification of the liposome constituents. In addition, this method can be used for the quantification of various neoglycolipids with different carbohydrate structures.

Enhancement of mucosal immune response against HIV-1 Gag by DNA immunization.

In order to examine the feasibility of Gag-expression DNA as a potential candidate for HIV vaccine using a mouse model, we injected DNA into mice either intramuscularly or by using a gene gun. Both methods induced a low level of antibody production. However, after booster immunization with p24 protein emulsified with complete Freund's adjuvant via a footpad, we found that only the preceding intramuscular DNA immunization induced an anti-Gag Th1-type (IgG(2a)) antibody response, in addition to the enhancement of a Th2-type (IgG(1)) antibody response. Importantly, when mice were boosted intranasally with p24 and cholera toxin, intramuscular DNA injection was found to enhance both systemic and mucosal Gag-specific immune responses. These results indicate that intramuscular DNA immunization confers the inducibility of memory cells, which circulate around various mucosal tissues. Therefore, intramuscular DNA priming, followed by a mucosal booster immunization, could be considered as a regimen applicable to HIV vaccine.

Cloning and characterization of novel ficolins from the solitary ascidian, Halocynthia roretzi.

Ficolins are animal lectins with collagen-like and fibrinogen-like domains. They are involved in the first line of host defense against pathogens. Human ficolin/P35 as well as mannose-binding lectin (MBL) activates the complement lectin pathway in association with MBL-associated serine proteases. To elucidate the origin and evolution of ficolins, we separated approximately 40 kDa (p40) and approximately 50 kDa (p50) N-acetylglucosamine-binding lectins from hemolymph plasma of the solitary ascidian. Binding assays revealed that p40 recognizes N-acetyl groups in association with a pyranose ring and that p50 recognizes N-acetylglucosamine alone. Based on the amino acid sequences of the proteins, we isolated two clones each of p40 and p50 from the ascidian hepatopancreas cDNA and determined the entire coding sequences of these clones. Because all of the clones contained both collagen-like and fibrinogen-like domains, we concluded that these were homologs of the mammalian ficolin family and designated ascidian ficolins (AsFCNs). The fibrinogen-like domain of the AsFCNs shows 45.4-52.4% amino acid sequence identity with the mammalian ficolin family. A phylogenetic tree of the fibrinogen-like sequences shows that all the fibrinogen-like domains may have evolved from a common ancestor that branched off an authentic fibrinogen. These results suggest that AsFCNs play an important role with respect to ascidian hemolymph lectin activity and the correlation of different functions with binding specificity.

Abnormal IgG galactosylation in MRL-lpr/lpr mice: pathogenic role in the development of arthritis.

MRL-lpr/lpr (MRL/lpr) mice spontaneously develop arthritis by an increase in the incidence of agalactosylated oligosaccharides in serum IgG, similar to rheumatoid arthritis patients. However, whether this association has a pathogenic significance is still unknown. In this study, we analyzed the oligosaccharide structure of serum IgG in various MRL mice with or without arthritis, to clarify the relationship between the oligosaccharide abnormality and the development of arthritis. The level of agalactosylation in serum IgG was comparable in both arthritis-free MRL/lpr and MRL-+/+ (MRL/+) mice at 6 weeks of age. In contrast, the incidence of IgG lacking galactose markedly increased in MRL/lpr mice at 6 months of age (the age at which arthritis occurred), compared with that from age-matched MRL/+ mice without arthritis. However, the proportion of agalactosylated IgG increased similarly in anti-CD4 monoclonal antibody-treated MRL/lpr mice at 6 months of age, despite the absence of the development of arthritis, because of depletion of CD4+ T cells. These results suggest that the abnormality in IgG galactosylation of MRL/lpr mice developed in an age-dependent manner, but it did so independently of CD4+ T cell-dependent B-cell activation and is not a consequence of the development of arthritis.

Kinetic analysis of interaction of different types of rheumatoid factors with immobilized IgG using surface plasmon resonance.

Rheumatoid factors (RFs) are autoantibodies, which recognize antigens on a constant region of immunoglobulin G (IgG). Among various RF classes, RF of the IgG class (IgGRF) forms immune complexes in rheumatoid joints and is implicated in the pathogenesis of rheumatoid arthritis (RA). To characterize the formation of IgGRF immune complexes, in the present study, IgGRF was isolated from sera of RA patients, and its interaction with immobilized IgG was analyzed and compared to that of IgMRF or IgARF by means of surface plasmon resonance. On gel filtration, the IgGRF was eluted as a single peak corresponding to IgG, excluding the possible formation of self-associating IgGRF complexes in solution. Sensorgrams of the interaction of IgGRF with immobilized IgG revealed that it clearly bound to the IgG at 6 degrees C, but not at 30 degrees C. The degree of interaction decreased inversely with an increase in temperature, suggesting that IgGRF is much more reactive at lower temperatures. In contrast, the interaction of IgARF and IgMRF with IgG at 6 degrees C was similar to that at 30 degrees C. The association rate constant (k(a)) of IgGRF decreased with an increase in temperature, while those of IgARF and IgMRF were similar under various thermal conditions. The dissociation rate constant (k(d)) of IgGRF was greatly reduced at 25 degrees C, but those of IgARF and IgMRF slightly increased with an increase in temperature. These results suggested that the mode of interaction of IgGRF with IgG differed from in the cases of IgMRF and IgARF. The kinetic properties of the IgGRF-IgG interaction may facilitate elucidation of the IgGRF immune complex formation in rheumatoid joints.

Pepstatin A-sensitive aspartic proteases in lysosome are involved in degradation of the invariant chain and antigen-processing in antigen presenting cells of mice infected with Leishmania major.

We previously reported that CA074, a specific inhibitor of cathepsin B, significantly deviated immune responses from the disease-promoting Th2 type to the protective Th1 type in BALB/c mice infected with Leishmania major. Herein, we found that pepstatin A-sensitive aspartic proteases (PSAP) in lysosomes seem to play a different role from that of cathepsin B in antigen-processing and Ii-degradation. That is, cathepsin B appears to digest 16-, 28-, and 31-kDa peptides of soluble leishmania antigen (SLA), whereas PSAP seems to process mainly 28-kDa peptides. Furthermore, the latter protease contributed to the degradation of Ii but cathepsin B did not. Following treatment with pepstatin A, both Th1 and Th2 responses were profoundly suppressed in resistant DBA/2 mice (H-2(d)) and in susceptible BALB/c mice (H-2(d)), and both strains of mice became markedly susceptible compared with the untreated groups, probably owing to failure in degradation of Ii and partly to failure in digestion of 28-kDa peptide.

Analysis of cytokine production in the colon of nude mice with experimental colitis induced by adoptive transfer of immunocompetent cells from mice infected with a murine retrovirus.

LP-BM5 murine leukemia virus (MuLV) is known to induce murine AIDS (MAIDS). We have shown that Sjögren's syndrome (SjS)-like exocrinopathy can be induced in mice with MAIDS and that adoptive transfer of spleen cells from MAIDS mice can induce inflammatory bowel disease-like colitis as well as SjS-like exocrinopathy in nude mice. To assess the role of interferon (IFN)-gamma and interleukin (IL)-10 in the pathogenesis of our experimental model, we tried to identify the cells producing these cytokines and their localization in the colitis lesions in situ. Expression of mRNA for IFN-gamma and IL-10 was assessed by RT-PCR, and protein expression of these cytokines was also analyzed in frozen sections of colon by double-color-staining immunofluorescence (IF). An increase of IFN-gamma and IL-10 mRNA was detected in the colon of mice with colitis, but not in that of control mice. Double-color IF showed that Mac-1(+) cells were positive for IFN-gamma or IL-10 and that most CD4(+) T cells were positive for IL-10, although the population of IFN-gamma-positive CD4(+) T cells was low. In our experimental colitis model, Mac-1(+) macrophages that produce both IFN-gamma and IL-10 might play a crucial role in the pathogenesis of colitis in combination with CD4(+) T cells.

Angiotensin-converting enzyme inhibitor captopril prevents activation-induced apoptosis by interfering with T cell activation signals.

Captopril is an orally active inhibitor of angiotensin-converting enzyme (ACE) which is widely used as an anti-hypertensive agent. In addition to its ability to reduce blood pressure, captopril has a number of other biological activities. Recently the drug was shown to inhibit Fas-induced apoptosis in human activated peripheral T cells and human lung epithelial cells. In this study, we investigated whether captopril blocks activation-induced apoptosis in murine T cell hybridomas, and found that captopril inhibited IL-2 synthesis and apoptotic cell death upon activation with anti-CD3 antibody. In addition, captopril inhibited an inducible caspase-3-like activity during activation-induced apoptosis. On the other hand, captopril did not interfere with Fas signalling, since anti-Fas antibody-induced apoptosis in Fas+ Jurkat cells was unaffected by the drug. Furthermore, we examined whether captopril blocks activation-induced apoptosis by interfering with expression of Fas, Fas ligand (FasL), or both on T cell hybridomas. FasL expression on activated T cells was significantly inhibited by captopril, whereas up-expression of Fas was partially inhibited, as assessed by cell surface staining. Taking all data together, we conclude that captopril prevents activation-induced apoptosis in T cell hybridomas by interfering with T cell activation signals. Captopril has been reported to induce systemic lupus erythematosus syndrome, and our findings may be useful for elucidating the mechanism of captopril-induced autoimmunity.

Genotyping of Pneumocystis carinii f. sp. hominis isolates in Japan based on nucleotide sequence variations in internal transcribed spacer regions of rRNA genes.

Genotyping of Pneumocystis carinii (Pc) isolated from 24 bronchoalveolar lavage (BAL) fluid specimens in Japan was examined based on nucleotide sequence variations in internal transcribed spacer regions 1 and 2 (ITS1 and ITS2, respectively) of rRNA genes. We found 11 ITS1 genotypes including 2 novel ones and 11 ITS2 genotypes including 3 new ones. Combining the ITS1 and ITS2 genotypes resulted in 30 ITS genotypes, of which 10 are newly described in this report. Two or more genotypes in ITS regions in a specimen were observed in 16 of 24 patients. Our results will be of help for the epidemiological investigation of Pc infection.

Autoantibody activity of IgG rheumatoid factor increases with decreasing levels of galactosylation and sialylation.

The occurrence of N-linked oligosaccharides lacking galactose is significantly higher than normal in serum IgG of patients with rheumatoid arthritis (RA) in whom rheumatoid factor (RF), an autoantibody against autologous IgG, has been detected. In the present study, IgGs with and without RF activity (IgGRF and non-RF IgG, respectively) were prepared from sera of RA patients, and their oligosaccharide structures were characterized in order to investigate the relationship between RF activity and glycosylation. Three IgGRF fractions and a non-RF IgG fraction were obtained based on their ability to bind to an IgG-Sepharose column. The specific RF activity, as measured by immunoassays, was highest in the IgGRF fraction, which bound most avidly to the IgG-Sepharose. When the oligosaccharides were released by hydrazinolysis, and analyzed by MALDI-TOF mass spectrometry and HPLC, in combination with sequential exoglycosidase treatment, all the IgG samples were found to contain a series of biantennary complex-type oligosaccharides. The incidence of galactose-free oligosaccharides was significantly higher in both IgGRFs and non-RF IgG from RA patients compared with IgG from healthy individuals. In all IgGRFs, the levels of sialylation and galactosylation were lower than those in non-RF IgG from RA patients; the sialylation of non-RF IgG was the same as that of IgG from healthy individuals. In addition, the decreases in galactosylation and sialylation of oligosaccharides in IgGRF correlated well with the increase in RF activity. These findings could contribute to our understanding of the mechanisms of IgG-IgG complex formation and the pathogenicity of these complexes in RA patients.

Generation of both MHC class I- and class II-restricted antigenic peptides from exogenously added ovalbumin in murine phagosomes.

The phagosome fraction derived from a murine macrophage cell line (J774.1), which had internalized ovalbumin (OVA)-coated latex beads, was isolated. The peptides recovered from the phagosome fraction were separated on reverse phase HPLC and each fraction was analyzed for the content of either major histocompatibility complex (MHC) class I- or class II-restricted OVA-derived peptide. Both peptides were detected in the phagosome fraction after less than 15 min of internalization. It was also indicated that phagosomes degrade OVA protein into both MHC class I- and class II-restricted antigenic peptides by employing the same types of cathepsins. Furthermore, the results suggest that the MHC class I-restricted peptide rapidly exits from the phagosome to the cytosol. These findings illustrate a potential role for phagosomes not only in MHC class II-restricted but also in MHC class I-restricted exogenous antigen presentation pathways. Our results also point to the vital role of phagosomes in non-cytosolic antigen presentation pathway, in which further degradation of antigens by the proteasome is dispensable.

Ras mediates effector pathways responsible for pre-B cell survival, which is essential for the developmental progression to the late pre-B cell stage.

Ras is essential for the transition from early B cell precursors to the pro-B stage, and is considered to be involved in the signal cascade mediated by pre-B cell antigen receptors. To examine the role of p21(ras) in the late stage of B cell differentiation, we established transgenic mice (TG) expressing a dominant-inhibitory mutant of Ha-ras (Asn-17 Ha-ras) in B lineage cells at high levels after the early B cell precursor stage. Expression of p21(Asn-17) (Ha-ras) was associated with a prominent reduction in the number of late pre-B cells, but had little effect on proliferation of early pre-B cells. Inhibition of p21(ras) activity markedly reduced the life span of pre-B cells, due, at least in part, to downregulation of the expression of an antiapoptotic protein, Bcl-xL. Thus, the apparent role for p21(ras) activity in pre-B cell survival may explain the decreased numbers of late pre-B cells in Asn-17 Ha-ras TG. Consistent with this possibility, overexpression of Bcl-2 in Asn-17 Ha-ras TG reversed the reduction in the number of late pre-B cells undergoing immunoglobulin light chain gene (IgL) rearrangement and progressing to immature B cells. These results suggest that p21(ras) mediates effector pathways responsible for pre-B cell survival, which is essential for progression to the late pre-B and immature B stages.