Analysis of the first two waves of thymus homing stem cells and their T cell progeny in chick-quail chimeras.

Chick-quail chimeras were used to study precursor/progeny relationships of hemopoietic stem cells (SC) that enter the embryonic thymus in waves to give rise sequentially to the TCR-1+, TCR-2+, and TCR-3+ lineages of T cells. The first wave of SC and their progeny were examined by grafting thymus from 9-d chick embryos (E9) into E3 quails. mAbs specific for chick T cell antigens were used to trace the development of T cells in the recipients. All three lineages of TCR-bearing cells were generated from the first wave of SC. The cortico-medullary transit time was several day shorter for the TCR-1 subpopulation than for the TCR-2 subpopulation, and the peripheral seeding of TCR-2 cells also occurred later in development. The duration of thymocyte production from the first wave of SC that entered the thymus was approximately 3 wk, during which gradual cortical to medullary replacement by second wave SC progeny occurred. When the latter was examined, after transplantation of E7 quail thymus into E3 chick embryos, a sequential generation pattern for the TCR-1 and TCR-2 cell progeny was not evident. Finally, recirculation of T cells to the thymus medulla was defined in this avian model.

Lack of alloantigen-specific cytotoxic T cell activity in the TCR-gamma delta T cell subpopulation of alloantigen-immune chickens.

We examined the in vivo generation of alloantigen-specific cytotoxic T lymphocytes in chicken TCR-alpha beta and TCR-gamma delta T cells. The TCR-alpha beta and TCR-gamma delta subpopulations were separated from spleen of alloantigen-immune chickens using a negative selection method. The separated cells were examined for alloantigen-specific CTL activity in a 51Cr release assay. While negatively selected TCR-alpha beta cells exerted alloantigen-specific CTL activity, no cytotoxic activity could be detected with TCR-gamma delta cells.

Chicken egg antibodies for immunohistochemical labeling of growth hormone and prolactin in bovine pituitary gland.

We describe the production of polyclonal chicken antibodies specific for bovine growth hormone (bGH) and prolactin (PRL). Antibodies were generated by immunization of laying hens with recombinant bGH (rbGH), pituitary derived bGH (pbGH), and ovine PRL (oPRL). After the lipoprotein fraction was removed by dextran sulfate precipitation the antibodies were isolated from the egg yolks by ammonium sulfate precipitation. Immunization with rbGH and oPRL generated large amounts of specific antibodies, as revealed by ELISA and Western blot analysis. Antibodies against pbGH showed pronounced crossreactions with oPRL. The antibodies against rbGH and oPRL were well suited for sensitive and specific labeling of the GH- and PRL-synthesizing cells in bovine pituitary glands by immunohistochemistry. In addition, a quick and sensitive procedure for demonstration of both bGH- and PRL-synthesizing cells in a single paraffin section by double immunohistochemistry is presented. The chicken anti-bGH antibodies showed excellent results in combination with rabbit anti-PRL antibodies. The main advantage of avian antibodies in double immunostaining methods is the lack of crossreactions between avian antibodies and mammalian immunoglobulins and receptors which bind to the crystalline fragment of mammalian immunoglobulins (Fc receptors).

Characterization of new monoclonal antibodies identifying avian T lymphocyte antigens.

Six monoclonal antibodies (mAb) were produced to identify and characterize surface antigens of chicken T cells. Determination of their reactivity with different lymphatic cells using immunofluorescence analysis demonstrates that mAb KH8, NA6, PD4 and TH8 stained 32-43% blood lymphocytes, 72-77% thymocytes and 19-27% spleen cells, mAb OC5 approximately 99% thymocytes and 55% blood and spleen lymphocytes each, and mAb OC2 36% blood lymphocytes, 79% thymocytes and 62% spleen cells. The KH8, NA6, PD4 and TH8 antibodies immunoprecipitated from lysates of surface-labeled chicken thymocytes a polypeptide of M(r) 60,000 under non-reducing conditions and the OC5 antibody a glycoprotein of M(r) 68,000 under reducing conditions. MAb OC2 precipitated a single polypeptide of M(r) 40,000 under both conditions. The mAb KH8, NA6, PD4, TH8 and OC2 inhibited ConA-induced proliferative responses of blood T cells in vitro. However, sepharose-bound or soluble OC5 antibody was able to increase DNA synthesis significantly. These results indicate that (a) the mAb KH8, NA6, PD4 and TH8 identify the avian homologue of the mammalian CD4 molecule, (b) the mAb OC2 detects the avian CD2 antigen, and (c) the mAb OC5 recognizes the putative avian CD5 homologue.

The cytotoxic T lymphocyte response in reticuloendotheliosis virus-infected chickens is mediated by alpha beta and not by gamma delta T cells.

We induced a virus-specific cytotoxic T lymphocyte (CTL) response in B2 chickens by i.v. inoculation with 100 TCID50 of the reticuloendotheliosis virus (REV). Chickens were sacrificed 7 days after the infection and cytotoxic activity of the spleen cells against various target cells was assayed in a 4 h 51Cr-release assay at an effector to target ratio of 100:1. In addition, T cell receptor (TCR) alpha beta and TCR gamma delta cells were negatively selected from the REV-immune spleen cells and used as effector cells against REV-infected B2 target cells. (On average 40% of spleen T cells express TCR gamma delta in the chicken.) By inhibition of the cytotoxic activity of the immune spleen cells against REV-infected syngeneic target cells with monoclonal antibodies specific for chicken CD3 and CD8 molecules, the effector cells could be identified as CD8+ T cells. The cytotoxic activity was MHC-restricted, as only syngeneic but not allogeneic REV-infected target cells were lysed by REV-immune spleen cells, and virus-specific, as no cytotoxic activity could be found using uninfected syngeneic target cells. When assaying the activity of the negatively selected, > 98% pure alpha beta and gamma delta T cells, it was found that alpha beta T cells exerted virus-specific CTL activity ranging from 26 to 62% specific 51Cr-release, while gamma delta T cells showed only 2-4% 51Cr-release. These data indicate that REV-specific CTL response is mediated by alpha beta T cells and that gamma delta T cells are not involved in virus-specific CTL activity in the spleen of REV-infected chickens.

Course of serum-Ig concentrations in B12 chickens of the UM line.

The University of Munich chicken line ("UM") is isogenic in respect to the MHC and divides into normogammaglobulinemic, permanent and transient dysgammaglobulinemic individuals. Hence the immune defect is independent of the MHC. Continual analysis of the immunoglobulins until the 50th week of life revealed: one group of dysgammaglobulinemic individuals showed an initial IgG peak between the third and sixth week of life. Unusually high IgM and IgA levels occur in permanent and transient dysgammaglobulinemic individuals previous to the appearance of the IgG deficit and previous to a possible initial IgG peak. These high levels remain throughout the life of the chicken, possibly due to a missing negative feedback mechanism. Transient dysgammaglobulinemic chickens also exhibited increased IgM and IgA values after IgG normalization. Based upon our results, we postulate that the dysgammaglobulinemia defect is already preprogrammed during late embryonic development. The prevalence of a B or T-cell defect is still under discussion.

Kinetics of delayed-type hypersensitivity (DTH) reactions and Ig levels in FCA-induced dysgammaglobulinemic B 12 chickens.

Kinetic studies on delayed-type hypersensitivity (DTH) to Freund's complete adjuvant (FCA) have been done in spontaneous dysgammaglobulinemic chickens, which have drastically reduced serum IgG levels and highly elevated IgM and IgA levels - in comparison to chickens with normal immunoglobulin (Ig) levels. For a period of five weeks, serum Ig levels, leukocyte migration inhibition (LMIT), and wattle reaction were examined once a week. In normal chickens, FCA treatment resulted in stimulation of IgG, but did not affect IgM synthesis, whereas in spontaneous dysgammaglobulinemic chickens, FCA stimulated only IgM synthesis. Spontaneous dysgammaglobulinemic chickens could produce LMIT and wattle reactions as well as normal birds. Whereas in normal birds, both types of DTH reactions declined continuously about the third or fourth week, in immunodeficient chickens, further increments of LMIT and wattle reactions up to the fifth week persisted as evidenced by LMIT and wattle reactions even 15 weeks after sensitization. In contrast, only minimal signs of reactivity were seen in normal birds. Spontaneous dysgammaglobulinemic chickens, nearly unable to synthesize IgG even after FCA stimulation in vivo, lack suppressive mechanisms regulating the course of DTH reaction to FCA. The possible B-cell nature of the regulatory cell population is discussed.

Helper activity of chicken V beta 1+ and V beta 2+ alpha beta T cells for in vitro IgA antibody synthesis.

Chickens have only two T cell receptor variable beta gene families: V beta 1 and V beta 2 (1). In our previous work we found that IgA production was almost completely suppressed in chickens depleted of V beta 1+ alpha beta T cells by treatment with a TCR V beta 1-specific monoclonal antibody (2), while IgM and IgG production was not affected. Our present results indicate that, in vitro, both V beta 1+ and V beta 2+ chicken cecal tonsil T cells provide help for the differentiation of cecal tonsil IgA B cells, suggesting that the failure of V beta 1+ T cell-depleted chickens to produce IgA is not caused by the inability of V beta 2+ T cells to provide help for IgA production by B cells, but rather by the scarcity of these T cells in mucosal tissues (3), where most IgA responses are induced (4).

Isolation of immunogenic and lethal peptides of alpha-toxin from Clostridium novyi type B.

The lethal alpha-toxin was isolated from the culture filtrate of Clostridium novyi type B using ammonium sulfate precipitation and ion exchange chromatography. The alpha-toxin has a mol. wt of 190,000 and does not contain any disulfide cross-linkages. It consists of a single polypeptide chain. The peptide fragments resulting from the cyanogen-bromide cleavage were isolated using reversed phase and gel filtration HPLC. The immunogenic actions of these peptides and peptide mixtures were studied in Balb/c mice. Three polyclonal antisera recognizing the uncleaved native toxin could be found using an ELISA test (Br3, Bro2, Bro5). One peptide mixture (Tx5), which was proved lethal in shell-less quail eggs (in vitro), was rechromatographed with gel filtration HPLC that resulted in one peptide with mol. wt 3000 (Txleth), which again proved lethal in the shell-less quail egg lethality test. The immunogenic peptides differ from the lethal one, therefore we assumed different locations on the polypeptide chain. The separation of the immunogenic, non-toxic fragment from the lethal one may allow the production of a highly specific non-toxic vaccine. By using synthetically produced immunogenic peptides, time-consuming purification methods and working with the whole toxin will become unnecessary.

Isolation of cold agglutinins in Eperythrozoon suis-infected pigs.

A simple and reliable procedure is described for isolating the cold agglutinins associated with Eperythrozoon suis-infection in swine. After initiating microagglutination by cooling the blood samples, the erythrocytes were separated by density-gradient centrifugation using Ficoll-Paque, resuspended in RPMI-medium and warmed to 40 degrees C. The cold agglutinins could be removed from the supernatant by a subsequent centrifugation. Double immunodiffusion, polyacrylamide gel electrophoresis and immunoblotting showed that the cold agglutinins isolated consisted of IgM antibodies exclusively. Their ability to induce agglutination in cooled erythrocytes from healthy pigs confirmed that they were genuine cold agglutinins. The method paves the way for more detailed investigation into the mechanisms of this agglutinating disease.

Attempts to localize the defect in dysgammaglobulinemia of UM-B19 chickens by studying the effect of immunomodulating substances on immunoglobulin and antibody production.

The extreme decreased levels of IgG in dysgammaglobulinemic UM-B19 chickens are linked with decreased antibody activity. Antibody activity to T-dependent (although diminished) and T-independent antigens is present but is restricted to IgM and IgA antibodies. Complete Freund's adjuvant enhances the existing isotype pattern of serum immunoglobulins and antibodies. The antibody response to a "T-independent" antigen (B. abortus) is greatly increased by CFA in dysgammaglobulinemic chickens. Beside the appearance of high levels of IgG in dysgammaglobulinemic chickens during the first weeks of life and in transitory dysgammaglobulinemia, remarkable IgG synthesis can be temporarily induced by the mitogenic activity of LPS and even more by the regulatory function of Levamisole. Furthermore, LPS and Levamisole induce IgG antibody synthesis to concomitantly administered antigen, the IgG antibodies appearing within the normal time. Contrary to a missing feedback inhibition from total IgG to IgM serum immunoglobulins, a feedback inhibition from IgG to IgM antibodies is found. No correlation can be found between Levamisole-induced IgG immunoglobulin concentrations, and IgG antibodies. Germfree rearing for one week or longer prevents the dysgammaglobulinemic defect. The following conclusions are drawn: Early antigenic stimulation seems to be the inducing factor for dysgammaglobulinemia in UM-B19 chickens. A BG cell pool is still present in adult dysgammaglobulinemic chickens. This BG cell pool is probably diminished to a varying extent. T cell helper functions seem to be present (albeit they may be disturbed) and can be stimulated. IgG specific T cell suppression is probable. From these conclusions the etiology of the dysgammaglobulinemia in UM-B19 chickens is hypothesized to be primarily due to delayed bursal development: Immature BG cells are eliminated by environmental antigens during the neonatal period in a process similar to tolerance induction. This event, in turn, induces suppressor mechanism(s) or disturbance in helper mechanism(s).

Coordinate induction of tetrahydrobiopterin synthesis and nitric oxide synthase activity in chicken macrophages: upregulation of GTP-cyclohydrolase I activity.

Biosynthesis of nitric oxide (NO) and tetrahydrobiopterin (BH4) was investigated during cytokine-mediated activation of chicken macrophages. Monocyte derived macrophages and HD11 cells, a chicken macrophage cell line, constitutively synthesize BH4. Treatment of these cells with chicken macrophage activation factor (ChMAF) causes up to 10-fold increases of intracellular BH4 and of nitrite concentrations in the cell culture supernatant. Elevated BH4 levels correlate with an increase in GTP-cyclohydrolase I (GTP-CH) activity. Kinetic studies show a joint upregulation of GTP-CH activity and NO synthase activity first detectable 4 hr after stimulation. A corresponding increase in the mRNA for GTP-CH was detected by Northern blot analysis with a chicken GTP-CH specific cDNA probe. These results demonstrate that cytokine-induced BH4 synthesis by chicken macrophages is at least partially regulated through increased GTP-CH gene expression. The functional relevance of BH4 formation for NO production is shown by experiments using 2,4-diamino-6-hydroxypyrimidine (DAHP) as a specific inhibitor of GTP-CH. Monocyte derived macrophages stimulated in the presence of DAHP show a significant decrease in NO synthesis. The effect of DAHP was reversed by adding sepiapterin, which allows synthesis of BH4 through a salvage pathway.

Establishment of a B-compatible chicken line with normogammaglobulinaemia and dysgammaglobulinaemia (IgM/IgG).

A dysgammaglobulinaemic line of chickens homozygous at the major histocompatibility complex has been established. In these chickens the IgG levels are decreased 10 to 100 times and IgM levels are increased 2 to 10 times when compared to those of normal birds. Dysgammaglobulinaemia is genetically determined. No linkage with the major histocompatibility complex of the chicken has been found.

Immunological identification of avian monomeric and polymeric immunoglobulin M and immunoglobulin A after fractionation on sodium dodecylsulfate pore gradient polyacrylamide gels.

Naturally existing variants of Immunoglobulin M (IgM) and Immunoglobulin A (IgA) in the serum of immunologically competent, as well as immunologically defective UM-B19 chickens can be detected without time consuming purification. The different molecular forms of IgM and IgA were separated by gel filtration. Pentameric IgM and dimeric IgA were well separated from the 7S-peak that contained monomeric IgM and IgA. A method for immunological identification of the protein components is described. Pore gradient gel electrophoresis was combined with antigen-antibody crossed electrophoresis in horizontal agarose slabs. The principle of the method is that IgM and IgA in their different molecular forms in the mixtures to be assayed after gel filtration are first separated with high resolution by sodium dodecylsulfate (SDS) polyacrylamide gel electrophoresis using a pore gradient of 3 to 15%. In the second step, a slab of polyacrylamide gel with the separated proteins in transferred onto agarose layers containing appropriate anti- mu- and anti-alpha antiserum and SDS in a concentration of .01%. The technique is easy to perform and gives reproducible results. In chicken serum, the monomeric state of IgM (184,000 d), along with the pentameric state (920,000 d) were identified. Serum IgA exists in dimeric (340,000 d) and in monomeric (170,000 d) states. No differences between the commercial Leghorn and dysgammaglobulinemic and normogammaglobulinemic lines were found.

A competitive inhibition enzyme immunoassay for detection and quantification of organophosphorus compounds.

A sensitive and specific method for detection and quantification of methyl phosphonic acid, p-aminophenyl 1,2,2-trimethyl-propyl diester (MATP) as a model substance for organophosphorus compounds is described. Different procedures for coupling the haptenic group for immunization, purification and immobilization allowed the detection of hapten-specific antibodies. The competitive inhibition enzyme immunoassay (CIEIA), using purified chicken and rabbit IgG-antibodies, was able to detect MATP-concentrations as low as 10(-10) mol/l. Based upon our results, we postulate that the CIEIA represents a good alternative to the customary diagnosis of organophosphate intoxications, measuring blood cholinesterase activity.

Granulocyte isolation from whole blood of goat, sheep, cattle, horse, dog, pig, and man.

A simple two step procedure for the isolation of caprine, ovine, bovine, equine, canine, porcine and human peripheral blood granulocytes is described. After enrichment of granulocytes by centrifugation, contaminating erythrocytes are lysed hypotonically. Recovery, purity, and viability of the granulocyte suspensions are determined. FACScan analysis of the cell suspensions measuring cellular size by forward and sideward light scatter is compared with the corresponding analysis of whole blood leukocytes. Constituencies of the isolated cell suspensions and loss of granulocyte subpopulations through isolation procedure is discussed with regard to granulocyte function assays.

Quantitative characterization of yeast cell incorporation in phagocytes: microscopic methods for differentiation between adherent and ingested particles.

A light- and a fluorescence-microscopic method for quantitative assessment of yeast cell incorporation in phagocytes were developed. The light-microscopic method offers methylene blue prestained yeast cells as phagocytosis particles and counterstains nonincorporated yeasts with eosine. The fluorescence-microscopic method works by acridine orange staining of phagocytosis assays. Fluorescence of nonincorporated yeast cells is suppressed by addition of methylene blue. Different ways of evaluating the results of microscopic quantitation of phagocytosis are discussed.

Complement-dependence of polymorphonuclear yeast cell phagocytosis: a comparison between man and various domestic animals.

The complement-dependence of polymorphonuclear yeast cell phagocytosis of sheep, goat, cattle, horse, dog, pig and man is determined by comparing the opsonizing abilities of untreated sera with complement-inactivated autologous sera at a serum concentration of 2.5% and at different phagocytosis periods. The performed phagocytosis assays only detects incorporated particles and allows for the differentiation of granulocytes with different quantities of phagocytosed particles. In sheep, horse and man the addition of heat-inactivated serum reduces the phagocytosis index to less than -80% of the value obtained at untreated serum addition at a phagocytosis period of 60 min. The other species show a smaller reduction ranging from -66.9% (dog) to 41.4% (cattle). The evaluation of the distribution pattern of granulocytes incorporating a certain number of yeasts offers significant differences in the investigated species.

Distribution of immunoglobulins during embryogenesis in the chicken.

Whereas the yolk of freshly-laid eggs contains only IgG, apart from traces of IgA, we were able to measure, on average, 0.145 mg/ml IgM and 0.207 mg/ml IgA in the yolk sac contents of 21-day-old chicken embryos. Up to the 14th embryonic day, IgG is exclusively contained in the yolk sac contents. It was not possible to demonstrate an increase in the amount of immunoglobulins present by comparing the amounts of IgM and IgA in freshly-laid eggs and in the yolk sac contents of 21-day-old embryos. The offspring of hens with experimentally-induced agammaglobulinemia did not begin with the production of the immunoglobulin isotypes IgG, IgM and IgA until after hatching. From these results the conclusion can be drawn that the immunoglobulins IgM and IgA are transferred from the egg white to the yolk sac contents during the last third of embryonic development. Embryonic synthesis of these immunoglobulins can be discounted as a result of this study. Synthesis was found to be initiated between the 2nd and 7th day of life for IgG, between the 2nd and 4th day of life for IgM and between the 6th and 13th day of life for IgA.