Comparative immunochemical and physicochemical study of the neurospecific antigen 10-40-4 from human and rat brains

Immunological non-identity of neurospecific protein 10-40-4 from various mammalian species has been demonstrated. Significant immunochemical differences were found between the protein 10-40-4 from the brain of rat, guinea pig and man. With respect to their physicochemical properties, no significant differences were found between individual neurospecific proteins 10-40-4 from the brain of man and rat. Therefore, immunological differences in the structure of a neurospecific determinant together with identical physicochemical properties of the proteins investigated imply that the latter are presented by species variations of the same protein.     

Isolation and some physico-chemical properties of a new neurospecific protein 10-40-4 from human brain

A method for isolation of a neurospecific protein 10-40-4 from human brain has been elaborated. This procedure includes immunoaffinity chromatography of a Sepharose 4B-IgG fraction of rabbit antisera against the protein fraction containing the antigen. The isolated protein cannot be detected in protein extracts of various organs and human blood serum by immunochemical methods. This indicates that the protein is specific for nervous tissue. The values of molecular weight (74 000) and pI (4.7) of the isolated protein suggest that the protein does not contain the carbohydrate component and reveals limited tissue specificity. The properties of protein 10-40-4 differ from those of the well-known neurospecific proteins, such as S-100, enolase 14-3-2 and glial fibrillar acid protein GFA.     

Cellular and regional localization of the neurospecific antigen 10-40-4 in the human brain

Cellular and regional localization of neurospecific protein 10-40-4 in human brain was studied by immunofluorescent staining and immunoenzymatic assay. Intense fluorescence of perikaryons of the medulla oblongata, thalamus and pons neurons was demonstrated. The same structures showed the maximal concentration of the protein (15-18% of water-soluble proteins). In the cortex of the hemispheres, in the cerebellum and hypothalamus the fluorescence intensity was not different from the background level. The concentration of the protein in these structures was minimal (1-4% of water-soluble proteins).     

聚合牛血红蛋白抗原性的初步研究

对乙醇醛聚合的牛血红蛋白的抗原性进行了研究。将聚合的牛血红蛋白桉兔血量的10％和20％分别输入兔耳缘静脉,间隔7天后重复输液,共输注3次。ELISA检测未显示抗体产生。将聚合的牛血红蛋白、人血红蛋白和兔血红蛋白分别与免疫佐剂混合常规免疫家兔3次,并用上述抗原包被聚乙烯板,用ELISA方法检测抗体滴度,结果显示聚合牛血红蛋白和人血红蛋白加佐剂免疫家兔后均产生抗体,且有交叉反应,表明这两种血红蛋白有同源性,存在相似的抗厚决定簇。兔血红蛋白免疫家兔后无抗体产生,且无交叉反应,表明机体对自体蛋白有天然的耐受。     

LPS-based conjugate vaccines composed of O-polysaccharide from Pseudomonas aeruginosa IATS 6 and 11 bound to a carrier protein

Lipopolysaccharide (LPS) is the major surface antigen of Pseudomonas aeruginosa and elicits protective antibodies in animals. No cross reaction was observed between LPSs of P. aeruginosa International Antigenic Typing Scheme (IATS) 6 and 11 strains using rabbit polyclonal antibodies raised against the whole cells. The O-polysaccharides (O-PSs) from IATS 6 and 11, the antigenic determinant of LPS, were directly coupled to bovine serum albumin (BSA) by carbodiimide mediated condensation reaction. The molar ratios of saccharide repeating units to BSA in the prepared conjugates were 15:1 and 26:1 for IATS 6 and 11 conjugates, respectively. Mice were immunized with the conjugates emulsified with monophosphoryl lipid A (MPL), Freund, and Alum adjuvants. The conjugates emulsified with MPL adjuvant elicited the highest IgM antibody, followed by Freund. While both MPL and Freund adjuvants elicited high IgG antibody. Good correlation was observed between the IgG and IgM levels with the bactericidal activities of the sera against homologous strains. In addition, immunization of mice with the prepared conjugates emulsified with MPL and Freund adjuvants provided high protection against ten times the LD₅₀ of P. aeruginsoa IATS 6 and 11, which showed a good correlations with the IgG titer.     

磷酸酪氨酸蛋白专一抗体的制备和纯化

以偶联磷酸化酪氨酸的牛血清白蛋白（ＢＳＡ）作为免疫原免疫兔获得抗血清．自抗血清中分离获得抗体．自酪胺合成磷酸酪胺,并偶联到溴化氰活化的Ｓｅｐｈａｒｏｓｅ４Ｂ上．抗体经磷酸酪胺－Ｓｅｐｈａｒｏｓｅ４Ｂ亲和柱纯化,所得抗体专一性强,Ｄｏｔｂｌｏｔ显示：抗体仅对酪氨酸磷酸化的蛋白质包括酪氨酸磷酸化的血清白蛋白,溶菌酶,卵清蛋白起抗原抗体反应,而不识别非酪氨酸磷酸化的溶菌酶,卵清蛋白,也不识别作为免疫原的骨架成分ＢＳＡ,也不识别丝氨酸磷酸化的卵清蛋白和苏氨酸磷酸化的卵清蛋白．     

Spatial learning induces predominant downregulation of cytosolic proteins in the rat hippocampus

Spatial learning is known to depend on protein synthesis in the hippocampus. Whereas the role of the hippocampus in spatial memory is established, the biochemical and molecular mechanisms underlying this process are poorly understood. To comprehend the complex pattern of protein expression induced by spatial learning, we analyzed alterations in the rat hippocampus proteome after 7 days of spatial learning in the Morris water maze. Forty Wistar rats were randomized into two groups. Animals of group A learned to localize a hidden platform in the water maze. Animals of group B served as controls and spent exactly the same time in the water maze as animals of group A. However, no platform was used in this test and the rats could not learn to localize the target. After the last trial, hydrophilic proteins from the hippocampus were isolated. A proteome-wide study was performed, based on two-dimensional gel electrophoresis and mass spectrometry. Compared with non-learning animals, 53 (70%) proteins were downregulated and 23 (30%) proteins were upregulated after 7 days in rats with spatial learning. The overall changes in protein expression, as quantified by the induction factor, ranged from -1.62 (downregulation to 62%) to 2.10 (upregulation by 110%) compared with controls (100%). Most identified proteins exhibit known functions in vesicle transport, cytoskeletal architecture, and metabolism as well as neurogenesis. These findings indicate that learning in the Morris water maze has a morphological correlate on the proteome level in the hippocampus.     

Effect of protein S-100 on phosphorylation of nuclear proteins of cells of rat brain and liver

The effect of acidic neurospecific protein S-100 on the phosphorylation of brain and liver nuclear proteins with 1 and 10 microM ATP was investigated. It was shown that protein S-100 increases the phosphorylation of brain nuclear proteins, while antigen D, another acidic neurospecific protein half-identical to 14-3-2 protein, inhibits this process. Ca2+ and cAMP at concentration of 10(-6) M do not affect the phosphorylation of brain nuclear proteins. In control assays the tracer 32P is presumably incorporated into high molecular weight nuclear protein fractions (Mr greater than 40000). After addition of protein S-100 the tracer is mainly incorporated into these proteins as well independently of ATP concentration (1 or 10 microM). The phosphorylation of nuclear proteins with molecular weights above 100000 is mostly increased in this case. At ATP concentration of 1 microM protein S-100 decreases histone phosphorylation 2.3 times but does not affect that of non-histone proteins. However, at 10 microM ATP the inhibitory action of this protein on histone phosphorylation is absent. The possible mechanisms of protein S-100 action on nuclear proteins phosphorylation are discussed.     

Vaginal candidosis

Enzyme linked immunosorbent assay was found to be a convenient method for the investigation of antibodies in mice immunized with Candida albicans ribosomes. Antibodies against the ribosomal antigen were detected in all the sera of mice (ICR and BALB/ c) immunized with ribosomes and incomplete Freund's adjuvant and in some of the sera of mice immunized with ribosomes only; the titer of antibodies varied from 1320 to 110 240. Vaccination of mice with ribosomal protein and IFA resulted in a high titer of antiribosomal antibodies. Treatment of ribosomes with pronase abrogated the capacity of the ribosomes to elicit anti ribosomal humoral responses, suggesting that the antibodies detected were directed against the protein moiety of the ribosomes. The presence of antibodies in sera of immunized mice could not be correlated with the protection afforded by the ribosomal vaccination.     

Forms of goal-directed behavior as affected by induced changes in the level of endogenous beta-endorphin in rats

Marked alterations in feeding and defense behaviour and motor activity partly resembling the effects of exogenous beta-endorphin administration were demonstrated in the experiments on rats. These alterations were observed after immunization with beta-endorphin--bovine serum albumin conjugate (two subcutaneous injections at a 7-day interval at a dose of 75 micrograms, 1 mole BSA/6 moles beta-endorphin mixed with complete Freund's adjuvant). A decrease in beta-endorphin content in some brain structures was noted. Unlike control animals, the immunized rats revealed within 3-4 weeks an increase in food intake without any rise in body weight and practically no response to handling.     

Detection of the neurospecific protein Sy-1 in the human brain and its regional localization in nerve tissue

Using affinity chromatography, a neurospecific protein was isolated from human and rat brain. Both proteins were shown to possess identical physico-chemical properties. However, antisera raised against these proteins exhibited no cross reactions with the corresponding proteins, thus suggesting their species specificity. Immunochemical analysis revealed that the neurospecific protein from rat brain is identical to protein Sy-1 specific for rat brain. Since the human brain protein is analogous to its rat brain counterpart, it may also be considered as protein Sy-1. The uneven distribution of the protein in various structures of human brain was demonstrated by the immunoenzymatic method. Protein Sy-1 is found at relatively low concentrations in the cortex, caudate nucleus and claustrum (2.5-5 micrograms/mg of water-soluble protein), but is predominant in the brain stem (20 micrograms/mg).     

Relation between the effects of the memory neuromodulator arginine-vasopressin and the levels of activity of the serotonin-, dopamine- and noradrenergic systems of the brain

The effect of vasopressin on learning, habituation and habit recovery in rat before and after electrolytic ablation of locus coeruleus, nuclei dorsalis et medialis raphe and pars compacta substantiae nigrae was studied by behavioural methods. The rats were learned with positive reinforcement in a T-shaped maze and with negative reinforcement in a U-shaped maze. It was found that improvement of trace consolidation and acceleration of learning were in many cases dependent on the activity level of the dophaminergic, noradrenergic and to a lesser degree on that of serotoninergic brain systems.     

Identification of Fasciola hepatica recombinant 15-kDa fatty acid-binding protein T-cell epitopes that protect against experimental fascioliasis in rabbits and mice

Vaccination with fatty acid-binding proteins (FABPs) from Fasciola hepatica has been shown to confer significant levels of protection against challenge infection in mice, rabbits, and sheep. A recombinant 15-kDa FABP (rFh15) has been purified and also shown to be an immunoprotective molecule. From the rFh15 molecule sequence 2, 12- and 10-mer putative T-cell epitopes were identified, the first an Fh15Ta of amino acid sequence IKMVSSLKTKIT, and the second an Fh15Tb of amino acid sequence VKAVTTLLKA. The synthesized oligonucleotides were cloned individually into a pGEX-2TK expression vector. The overexpressed fusion protein was affinity purified using glutathione S-transferase (GST) by competitive elution with excess reduced glutathione. These GST fusion proteins were emulsified in Freund adjuvant for rabbit immunizations or further purified as peptides after digestion with thrombin. The purified 12- and 10-mer peptides were either emulsified in Freund adjuvant for immunizations in rabbits or used in an adjuvant-adaptation (ADAD) system, followed by challenge infection with F. hepatica metacercariae in mice and rabbits. In vaccinated-challenged rabbits, the highest levels of protection were found in those treated with GST-epitopes (Fh15Ta 48.2% and Fh15Tb 59.1% reduction, respectively), as compared to GST-immunized controls. Moreover, those immunized with Fh15Ta had higher (84%) numbers of immature flukes as compared with Fh15Tb (41%) or GST alone (64%). The rabbits immunized with the putative T-cell epitopes in adjuvant had a 13% reduction in flukes in those with Fh15Ta and also were highest with immature flukes (46%). In vaccinated mice challenged with a lethal number of metacercariae, both CD-1 and BALB/c mice treated with complete ADAD-GST-Ta had the highest (40%) survival rates of all groups by 47 days postinfection. Thus the Fh15Ta and Fh15Tb polypeptide epitopes warrant further study as a potential vaccine against F. hepatica. Antibody isotype studies in mice revealed a mixed Thl/Th2 response to vaccination.     

Contraceptive potential of porcine zona pellucida in cats

The contraceptive potential of solubilized porcine zona pellucida (spZP) was studied in 2 groups of cats after active immunization using slightly different protocols. Cats from Group 1 (n = 3) were immunized with a total of 300 8g spZP divided in 4 s.c. multisite injections (each of 37.5 8g) given at 10 day intervals followed by a booster 150 days after the initial immunization. Cats from Group 2 (n = 5) were immunized with a total of 400 8g spZP divided in 4 i.m. injections (each of 50 8g) given at 2 wk intervals followed by a booster 92 days after initial immunization. Immunogen was emulsified in Complete Freund Adjuvant for the first dose and in Incomplete Freund Adjuvant for the following 3 doses. The respective controls were immunized in the same manner using only adjuvant and PBS. Immunofluorescence studies showed intense fluorescence on the zona pellucida (ZP) of the oocytes isolated from immunized cats, as well as on the ZP of porcine and cat oocytes preincubated in immune sera. Sera from cats immunized with spZP inhibited in vitro binding was demonstrated in oocytes isolated from immunized group 1 cats. In vivo fertility data in Group 2 cats revealed that only 1 of 5 cats became pregnant, the one with the lowest anti-spZP titer. The results from the experiments reported above, suggest that in this preliminary study spZP can elicit antibodies with contraceptive potential in actively immunized female cats.     

Characterization of two proteins from Leishmania donovani and their use for vaccination against visceral leishmaniasis

Two proteins from Leishmania donovani, dp72 and gp70-2, have been previously utilized to specifically serodiagnose patients with visceral leishmaniasis. The proteins were shown by ELISA and Western blotting with monoclonal and polyclonal antibodies to be present in both stages of the parasite. Antibodies to gp70-2 recognize in promastigotes multiple discrete bands of similar m.w. which are common to several isolates of L. donovani. The total amount of Ag and number of bands observed per isolate is not constant. Lectin blots with Con A show gp70-2 to be a glycoprotein. Dp72 shows pronounced microheterogeneity between isolates of L. donovani. The Brazilian isolates examined appear to possess a lower m.w. form (64,000 or 68,000) of this molecule. No reactions were observed with dp72 and lectins in Western blots; and neither tunicamycin, N-glycanase, endoglycosidase H nor F affected the migration of [35S]-methionine-labeled protein on SDS-PAGE. A mAb against dp72 also cross-reacted in Western blots with a 60-kDa protein in Leishmania major, Leishmania aethiopica, and Leishmania tropica. No reaction was observed between the purified promastigote surface protease (gp63) and either monoclonal or polyclonal antibodies produced to dp72 or gp70-2. The ability of the pure proteins to provide protection against a challenge by L. donovani amastigotes was examined. BALB/c mice were immunized with gp70-2 and/or dp72 by using Corynebacterium parvum as an adjuvant. Mice immunized with gp70-2 were not protected; however, mice receiving dp72 showed a 81.1% reduction in the liver parasitemia compared with the adjuvant controls.     

Further studies on amplification of cell-associated immunological memory by secondary antigenic stimulus.

Using the hapten-carrier system in which the dinitrophenyl group (DNP) served as a B cell reactive hapten and bovine serum albumin (BSA) or human gammaglobulin (HGG) as a T cell reactive carrier, changes in the hapten-specific memory (B cell-associated memory) and the carrier-specific memory (T cell-associated memory) after a secondary antigenic stimulus were analyzed in mice. Since an immunological adjuvant was indispensable in the induction of the primary increase in memory, antigen used for the primary antigenic stimulus was injected together with the capsular polysaccharide of Klebsiella pneumoniae (CPS-K) which has already been shown to exhibit a potent adjuvant effect. With the cell-transfer technique, it was found that the cell-associated hapten-specific memory for anti-DNP antibody response to DNP-BSA was truly amplified by the secondary injection of DNP-HGG into mice primed with DNP-HGG, and that the cell-associated carrier-specific memory as judged by the helper effect on anti-DNP response to DNP-BSA was also truly amplified by the secondary injection of BSA into mice primed with BSA. However, when memory was assessed in actively immunized mice, the secondary injection of BSA into mice primed with DNP-BSA and HGG decreased anti-DNP responsiveness to the tertiary injection of DNP-BSA, whereas the secondary injection of DNP-HGG secondarily increased anti-DNP responsiveness. In mice primed with DNP-BSA the titers of serum antibodies to BSA increased after the secondary injection of DNP-BSA or BSA. From these results it has been concluded that, like B cell-associated memory, T cell-associated memory is also amplified by a secondary antigenic stimulus, although its expression is inhibited in actively immunized mice through negative control by their antibodies.     

髓鞘碱性蛋白片段69-85诱导建立大鼠自身免疫性脑脊髓炎模型

目的探索以大鼠髓鞘碱性蛋白片段69-85（MBP69-85）为单一抗原,建立Wistar大鼠实验性自身免疫性脑脊髓炎（EAE）模型,并观察其病理改变。方法MBP69-85以生理盐水溶解,与完全福氏佐剂（CFA）充分混匀制备免疫乳剂;雌性Wistar大鼠70只,留取10只作为正常对照组,余者根据免疫乳剂组分的不同随机分为A、B、C组（n=20）。A组：MBP69-85每只50μg＋CFA（含卡介苗6 mg）;B组：MBP69-85每只25μg＋CFA（含卡介苗6 mg）;C组：MBP69-85每只25μg＋CFA（含卡介苗12 mg）。脑和脊髓组织切片进行HE染色,MBP及神经微丝（neurofilament,NF）免疫组化检测,观察神经组织的病理改变。结果A组内部分大鼠在免疫后第12-16天发病,表现为尾部及四肢无力或麻痹、斜颈等,平均临床症状评分为2.38±1.89;B、C组均未见发病;HE染色可见神经组织内炎细胞浸润,血管＂袖套＂样病灶形成;MBP及NF免疫组化染色显示病变组织内白质脱髓鞘及轴突损伤明显。结论EAE模型建立与免疫抗原的剂量有一定的依赖关系;佐剂中的卡介苗含量不是诱导大鼠发病的主要原因;本模型具有多发性硬化的典型临床表现及病理改变,是研究多发性硬化发病机制及治疗的可靠的动物模型。     

Molecular analysis of the antigenicity and immunogenicity of recombinant zona pellucida antigens in a primate model.

The studies reported here are the first to demonstrate that recombinant zona pellucida (ZP) proteins will elicit a humoral immune response that recognizes native ZP proteins. Three cDNAs encoding rabbit ZP protein antigens expressed in bacteria were used to immunize cynomolgus monkeys. Four groups of six monkeys each were immunized with bacterially expressed cro-beta-galactosidase recombinant proteins encoded by a full-length cDNA (rc55) encoding the 55-kDa rabbit ZP recombinant protein (rec55), two partial cDNAs (rc75a and rc75b) encoding two recombinant peptides (rec75a and rec75b) of the 75-kDa rabbit ZP protein, and the plasmid-encoded cro-beta-galactosidase control protein. Initial immunizations with these fusion proteins using the muramyl dipeptide adjuvant did not elicit significant levels of antibodies to native or recombinant ZP proteins. Further immunizations were therefore carried out using recombinant ZP proteins conjugated to either protein A or keyhole limpet hemocyanin. Antibodies were detected in the groups immunized with the rec55 and rec75a; however, no antibodies were generated against the rec75b protein. These antibodies have been characterized by two-dimensional PAGE immunoblotting and shown to recognize antigenic domains associated with two of the native rabbit ZP proteins. Reprobes of these immunoblots with sheep anti-total native rabbit ZP proteins, affinity-purified on pig ZP, further demonstrate that a fourth distinct rabbit ZP antigen may be present. The characterization of species-conserved antigenic domains of mammalian ZP proteins is important for studies of the functional regions of ZP proteins and is critical for the design of safe and effective contraceptive vaccines.     

Infertility induced in rats by immunization with synthetic peptide segments of a sperm protein.

Three peptide segments (YAL-198, YAL-201 and YAL-212) corresponding to the extracellular domain of a human sperm protein designated as YWK-II antigen were synthesized as multiple antigen peptide (MAP). Male and female rats were immunized with the YWK-II-MAPs and fertility determined. In a group of 12 female rats immunized with YAL-198, seven animals were infertile and two animals were subfertile. When immunized with YAL-201 and YAL-212, 4 and 2 animals were infertile, respectively. In a group of 15 males immunized with YAL-198, 2 animals were infertile and 6 were subfertile. Two animals immunized with YAL-201 were subfertile. All control male and female rats immunized with bovine serum albumin and adjuvant were fertile. Sera obtained from infertile rats immunized with YAL-198 contained higher titers of antibodies compared to those obtained from fertile animals. The present study shows that immunization with synthetic peptide segments of a sperm protein can effectively reduce fertility.     

Influence of active and passive immunity on the disposition of dihydromorphine-H3

The effect of circulating antibodies on the plasma and brain concentration of dihydromorphine-³H was examined. Rabbits immunized with the morphine antigen 3-0-carboxymethyl morphine coupled to bovine serum albumin served as the source of the antibodies which were used for passive immunization of mice and rats. The 45 minute plasma concentration of the narcotic was increased 90–100 fold in passively immunized animals whereas the brain concentration decreased by at least 75 percent. Mice were also actively immunized with the morphine antigen. Compared to non-immunized mice, plasma levels of dihydromorphine-³H were increased in the immunized mice from 7–30 fold at all intervals measured for at least 4 days. The plasma half life was markedly slowed in immunized mice. The narcotic bound to the antibody in the plasma $\text{in vivo}$ could be displaced by the administration of morphine. The consequence of active immunization on brain narcotic content varied with the dose of the drug and time interval studied. We suggest the possibility that the antibodies may initially act to sequester the narcotic but with time as the narcotic is slowly released from the antibodies that they may also act as a circulating source of the drug. It is apparent that the presence of circulating antibodies can have marked effects on the disposition of narcotics.