Injection Of Sheep Erythrocytes Research Articles

Humoral immune response in Oreochromis niloticus: modified direct agglutination test

Understanding the functional mechanisms of the humoral and cellular immune responses among teleost broadens the possibilities for intervention and improvement of vaccination methods. The aim of this study was to evaluate the dynamics of the humoral immune response, hematological biochemical changes, and electrophoretic profile of proteins in Oreochromis niloticus, when submitted to antigen stimulation using sheep erythrocytes in a single dose and with booster doses. Seventy O. niloticus were distributed in two groups (n = 35): one received a single dose and the other received two doses of antigen. Evaluations were made at the following predetermined times: zero (pre-immune, i.e., before inoculation with the first injection of sheep erythrocytes, negative control) and on 7th, 14th, 21st, and 28th days after the first immunization to evaluate the hemogram, biochemistry, and electrophoresis of proteins and direct hemagglutination. Twenty-eight days after antigen inoculation, it was observed that there had been an increase in the serum antibody titer and fractions gamma globulins after second antigen stimulation. The results suggest that teleost fish have the capacity to produce antibodies from a single stimulation, and a second antibody production wave after second stimulation.

Relative Sensitivities of Japanese Quail to Foreign Red Blood Cell Challenges for Immunotoxicity Testing

Given evidence that the immune system is sensitive to environmental contaminants, evaluating immunocompetence in toxicology studies is increasingly important. By incorporating a test of humoral response into controlled reproductive and developmental studies, more comprehensive results can be gathered to assess the potential for disease. The foreign red blood cells (RBC) challenge is a minimally invasive method for evaluating humoral responses to a foreign antigen. Typically, antibody response is assessed following injections of sheep erythrocytes; however, Japanese quail (Coturnix japonica) are only minimally sensitive to sheep RBC. In the present study, adult Japanese quail were treated with 5% solutions of RBC from goose, goat, cow, donkey, or pig in primary and secondary challenges. After each treatment, plasma samples were taken and antibody responses were measured for total immunoglobulins (Ig), IgG, and IgM. Overall, goose RBC generated the poorest responses in both primary and secondary challenges, while the strongest antibody responses were to pig and donkey RBC. Therefore, pig RBC appear to be a superior antigen for testing humoral response in Japanese quail.

Regeneration of Autotransplanted Avascular Lymph Nodes in the Rat Is Improved by Platelet-Rich Plasma

The aim of this study was to verify that subcutaneous lymph node transplantation inducing lymphatic regeneration is possible in healthy adult rats, in analogy to results obtained in other species. This rat model was used to determine the effects of lymph node fragmentation as well as sheep erythrocytes and platelet-rich plasma injection on the regeneration of the transplanted lymph nodes. The results show for the first time that the rat is an adequate model to study the regeneration of transplanted lymph nodes. Lymph node fragmentation seems to affect transplant regeneration negatively. An immune challenge by injection of sheep erythrocytes in the drainage area of the transplanted lymph nodes does not improve fragment regeneration. However, injection of syngeneic platelet-rich plasma containing several growth factors resulted in an improvement in regeneration. Lymph node fragment regeneration, although still experimental, could be relevant for lymphedema prevention. Acquired lymphedema has a high prevalence in developed countries as a consequence of the removal and/or radiotherapy of tumor-draining lymph nodes in cancer patients. This disease causes lifelong disability due to chronic swelling and increased risk of infections. It currently lacks an effective treatment.

Antigen-Induced Changes in the Endocrine Function of the Thymus in CBA Mice during Aging: Role of Peptide Factors Released by the Pineal Gland

The titer of thymic serum factor was measured in adult and old CBA females 15, 30, and 60 min, 24, 48, 72, and 96 h after injection of 3% suspension of sheep erythrocytes and changes of this parameter under the effect of epithalamin were studied in old immunized mice. The titer of thymic serum factor increased appreciably in adult mice virtually at all periods of the study after immunization (a drop was observed only 72 h after immunization). In old mice the titer of thymic serum factor virtually did not change after injection of sheep erythrocytes, while immunization of old mice preinjected with epithalamin significantly increased this parameter. Not only the values, but their dynamics in old mice injected with epithalamin corresponded to those in immunized adult animals.

Pharmacotoxicological aspects of nitrate and nitrite in domestic fowls

1. The effects of nitrates and nitrites on growth, erythrocytic count, liver and kidney functions, humoral and cell mediated immune responses in cockerels were investigated. 2. Sodium nitrate (4.2 g/kg diet) and sodium nitrite (1.7 g/kg) retarded growth, caused methaemoglobinaemia and changes in erythrocytic count, serum concentrations of glutamic-pyruvic transaminase, creatinine and urea. 3. Cockerels given nitrate or nitrite in the food had reduced haemagglutination responses after injection of sheep erythrocytes and a reduced delayed hypersensitivity reaction to purified protein derivative tuberculin following sensitisation to Bacille Calmette Guerin. 4. Nitrates and nitrites are environmental pollutants present in food and water and they may contribute to the aetiology of liver and kidney diseases and problems related to failure of immunity in domestic fowls.

Cell surface changes of in situ macrophages induced by superimposed antigen

Mice received an injection of sheep erythrocytes (SRBC) into the footpad " prepared" or "not prepared" with a 7-day-prior injection of a protein-bound polysaccharide from Coriolus versicolor (PSK; Krestin), and the ultrastructure of in situ macrophages was studied at various intervals after the injection. A single SRBC injection into the footpad induced linear cell arrangements of several macrophages. The macrophages showed no prominent morphological alterations after SRBC digestion. When PSK-stimulated subcutaneous macrophages were challenged by SRBC, they rapidly sent out numerous long cytoplasmic projections which radiated in all directions. Such projections of neighboring macrophages tended to contact one another. At the following stage, a pronounced sequential alteration was noted, characterized by the interlocking of elongated projections. This provided massive aggregations of "activated" macrophages. These observations suggest the possibility that intercellular communication among "activated" macrophages was elicited, particularly in the subcutaneous region, and maintained through an intensive interaction of cytoplasmic projections. Further, the present results histologically support our previous report which shows that the "PSK-prepared" footpad site but not the "prepared" one supports development of a splenic humoral immune response following injection of superimposed SRBC.

Cellular aspects of Holothuria polii immune response

The cellular events in Holothuria polii after injection of sheep erythrocytes into the coelomic cavity are various and include recruitment of a large number of coelomocytes from hemopoietic areas, phagocytosis by amoebocytes, and the formation of a complex, active structure, the brown mass, through which the phagocytic residual products are cleared. Of interest is the finding that brown masses are positive to Schmorl's reaction, suggesting a possible melanin presence. The coelomocyte lysates are also able to oxidize DOPA, an intermediate product of one melanin synthesis pathway.

Non‐Determinant Specificity of Feedback Immunosuppression by IgG Antibodies Injected after the Antigen

The determinant specificity of the IgG-mediated suppression of the humoral immune response in mice was studied. One hour before or 2.5, 6, 12, or 24 h after the injection of sheep erythrocytes (SRBC) or SRBC-TNP, CBA/Ca mice received SRBC-specific monoclonal IgG antibodies. The antibodies did not cross-react with TNP or goat erythrocytes, the latter an antigen which shows 30% cross-reactivity with SRBC. Five days later the determinant-specific plaque-forming cell response against SRBC and the non-determinant-specific response against goat erythrocytes and TNP were determined. Regardless of whether the antibodies were injected before or after the antigen, they suppressed not only the response to the antigenic determinant they bound to, but also the response to other epitopes on the same antigen. This shows that Fc parts of the IgG molecules play a crucial part in suppression of the in vivo antibody response even when, as in a natural situation, the antigen is presented to the immune system before the antibody.

Haemolytic activity in the serum of brown trout, Salmo trutta

Brown trout serum contains a natural, spontaneous, antibody‐independent lytic activity and a haemolysin antibody complement‐mediated lytic activity against unsensitized and trout antibody‐sensitized sheep erythrocytes, respectively. The use of various activators and inactivators of the mammalian complement system demonstrated that trout serum possesses complement or complement‐like components similar in activity to those present in the classical and alternative pathways found in mammals. A single injection of trout with sheep erythrocytes stimulated the production of antibody‐secreting cells in lymphoid organs and increased the levels of natural haemolysins. A second injection of sheep erythrocytes further raised the haemolysin values and antibody‐secreting cell counts. Serum complement from homologous or closely related fish species was more effective for use in the haemolysin and antibody‐secreting cell assays than that from heterologous sources, except guinea pig. Based on physico‐chemical properties, gel filtration and immunoelectrophoretic studies, natural and induced anti‐sheep erythrocyte haemolysins were found to be similar molecules and are possibly high molecular weight IgM antibodies.

Enhancement by α-1-antichymotrypsin of antibody response in vivo

Summary The immunological effect of α-1-antichymotrypsin on the antibody response in vivo was studied in mice. The plaque-forming cell response to sheep erythrocytes was enhanced by an intravenous injection of α-1-antichymotrypsin. α-1-antichymotrypsin was effective if injected 2 days before or simultaneously with the injection of sheep erythrocytes. Immunoadsorption with an anti α-1-antichymotrypsin-Sepharose column indicated that α-1-antichymotrypsin itself was responsible for enhancement of plaque-forming cell response.

Cadmium, a metallic inhibitor of antibody-mediated immunity in mice

Chronic administration of cadmium chloride to B10-A-2R mice was discovered to severely depress the numbers and to delay the onset of appearance of splenic IgG and IgM plaque-forming cells (PFC) following injection of sheep erythrocytes. A recovery period of at least 1 month following cessation of administration of CdCl 2 resulted in no increase in IgM PFC and only a minimal increase in IgG PFC. An apparent cadmium-induced splenomegaly was also noted in the intoxicated mice. Application of immune adherence and rosetting techniques as well as immunofluorescence to study the cellular morphology of these spleens indicated that the cell type most responsible for the increased spleen size had Fc and complement receptors as well as surface or cytoplasmic immunoglobulins. Populations of polymorphs and macrophages were not found to significantly contribute to the hyperplasia observed.

STUDIES ON IMMUNE RESPONSES TO LARVAL CESTODES IN MICE:

After intraperitoneal (i.p.) injection of sheep erythrocytes (SRBC) or dinitrophenylated Ficoll (DNP-Ficoll), mice infected with the larval cestode, Mesocestoides corti, contain at least 20x fewer antibody-secreting cells (PFC) in their spleens (or spleens plus lymph nodes) than uninfected mice. By contrast, intravenous injection of antigen leads to normal PFC responses. Results of studies on the fate of labelled syngeneic erythrocytes and foreign proteins suggest that i.p. injected materials are retained in the inflamed peritoneal cavity. Sequestration of antigen, and its subsequent local destruction, presumably accounts for the markedly suppressed systemic immune responses induced by i.p. injected antigens in M. corti-infected mice.

Immunological memory function of the T and B cell types: distribution over mouse spleen and lymph nodes.

Spleens from LAF1 mice injected intravenously with sheep erythrocytes (SE) are relatively rich in memory T cells early in the immune response (1 to 3 days) and rich in memory B cells as the response progresses (2 weeks or more). Marked cooperation for the secondary immune response in vitro was obtained by combining 10(6) spleen cells from LAF1 mice, taken 2 days after intravenous priming with SE, with 10(7) spleen cells from day 14 primed mice. The results indicate relative deficiencies in the spleen for B memory cells on days 1 to 2 and for T memory cells on day 14 after priming. Day -14, but not day -2, immune lymph node (LN) cells could replace the day -2 spleen cells (anti-Thy 1.2 sensitive) in the in vitro cooperation with day -14 immune spleen cells. Immune spleen cells taken 4 to 7 days after priming contain more equivalent numbers of B and T memory cells, but 10 to 7 days after transfer of such immune spleen cells without SE into irradiated recipients the T memory cells were again more prominent in lymph node and the B memory cells in spleen as shown by in vitro cooperation studies. These results suggest that during the second week after intravenous injection of SE relatively more T than B memory cells migrate from spleen to lymph node, resulting in an imbalance in the splenic memory cell population favoring B memory cell function.

Enhancement and Retardation of Spontaneous Reticulum Cell Neoplasm Development in SJL/J Mice<xref ref-type="fn" rid="fn2">2</xref>

The enhancement or delay in the appearance of spontaneous reticulum cell neoplasms (RCN-B) in SJL/J mice was tested. Immunosuppressive treatment with antithymocyte serum and prolonged antigenic stimulation by repeated injections of sheep erythrocytes, leukemogenic cell-free centrifugates, and multichain synthetic polypeptides had no effect on the incidence or latency of spontaneous tumor appearance. Treatment with mineral oil or incomplete Freund adjuvant markedly enhanced tumor appearance (though the abundance of plasma cells induced by these treatments did not evoke the development of plasmacytomas, nor did it affect the incidence of serum paraproteinemia among mice with spontaneous tumors). Treatment with cortisone acetate, however, markedly retarded spontaneous tumor development. Tumors occurring early due to mineral oil and adjuvant treatment all had the characteristics of "RCN-B plasma cell type," and tumors occurring late due to cortisone treatment were all of the "RCN-B reticulum cell type." The possible involvement of the stem cell pool size in the enhancement or delay of spontaneous tumor development was discussed.

Antibody responses in athymic ('nude') mice implanted with neonatal or adult allogeneic thymus.

SummaryIt has been reported that antibody-forming capacity in athymic (nude) mice can be restored only by transplantation of thymus cells from histocompatible donors. In experiments reported here, nude mice received, 4 weeks prior to the injection of sheep erythrocytes, solid thymus grafts from neonatal or 3-week-old allogeneic donors. Restoration of antibody-forming capacity and persistence of transplants at the graft site were observed in animals receiving neonatal but not in animals receiving 3-week-old thymus tissue.We gratefully acknowledge the skillful technical assistance of Miss Lesley Ann Radov.

Rate of Hemolytic Antibody Production by Single Cells in Vivo in Rabbits

Abstract Published estimates of the rate of synthesis and secretion of antibody molecules by antibody-forming tissues in vitro range from 0.5 to 5000 molecules per cell per second. In the present paper we describe an experimental approach to the estimation of the absolute rate of antibody secretion per cell by undisturbed antibody-forming cells in vivo. There is evidence that most of the serum antibody appearing during the rapid rise to peak titer after a single intravenous (i. v.) injection of sheep erythrocytes into rabbits is made in the spleen. Plots of serum antibody titers typically exhibit an initial exponential rise followed by an arithmetic rise during which most of the antibody is produced and from which a constant rate of antibody production can be calculated. Rabbits immunized with a single i. v. injection of boiled sheep erythrocyte stromata were bled frequently, then sacrificed during the period of constant arithmetic antibody production and their spleen cells assayed by the liquid plaque assay technique to detect plaque-forming cells (PFC) making IgM hemolysin. Total splenic PFC were calculated based on spleen weights. The number of extrasplenic PFC was also taken into consideration and an allowance was made for PFC in the liver and blood. The rate of antibody production per milliliter in each animal was obtained from computer analysis of data for photometric 50% hemolysin units vs time and was converted to the rate of total hemolysin production based on total serum volume for each animal. This data, together with a literature value for molecules per RBC for 50% hemolysis, led to values for molecules of hemolytic antibody per PFC per second ranging from 8000 to 20,000 with a mean of 12,000 for seven rabbits. Our estimation of the protein-synthesizing machinery required for a cell to manufacture IgM antibody at this rate leads us to conclude that an average-sized plasma cell could do this if the messenger RNA is relatively stable or if gene expansion occurs to provide rapid transcription to maintain the level of messenger required.

The effect of x-irradiation of the appearance of antibody-forming cells in the mouse spleen following the injection of sheep erythrocytes.

AbstractThe effects of ionizing irradiation on the formation of plaque‐forming cells (PFC) against sheep red blood cells (SRBC) in the mouse spleen were investigated. The radiation effect is expressed as the reduction in numbers of PFC in irradiated mice as compared to the number of these cells in the control mice.A simple negative exponential radiation dose‐response relation was obtained with mice which were injected with SRBC 24 h after irradiation. The D37 for both the primary and the secondary IgM and IgG responses under these conditions was about 90 rad. Antigen injection in a period extending from 24 h before, to 2 h after irradiation resulted in a dose‐response curve with a shoulder, both for the IgM and IgG response.The doubling time of the number of PFC in the spleen during the first four days after antigen injection was not influenced by irradiation with 186 rad or less. With 279 rad, however, the doubling time increased.An explanation for the shoulder in the dose response curve is presented, which is an alternative to the usual explanation denoted by the “multi‐hit” effect of irradiation as a causative mechanism. It is postulated that the shoulder is a consequence of the masking of the irradiation effect by a process which normally regulates the extent of the immune response.

Uptake of (3H) corticosterone and the immune response in growth-retarded rats.

Extract: Maternal rats were underfed during pregnancy and lactation. At 7 days of age their offspring received one injection of sheep erythrocytes. The immune response (hemolytic plaque-forming cells in spleen and thymus) to this antigen showed a peak at 15 days of age in undernourished rats, 2 days later than in normal animals. However, the size of this response at its peak was, in the undernourished group, at least appropriate for body weight. Growth retardation during the fetal and suckling periods resulted also in an increased rate of uptake of 3H-corticosterone into spleen and thymus in vivo.Speculation: High rates of corticosteroid entry into spleen and thymus in undernutrition may tend to depress the immune functions of these organs independently of direct nutritional factors. During growth retardation the rate of certain immune responses may be impaired at least as much as their extent.

Immunocompetent cells in the chicken: II. Synergism between thymus cells and either bursa or bone marrow cells in the humoral immune response to sheep erythrocytes

Newly hatched F 1 hybrid chicks isogenic for the strong B histocompatibility locus were rendered immunologically incompetent by cyclophosphamide treatment and x-irradiation. They were then injected intravenously with thymus, bone marrow, or bursa cells together with sheep erythrocytes (SE) and received another iv injection of SE 3 days later. Splenic plaque-forming cells (PFC) and serum hemagglutinins were assayed 7 days after transfer. At donor ages of 14–26 days, cells from thymus (T) and bone marrow (BM) showed synergism when injected together, as indicated by a significantly higher geometric mean of PFC per recipient spleen in the BM + T group than in the BM group. The response of the T group was extremely low. With thymus and bursa cells from 6- to 28-day-old donors, significant synergism was demonstrated in 3 of 9 individual experiments. However, almost all the other 6 experiments showed marked differences in the same direction, and the combined probability for all experiments was < 0.001. The most striking demonstration of thymus + bursa synergism was made in 2 experiments using 1-week-old donors. Bone marrow cells from 1-week-old donors failed to cooperate with thymus, as did BM cells from older bursectomized agammaglobulinemic donors. This suggests that B cells from bone marrow originate in the bursa. Thymus-bursa cooperation was somewhat difficult to demonstrate in individual experiments using donors over 1 week of age, owing to the occurrence of some responses with bursal cells alone and to variability of response within bursa or bursa + thymus recipient groups. Synergism between thymus and bursa cells was more consistently demonstrable when irradiated normal spleen or low doses of bone marrow cells were added. These additions led to an increased response and a lowered coefficient of variation in the thymus + bursa recipient groups. The ‘third’ cell type needed for optimal response by the thymus and bursa cells together was tentatively identified as a macrophage.

Immunologic competence of alveolar cells. II. Modification of the plaque-forming response by inhibitors, tolerance, and chronic stimulation.

SUMMARy _ After intranasal immunization of rabbits with either sheep erythrocytes or human serum albumin, plaque-forming cells were detected among cells collected by alveolar lavage. Direct plaque formation was inhibited by 10 JLg of actinomycin D per ml, 10 JLg of puromycin per ml, 0.02 M 2-mercapto­ ethanol, guinea pig anti-rabbit immunoglobulin ~I serum, and antilymphocyte serum. Thus, direct plaque formation required protein synthesis and was mediated by immunoglobulin M antibody. During a primary response to human serum albumin, 75 per cent of the plaque-forming cells could be detected with the direct plaque assay and 25 per cent required anti-rabbit immunoglobulin G serum. After prolonged immunization, two thirds of the plaque-forming cells required the facili­ tating anti-immunoglobulin G serum. Intraperitoneal injection of sheep erythrocytes into newborn rabbits produced a state of tolerance accompanied by inhibition of plaque formation after subse­ quent intranasal immunization. Alveolar exudates contain cells that synthesize immunoglobulin G and immunoglobulinM anti­ bodies. These cells are a likely source of some of the immunoglobulins in respiratory secretions.