Contribution of glycolipids to species-specific antigens on erythrocytes of several animal species as to recognition of antigens with rabbit anti-glycolipids and anti-erythrocyte antisera

Because anti-glycolipid antibodies are involved in the onset of several neurological diseases, the reactivity of glycolipids on erythrocytes and the probability of generating the antibodies were determined to clarify the contribution of glycolipids as antigens. Anti-erythrocyte antisera reacted with the following glycolipids in a species-specific manner, i.e. blood group A-active glycolipid for man, Forssman glycolipid for sheep, Gg₃Cer for guinea pig, and Gg₄Cer and fucosyl GM1 for rat, and the hemolytic activities of the anti-erythrocyte antisera were attenuated by absorption of the antisera with liposomes prepared from the lipids of erythrocytes to the following levels, 94.5% for man, 24.5% for sheep, 17.5% for guinea pig, and 54.5% for rat. These species-specific glycolipids on erythrocytes reacted well with the respective anti-glycolipid antisera, but Gb₄Cer in man and GM1 in rat were shown to be cryptic on immunization with erythrocytes, indicating that the contribution of glycolipids as erythrocyte antigens differs among animal species. The glycolipid nomenclature is based on the recommendations of the IUPAC-IUB Commission on Biochemical Nomenclature [1]. The ganglioside nomenclature of Svennerholm is employed throughout [2].     

Induction of CD1-restricted immune responses in guinea pigs by immunization with mycobacterial lipid antigens

Group 1 CD1 molecules have been shown to present lipid and glycolipid Ags of mycobacteria to human T cells. However, a suitable animal model for the investigation of this component of antimycobacterial immunity has not yet been established. Previously, we found that guinea pigs express multiple isoforms of group 1 CD1 proteins that are homologous to human CD1b and CD1c. In this study, we show that CD1-restricted T cell responses can be generated in guinea pigs following immunization with lipid Ags from Mycobacterium tuberculosis. Splenic T cells from lipid Ag-immunized guinea pigs showed strong proliferative responses to total lipid Ags and partially purified glycolipid fractions from M. tuberculosis. These lipid Ag-reactive T cells were enriched in CD4-negative T cell fractions and showed cytotoxic activity against CD1-expressing guinea pig bone marrow-derived dendritic cells pulsed with M. tuberculosis lipid Ags. Using guinea pig cell lines transfected with individual CD1 isoforms as target cells in cytotoxic T cell assays, we found that guinea pig CD1b and CD1c molecules presented M. tuberculosis glycolipid Ags to T cells raised by mycobacterial lipid immunization. These results were confirmed using a T cell line derived from M. tuberculosis lipid Ag-immunized guinea pigs, which also showed CD1-restricted responses and cytolytic activity. Our results demonstrate that CD1-restricted responses against microbial glycolipid Ags can be generated in vivo by specific immunization and provide support for the use of the guinea pig as a relevant small animal model for the study of CD1-restricted immune responses to mycobacterial pathogens.     

Monoclonal antibodies directed to chemically synthesized lactogangliotetraosylceramide, a leukemia-associated antigen having a novel branching structure

Murine leukemia cells (M1), in their undifferentiated state, have been characterized by the presence of cancer-associated lactoganglio-series glycolipids, one of which was identified as lactogangliotetraosylceramide (LcGg4) having a novel branching at the II-Gal of lactosylceramide through GlcNAc beta 1----3 and GalNAc beta 1----4 linkage, as shown below (Kannagi, R., Levery, S.B., and Hakomori, S. (1984) J. Biol. Chem., 259, 8444-8451): GalNAc beta 1----4 Gal beta 1----4Glc beta 1----1Cer GlcNac beta 1----3 Since this glycolipid is a very minor component, it has been difficult to obtain enough of the purified glycolipid for the preparation of a monoclonal antibody. We developed a method to chemically synthesize this glycolipid using a lactose unit, a ceramide unit, and two hexosamine donors as synthons and made the synthetic glycolipid available as an immunogen. The two monoclonal antibodies we obtained (YI328-18 and YI328-51, both IgG3) specifically recognized the novel branching structure and had no cross-reactivity with gangliotriaosylceramide or lactotriaosylceramide. Thus, the antibodies were found to be useful probes to detect lactogangliotetraosylceramide expressed in undifferentiated M1 leukemia cells, which disappears on induced differentiation. The results of this study indicate a new strategy to establish monoclonal antibody directed to novel minor glycolipid markers or their artificially designed analogs, employing chemically synthesized glycolipid antigens.     

Further characterization of the putative glycolipid receptor for mif: Role of fucose associated with an acidic glycolipid

Water soluble glycolipids were extracted from guinea pig macrophages. These glycolipids, when incubated with macrophages, augment the cells' response to migration inhibitory factor. The glycolipids were fractionated by diethylaminoethyl-Sephadex ion exchange chromatography into neutral and acidic fractions. Only the acidic glycolipid fraction was able to enhance the responsiveness of macrophages to migration inhibitory factor. Additional studies indicate that the enhancing activity of these glycolipid preparations can be abrogated by the removal of terminal fucose residues with α-L-fucosidase. The possibility that fucose functions as an essential component of a macrophage glycolipid receptor for migration inhibitory factor is discussed.     

Cell surface receptors for lymphokines. I. The possible role of glycolipids as receptors for macrophage migration inhibitory factor (MIF) and macrophage activation factor (MAF).

Incubation of culture supernatants from concanavalin A-stimulated guinea pig and rat lymphocytes with protein-free preparations of bovine brain gangliosides abolished their macrophage migration inhibitory factor (MIF) and macrophage activation factor (MAF) activity. The identity of the MIF/MAF-binding component(s) present in these glycolipid mixtures has yet to be established, but adsorption experiments using purified preparations of mono- (GM₁, GM₂, and GM₃), di- (GD_1a), and trisialogangliosides (GT₁) were negative. Since these gangliosides account for over 90% of the glycolipid content in brain ganglioside mixtures it appears that the MIF-binding component(s) is present only in very small amounts. Treatment of guinea pig peritoneal macrophages with liposomes containing similar brain gangliosides or water-soluble glycolipids extracted from guinea pig macrophages enhanced their responsiveness to MIF. The enhanced response to MIF of liposome-treated macrophages was abolished by incubation of the treated macrophages with fucose-binding lectins (Lotus agglutinin and Ulex europaeus agglutinin I) before exposure to MIF, suggesting that the MIF-binding component donated by the liposomes may be a fucose-containing glycolipid. The possible role of glycolipids as surface receptors for MIF and MAF is discussed.     

A highly sensitive enzyme immunoassay of anti-insulin antibodies in guinea pig serum

A highly sensitive enzyme immunoassay of anti-insulin antibodies in guinea pig serum is described. Guinea pig anti-insulin serum was diluted to various extents with nonspecific guinea pig serum and incubated with insulin. After incubation, free insulin was separated from insulin-anti-insulin antibody complex by treatment with dextran-charcoal. Anti-insulin antibodies in the complex were dissociated from insulin by incubation with 0.23 M HCl and inactivated. The amount of dissociated insulin was measured by sandwich enzyme immunoassay using anti-insulin IgG-coated polystyrene balls and affinity-purified anti-insulin Fab'-horseradish peroxidase conjugate. The detection limit of anti-insulin antibodies in guinea pig serum was 6.7 pg/assay or 150 ng/liter of serum. The present enzyme immunoassay was 10,000-fold more sensitive than the previously described enzyme immunoassay, in which insulin-coated polystyrene balls were incubated with diluted guinea pig anti-insulin serum and subsequently with rabbit (anti-guinea pig IgG) Fab'-horseradish peroxidase conjugate.     

A new type of blood group B active glycosphingolipid in rat bone marrow cells. Occurrence of the glycolipid in rat immunocytes and ascites hepatoma

A major glycosphingolipid in rat bone marrow cells was purified, and its structure was studied. The glycolipid was found to exhibit blood group B activity by the hemagglutination inhibition test. The structure was determined to be (formula; see text) by studies of nuclear magnetic resonance, sequential hydrolysis by exoglycosidases, linkage analysis of methylated sugars by gas chromatography-mass spectrometry, and immunological tests. The blood group B active glycolipid was detected not only in the bone marrow cells but also in spleen, thymus, and rat ascites hepatoma AH 7974F cells. Besides the glycolipid, gangliotriaosylceramide, gangliotetraosylceramide, and fucogangliotetraosylceramide were commonly detected in these cells. The similarity between the glycolipid species on the cell surfaces of the immunocytes and the tumor cells is discussed with the respect to an escape mechanism of the tumor cells from the immunosurveillance system.     

An interspecies comparison of gangliosides and neutral glycolipids in adrenal glands

Gangliosides and neutral glycolipids of adrenal glands of mouse, rat, guinea pig, rabbit, cat, pig, cow, monkey, and chicken were analyzed by thin layer chromatography (TLC). The major gangliosides common to all species had lactosylceramide in their core structure. GM3 containing N-acetylneuraminic acid (NeuAc) was the major ganglioside in rat, guinea pig, rabbit, and cat, whereas GM3 containing N-glycolylneuraminic acid (NeuGc) was the major one in mouse, cow, and monkey. GD3 was also detected in all species except mouse and GD3(NeuAc)2 was the major in pig adrenal gland. GM4(NeuAc) was detected in the adrenal glands of guinea pig and chicken but not in those of the other species. In the neutral glycolipid fractions, galactosylceramide, glucosylceramide, lactosylceramide, globotriaosylceramide and globotetraosylceramide were detected and the proportions of these glycolipids varied among the species. Guinea pig and chicken adrenal glands contained large amounts of galactosylceramide, this being consistent with the presence of GM4 in these two species. Globopentaosylceramide was detected in mouse, guinea pig, cat, and chicken by the TLC-immunostaining procedure.     

Guinea pig complement potently measures vibriocidal activity of human antibodies in response to cholera vaccines

The vibriocidal assay using guinea pig complement is widely used for the evaluation of immune responses to cholera vaccines in human clinical trials. However, it is unclear why guinea pig complement has been used over human complement in the measurement of vibriocidal activity of human sera and there have not been comparison studies for the use of guinea pig complement over those from other species. Therefore, we comparatively investigated the effects of complements derived from human, guinea pig, rabbit, and sheep on vibriocidal activity. Complements from guinea pig, rabbit, and human showed concentration-dependent vibriocidal activity in the presence of quality control serum antibodies. Of these complements, guinea pig complement was the most sensitive and effective over a wide concentration range. When the vibriocidal activity of complements was measured in the absence of serum antibodies, human, sheep, and guinea pig complements showed vibriocidal activity up to 40-fold, 20-fold, and 1-fold dilution, respectively. For human pre- and post-vaccination sera, the most potent vibriocidal activity was observed when guinea pig complement was used. In addition, the highest fold-increases between pre- and post- vaccinated sera were obtained with guinea pig complement. Furthermore, human complement contained a higher amount of V. cholerae- and its lipopolysaccharide-specific antibodies than guinea pig complement. Collectively, these results suggest that guinea pig complements are suitable for vibriocidal assays due to their high sensitivity and effectiveness to human sera.     

The red blood cells in growing guinea pigs (Cavia cobaya). I. Communication: pre- and perinatal changes in bone marrow and blood

118 blood samples, 98 bone-marrow smears of guinea pig fetuses, 156 blood samples of spontaneously born guinea pigs 1 to 5 days of age, and 119 bone-marrow smears of the same age group were analysed quantitatively. The normoblasts show significant differences in pre- and early postnatal animals: a peak of the cell count is found in the late fetus which decreases immediately after birth. The prenatal development of the red blood picture is characterised by an increase of erythrocyte count and hematocrit and a decrease of cell size, and reticulocyte count. This development continues postnatally. The perinatal bone-marrow and blood picture findings suggest a true postnatal increase of the erythrocyte count in the guinea pig during the first 3 to 5 days of life which likely is due to the grown demand for oxygen transport capacity.     

Characterization of monoclonal antibodies that recognize band 4.5 polypeptides associated with nucleoside transport in pig erythrocytes.

Three monoclonal antibodies have been raised against partially purified band 4.5 polypeptides [Steck (1974) J. Cell Biol. 62, 1-19] from pig erythrocyte membranes. The antibodies were capable of binding to both intact pig erythrocytes and protein-depleted membrane preparations and recognized detergent-solubilized polypeptides from adult and neonatal pig erythrocytes that were photolabelled with [G-3H]nitrobenzylthioinosine (NBMPR), a potent specific inhibitor of nucleoside transport. The antibodies did not recognize polypeptides from neonatal pig erythrocytes that were photolabelled with the glucose-transport inhibitor [3H]cytochalasin B. Reactivity with polypeptides of apparent Mr 64,000 [10% (w/v) acrylamide gels] was demonstrated by Western-blot analysis. The antibodies recognized pig band 4.5 polypeptides after prolonged treatment with endoglycosidase F, a finding consistent with reactivity against polypeptide, rather than carbohydrate, determinants. Trypsin digestion of NBMPR-labelled protein-depleted pig erythrocyte membranes generated two labelled polypeptide fragments (Mr 43,000 and 26,000). Two of the antibodies recognized both fragments on Western blots, whereas the third bound to the larger, but not to the smaller, fragment. The antibodies had no significant effect on reversible binding of NBMPR to protein-depleted pig erythrocyte membranes and did not bind to NBMPR-labelled polypeptides in human, rabbit or mouse erythrocytes.     

Species specificity of hybridoma antibodies to surface antigens of guinea pig sperm

The species specificity of hybridoma antibodies to sperm surface antigens was studied. A collection of over 50 hybridoma antibodies that bind to the guinea pig sperm surface was tested for binding to mouse, rat, hamster, and human sperm by indirect immunofluorescence. None of the antibodies bind to mouse sperm. rat sperm, or human sperm. All but three of the antibodies also fail to bind to hamster sperm. AH-30, AH-31, and AH-1032, the three antibodies that crossreact with hamster sperm, show a different topographical localization on hamster sperm from that seen on guinea pig sperm. The three antibodies do not precipitate a ¹²⁵I surface-labeled antigen from hamster sperm extracts. However, from guinea pig sperm extracts, all three antibodies precipitate ¹²⁵I surface-labeled polypeptides with molecular weights (M_r) of 62,000, 52,000, and 38,000. This result suggests that the crossreacting antibodies may be recognizing different antigens on hamster and guinea pig sperm.     

三个品种豚鼠血液蛋白多态性的比较分析

目的比较分析白毛黑眼（WHBE）豚鼠和DHP豚鼠、花色豚鼠三个品种豚鼠在13个血液蛋白位点上的多态性。方法采用垂直板浓度和pH均不连续的聚丙烯酰胺凝胶电泳法对WHBE豚鼠、DHP豚鼠和花色豚鼠的66只个体的后白蛋白（Po）、前转铁蛋白1（Prt1）、前转铁蛋白2（Prt2）、转铁蛋白1（Tf1）、转铁蛋白2（Tf2）、后转铁蛋白（Ptf）、慢α球蛋白（Sag）、红细胞酯酶（Es）、血清酯酶1（Est1）、血清酯酶3（Est3）、血红蛋白α（Hbα）、血红蛋白β（Hbβ）和白蛋白（Alb）共13个蛋白位点进行了电泳及染色,再利用电泳图谱对各蛋白位点基因频率、平均杂合度和遗传距离进行计算,然后结合聚类分析。结果 Tf1、Tf2、Ptf、Est1和Es在三个豚鼠品种中表现为多态,其中Tf1可作为识别WHBE豚鼠的遗传标记。Po、Prt1、Prt2、Sag、Est3、Hbα、Hbβ和Alb等位点在三个豚鼠品种中的表型一致。Hardy-Weinberg平衡状态分析表明,Es为DHP豚鼠的高度不平衡位点。Ptf为花色豚鼠的高度不平衡位点。在WHBE豚鼠中,Tf1为高度不平衡位点,Est1为不平衡位点。在三个豚鼠品种中,所检测的13个蛋白位点的平均杂合度的排列顺序为：花色豚鼠（0.350 1）〉WHBE豚鼠（0.339 0）〉DHP豚鼠（0.313 5）。聚类分析结果表明,花色豚鼠和WHBE豚鼠的遗传遗传距离最近（0.064 3）,DHP豚鼠与花色豚鼠的遗传距离最远（0.179 2）。结论利用这些蛋白位点可以有效鉴别WHBE豚鼠、DHP豚鼠和花色豚鼠血液蛋白的遗传多态性。     

The Effect of Erythrocyte Lysis in Selected Animals on the Absorption Spectra of Oxyhemoglobin

Biophysics - Abstract—The absorption spectra of lysed and non-lysed erythrocyte samples isolated from chicken, goose, and guinea pig blood were studied. It was found that the position of the...     

3-Mercaptopyruvate sulfurtransferase activity in guinea pig and rat tissues

Activities of 3-mercaptopyruvate sulfurtransferase (EC 2.8.1.2) in guinea pig tissues were determined and compared with those in the corresponding rat tissues. The activities in guinea pig tissues were found to be very low. The activity in the liver was 145.9 mumol pyruvate formed per g of fresh tissue per 15 min. This was 1/50 of the activity in the rat liver. Activities in the kidney and brain were 1/100 of the corresponding rat tissues. Those in the erythrocyte and heart were negligibly low and far less than 1/1000 of these tissues in the rat. Similarities between the guinea pig and patients with beta-mercaptolactate-cysteine disulfiduria are discussed.     

Synthetic guinea pig insulin B-chain C-terminal decapeptide: a novel immunogen for generating immunocytochemical-grade antisera

Antisera to guinea pig insulin are not commonly available, largely because of the short supply and limited immunogenicity of the intact hormone. To overcome these problems we have employed a novel reagent, synthetic guinea pig insulin B-chain C-terminal decapeptide, as a hapten for raising antibodies that react with intact guinea pig insulin. The decapeptide, coupled to bovine serum albumin, was successfully used as an immunogen in rabbits. The resulting anti-serum was employed for immunocytochemical staining of guinea pig insulin in pancreatic sections. The specificity of the staining was verified by both pre-absorption and pre-immune serum controls. The utility of this new antiserum for investigations of guinea pig insulin physiology is discussed.     

Composition and synthesis of glycolipids in megakaryocytes and platelets: differences in synthesis in megakaryocytes at different stages of maturation

The composition and synthesis of megakaryocyte and platelet glycolipids were compared since these lipids are thought to be important for biologic activities such as adhesion and maturation. Highly purified guinea pig megakaryocytes at different stages of maturation and platelets were studied. Glycolipids and gangliosides were extracted, separated by thin-layer chromatography, and the carbohydrate content was analyzed by gas-liquid chromatography (GLC). Synthesis of ceramides and glycolipids was determined by the incubation of megakaryocytes with [14C]acetate, [3H]palmitic acid, and [3H]galactose. A major neutral glycolipid present in guinea pig megakaryocytes and platelets was identified as asialoGM2 by selective enzymatic hydrolysis with beta-N-acetylhexosaminidase, alpha-galactosidase and endo-beta-galactosidase, and carbohydrate analysis by GLC. Trace amounts of asialoGM1 were detected immunologically. The cells also contained glucosyl ceramide and lactosyl ceramide. Several ganglosides were detected of which one was identified as GM1 by its reaction with the beta-subunit of cholera toxin and by the identification of an asialoGM1 core with anti-asialoGM1 antibody after desialylation. The synthesis of ceramides from palmitic acid and acetate was 5 and 10 times greater, respectively, in megakaryocytes than in platelets. Ceramide and glycolipid synthesis from palmitic acid occurred primarily in immature megakaryocytes while synthesis from acetate occurred primarily in more mature megakaryocytes. The glycosylation of ceramides from galactose was 42 times greater in megakaryocytes than in platelets. Thus, ceramides and glycolipids are primarily synthesized in megakaryocytes, but platelets retain the capacity to synthesize significant amounts of free ceramides. The glycosylation of free ceramides occurs almost exclusively in megakaryocytes and only in trace amounts in platelets. These data indicate that megakaryocytes determine the composition of glycolipids in platelets and that there is considerable compartmentalization of glycolipid synthesis and membrane assembly at various stages of megakaryocytes development.     

Immunoluminescent detection of antibodies to testicular antigens in experimental autoimmune orchitis]

Antibodies interacting with spermatid structures in the testis sections and with acrosome and caudal regions of spermatozoa in the smears of sperm were determined in the sera of guinea pigs and mice with experimental autoimmune orchitis by the indirect immunofluorescent method. The antibodies were found in mice from the 14th to the 30th day and in guinea pigs from the 14th to the 60th day after immunization. Circulation time and the level of the fluorescent antibodies were significantly decreased in immunized female guinea pigs and mice. Fluorescent antibodies did not show cytotoxic effect and belonged to the IgG fraction. The fluorescent antibodies of guinea pigs did not react with the mouse testicular antigens in cross reaction. The analogous results were seen in the cross reaction of the mouse antibodies with the guinea pig antigens.     

Immunohistochemical demonstration of airway epithelial cell markers of guinea pig

The guinea pig (Cavea porcellus) is a mammalian non-rodent species in the Caviidae family. The sensitivity of the respiratory system and the susceptibility to infectious diseases allows the guinea pig to be a useful model for both infectious and non-infectious lung diseases such as asthma and tuberculosis. In this report, we demonstrated for the first time, the major cell types and composition in the guinea pig airway epithelium, using cell type-specific markers by immunohistochemical staining using the commercial available immunological reagents that cross-react with guinea pig. Our results revealed the availability of antibodies cross-reacting with airway epithelial cell types of basal, non-ciliated columnar, ciliated, Clara, goblet and alveolar type II cells, as well as those cells expressing Mucin 5AC, Mucin 2, Aquaporin 4 and Calcitonin Gene Related Peptide. The distribution of these various cell types were quantified in the guinea pig airway by immunohistochemical staining and were comparable with morphometric studies using an electron microscopy assay. Moreover, this study also demonstrated that goblet cells are the main secretory cell type in the guinea pig's airway, distinguishing this species from rats and mice. These results provide useful information for the understanding of airway epithelial cell biology and mechanisms of epithelial–immune integration in guinea pig models.     

Detection of guinea pig macrophages by a new CD68 monoclonal antibody, PM-1K

A new monoclonal antibody, PM-1K, was raised against 24-h cultured human peritoneal macrophages. In immunohistochemical assays, PM-1K recognized freshly isolated blood monocytes and most tissue macrophages as well as myeloid dendritic cells such as Langerhans cells and interdigitating cells. The molecular size of the antigen recognized by PM-1K was determined to be 110 kD by means of immunoaffinity purification. Because this affinity-purified antigen recognized by PM-1K was also recognized by anti-CD68 antibodies, it is believed to be one of the heterogeneous molecules of the CD68 antigen. Analysis showed interspecies reactivity of PM-1K with macrophages from guinea pigs, pigs, bovine species, and monkeys. Among these macrophages, those of the guinea pig reacted strongly with PM-1K. Patterns of PM-1K immunostaining in guinea pig tissues were similar to those found in human tissues. Studies with the immunoelectron microscope revealed reaction products of PM-1K in the cytoplasm, especially around endosomes. Since only a few antibodies are available to label guinea pig macrophages, PM-1K is considered to be one of the most suitable antibodies to examine macrophages in experimental guinea pig models.