Gangliosides as markers for murine lymphocyte subpopulations.

Antibodies to GM1 ganglioside were used to study murine lymphocyte populations. In A, AKR, and BALB/c mice, anti-GM1 reacts with thymocytes and peripheral T cells. This reactivity of anti-GM1, studied by immunofluorescence, is independent of Thy-1 type and appears to be related to the reactivity of cross-reacting antibodies to asialo GM1 and GD1b, rather than GM1 itself. In addition, a subpopulation of lymphocytes reacting with anti-GM1 and anti-immunoglobulin has been found in approximately 26% of the peripheral lymphocytes of C3H mice, nude mice, and nude heterozygotes. This subpopulation is found in small numbers in A, AKR, and BALB/c mice. These studies demonstrate that antibodies to a chemically defined antigen can be used to identify T cells in many strains of mice and may delineate previously unrecognized lymphocyte subpopulations.

Hippocampal volumes and diffusion-weighted image findings in children with prolonged febrile seizures.

OBJECTIVES: To assess hippocampal volumes (HV) and signal changes on diffusion-weighted imaging (DWI) within 5 days of prolonged febrile seizures (PFS) and compare them with the PFS duration and EEG. METHODS: We studied 12 children (mean age: 32 +/- 21 months, range 10 months-5 years) within 5 days of a first episode of PFS (a seizure or series of seizures lasting for 30 min or longer, without return of consciousness between the seizures). The HV measurements were carried out using high-resolution magnetic resonance imaging and signal intensity abnormalities were evaluated visually on DWI. HV in patients were compared with those of 13 neurologically normal controls (mean age 31 +/- 16 months, range 15 months-5 years). HV abnormalities correlated with PFS duration. HV and DWI abnormalities were compared with EEG abnormalities. RESULTS: Seizure duration ranged from 40 to 95 min. In seven out of twelve patients, seizures were refractory and lasted for 60 min or longer despite intravenous infusion of diazepam. In the patients with PFS for 60 min or longer, HV were significantly larger than that of controls. In all patients, there was a positive correlation between HV and seizure duration. DWI showed hyperintensity in unilateral hippocampus in three patients with intractable seizures, ipsilateral thalamus in two, and cingulate in one. EEG showed abnormalities in temporal areas ipsilateral to the DWI abnormalities in these patients. CONCLUSIONS: Large HV and hippocampal hyperintensity on DWI were seen in patients with refractory PFS. Our results suggest that medically refractory PFS lasting for 60 min or longer may cause structural changes in limbic structures that could promote later epileptogenesis.

Hippocampal volumes and diffusion-weighted image findings in children with prolonged febrile seizures.

OBJECTIVES: To assess hippocampal volumes (HV) and signal changes on diffusion-weighted imaging (DWI) within 5 days of prolonged febrile seizures (PFS) and compare them with the PFS duration and EEG. METHODS: We studied 12 children (mean age: 32 +/- 21 months, range 10 months-5 years) within 5 days of a first episode of PFS (a seizure or series of seizures lasting for 30 min or longer, without return of consciousness between the seizures). The HV measurements were carried out using high-resolution magnetic resonance imaging and signal intensity abnormalities were evaluated visually on DWI. HV in patients were compared with those of 13 neurologically normal controls (mean age 31 +/- 16 months, range 15 months-5 years). HV abnormalities correlated with PFS duration. HV and DWI abnormalities were compared with EEG abnormalities. RESULTS: Seizure duration ranged from 40 to 95 min. In seven out of twelve patients, seizures were refractory and lasted for 60 min or longer despite intravenous infusion of diazepam. In the patients with PFS for 60 min or longer, HV were significantly larger than that of controls. In all patients, there was a positive correlation between HV and seizure duration. DWI showed hyperintensity in unilateral hippocampus in three patients with intractable seizures, ipsilateral thalamus in two, and cingulate in one. EEG showed abnormalities in temporal areas ipsilateral to the DWI abnormalities in these patients. CONCLUSIONS: Large HV and hippocampal hyperintensity on DWI were seen in patients with refractory PFS. Our results suggest that medically refractory PFS lasting for 60 min or longer may cause structural changes in limbic structures that could promote later epileptogenesis.

Quantitative determination of N-glycolylneuraminic acid expression in human cancerous tissues and avian lymphoma cell lines as a tumor-associated sialic acid by gas chromatography-mass spectrometry.

N-Glycolylneuraminic acid (NeuGc) is distributed in most animals except humans and chickens. However, human and chicken cancerous tissues often synthesize this heterophilic sialic acid as a tumor-associated Hanganutziu-Deicher antigen [M. Naiki and H. Higashi, Adv. Exp. Med. Biol., 152: 445-456, 1982; H. Higashi et al., Cancer Res., 45: 3796-3802, 1985]. In this paper, NeuGc in human cancerous tissues and chicken Marek's disease lymphoma cell lines was determined quantitatively with gas chromatography-mass spectrometry analysis using mass fragmentography. The detectable limit of NeuGc was 40 pg (0.12 pmol) in each injection using 5 ng of trideuteriomethyl ester trideuteriomethyl glycoside of the sialic acid as an internal standard sample when a pair of ions at m/e 386 and 389 was chosen for ion monitoring. NeuGc was detected in ganglioside-rich fractions of various human cancerous tissues from 5 of 8 patients examined but was not detected in glycosphingolipids of normal human tissues. The contents of NeuGc in these cancerous tissues ranged from 0.02 to 0.5% of the total sialic acid content. NeuGc was also detected in freeze-dried samples of 5 different cell lines from chicken Marek's disease lymphomas but was not detected in a cell line from chicken lymphoid leukosis lymphoma and normal chicken skeletal muscle tissue. The contents of NeuGc in the positive cell lines ranged from 0.03 to 0.11% of the total sialic acid content. These results indicate that NeuGc can be synthesized in both humans and chickens in some cancers.

Characterization of N-glycolylneuraminic acid-containing gangliosides as tumor-associated Hanganutziu-Deicher antigen in human colon cancer.

Hanganutziu-Deicher (HD) antigen-active N-glycolylneuraminic acid (NeuGc)-containing gangliosides were isolated and characterized from human colon cancer tissues. The antigenic gangliosides were detected by thin-layer chromatography by our newly developed method (H. Higashi, Y. Fukui, S. Ueda, S. Kato, Y. Hirabayashi, M. Matsumoto, and M. Naiki. J. Biochem., 95: 1517-1520, 1984) of enzyme immunostaining using affinity-purified chicken antibody against hematoside containing NeuGc (II3NeuGc-LacCer) and horseradish peroxidase-conjugated rabbit anti-chicken lgG. One to six species of the antigenic gangliosides were isolated from seven of 16 cases of colon cancer, whereas no antigenic compound was detected in all apparently normal colorectal tissues from 17 individuals without colorectal cancer. Tissues from different patients showed different patterns of molecular species of the antigenic gangliosides. Densitometric determination indicated that HD antigenic sialic acid, NeuGc, accounted for about 1% or less of the total lipid-bound sialic acids. Four species of antigenic gangliosides were identified as hematoside and hematoside-containing O-acyl ester (II3NeuGc-LacCer and II3 4- or 7-O-acyl-NeuGc-LacCer), GM2-containing NeuGc (II3NeuGc-GgOse3Cer), and sialylparagloboside (IV3NeuGc-nLcOse4Cer) by their behaviors on 2-dimensional thin-layer chromatography, and the effects of mild alkaline treatment, sialidase treatment, periodate oxidation, and endo-beta-galactosidase treatment.

Enhanced immunogenicity of the cultured rat fibrosarcoma KMT-17 by cultivation in a low concentration of fetal calf serum.

We examined in different culture conditions alterations in the tumorigenicity and immunogenicity of an A3 clone that had been derived from a rat fibrosarcoma KMT-17. When a fetal calf serum concentration in a culture medium was lowered from 10 to 1%, the tumorigenicity was diminished while the immunogenicity was enhanced in a reversible manner; this was accompanied by a reversible prolongation of the in vitro doubling time. These phenomena were not due to an increase in the quantities of the original tumor-associated antigen and/or of the rat major histocompatibility complex (RT1) but seemed to be due to the appearance of a unique antigen(s) that was detected by an antibody taken from rats immunized with A3 tumor cells cultured in the low fetal calf serum concentration; this antigen(s) may consist of glycoprotein and exist as a crypt antigen(s). These phenomena were measured by an absorption test and flow cytometric analysis. Our observations suggest that the in vitro culture condition of tumor cells, in particular their culturing in the low fetal calf serum concentration medium, modifies the surface of tumor cells and causes a diminishment in their tumorigenicity and an enhancement of their immunogenicity.

Detection of 4-O-acetyl-N-glycolylneuraminyl lactosylceramide as one of tumor-associated antigens in human colon cancer tissues by specific antibody.

Antibody to 4-O-acetyl-N-glycolylneuraminyl lactosylceramide [GM3(Neu4AcGc)] was prepared by immunizing chicken with the glycosphingolipid antigen. The specific antibody was purified by affinity chromatography columns of Octyl-Sepharose linked to the homologous immunogen and its deacetylated analogue [N-glycolylneuraminyl lactosylceramide, GM3(NeuGc)], respectively. The specificity of the purified antibody was confirmed by enzyme-linked immunosorbent assay (ELISA) and inhibition of equine erythrocyte hemagglutination using authentic glycosphingolipids as antigens. The results indicated that the antibody recognized both 4-O-acetyl and N-glycolyl groups of terminal sialic acid residue as the immunodeterminants. The purified specific antibody was applied in the confirmation of the presence of GM3(Neu4AcGc) in ganglioside fractions of human colon cancer tissues, which were suspected to have this antigen by studies of alkaline, periodate or neuraminidase treatment [Higashi et al. (1985) Cancer Res. 45, 3796-3802.], by thin-layer chromatography (TLC)-immunostaining technique.

Specificities of human heterophilic Hanganutziu and Deicher (H-D) antibodies and avian antisera against H-D antigen-active glycosphingolipids.

The precise specificities H-D antibodies and newly obtained avian antisera to the H-D antigen-active glycosphingolipids. N-glycolylneuraminyl (alpha 2-3) lactosyl ceramide (hematoside) and N-glycolylneuraminyl (alpha 2-3) lactoneotetraosyl ceramide (sialosylparagloboside) were compared by gel precipitation and hemagglutination inhibition using other gangliosides with a related structure and chemically modified hematoside derivatives and the oligosaccharides from both the antigenic glycosphingolipids. Human H-D antibodies were found to have the same affinity for both antigenic glycosphingolipids; however, the avian anti-hematoside or anti-sialosylparaglobosides showed a higher affinity for the homologous antigen. Human H-D antibodies were directed more exactly toward the terminal sialic acid residue and more roughly to the hydrophobic portion of both compounds; however, the two avian antibodies were directed more specificity to the whole structure of the homologous antigen glycosphingolipid.

In vitro immunization of rat spleen lymphocytes with Forssman glycosphingolipid antigen and the generation of a monoclonal antibody.

A procedure for in vitro immunization of splenic lymphocytes with a glycolipid antigen is described. Culture medium supernatant of ConA- and PHA-stimulated spleen cells and that of Con A-stimulated human Jurkat T cell line (IL-2-rich medium) were used as sources of cytokines to support T and B cell stimulation, and anti-mu was used to support B cell differentiation. Unprimed rat spleen cells (2 x 10(6)/ml) were stimulated with 2 micrograms/ml Forssman glycolipid antigen coupled to Sepharose for 4 days. The cells were fused with a mouse myeloma cell line P3-X63-Ag8-U1. At initial screening, 12% of the colony forming wells were secreting specific antibody. After cloning, a stable hybridoma cell line (designated 4C3) was established which secreted a monoclonal IgM antibody directed against the carbohydrate moiety of Forssman glycosphingolipid (GalNAc alpha 1-3GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc-ceramide).

A specific enzyme-linked immunosorbent assay (ELISA) procedure for detection of heterophile Hanganutziu and Deicher (HD) antibodies.

A specific, relatively sensitive, quantitative and standardized enzyme-linked immunosorbent assay (ELISA) procedure was developed for the detection of heterophile Hanganutziu and Deicher (HD) antibodies which are occasionally elevated in pathologic human sera. The HD antigen-active molecule used for the assay was a ganglioside (N-glycolylneuraminyllactosylceramide, abbreviated as NeuGc-LacCer) previously purified from horse erythrocyte membranes. The test used antigen-coated plastic microtiter plates and anti-human immunoglobulin G (IgG, Fab fragment) conjugated with alkaline phosphatase. Fifty-four normal human sera gave ELISA values ranging from -2 to 2%. Random sera from hospitalized patients were first screened by the horse erythrocyte hemagglutination (HA) test, whereby 5.7% (76 cases) gave abnormal HA titers of 128-4096 compared to titers in normal sera equal to or less than 64. Ninety-seven % of the patients' sera gave abnormal ELISA values (3-200%). They were classified into 3 groups: cancer (42 cases), infection (10 cases), and others (24 cases). The potential value of this ELISA method is discussed.

A potential radioimmunoassay system for detection of Hanganutziu-Deicher type heterophile antigen(s) and antibodies in tissues and fluids.

A relatively simple, specific and sensitive radioimmunoassay system has been developed for the detection of heterophile Hanganutziu-Deicher (H-D) antigen(s) and antibodies. The 125I-labeled H-D antigen-active molecule used for the assay is a bovine erythrocyte major glycoprotein previously found to have a strong H-D antigen potency. The antigen-antibody complex was precipitated with normal human serum as the carrier protein, followed by the addition of rabbit anti-human IgG F(ab')2 serum. With this method, different H-D antigen-active molecules were compared for heterophile H-D antigen potency with reasonable sensitivity detecting about 0.3 ng of cold glycoprotein. 8 different lung cancer tissues were assayed for H-D antigen. The sera from the 8 lung cancer patients were also screened by ELISA and RIA in an attempt to correlate expression of H-D antigen on tissues with elevation of H-D antibodies. The results showed that all patients' tissues expressed the antigen(s) but only 3 of them had abnormal levels of H-D antibodies. This could have been due to excess antigens in circulation or immune complexes.

Suppression of acute experimental allergic encephalomyelitis by neurotropin: clinical, histopathologic, immunologic and immunohistochemical studies.

The effect of neurotropin, an extract isolated from the inflamed skin of rabbits inoculated with Vaccinia virus was examined on acute experimental allergic encephalomyelitis (EAE) in Lewis rats. A dose of 40 mg per kg body weight of neurotropin was administered intraperitoneally for 7 days post-inoculation. The severity of clinical signs of acute EAE was decreased by the administration of neurotropin. Histopathologic evaluation showed that lesion severity of EAE in neurotropin-treated rats was less than that seen in untreated rats. Blood lymphocyte subset analysis revealed that in comparison to untreated EAE rats, in neurotropin-treated rats, the percentage of OX6+ (Ia antigen) cells was lower and the W3/25+ (helper T cell): OX8+ (suppressor/cytotoxic T cell) cell ratio was greater during the period of peak inflammation. Immunohistochemical examination of neurotropin-treated rats demonstrated that OX6+ and W3/25+ cells within EAE lesions were fewer and that OX8+ cells in lesions occurred in greater numbers than those in untreated rats. These findings suggest that the OX8+ cells in the inflammatory lesions may have been induced by neurotropin treatment and that the suppressive effects on the disease may have been causally related to their presence.

Effect of an autoreactive T cell clone from (NZB x NZW)F1 mice on the production of anti-DNA antibodies in vivo and in vitro.

An autoreactive T lymphocyte clone, designated as F1C4 was established from an autoimmune mouse strain (NZB/NZW)F1. This clone proliferated in the presence of mitomycin C-treated splenic adherent cells (MMC-SAC) from syngeneic mice. This response was dependent on the numbers of MMC-SAC. The specificity of F1C4 for I-A was determined by an inhibition test carried out with monoclonal anti-Ia sera. Furthermore, the F1C4 cells did not exhibit alloreactivity in a proliferation assay and did not react to foreign antigens such as fetal calf serum (FCA) used in the culture medium. When F1C4 cells were cultured with autologous non-T cells in the absence of antigen, they strongly enhanced IgM class anti-ssDNA production from non-T cells of both young and old B/W F1 mice at appropriate cell numbers in vitro. Furthermore, the production of IgG class anti-ssDNA from non-T cells of old B/W F1 mice was also enhanced. The adoptive transfer of F1C4 cells enhanced the levels of both IgM and IgG anti-ssDNA antibodies in the serum of aged B/W F1 mice. Moreover, the serum levels of anti-ssDNA of IgG2a and IgG2b subclasses were readily enhanced by the transfer of F1C4 in vivo.

Microstructure of the sialic acid moiety of N-glycolylneuraminyllactosylceramide and the elucidation of Hanganutziu and Deicher (HD) antigenicity.

The carboxyl (-COOH) group of heterophile Hanganutziu and Deicher (HD) antigen-active ganglioside (N-glycolylneuraminyllactosylceramide) was esterified (-CO2CH3) by methyl iodide and then reduced to a primary alcohol (-CH2OH) by sodium borohydride. The intact molecule (commonly known as HD3) as well as its two derivatives were tested for HD antigen potency using four human pathologic sera containing HD antibodies. The methyl ester derivative (1-methyl-HD3) gave the same inhibition potency as HD3, but the reduced HD3 gave poor inhibition (1/66) compared to the intact HD3. The results show that reduction of the carboxyl group diminishes the inhibitory potency of HD3. This suggests that although the N-glycolyl (-CH2OH) group of HD3 is the most important determinant for manifestation of HD antigenicity, it is likely that the antibody recognizes both the N-glycolyl and carboxyl groups together when they form a hydrogen bond (-CH2OH---OOC-), aided by their possible proximity, and that substitution of either group therefore reduces the reaction of HD3 with HD antibody dramatically.

Isolation and characterization of rainbow trout C-reactive protein.

An acute phase serum component, C-reactive protein (CRP), was isolated from the sera of rainbow trout (Salmo gairdneri). The isolation was based on its calcium-dependent binding affinity for pneumococcal C-polysaccharide (CPS) according to the isolation procedure of human C-reactive protein. In SDS-PAGE, the nonreduced CRP showed two subunits with molecular weights of 43,700 and 26,600, respectively, at a molar ratio of 1:1. The reduced CRP showed a single subunit of 26,600. The molecular weight of the native protein was estimated as 66,000 by native gradient PAGE and 81,400 by sedimentation equilibrium analysis using ultracentrifugation. The antigenic determinant on CPS-reactive site was destroyed by periodate oxidation, indicating that rainbow trout CRP is a glycoprotein. CRP levels in rainbow trout serum measured by the CPS-ELISA procedure showed that the rainbow trout CRP could behave as an acute phase reactant, following experimental infection with the fish pathogen Vibrio anguillarum.

A monoclonal antibody reacting with the zona pellucida of the oviductal egg but not with that of the ovarian egg of the golden hamster.

A monoclonal antibody (mAb) designated AZPO-8 was produced by hybridizing a mouse myeloma with spleen cells from BALB/c mice immunized with materials obtained from the hamster oviduct. With an immunofluorescence test, AZPO-8 reacted with the zona pellucida (ZP) of ovulated eggs in the oviduct (ZP-OVI) but not with the zona pellucida of eggs in the ovary (ZP-OVA). Using indirect enzyme immunostaining, this mAb reacted with epithelial cells of the oviduct, the uterus (especially the cervical epithelium) and the gastric mucosa, but not with other hamster tissues examined. The reactivity of antigen-positive tissues was abrogated by pretreatment of the tissues with periodic acid. Western blotting analysis revealed that AZPO-8 reacted with substances of broad molecular weight range, and the strongest reactivity was detected at a molecular weight of approximately 200,000 in both cases when extract of ZP-OVI or the hamster oviduct was applied on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel. AZPO-8 showed strong hemagglutination activity only to group A human red blood cells. These results indicated that (1) ZP-OVI had an antigen that was not detected on ZP-OVA, (2) ZP-OVI and the oviduct shared the same antigenicity, and (3) the antigenic determinant reactive with the mAb might be carbohydrate in nature. A possible role of this antigen in fertilization was discussed.

Heterophile antibodies to rabbit erythrocytes in human sera and identification of the antigen as a glycolipid.

Normal human sera contain heterophile hemagglutinins to rabbit erythrocytes which are different from anti-B isoantibody and other heterophile antibodies such as Hanganutziu-Deicher antibody or Paul-Bunnell antibody. The antigen to this antibody was purified from rabbit erythrocyte stroma, and identified as pentaglycosyl ceramide, Gal(alpha 1-3)Gal(beta 1-4)GlcNAc(beta 1-3)Gal(beta 1-4)Glc-Cer.

Establishment of lymphoid cell lines from persons with different P blood group phenotypes and the biosynthesis of the antigenic glycosphingolipids.

Human lymphoid cell lines were established from the peripheral lymphocytes of persons with different P blood group phenotypes by in vitro transformation with EB virus. The glycosphingolipid compositions of these cell lines were examined by TLC. The results show that P1k and P2k phenotype cells lack globoside (P antigen) and that two cell lines established from persons with the p phenotype lack both globoside and CTH (Pk antigen), while both the glycosphingolipids were detected in two cell lines established from persons with usual phenotypes (P1 or P2 phenotype). CTH synthetase activity was detected at decreased levels in two p phenotype cell lines, however, globoside synthetase activities could not be significantly detected in all the P phenotype cells.

Avian antisera to various gangliosides: detection by enzyme immunoassay.

We attempted to produce specific antibodies to various gangliosides by immunizing chickens. Antibody activity was determined by enzyme immunoassay (EIA). The optimal conditions for EIA were examined by using chicken anti-GM1 ganglioside (GM1) serum and the final procedure was as follows. Fifty microliters of ethanol solution containing 2.5 micrograms of the glycolipid antigen and 50 micrograms of taurodeoxycholate were added to each well of an EIA microtitration plate and dried at 37 degrees C for 2 h. Nonspecific sites were blocked by incubation with 1% gelatin-containing buffer, then titration of the chicken antisera was carried out in the antigen-coated plate by incubation at 4 degrees C for 12 h. Next, alkaline phosphatase-labeled anti-chicken IgG (specific antibody, 15 ng; enzyme, 0.054 units) was allowed to react at 37 degrees C for 2 h. The enzyme activity which was bound to the plate was assayed with p-nitrophenol phosphate as substrate. Under the above conditions, anti-GM1 serum reacted strongly with GM1 and asialo GM1, and anti-GM2 serum indicated a considerable specificity for GM2. However, we failed to elicit any antibody to GD1a, GD1b, and GT1b. Anti-NeuAc-sialosylparagloboside and anti-NeuGc-sialosylparagloboside sera showed a high specificity for the homologous ganglioside. However, anti-NeuGc-hematoside serum reacted equally with both the homologous antigen and NeuGc-sialosylparagloboside, and anti-NeuAc-hematoside (GM3) serum cross-reacted with both N-acetyl and N-glycolyl types of hematoside and sialosylparagloboside.

Autoradiography of ganglioside antigens separated by high-performance thin-layer chromatography with their antibodies.

An improved method is described for the detection of ganglioside antigens with the antibodies on thin-layer chromatograms. The method involves the following steps: Separation of gangliosides on a high-performance thin-layer chromatographic plate, application of antibody solution, and detection of bound antibody with [125I]staphylococcal protein A. Using specific antibodies against gangliosides GM4, GM1, GD3, and asialo GM1 ganglioside, the method allowed positive identification of these antigens on thin-layer plates. It also provides a convenient means of assessing the specificity of an anti-glycolipid antibody.