An extracellular 5'-nucleotidase with both monoesterase and diesterase activity from Micrococcus sodonensis.

Bovine γ-globulin was separated into four fractions by chromatography on cellulose phosphate. The chromatographic distribution was similar to that reported for human and dog γ-globulin. More than 80% of a nonspecific phagocytosis stimulating factor (leucokinin) present in the serum was isolated in γ-globulin fraction IV. Bocine red blood cells and polymorphonuclear leukocytes bind γ-globulin without appreciable selectivity for any of the four chromatographic fractions, but they do selectively bind the phagocytosis stimulating factor. Splenectomy caused no observable change in either the chromatographic distribution or phagocytosis stimulating activity of bovine serum γ-globulin. The tetrapeptide tuftsin stimulates phagocytosis by bovine neutrophiles, but on a molar basis the activity of tuftsin was only 10% that of the phagocytosis stimulating factor. If the factor exerts its effect, as has been proposed, by having a phagocytosis stimulating peptide cleaved from it by an enzyme on the leukocyte membrane, that peptide must differ in structure from tuftsin. This conclusion is supported by the inability of trypsin to liberate an active peptide from bovine serum.     

Antigen-induced in vitro inhibition of immune responsiveness

Addition of the dinitrophenyl derivative of the copolymer of d-glutamic acid and d-lysine (DNP-d-GL) or dinitrophenyl bovine γ-globulin (DNP-BGG) to spleen cell cultures specifically inhibited their capacity to produce an anti-DNP plaque-forming cell (PFC) response to the T-independent antigen dinitrophenylated polyacrylamide beads (DNP-PAA) or to the T-dependent antigen TNP-burro erythrocytes. The degree of unresponsiveness was dependent upon the tolerogen concentration and the duration over which the tolerogen was present in the culture. Treatment with rabbit anti-mouse brain antiserum and complement did not alter the induction of unresponsiveness suggesting a state of B-cell tolerance. Culture of spleen cells for 4 days in the absence of antigen led to the appearance of nonspecific suppressor activity which was demonstrable by its effect on the response of fresh spleen cells to antigen. Preculture in the presence of the immunogen DNP-PAA induced both nonspecific and specific suppressor activities. Induction of specific suppressor activity was not prevented by the presence of the tolerogen DNP-D-GL in the culture. The suppressor activity resided in an adherent T-cell population and did not appear to require macrophages for its induction.     

Comparison of heterogeneities of antitrinitrophenyl antibodies in strains of mice immunized by various methods.

Heterogeneity of antibodies directed against the trinitrophenyl (TNP) determinant in immunized mice was analyzed by investigating the band groups of antibodies formed by thin layer isoelectric focusing of serum. The degree of heterogeneity in C57BL/6 mice was markedly higher than that in CBA mice under the following conditions of immunization: immunization with TNP conjugated with bovine gamma-globulin or ovalbumin, interchange of these carrier proteins at the first and second injections, change of epitope density of the antigens, replacement of the hapten by dinitrophenyl group on the antigen for the secondary stimulation, and change of intervals of these injections from 15 days to five months. The degree of heterogeneity within a strain also varied with these immunizing conditions. Furthermore, the heterogeneity in C57BL/6 mice of any immunization group was greater than that in CBA mice in any group. This was also true when the heterogeneity was examined with the immune sera diluted to the same titer. These results indicate that the number of predominant clones of cells producing anti-TNP antibody after immunization is larger in C57BL/6 than in CBA mice.     

Studies of Electrophoresis on Cellulose Acetate Membrane of Serum Proteins from Normal Horses,Sheep and Pigs

A method for the rapid electrophoresis on a cellulose acetate membrane of serum proteins from horses, sheep and pigs is discussed. The various main globulin fractions in the serum of these animals were experimentally identified. Normal values for the percentage composition of serum from normal horses, sheep and pigs were calculated. In the horse there was great individual variation in the shape of the β-fraction, assumed to be due to different transferrin types. The mean value for β-globulin of 19.5 % in the horse was higher than for the other two species. The albumin percentage was highest in the sheep and lowest in the pig, 48.5 % and 43.2 % respectively. The sheep had the highest γ-globulin percentage, 22.8 %, while the horse had the lowest with 19.0 %. Finally the values were compared with corresponding figures reported by other authors and the results discussed.     

Laboratory Diagnosis of Canine Pyometra

The principal laboratory test used to confirm the pyometra diagnosis in the bitch has been the determination of the total white blood cell count in venous blood. A marked elevation is known to be a characteristic of the disease. In the present study, the white blood cell count was determined as well as the γ-globulin level. An elevation of the γ-globulin level and the total white blood cell count was very characteristic to the pyometra patients. The increase in the number of white blood cells nor the high γ-globulin level cannot be regarded specific for pyometra, therefore it was regarded important to find out a more specific test for pyometra. When sonicated E. coli bacteria were tested against sera from pyometra patients in electroimmunodiffusion, the precipitation was almost always detected when E. coli had been isolated from the uterus. This technique provides a quick method in detecting the causative E. coli infection. The present study suggests that whenever laboratory tests are used to confirm the pyometra diagnosis by the total white blood cell count, it is advantageous to analyze the total γ-globulin level in the serum as well as specific antibodies against a common E. coli antigen. Because of the reliability of the glutaraldehyde coagulation test and the simple technique, this can be suggested as the method of choice for an average small animal practice.     

Some Effects of Protein Deficiency in Young Growing Pigs

No significant differences between the serum protein concentrations of the totally starved pigs and control animals given a milk protein supplemented diet were found. Serum lipid levels rose in the totally starved group. During the first 42 days of the experimental period sharp falls in serum protein concentrations were noted in the protein deprived pigs. The losses were greatest in the albumin and two β-globulin fractions, γ-globulin levels rose but at a much slower rate than in the control group. In the pigs which were refed a protein containing diet during the second 42 day period, the concentrations of all the serum protein fractions except for γ-globulin had reached control levels by the end of the investigation. The pigs which continued with the protein free diet showed further losses of albumin, a reduction in some α-globulin fractions and a cessation of net γ-globulin synthesis. The trends observed by measurements of protein bound carbohydrate were similar to those obtained from polypeptide determinations indicating quantitative rather than qualitative changes in the serum proteins. The reduction in the serum lipid concentrations of the pigs undergoing protein starvation was largely accounted for by losses of α- and β-lipoproteins. The protein deprived pigs maintained their initial body weight, while a continuous fall in weight was found in the totally starved group and a continuous increase in the control pigs.     

Thermodynamics of antibody-antigen reactions. 2. The binding of bivalent synthetic random coil antigens to antibodies having different antigen precipitating properties

The objects of this study were the equine IgG and IgG(T) classes of antibodies with immunologic specificity for the dinitrophenyl group and bivalent antigens consisting of linear poly(ethylene glycol) polymers which terminated at both ends in dinitrophenyl groups. Complex formation between antibodies of both classes and one of several sharp fractions of antigen having number average molecular weights in the range 25 000 to 75 000 were studied by measuring the light scattered from solutions containing equimolar amounts (approximately 5 x 10(-6) mol/L) of one of the antibodies and one size fraction of antigen, and variable amounts of monovalent hapten. The data were analyzed in the context of a model that accounted for the formation of linear and cyclic complexes of all extents of aggregation. Two parameters in addition to the intrinsic antibody-dinitrophenyl group association constant were found to be necessary in the assumed equilibrium model to account for the behavior of the system. One of these accounted for the looses in configuration entropy that resulted when a random-coil polymer became bound at one end to a space-occupying antibody. The other was a ring closure factor for the formation of cyclic complexes. Ring closure factors for the formation of larger cyclic complexes (present in only small amounts under the conditions studied) were related to the ring closure factor for the formation of the smallest, which was found to increase as antigen size decreased, and for each antigen size to be consistently higher for IgG(T) antibody than for IgG antibody. Comparison of the theoretically estimated values of the two parameters within their measured values indicated that the average conformation of IgG antibodies in solution is open ("T" shaped) but the average inter-Fab are angle in IgG(T) antibodies is approximately 60 degrees or less.     

A novel anti-peroxiredoxin autoantibody in patients with Kawasaki disease

Antibodies to the anti-oxidative peroxiredoxin (Prx) enzymes occur in both systemic autoimmune disease and vasculitis in adulthood. Because increased oxidative stress induces vasculitis in Kawasaki disease (KD), autoimmunity to Prxs in patients with KD was investigated. The presence of antibodies to Prx 1, 2 and 4 was analyzed by ELISA and Western blot. Of 30 patients with KD, 13 (43.3%) possessed antibodies to Prx 2, whereas these antibodies were present in only 1 of 10 patients (10.0%) with sepsis (4 with purulent meningitis and 6 with septicemia). In contrast, antibodies to Prx 1 and 4 were not detected in either group. There was no significant correlation among the titers of the three antibodies. Clinical parameters were compared between anti-Prx 2-positive and -negative patients. The presence of anti-Prx 2 antibodies correlated with a longer period of fever and poor response to high-dose γ-globulin therapy in patients with KD. Anti-Prx 2-positive patients had significantly greater excretion of urinary 8-isoprostaglandin than did anti-Prx 2-negative patients. These results provide the first evidence for an antibody to Prx 2 in patients with KD. They also suggest that this antibody might serve as a marker of disease severity and be involved in the pathophysiology of vasculitis in some patients with KD.     

Specific monoclonal antibodies to Opisthorchis viverrini.

A Balb/c mouse was immunized with a crude soluble antigen of Opisthorchis viverrini adult worms (OVAA) over a period of 7 months. Spleen cells from the immune mouse were fused with Sp2/0 myeloma cells. Among the 264 tissue culture wells containing the fused cells, cells of 96 wells (36%) produced antibodies to the immunizing agent. Antibodies produced by cells in several wells reacted with antigens from other species of parasite. Cells of 17 wells produced antibodies specific only to OVAA, thus cells from three representative wells were cloned by limiting dilution. Hybrids obtained produced antibodies which could be classified according to their tissue specificities into three groups. The first group of antibodies reacted strongly to the worm integument and weakly with the muscles while those belonging to the second group reacted only to muscles of the worms. The monoclonal antibodies of the third group gave a positive reaction to both muscles and tegument.     

Grafting of different glycosides on the surface of liposomes and its effect on the tissue distribution of 125I-labelled γ-globulin encapsulated in liposomes

Different glycosides were grafted on the surface of liposomes containing ¹²⁵I-labelled γ-globulin by two ways: (1) by using glycolipid and (2) by covalent coupling of $\text{p-}\text{aminophenyl-}\text{d}\text{-glycosides}$ to phosphatidylethanolamine liposomes using glutaraldehyde. The distribution of ¹²⁵I-labelled γ-globulin was determined in mouse tissues from 5–60 min after a single injection of these liposomes. The liver uptake of encapsulated ¹²⁵I-labelled γ-globulin was highest from liposomes having galactose and mannose on the surface. Competition experiments and cross-inhibition studies indicate that this uptake are mediated by specific recognition of the surface galactose and mannose residues of liposomes by the receptors present on the plasma membrane of liver cells. Stearylamine-containing liposomes were found to be more efficient in mediating the uptake of ¹²⁵I-labelled γ-globulin by the lung, whereas in the case of spleen, phosphatidylethanolamine liposomes were more efficient. The extent of uptake of ¹²⁵I-labelled γ-globulin from all types of liposome decreases as the amount of given liposomes increases. The uptake of ¹²⁵I-labelled γ-globulin from liposomes containing asialogangliosides depends upon the phospholipid/ glycolipid ratio. These experiments clearly demonstrate that enhanced liposome uptake by liver cells could be achieved by grafting galactose and mannose on the liposomal surface.     

Immunochemical Isolation of γ-Globulin mRNA and Estimation of Immunoglobulin Gene Reiteration

The polyribosomes synthesizing γ-globulin have been isolated by the achievement of specific precipitation using bentonite-treated anti-IgG antibody. The RNA extracted from the immunochemically precipitated polysomes was tested for its ability to direct the synthesis of proteins in a cell-free system. The specific γ-globulin-synthesizing activity (cpm of γ-globulin synthesized/μg RNA) of this RNA was 10-fold greater than that from total polysomes. γ-globulin mRNA (messenger RNA) isolated by immunoprecipitation was more than 89% pure with respect to contamination by other species of mRNA. The products synthesized by the cell-free system were also analyzed by sodium dodecyl sulphate(SDS)-polyacrylamide gel electrophoresis. This RNA has been hybridized with mouse myeloma DNA. The estimation of immunoglobulin gene reiteration was carried out using hybridization kinetics with consideration given to the DNA/RNA ratio since the estimation from the “half Cot value” is not accurate. The results suggest that in the mouse there are about 20 copies per subgroup of genes coding for the variable region of the H and L chains.     

Studies of Enteric Pathogens and γ-Globulin Levels of Neonatal Calves in Sweden

Faecal and blood samples were taken from 10-30% of calves, 36 hours to 14 days old, in 47 dairy herds in different regions of Sweden from September 1987 to October 1988 (Olsson et al 1993). Faecal samples from 279 calves were analysed for the presence of Escherichia coli (K99+), rotavirus and Cryptosporidium sp. Twenty (7.2%) of these samples were from diarrhoeic calves. An ELISA was developed and used for the rotavirus analysis. E. coli K99+ was detected in 11.5%, Cryptosporidium sp. in 6.1% and rotavirus in 5.4% of the faecal samples. The presence of rotavirus alone and the combination rotavirus and E. coli (K99+) was found to be associated with diarrhoea (p<0.001 and p<0.01 respectively). Blood samples from 327 calves were analysed for the level of total protein and γ-globulin. In 43 of these samples (13%) γ-globulin did not separate from the ß2-region by electrophoresis. The mean total protein concentration was 53.6 g/1 in calves free from diarrhoea. The mean γ-globulin concentration, adjusted to 7 days age was 5.9 g/1. The 20 diarrhoeic calves had lower levels of both total protein and γ-globulin, compared with calves without diarrhoea, but the difference was not significant. One litre more of colostrum at the first feed increased the level of total protein of the calves’ sera by 1.4 g/1 (p = 0.05). Calves born between May and September had a 2.0 g/1 higher (p<0.001) serum concentration of γ-globulin than calves born between October and April.     

Studies on γM and γG Antibody Response of Mice to Bovine Serum Albumin

Response of CBA mice with γM and γG antibodies to bovine serum albumin (BSA) was studied in relation to a variety of conditions of antigen administration. The variables in the conditions were doses and physical forms of antigen, and injection routes. It was realized that γG antibody response to soluble BSA and both γM and γG antibody responses to particulate forms of BSA were augmented as the dose was increased. The γM response to soluble BSA was not elevated by an increase in the amount of antigen up to 1 mg. The soluble form was not so immunogenic as the particulate forms, in which alum-precipitated BSA was capable of inducing both γM and γG antibodies to high titers, and heat-denatured BSA elicited preferably antibody. Alum-precipitated BSA and the emulsified BSA were strong inducers for γG antibody response when injected subcutaneously. In any antigen form, γM response was markedly influenced by changing the injection route, the order of decreasing efficiency for antibody being intravenous, intraperitoneal and subcutaneous. The γG antibody response was hardly affected by the injection route. The effect of a single intravenous injection of 0.01 mg of endotoxin, given 1 to 2 hr after antigen injection, on γM and γG antibody production differed according to the antigen administration procedures. Generally speaking, this agent had an enhancing effect when the antigen was given in the particulate forms, and it depressed the response when the antigen was given in the soluble form.     

Modulation of the IgE immune response to BSA by fragments of the antigen. I. Suppression by free fragments and by fragments conjugated to homologous gamma-globulin

Nonimmunogenic peptic fragments of bovine serum albumin (BSA), Fraction Ia, suppressed immune response to BSA in mice. Splenic T lymphocytes from mice treated with these fragments suppressed the anti-DNP response in irradiated mice reconstituted with DNP-BSA-primed cells, indicating carrier-specific suppression. The conjugate of Fraction Ia with mouse γ-globulin (MGG) was found to be an effective suppressive substance but it did not induce suppressor T cells. B cells from mice given Ia-MGG were unresponsive to BSA when transferred to irradiated recipients along with either normal or BSA-primed T cells. Thus, unresponsiveness to BSA was mediated by either T or B lymphocytes, depending whether the inducing substance was a free fragment of the antigen or fragments conjugated to homologous γ-globulin.     

Heterogeneity of antibodies produced by single hemolytic foci

Lethally irradiated male BDF₁ mice were injected with 10⁷ bone marrow cells, 8 × 10⁵ spleen cells, and sheep or goat red blood cells as antigens. The cross-reactivity between these red blood cells is approximately 40%, as determined by injecting normal mice with either sheep or goat red blood cells, removing their spleens 4 days later, and assaying all spleens for both antisheep and antigoat plaque-forming cells. Eight days after injection of bone marrow, spleen, and antigen, the spleens of the irradiated recipients were removed and assayed for the presence of hemolytic foci by the Playfair technique, and were found to contain an average of 0.7 foci/spleen. Earlier studies had demonstrated that such foci could contain plaque-forming cells derived from more than one precursor cell. The positive pieces comprising a single hemolytic focus were removed, pooled, and aliquots were assayed for direct and indirect plaque-forming cells to the immunizing antigen. Several foci were found to contain 17–82% as many plaque-forming cells lysing the cross-reacting antigen as lysed the immunizing antigen. These data indicate that these foci contained plaque-forming cells of two specificities: (1) plaque-forming cells which responded to determinants on the immunizing antigen only, and (2) plaque-forming cells which responded to determinants on both the immunizing and cross-reacting antigens. In addition, single foci were found which contained both direct and indirect plaque-forming cells lysing the immunizing antigen. Sixteen of 39 foci assayed contained more than twice as many indirect as direct plaque-forming cells. We concluded that a single hemolytic focus, probably derived from more than one precursor cell, could contain plaque-forming cells producing antibodies of more than one set of specificities and immunoglobulins of more than one type.     

Preliminary studies on allotypes in cattle1

This paper presents evidence of five isoimmune sera, which identified five antigens of cattle blood serum. These antigens were examined by means of staining, immuno-electrophoresis and Sephadex G 200 column chromatography. It was established that the BA-2, BA-3, BA-4 and BA-4′ antigens were proteins, whilst the BA-1 antigen was a lipoprotein. The BA-3 antigen migrated in the γ-globulin position; the remaining four antigens migrated in the α-globulin region. The antigens under examination were already present in the blood serum during the first few days of life and appeared to be of a stable character. The results of genetic analysis suggest that these antigens were transmitted according to Mendelism.     

Evidence that anti-idiotype induced immunity to experimental African trypanosomiasis is genetically restricted and requires recognition of combining site-related idiotopes

The ability of anti-idiotypic (anti-Id) antibodies to immunize mice against African trypanosomiasis independent of antigen has been confirmed. Of three allogeneic anti-Id antibodies raised against three protective monoclonal antibodies, each with specificity for the variant surface antigen of a clone of Trypanosoma rhodesiense, only one (anti-7H11 Id) was effective in immunizing BALB/c mice against homologous challenge. The immunity was associated with the more rapid and enhanced expression of the corresponding Id after infection. The immunity was restricted to mice bearing genes linked to Igh-Ca, which appeared to control expression of this Id both in response to infection and anti-Id treatment. Another Id, 11D5, appeared to be under similar genetic control. Anti-11D5 Id, however, was ineffective in immunizing mice against infection despite inducing high levels of Id bearing molecules before challenge. The immunizing potential of the respective anti-Id antibodies appeared to be related to the relative concentrations of antibodies reactive with idiotopes near to or within the antigen-combining site, which, in turn, determined the relative proportion of Id-bearing clones activated that had antigen binding activity.     

BCG-CpG-DNA对重组HBsAg免疫原性的影响

为了研究BCG-CpG-DNA对重组HBsAg免疫原性的影响,了解其佐剂功能。采用不同剂量BCG-CpG-DNA与不同剂量重组(汉逊酵母)表达的HBsAg或Al-HBsAg混合免疫小鼠,与同剂量铝佐剂疫苗对比,以放射免疫法检测抗-HBs中和抗体水平和抗体持续时间,ELISA法检测抗体亚类。结果显示,BCG-CpG-DNA和HBsAg混合,抗-HBs的中和抗体水平达到或高于同剂量铝佐剂疫苗;BCG-CpG-DNA和Al-HBsAg混合,抗-HBs的中和抗体水平高于同剂量铝佐剂疫苗,并有统计学显著意义(P<0.05);添加BCG-CpG-DNA组抗体水平4周时增加不明显,到10周则显著地高于同剂量铝佐剂疫苗组,IgG2a抗体水平也高于相应的对照组。实验结果表明BCG-CpG-DNA对HBsAg有较好的免疫佐剂作用,并和AL佐剂有协同作用。     

Inhibition of antigen-induced T-cell proliferation by antibodies to endogenous AKR-MuLV

Lymph node cells from BALB/c mice immunized with ovalbumin or human γ-globulin were restimulated in vitro with these antigens and assayed for antigen-induced proliferation. The proliferative response was shown to be antigen specific and T cell dependent. A rabbit antiserum to envelope and core proteins of AKR murine leukemia virus was found to inhibit antigen-induced T-cell proliferation. The IgG fraction and F(ab′)₂ fragments of the antiserum were also inhibitory. The inhibition occurred after the initial step of antigen-T cell interaction and viral absorption studies showed the inhibition to be specific for anti-AKR virus antibodies. A hypothesis for the mechanism of inhibition is discussed in relation to a functional role for endogenous murine leukemia virus.     

人肝脏特异的F抗原分离纯化及其抗血清制备

取新鲜的人肝脏,充分洗涤,制备匀浆,上清液经Sephadex-G200、DE52层析,聚丙烯酰胺凝胶电泳,得到电泳纯Ｆ抗原,并免疫制备抗血清.