Serum protein changes in brown trout (Salmo trutta L.) after single injections of soluble and cellular antigens

Brown trout sera were examined by cellulose acetate (CAE) and polyacrylamide electrophoresis (PAE) and the protein concentrations measured by the Lowry method, before and after stimulation with haemocyanin, horse serum, Salmonella typhi 'H' and human group 'O' erythrocytes. Uninjected fish contained 5.l1±0.71 g protein/100 ml. Protein concentration was unaffected by haemocyanin and horse serum but was significantly lowered by S. typhi and human group 'O' erythrocytes. Male and female fish gave similar responses. Uninjected and injected fish showed 8 components on CAE and 16 on PAE. Significant differences were found between injected and uninjected control fish and between antigen stimulated and uninjected controls.     

Immune response of the prawn, Macrobrachium rosenbergii, to bacterial infection

Serum agglutinins of Macrobrachium rosenbergii found in normal serum were reactive toward eight bacterial species and type A human red blood cells. Absorption studies indicated the ability of the agglutinins to distinguish between different bacterial species as well as between certain bacteria and red blood cells. Agglutinin titers were approximately the same for the bacteria, whereas those for red blood cells were significantly higher. A virulent strain of Vibrio anguillarum was used in infectivity experiments. An LD₅₀ value was determined between 5 × 10⁶ and 10⁷ cells/animal, and an attempt was made to immunize the animals using formalin-killed cells. The animals did not respond to the vaccination, as there was neither an increase in the level of circulating agglutinins nor the LD₅₀ level 6 days after injection. Structural and functional traits of serum agglutinins are markedly different from vertebrate antibodies.     

Piezoelectrically driven vertical cavity acoustic transducers for the convective transport and rapid detection of DNA and protein binding to DNA microarrays with SPR imaging--a parametric study

The identification of pathogenic bacteria in water is important for addressing preventive and treatment issues regarding health and safety. A highly sensitive and specific solid-phase sandwich ELISA procedure was developed for the detection of typhoid causing extremely lethal water borne pathogen Salmonella typhi (S. typhi) on modified isopore polycarbonate (PC) black membranes. PC membranes were chemically derivatized to generate amino groups on the surface maintaining their pysico-optico properties. Surface modified PC membranes were characterized by ATR-FTIR spectrometer, goniometer and scanning electron microscope. Polyclonal somatic 'O' type antibodies (Abs) against whole cell S. typhi were immobilized on them by following the amine glutaraldehyde chemistry. Antibody immobilized membranes captured S. typhi from buffer solution and this complex was detected colourimetrically using HRP labelled S. typhi Ab. A detection limit of 2×10(3)cells/ml of bacteria was achieved with the modified PC membranes without any pre-enrichment step as against 10(6)-10(7)CFU/ml of bacteria by typical ELISA method. The assay was demonstrated to be specific for the target bacteria when compared with other cross-reactant water borne pathogens. The intra- and inter-assay precision for 10(4) and 10(5)cells/ml was 5.3-7.4 and 10.3-19.7% respectively. The developed immunoassay for the detection of S. typhi is simple, easy to handle, sensitive specific, reproducible and cost effective in comparison with the commercially existing immunochromatographic assays.     

Polysaccharides and their common proteinic carrier in the Vi antigens of Citrobacter ballerup and Salmonella typhi Ty2

Immunochemical analysis of Citrobacter ballerup and Salmonella typhi Ty2 showed that the strains share native and heat-resistant proteins that are, apparently, the carriers of a common polysaccharidic determinant present in their respective somatic antigens. After the classic acetic acid hydrolysis, the somatic antigen of C. ballerup reacted, in agar gel, against the homologous antiserum by two precipitation lines, one of which also precipitated against the anti S, typhi Ty2 serum; the hydrolysis of the S. typhi Ty2 somatic antigen demonstrated that, in addition to the 'O' polysaccharide, reacting against all the S. typhi antisera, it contains a polysaccharide that precipitated against the anti-C. ballerup serum. The elusiveness in the agglutinability of only freshly isolated bacterial authorizes some doubt concerning the responsibility of the antipolysaccharide antibodies in the agglutinating Vi sera; in order to induce anitpolysaccharides hyperimmunizations are needed while antiproteins are easily induced by short immunizations.     

Physicochemical and Biological Properties of Sonically Treated Vi Antigen

Electrophoretically purified Vi antigen from Citrobacter freundii 5396/38 was depolymerized by sonic treatment. The treatment caused an 80% reduction in specific viscosity and a reduction in molecular weight from 1.6 x 10(6) to 3.9 x 10(4). The O-acetyl and N-acetyl contents of the antigen and its infrared spectrum remained unchanged. The sonically treated antigen was only 1% as effective as the original antigen in eliciting protection in mice against challenge with Salmonella typhi. Sonically treated antigen also elicited lower antibody titers after single injections in mice and rabbits. No loss in ability to precipitate antibody or to sensitize red blood cells for hemagglutination was observed.     

Apoptosis of human keratinocytes after bacterial invasion

In this study, we examined the invasive capacity of Staphylococcus aureus and Salmonella typhi in human keratinocytes and monitored the number of viable intracellular bacteria at different post-infection times. The strains tested entered keratinocytes; both S. typhi and S. aureus were internalized within 30 min to 2 h after infection. No intracellular multiplication was observed, but S. typhi and S. aureus remained viable 72 h after infection. We also demonstrated that keratinocyte death following S. typhi and S. aureus invasion occurs by apoptosis as shown by DNA fragmentation. After 24 h of infection with S. typhi, the number of cells undergoing apoptosis were higher compared to infection with S. aureus. For prolonged infection times (48 h, 72 h) with both bacteria, there was no significant change in the number of cells undergoing apoptosis. The results demonstrated that viable intracellular S. typhi and S. aureus induced apoptosis in keratinocyte cells.     

Hemagglutinins of some Aspergilli

Hemagglutinins for human red blood cells have been found in hot-water soluble mycelial extracts of a strain of Aspergillus flavus and two mutant strains of A. parasiticus. The agglutinin from one strain of A. parasiticus was specific for blood group A cells while the other two agglutinins were non-specific. With the A. flavus strain, the greatest hemagglutination activity (HA) was found at 10 days for the mycelial extract, and at 12 days for culture fluid preparations. More agglutinin was produced by fungi grown on sucrose than on d-glucose as carbon source. Solubilities in ammonium sulfate solutions and protein and carbohydrate analyses show that the agglutinins from the mycelial extract and culture fluid preparation are different. The mycelial agglutinin was inhibited by a number of different sugars, many of which possess common stereochemical features.     

The serological specificities of Salmonella typhi antigens.

Sera prepared with two different strains of Salmonella typhi were analysed against all the soluble antigens isolated from S. typhi 0901, S. typhi Ty2 and S. typhi Vi. Agar-gel diffusion against individual sera showed that, in all the sera, antibodies were induced against somatic antigens and free proteins. Absorptions of the sera with polysaccharides, split from the somatic antigens, removed the antibodies induced against the polysaccharide and its proteinic carrier in most of the somatic antigens of S. typhi 0901. The antibodies left in the absorbed sera reacted against the proteinic moieties of more complex somatic antigens of S. typhi and against free proteins from all the analysed strains. Only the absorption with proteins removed all the precipitating antibodies from the sera. Moreover, in incomplete absorptions with proteins, the first antibodies removed are the antipolysaccharides, since antibodies are never induced against the haptenic polysaccharide but against somatic conjugates; in these the proteinic moiety eventually varies with every batch of bacteria. The sera exhausted of precipitins still agglutinate the bacteria, thus confirming the assumption that agglutinins and precipitins may be different antibodies.     

Carbohydrate-binding specificities of anti-erythrocyte lectins (haemagglutinins) in Anopheles gambiae gut extracts and haemolymph

Lectins that agglutinate red blood cells (RBC) were demonstrated in Anopheles gambiae mosquito haemolymph and gut extracts. No apparent differences in haemagglutinin titres were detected between male and female mosquitoes and overall agglutinin levels were not increased following a bloodmeal. Titres were highest in the haemolymph and midgut extracts versus human AB, horse, chicken and goat RBCs and in hindgut against human AB, chicken and sheep; foregut extract gave relatively low titres. Adsorption of haemolymph and gut extracts with selected RBCs coupled with carbohydrate inhibition and the use of enzyme-treated RBCs revealed the presence of multiple (hetero-) agglutinins. An.gambiae lectins were specific for (1-1)-, (1-4)- or (1-6)-linked glucose based disaccharides, glucose and its (1-2) or (1-3) linkages with fructose and, to a lesser extent, aminated or N-acetylated glucose, or galactose and its deoxy derivatives. This study presents the first report of the occurrence of heterogenous anti-RBC agglutinins in haemolymph and gut extracts of the mosquito An.gambiae, together with the sugar-binding specificities of these lectins.     

Detection of Agglutinins in Chickens Infected with JM Leukosis Virus

Hemagglutinins for sheep red blood cells were detected in sera from JM virus-infected single-comb White Leghorn (susceptible S line) chickens. The agglutinins were not sedimented at 97,000 x g and were not affected by freezing and thawing, possibly indicating a soluble hemagglutinating factor. Agglutination titers were read in a relatively short period, after 4 hr of incubation at 4 C. The usefulness of this test for quick and low-cost screening of a large number of samples is indicated.     

伤寒Vi多糖菌苗免疫血清的LPS抗体测定

以肠炎沙门氏菌脂多糖为抗原,用酶标法测定Ｖｉ多糖菌苗免疫血清的抗ＬＰＳ抗体。各Ｖｉ多糖菌苗组免疫后１月,６月的抗ＬＰＳ水平均显著高于免前（Ｐ＜０．０００１）,对照组无显著差异（ｐ＞０．１）。两３０μｇ菌苗组抗ＬＰＳ阳转率均约为１５％。提纯Ｖｉ多糖菌苗所含的微量伤寒ＬＰＳ,也可能为接种者提供一定的保护作用。     

Development of a one-step immunochromatographic strip test using gold nanoparticles for the rapid detection of Salmonella typhi in human serum

An immunochromatographic strip test using gold nanoparticles was developed for the rapid detection of Salmonella typhi (S. typhi) in human serum. The strip test based on the principle of sandwich immunoassay by the specific binding of antigens from S. typhi O901 and antibody of S. typhi O901 on a nitrocellulose membrane. Antibody-gold nanoparticle conjugate was used as the label and was coated onto a glass fiber membrane, which was used as a conjugate pad. To create a test and control zone, antibody of S. typhi O901 and an anti-IgG were dotted on the nitrocellulose membrane, respectively. Positive samples were displayed as red dots at the test and control zones of the nitrocellulose membrane, while negative samples resulted in a red dot only in the control zone. The limit of detection (LOD) was found to be 1.14×10(5) cfu mL(-1), which could be visually detected by the naked eye within 15 min. This strip test provided a lower detection limit and analysis time than a dot blot immunoassay (8.88×10(6) cfu mL(-1) for LOD and 110 min for reaction time). In addition, our immunochromatographic strip test was employed to detect S. typhi in human serum effectively, with high accuracy. This strip test offers great promise for a rapid, simple and low-cost analysis of S. typhi.     

The immune response of the brown trout Salmo trutta to lipopolysaccharide

Brown trout produced high molecular weight, thermostable, dithiothreitol sensitive, non-precipitating, complement-fixing antibodies and agglutinins to lipopolysaccharides after intramuscular injection with adjuvant. Antibodies were first detected on Day 14 and reached maximum titres after 56 to 63 days when a single injection was given. When either a second or a third injection was administered maximum titres occurred 34 to 40 days after the injection. After each injection the titres increased significantly, and the protein concentration of the sera was significantly decreased. In cellulose acetate electrophoresis experiments those bands which migrated in the β- to γ-globulin regions were increased.
Antibody-secreting and antigen-binding cells were detected on Days 8 and 4 respectively and maxima were reached between Day 16 and Day 18. The number of cells per 10⁶ lymphoid cells was higher in the spleen than in the kidney.     

Electrophoretic and immunologic studies of a hemagglutinin in the hemolymph of the decapod Macropipus puber

Serum agglutinins directed against antigens of the A-B-O blood groups of human erythrocytes have been detected in the blood of male Macropipus puber crabs. The hemagglutinating activity was constant in the specimens examined of which the intermolt stages ranged from C2 to D2. These hemagglutinins, which are not group specific, are proteinaceous and with an electrophoretic mobility similar to some human immunoglobulins. Their molecular weights approximate 300,000.     

Lectins (haemagglutinins) in the haemolymph of Glossina fuscipes fuscipes: Isolation,partial characterization,selected physico-chemical properties and carbohydrate-binding specificities

Glossina fuscipes fuscipes haemolymph contained agglutinins (lectins), titre range 2⁻¹¹–2⁻¹⁸, against red blood cells (RBC) of human ABO(H) blood group with highest values detected against “AB” RBC. The use of protease- and neuraminidase-treated RBC in many cases increased titres whilst treatment with galactosidases or glucosidases caused decreased levels. Haemolymph adsorption with “O” RBC reduced titres against “O” and “AB” but to a lesser extent anti-A or -B activity indicating lectin heterogeneity. The carbohydrate-binding specificities for human RBC were directed towards N-acetylated and deoxy derivatives of glucose and/or galactose. In addition the haemagglutinins were reactive against some oligosaccharides, ribose, deoxymannose, deoxygalactose, xylose and xylan with certain of the RBC types. The agglutinins were glycoprotein in nature, thermo-labile, affected by storage, freezing and thawing treatments and exposure to a high dosage of γ-radiation, possessed limited disulphide and hydrogen bonds, and depended upon slightly acid to neutral conditions for optimum agglutination. The haemag-glutinins did not require the presence of divalent cations (Ca²⁺, Mn²⁺ or Cu²⁺ ions) for activity although an elevated concentration of Mg²⁺ ions resulted in increased endpoint titres. However heavy metal ions (Pb²⁺ and Fe²⁺) in the buffer lowered agglutinin levels. The intact lectin molecule had an isoelectric point of 6.2, a relative molecular weight of 710 kDa and comprised approx. 70 kDa subunits.     

Cyclic kinetics and mathematical expression of the primary immune response

The kinetics of antibody-forming cells (AFC) in the spleen of rats immunized with Salmonella typhi O-antigen was investigated. The number of nucleated cells of spleen and blood serum antibody titres in passive haemagglutination were determined in parallel. Cyclic changes in the number of antibody-forming cells were detected as three peaks on the 4th, 9th, and 13th days following immunization. The fluctuations of their number were not related to the total number of nucleated cells of spleen. The antibody titres reached their peak on the 10th day following immunization, decreased by the 14th day and rose again on the 16th day after immunization. Repeated increases of the number of AFC were probably due to the regular, not accidental, recruitment of committed precursors cells (memory cells).     

Natural anti-bacterial activity against Salmonella typhi by human T4+ lymphocytes armed with IgA antibodies

Peripheral blood mononuclear cells from normal volunteers possess natural anti-bacterial (NA) activity against S. typhi that can be measured in a 2-hr in vitro assay. Employing fractionation on nylon wool columns, Percoll gradients, plastic adherence, and E rosetting, the effector cell of NA activity appeared to be a lymphocyte of the T lineage rather than a macrophage, a B lymphocyte, or a large granular cell. Moreover, complement-dependent killing with monoclonal antibodies such as OKM1, OKB7, OKT8, 5.9 and the anti-natural killer cells AB8.28 did not reduce NA activity. On the contrary, this was completely inhibited when OKT3, OKT11, or OKT4 antibodies and complement were used to pretreat the effector lymphocytes. Indeed, T4+ cells sorted with a FACS displayed an extremely high NA activity against S. typhi. By pretreatment of peripheral lymphocytes with F(ab')2 fragments against human IgA, the NA activity was blocked. It is therefore suggested that NA activity by human cells might be a mechanism of defense against infections, acting as antibody-dependent cellular cytotoxicity expressed by T4+ lymphocytes coated with preexisting anti-Salmonella IgA antibodies.     

Effect of purified recombinant human erythropoietin on anemia in rats with experimental renal failure induced by five-sixth nephrectomy

Recombinant human erythropoietin (rHuEPO) was purified from the conditioned media of Chinese hamster ovary cells with a transfected human erythropoietin gene. We investigated the effects of the rHuEPO in rats with renal anemia induced by partial nephrectomy. Five-sixth nephrectomy resulted in renal failure with anemia. Twenty-five days after the operation plasma urea nitrogen was increased about 2.5 times, and the red blood cell count, hematocrit, and hemoglobin concentration fell to 85% of normal. The reticulocyte count and plasma erythropoietin level did not change such as they do in patients with anemia due to chronic renal failure. Both total red blood cell volume and the plasma iron turnover rate were depressed in five-sixth nephrectomized rats compared with normal rats.The five-sixth nephrectomized rats were injected with rHuEPO (60 IU/kg) intravenously every second day for a total of six injections. After three injections of rHuEPO, circulation volume of total red blood cells was increased from 9.9 ml to 14.6 ml, and the plasma iron turnover rate was increased from 1.03 mg/kg/day to 2.12 mg/kg/day, and the reticulocyte count was also increased. After six injections, a marked increase of the red blood cell count, hematocrit, and hemoglobin concentration were observed. Plasma urea nitrogen and the creatinine levels as indications for renal function did not change after rHuEPO administration in both normal and five-sixth nephrectomized rats.In conclusion rHuEPO has a potent erythropoietic action and it is possible to cure the anemia caused by renal failure.     

Antigens of the Ii System on Lymphocytes

USUALLY Ii specificity is assigned to cold agglutinins on the basis of their reactions with the red cells of human adults (adult red cells) and newborn infants (cord red cells). Those which react more strongly with adult red cells are said to detect I antigen and are designated anti-I; those which react more strongly with cord red cells are said to detect i antigen and are designated anti-i^1,2. We have now found that I and i antigens can be easily detected on lymphocytes.     

Immunological Responses of the Dogfish (Scyliorhinus canicula L.) to Cellular Antigens

Humoral and cellular aspects of the immune response of the common dogfish (Scyliorhinus canicula L.) to a variety of cellular antigens have been examined. The fish made a relatively slow but positive antibody response to injected Salmonella typhi. Using the haemolytic plaque technique, antibody synthesis was shown to occur in the spleen after fish were challenged with sheep red blood cells, suggesting that this organ is a major site of antibody synthesis. Intraperitoneal and intravenous injections of allogeneic leucocytes stimulated marked histological changes in the spleens of recipients, indicative of a host-vs-graft reaction and suggesting that the fish are capable of alloimmune reactions.