Immune capacity of the chicken bursectomized at 60 hr of incubation: cytoplasmic immunoglobulins and histological findings

Chickens were surgically bursectomized at 60 hr of incubation, before the bursal anlage appears. Completeness of the bursectomy was confirmed at autopsy at 10 weeks of age. These embryonically bursectomized (Bx)3 chickens are known to produce immunoglobulins of IgM, IgG, and IgA classes but so far no specific antibodies have been observed even after heavy immunization. The Bx chickens had mature plasma cells in an almost normal frequency when studied at 10 weeks of age. The amount of germinal center formation in the spleen and cecal tonsils was markedly decreased when compared to the control (Co) chickens. Also, the frequency of cytoplasmic IgA-positive (c-IgA+) cells was severely decreased in the Bx animals, whereas the occurrence of c-IgG+ and c-IgM+ cells was not affected to the same extent. These findings support the hypothesis that heavy-chain class switch may occur without the bursal influence, and that the bursa of Fabricius is essential only for expansion or creation of the antibody repertoire.     

Immune capacity of the chicken bursectomized at 60 hours of incubation: transplantation of bone marrow cells of the bursectomized chickens into cyclophosphamide-treated newly hatched recipients

Chickens bursectomized at 60 h of incubation are known to be able to produce immunoglobulins (Ig) but not specific antibodies. In the present work bone marrow (BM) cells of 2- and 10-week-old embryonically bursectomized (Bx) chickens were transferred to newly hatched cyclophosphamide-treated chickens for the purpose of studying whether the transplanted cells home into the bursa and gain a capacity to produce specific antibodies. BM cells of normal 2- and 10-week-old chickens were transferred as controls. Cells of 2-week-old control (Co) and of 2- and 10-week-old Bx chickens could to some extent reconstitute serum Ig of the recipients, but were totally incapable of homing into the bursa and of restoring the specific antibody production. Only BM cells of 10-week-old Co chickens could restore the production of specific antibodies without homing into the bursa, indicating their postbursal nature. These findings indicate (a) that 2- and 10-week Bx BM cells have irreversibly bypassed the bursal phase of education, (b) that also the normal BM at the age of 2 weeks contains cells that are capable of Ig but not of specific antibody synthesis and (c) that bursal microenvironment is not necessary for isotype switch, but is essential for creation and expansion of the antibody repertoire.     

Bursa lymphocytes and IgM-containing cells in chicken embryos bursectomized at 52-64 hours of incubation

A technique of surgical removal of the bursal primordium ("bursectomy") of chicken embryos at stage 17, approximately 52-64 hours and 29-32 somites, is described. The survival rate of bursectomized (Bx) embryos approached a level of 50% on the 21st day. About 20% of correctly Bx embryos exhibited malformations of the anal sphincter and the large intestine. Using a rabbit anti-bursacyte serum, which did not react with thymocytes, the specific bursa-derived cell (Bu) marker was detected on the surface of bursa, spleen, bone marrow and thymus lymphocytes. Early embryonic bursectomy caused a moderate depletion of Bu marker-bearing and IgM-containing cells. It has been postulated that embryonic Bu cells can be recruited from sites other than the bursa and in the absence of the bursa.     

Expression of B-L and Bu-1 antigens in chickens bursectomized at 60 h of incubation

Differences in expression between B-L (chicken class II major histocompatibility complex antigen) and Bu-1 B cell antigens were found in normal animals by using monoclonal antibodies and flow cytometric immunofluorescence analysis. Fluorescence intensity profile was used in assaying cell surface density of antigen molecules. The density of B-L antigen on the cell surface is apparently low in immature and high in mature cells, whereas the density of Bu-1 antigen does not vary in cells at different maturational stages. The existence of B-L+ and Bu-1+ cells in chickens bursectomized at 60 h of embryonic development (Bx) is demonstrated, indicating that neither B-L or Bu-1 antigen is exclusively specific for cells differentiated in the bursa. The densities of B-L and Bu-1 molecules on lymphoid cells in Bx chickens are similar to those of controls. However, the number of B-L+ and Bu-1+ cells was decreased in Bx chickens. We conclude that the extrabursal site where Bx B cells mature has an ability similar to that of the bursa to induce and enhance the expression of B-L and Bu-1 antigens. However, only few B cells proliferate and/or are released into the circulation. Further, the extrabursal site unequivocally lacks the most important function of the bursa, the creation and expansion of antibody diversity.     

Immunological capacity of the chicken embryo. II. Humoral immune responses in embryos and young chickens bursectomized and sham-bursectomized at 52--64 h of incubation.

White Rock embryos surgically "bursectomized" at 52--64 h of incubation, and shambursectomized embryos were injected with 10(6) guinea-pig red blood cells on day 12 of incubation, and tested for plaque-forming cells and serum haemagglutinins 3, 5, 7, 10, 15 and 19 days after immunization, i.e. as 15- to 19-day-old embryos and 1- to 10-day-old chickens. The number of natural plaque-forming cells detected by direct or indirect techniques was small in nonimmunized shambursectomized and bursectomized embryos, but increased in very young chickens. The injection of guinea-pig red blood cells induced a significant increase in the number of direct and indirect plaque-forming cells in the spleen of bursectomized and sham-bursectomized embryos and chickens. Agglutination of papain-treated guinea-pig red blood cells and indirect anti-chicken globulin (Coombs) test revealed the presence of natural agglutinins for guinea-pig and for sheep erythrocytes in non-immunized sham-and bursectomized embryos. A small number of sera from nonimmunized bursectomized and sham-bursectomized embryos contained IgM. The immunization with guinea-pig red blood cells increased the antibody production in both bursectomized and sham-bursectomized embryos and chickens. Sham-bursectomized embryos responded better to antigenic stimulation than bursectomized embryos. The injection of guinea-pig red blood produced an enlargement of the spleen only in bursectomized embryos and chickens. The first plasma cells appeared in nonimmunized sham-and bursectomized 6-day-old chickens. The number of plasma cells increased in chickens immunized as embryos. Cytomorphological analysis of the thymus, bone marrow and liver did not reveal apparent differences between bursectomized, sham-bursectomized embryos and very young chickens. It has been postulated that the chicken embryo has an antibody-producing system composed of the bursal and the nonbursal (or accessory "bursal") microenvironment, the latter being bursa-independent. The final microenvironmental network for the formation of Bu lymphocytes is the result of coordinated activities of a variety of intrinsic cellular and humoral factors.     

Effect of antibodies against immunoglobulins and the theta antigen on the specific and non-specific stimulation of mouse spleen cells in vitro

Normal mouse spleen cells were stimulated in culture by phytohaemagglutinin (PHA), pokeweed mitogen (PWM), allogeneic cells; keyhole limpet haemocyanin (KLH) sensitized cells by the specific antigen. The stimulation of the cells was measured by [³H]thymidine incorporation into the TCA precipitable fraction of the cultures. In this system, the effect of treating the cells with an antibody against the theta antigen and an antibody against immunoglobulin, with or without complement, was investigated. Treatment of the cells with antisera and complement or without complement gave similar results. The secondary in vitro stimulation with soluble antigen KLH could be markedly reduced with both anti-θ and anti-immunoglobulin serum. The response to allogeneic cells in the mixed lymphocyte reaction was only reduced by anti-θ serum and not by anti-immunoglobulin serum. No definite effect could be demonstrated by either antibody on the non-specific stimulation by PHA or PWM.     

A simple method for detecting cells producing antibodies of specific immunoglobulin classes in the chicken

A simple plaque method for detecting cells producing antibodies of specific immunoglobulin class in the chicken is described. This method is based upon the inability of chicken antibody to activate guinea-pig complement. IgM- and IgG-specific plaque-forming cells in the spleen of chickens immunized with sheep red blood cells were detected using guinea-pig complement and rabbit anti-chicken-μ- or γ-chain serum. The specificity of the immunoglobulin class of plaques was confirmed by the abolition of class-specific hemolytic plaques after treatment of the lymphoid cells with rabbit antisera specific for chicken heavy chains in the presence of guinea-pig complement. The method is very simple and rapid, and IgM and IgG specific plaques are reliably detected.     

The effect of the dose of tumor tissue antigens on the production of specific antibodies

Summary The antigen dosage of the Ehrlich's ascitic tumor tissue employed, in immunization of rabbits has a considerable effect on the titer of specific antibodies. However, the antitumor antibody production depends directly on the dosate of the antigens only when administered up to a certain moment following which an increase of the dose does not promote the rise of the specific antibodyAfter cessation of immunization the antibody production has an undulating character. At one time it is the antitumor antibodies that prevail in the serum, while at the other, the antisplenic antibodies, By a constant serological control of dynamics of the antibody production it is possible to obtain a serum with a considerable prevalence of the antitumor antibodies.Presented by Active Member AMN SSSR N. N. Zhukov-Verezhnikov     

Changes in the synthesis of antibodies and non-specific immunoglobulins in the spleen perfused in vitro

The synthesis of antibodies and non-specific immunoglobulins by the rabbit spleen perfused with culture medium in vitro has been studied using the incorporation of [¹⁴C]glycine. Separate perfusion of the two halves of the same spleen provided an opportunity to determine the influence of several factors on the synthesis of these proteins.

It was shown that changes in the rate of synthesis of antibodies and non-specific immunoglobulins in the perfused spleen are in some cases similar to the changes observed in the whole organism, and in some cases they are not. Thus, the synthesis of antibodies in the spleen withdrawn 2 days after secondary immunization increased during the 2 days of perfusion and in the spleen taken 4 days after immunization it decreased in the same way as in the whole organism. However, in the spleen taken 3 days after secondary immunization, the synthesis of antibodies during the perfusion period remained unchanged. Another distinction from the processes occurring in the living body was that the increased rate of antibody synthesis in the perfused spleen was not associated with an increase in nonspecific immunoglobulins synthesis.

     

Concomitant synthesis, in separate cells, of non-reactive immunoglobulins and specific antibodies after immunization with tobacco mosaic virus

Injection of tobacco mosaic virus into rabbits induces the concomitant synthesis of specific antibodies and of non-reactive immunoglobulins. Labelling experiments show that the amount of non-specific immunoglobulins produced for a given amount of antibodies is much greater after a first stimulation than after a booster injection.

Immunofluorescence studies on the spleen of animals before and after immunization clearly indicate that the increase in concentration of non-specific immunoglobulins observed in the serum is correlated with a significant increase in the number of cells secreting non-reactive immunoglobulins simultaneously with the appearance of antibody producing cells.

Finally cell counts and in vivo and in vitro synthesis by spleen fragments of specific antibodies and non-reactive immunoglobulins provide a strong argument in favour of a synthesis of non-reactive immunoglobulins and antibodies in different plasma cells.

The possible significance of the induced synthesis of non-specific immunoglobulins after antigenic stimulation are discussed.

     

Expression of full-length immunoglobulins in Escherichia coli: rapid and efficient production of aglycosylated antibodies

Many research and clinical applications require large quantities of full-length antibodies with long circulating half-lives, and production of these complex multi-subunit proteins has in the past been restricted to eukaryotic hosts. In this report, we demonstrate that efficient secretion of heavy and light chains in a favorable ratio leads to the high-level expression and assembly of full-length IgGs in the Escherichia coli periplasm. The technology described offers a rapid, generally applicable and potentially inexpensive method for the production of full-length therapeutic antibodies, as verified by the expression of several humanized IgGs. One E. coli-derived antibody in particular, anti-tissue factor IgG1, has been thoroughly evaluated and has all of the expected properties of an aglycosylated antibody, including tight binding to antigen and the neonatal receptor. As predicted, the protein lacks binding to C1q and the FcgammaRI receptor, making it an ideal candidate for research purposes and therapeutic indications where effector functions are either not required or are actually detrimental. In addition, a limited chimpanzee study suggests that the E. coli-derived IgG1 retains the long circulating half-life of mammalian cell-derived antibodies.     

Heart rate and heart rate variability in chicken embryos at the end of incubation

Our immediate goal was to study heart rate variability (HRV) in chicken embryos in the egg. Instantaneous heart rate data were needed for this purpose, and accordingly an ECG recording method in the egg was developed. The aim of this work was to test the hypothesis that autonomic nervous cardiac modulation, as shown from HRV parameters, is present at the end of development and that it reaches a constant value during the last days of incubation. Embryonic chicken heart rate was obtained at the final incubation period (days 19 and 20) from ECG recordings. Tachograms were computed and time- and frequency-domain indices of HRV were determined. No significant differences were found between HRV indices from day 19 and day 20. The power spectra extended in two frequency bands with centre frequency around 0.6-0.7 Hz (low frequency (LF) component), and another around 1.2-1.5 Hz (high frequency (HF) component); the latter was shown to reflect respiratory sinus arrhythmia. A relation between mean RR interval and some HRV parameters (rMSSD, pNN5 and HF power) was shown. HRV results obtained from embryonic chickens, showed the presence of modulation of cardiovascular function by the autonomic nervous system. The results suggested that sympathetic and parasympathetic activities have already reached a constant level at day 19 of incubation. High frequency oscillations (0.78-2.5 Hz) were detected and are considered to reflect respiratory sinus arrhythmia.     

The effect of adherent peritoneal exudate cells (APECs) and their products on the RPC-5 plasmacytoma growth in vitro

The ability of peritoneal exudate cells (PECs) and their adherent (APECs) and nonadherent (NAPECs) fractions to enhance RPC-5 plasmacytoma growth in vitro was studied. The capability of these cells to bind RPC-5 cells and influence of the binding on cytolysis tumor cells by activated with C. parvum macrophages was also determined. The effector cells were harvested from mice injected i.p. with pristane, thioglicollate medium or C. parvum or from intact mice. The effect of supernatants from the in vitro cultured PECs, APECs or NAPECs on growth RPC-5 cells were also tested. It was found that the RPC-5 plasmacytoma growth was enhanced only by cells obtained from mice treated with pristane, or by supernatants from cultured PECs and APECs derived from pristane treated mice. The adherent cells from pristane treated mice were able to bind tumor cells. The tumor cells preexposed to adherent cells from pristane stimulated mice were resistant to lysis by activated with C. parvum macrophages.     

Modeling mechanosensing and its effect on the migration and proliferation of adherent cells

The behavior of normal adherent cells is influenced by the stiffness of the substrate they are anchored to. Cells are able to detect substrate mechanical properties by actively generating contractile forces and use this information to migrate and proliferate. In particular, the speed and direction of cell crawling, as well as the rate of cell proliferation, vary with the substrate compliance and prestrain. In this work, we present an active mechanosensing model based on an extension of the classical Hill’s model for skeletal muscle behavior. We also propose a thermodynamical approach to model cell migration regulated by mechanical stimuli and a proliferation theory also depending on the mechanical environment. These contributions give rise to a conceptually simple mathematical formulation with a straightforward and inexpensive computational implementation, yielding results consistent with numerous experiments. The model can be a useful tool for practical applications in biology and medicine in situations where cell–substrate interaction as well as substrate mechanical behavior play an important role, such as the design of tissue engineering applications.