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Chicken alpha beta T cells express either CD4 or CD8 accessory molecules, whereas most of the gamma delta T cells do not. The functional significance of the alpha beta T cells is relatively well understood. The CD4+ alpha beta T cells function as coordinators of the immune response, and CD8+ alpha beta T cells are the effector cells in cytotoxic responses, killing infected target cells. In comparison, the role of gamma delta T cells is so far poorly known. In chicken, the gamma delta T cells comprise a large lymphocyte subset. They can be induced to proliferate by various stimuli, but the proliferative response is dependent on CD4+ alpha beta T cells. The CD4+ T cells are also essential for the generation of antibody responses by providing help for the B cells and can influence cytotoxic responses as well. Thus, the CD4+ alpha beta T cells have a central role in the avian immune system, and their activation is a prerequisite for responses by other types of cells, including gamma delta T cells.

A mAb recognizing a 40- to 44-kDa monomeric molecule on the surface of chicken T cells was used to screen a cDNA expression library made from Con A-stimulated chicken spleen cells. The sequence of the cDNA obtained encoded a molecule having 50% amino acid sequence identity with mammalian CD28, but the cysteine residue involved in the inter-chain bridge of the mammalian CD28 homodimer was not conserved in the chicken sequence. The molecule produced in transfected COS-7 cells was also recognized by another mAb that had previously been thought to recognize an avian homologue of CD2. The sequence data establish that this molecule is a homologue of mammalian CD28 in the strict evolutionary sense.

This study was set up to evaluate the effects of propofol infusion anaesthesia on immunological function in minor surgery. Twenty-seven patients (median age 51 years, ASA 1-2) scheduled for minor breast surgery were randomly assigned to two groups. Anaesthesia was induced in group 1 with propofol 2.5 mg.kg-1 and maintained with propofol 12 mg.kg-1 x h-1 and 30% O2 in air, whereas in group 2 anaesthesia was induced with thiopentone 4 mg.kg-1 and maintained with 70% N2O in O2. Fentanyl and vecuronium were used in both groups. The percentages of T cells (p < 0.001), B cells (p < 0.01) and memory T cells increased (p < 0.01) in both groups. T helper cell percentages increased in the propofol but not in the thiopentone group (p < 0.05). The percentages of natural killer cells decreased from pre-induction values in both groups (p < 0.001). No changes were seen in lymphocyte proliferative responses. Minor breast surgery under propofol or conventional combined anaesthesia had only minor effects on the immune response. The higher percentage of T helper cells after propofol anaesthesia compared to conventional combined anaesthesia is beneficial, but its clinical importance remains to be determined.

We have created a panel of mouse monoclonal antibodies detecting different epitopes on avian CD4 molecule. Two-color immunofluorescence analysis shows that chicken peripheral alpha beta T cells are either CD4 or CD8 single positive whereas most gamma delta T cells are CD4-negative both in the thymus and peripheral tissues. Unlabeled antibody competition analysis by flow cytometry demonstrates that several different epitopes on chicken CD4 are recognized by these antibodies. Antibodies precipitate a monomeric glycoprotein from surface-labeled chicken thymocytes and T cells with relative molecular mass (M(r) of 64 kd as analyzed by SDS gel electrophoresis. Removal of N-linked carbohydrates by endoglycosidase-F increases the electrophoretic mobility and reveals the core protein size with M(r) of 45 kd. The anti-CD4 antibodies inhibit antigen-induced cellular proliferation of a keyhole limpet hemocyanin (KLH) -specific T cell line. They synergize in the blocking of T cell proliferation with anti-class II major histocompatibility complex (MHC)-specific antibodies indicating that chicken CD4 is involved in antigen recognition process by CD4+ T cells. We also show that chicken CD4 is down-modulated in a similar manner as its mammalian equivalent when thymocytes are stimulated in vitro with phorbol esters. Altogether these findings suggest functional and biochemical conservation of the CD4 molecule in evolution.

In chickens from congenic inbred lines CB and CC that differ only in the major histocompatibility complex (MHC), we observed significantly different percentages of CD4+ and CD8+ cells in peripheral blood lymphocytes (PBL) and spleen. Positive cells were detected by indirect immunofluorescence test as analysed by flow cytometry. In both PBL and spleen cell suspensions, the number of CD4+ cells was significantly higher in CB than in CC chickens, whereas in CC birds there was a higher percentage of CD8+ cells than in CB. These statistically significant differences were under the MHC control. We found no statistically significant influence of regressions or progression of Rous sarcoma virus-induced tumours on the percentage of peripheral T cells and on the interleukin-2 production in vitro.

Thirty-nine horses were given detomidine 10 micrograms/kg bodyweight (bwt) intravenously (iv) and six horses were given a corresponding volume of saline solution before minor procedures. Venous blood samples were collected for catecholamine and cortisol determination immediately before the detomidine or saline injection and 20 mins after it. The behaviour of the horse at the time of detomidine injection and the extent of sedation were evaluated. Plasma adrenaline, noradrenaline and the catecholamine metabolites, 3,4-dihydroxyphenylglycol (DHPG) and 3,4-dihydroxyphenylacetic acid (DOPAC), and cortisol concentrations decreased significantly after administration of detomidine, but they remained unchanged in the control group. A high plasma adrenaline concentration before detomidine injection, indicative of a high level of stress, seemed to correspond with a reduced sedative effect of detomidine. The extent of sedation also was related to the concentrations of adrenaline, noradrenaline and DHPG in plasma after the detomidine injection. The reductions in the plasma catecholamine concentrations may have been caused, in part, by decreased secretion of catecholamines due to the sedative effects of detomidine; but detomidine also influences the plasma catecholamine concentrations by reducing directly sympatho-adrenal activity.

Detomidine (10 micrograms/kg and 20 micrograms/kg) was administered to seven horses with and without epinephrine infusion (0.1 microgram/kg/min) from 5 minutes before to 5 minutes after detomidine injection. One or more single supraventricular premature heartbeats were observed in three horses after detomidine administration. Epinephrine infusion did not modify the incidence of cardiac arrhythmias in detomidine-treated horses at the doses tested. Relatively high momentary peak systolic pressures were registered in some horses after detomidine administration during epinephrine infusion. The highest systolic arterial blood pressure was 290 mm Hg, but this value was not higher than that reported in horses during maximum physical exercise. Epinephrine infusion did not alter blood gases, arterial pH, or base excess.

We describe two mouse mAb recognizing the putative chicken homologue of mammalian CD2 Ag and provide evidence for both structural and functional conservation between the avian and mammalian CD2 molecules. The antibodies were T cell-specific and immunoprecipitated a single diffuse band of Mr 40,000 from lysates of surface-labeled chicken thymocytes and peripheral T cells. Removal of N-linked carbohydrate with endo-beta-N-acetylglucosaminidase F revealed the core protein size of Mr 25,000. A rabbit antiserum raised against a synthetic peptide (CD2-300), composed of 18 amino acid residues of the conserved cytoplasmic domain of human, mouse, and rat CD2, precipitated an Ag similar to chicken CD2. Sequential precipitation with CD2-300 antiserum indicated the conservation of an avian and mammalian CD2 epitope. CD2 expression on thymocytes starts at day 11 of embryonic development, and, during subsequent development, thymic gamma delta cells are all CD2+, whereas most peripheral gamma delta-T cells lack CD2. Functional conservation between the chicken and mammalian CD2 molecules was demonstrated by the induction of DNA synthesis in chicken thymocytes and peripheral T cells with the combination of anti-CD2 mAb and PMA.

Embryonic chimeras were used to demonstrate an early separation of chicken T and B cell precursors. Genetically polymorphic cell surface antigens, Bu-1 and Ov, which are expressed on cells of the B and T lineage, respectively, are useful markers in adoptive cell transfer studies. Allelic products Bu-1a and Bu-1b can be detected with monoclonal antibodies (mAbs) L22 and 11G2, respectively, and the Ov antigen with mAb 11A9. Chimeric chickens were constructed by reconstituting irradiated 14-d Ov- H.B19 embryos with the sorted Bu-1+ or Bu-1- fractions of spleen cells from age-matched H.B19 Ov+ embryos. Chimeras were analyzed, 3-4 wk after hatching, for the presence of Ov+ cells in the bursa, thymus, spleen, and peripheral blood lymphocytes. T cell precursors giving rise to thymocytes and peripheral T cells were present only in the Bu-1-, but not in the Bu-1+, fraction. We previously demonstrated that, in contrast, all B cell precursors in spleen from 14-d embryos are exclusively present in the Bu-1+ fraction. We also analyzed the immunoglobulin light chain gene rearrangement in these populations by polymerase chain reaction. We show here that VJ recombination occurs in the Bu-1+, but not in the Bu-1-, fraction of spleen. These data demonstrate an early commitment to the B cell lineage, which occurs before the colonization of the bursa of Fabricius. Segregation of B cell precursors from the other hemopoietic precursors, and consequently separation of T and B cell precursors, occurs before the colonization of the primary lymphoid organs.

In birds, B-lymphocytes mature in a special immune organ, the Bursa Fabricii. This organ thus offers unique possibilities for the study of the microenvironment of B-lymphocyte differentiation. We previously reported tachykinin-, vasoactive intestinal peptide-, calcitonin gene-related peptide- and galanin-immunoreactive (ir) fibres in the chicken bursa. As judged from light microscopic studies, each of the peptides was found in fibres contacting B-lymphocytes. Vasoactive intestinal peptide-ir fibres contacted macrophages. Now, we demonstrate neuropeptide Y, indicating the sympathetic nervous system, in fibres associated with arteries, not entering the follicles. CD4- and CD8-positive T-lymphocytes were dispersed in bursal follicles and the connective tissue, most densely in subepithelial regions. We could not find close apposition of fibres with either T-cell subset. We conclude that the potential neuro-immune axis in the Bursa Fabricii may represent a neuro-B-cell-link with only indirect participation of T-lymphocytes. The sympathetic input may influence the bursal microenvironment primarily by regulating the blood supply.

Single doses of the alpha 2-adrenergic sedative-analgesic drug, detomidine (10 micrograms/kg, n = 7; 20 micrograms/kg, n = 9), were administered IV to adult horses. Plasma concentrations of adrenaline, noradrenaline, the catecholamine metabolites 3,4-dihydroxyphenylglycol (DHPG) and 3,4-dihydroxyphenylacetic acid (DOPAC), and cortisol were determined before the medication and 30 minutes after it. The plasma concentrations of noradrenaline and the catecholamine metabolites decreased significantly after administration of both 10 micrograms/kg and 20 micrograms/kg of detomidine. Plasma adrenaline level had a tendency to decrease, but plasma cortisol levels were not influenced. The findings suggest a reduction in sympatho-adrenal activity in horses treated with detomidine.

B-G antigens are a polymorphic multigene family of cell surface molecules encoded by the chicken major histocompatibility complex (MHC). They have previously been described only on cells of the erythroid lineage. By using flow cytometry, section staining, and immunoprecipitation with monoclonal antibodies and rabbit antisera to B-G molecules and by using Northern blots with B-G cDNA clones, we demonstrate here that B-G molecules and RNA are present in many other cell types: thrombocytes, peripheral B and T lymphocytes, bursal B cells and thymocytes, and stromal cells in the bursa, thymus, and caecal tonsil of the intestine. The reactions also identify at least one polymorphic B-G determinant encoded by the B-F/B-L region of the chicken MHC. The serology and tissue distribution of B-G molecules are as complex as those of mammalian MHC class I and class II molecules. These facts, taken with certain functional data, lead us to suggest that B-G molecules have an important role in the selection of B cells in the chicken bursa.

In the experiments to be described two congenic inbred lines CB and CC and two recombinant lines CB.R1 and CC.R1 were used. All four lines differ only in regard to the major histocompatibility complex (MHC). To determine the percentage distributions of the two cell subsets in peripheral blood lymphocytes (PBL) in these lines, monoclonal antibodies to these two antigens were used. By FACScan there were more CD4+PBL in CB and CB.R1 lines (share B-F/B-L region, controlling class I/class II antigens with line CB) than CC and CC.R1, while the reverse was true with CD8+ subsets. There were more CD8+ PBL in the CC and CC.R1 lines and less in CB and CB.R1 lines. The ratio of CD4+ to CD8+ in CB chickens was 3.4 +/- 0.2 and in CC chickens 1.6 +/- 0.1.

The effects of atipamezole, an alpha 2-antagonist, on six medetomidine-sedated laboratory beagles were studied in a randomised complete block design. The dogs were sedated with medetomidine (20, 40 or 80 micrograms/kg intramuscularly) and five- and 10-fold larger doses of atipamezole were administered intramuscularly 30 minutes later. Atipamezole significantly increased the medetomidine-depressed heart rate, respiratory frequency and arterial Po2. The drug also transiently decreased the mean arterial blood pressure but subsequently the blood pressure of the treated group did not differ from that of a group of dogs treated with a placebo.

beta 2-microglobulin (beta 2m) attracts hemopoietic precursors from chicken bone marrow cells in vitro. The cell population responding to beta 2m increases during the second period of thymus colonization, which takes place at days 12-14 of incubation. The precursors from 13.5 day old embryos were isolated after migration towards beta 2m in vitro and shown to be able to colonize a 13 day old thymus in ovo, where they subsequently acquire thymocyte markers. In contrast these beta 2m responsive precursors did not colonize embryonic bursa, i.e. differentiate into B lymphocytes. During chicken embryogenesis, peaks of beta 2m transcripts and of free beta 2m synthesis can only be detected in the thymus. The peak of free beta 2m synthesis in the thymus and the increase of beta 2m responding bone marrow cells both occur concomitantly with the second wave of thymus colonization in chicken embryo, facts which suggest that beta 2m mediated chemotaxis is involved in the second wave.

To study the changes in immune system during aging and their effects on patient survival, immune functions of 18 very old (92-107 years) hospital patients were tested. After three years follow-up, the survival of patients and the correlation of immune capacity to lifetime were determined. In the very old patients, the frequency of T lymphocytes was significantly lower than in young adults. This decrease was attributed more to the CD8-positive than to CD4-positive cell subset, leading to generally high CD4/CD8 ratios. The mitogenic responses to phytohemagglutinin (PHA) and concanavalin A (Con A), but not pokeweed mitogen (PWM), were decreased significantly in cultures with isolated lymphocytes, while in whole blood cultures the responses to PHA and PWM but not to Con A were reduced. In very advanced age, there was a significant decrease in the number of B cells, and the ability to produce IgM in vitro was decreased. During the follow-up time, 15 out of the 18 patients died. However, their lifetime was not in correlation with any single immune parameter.

Atipamezole reversed the sedative effect of medetomidine in twelve laboratory beagles. The dogs were sedated with medetomidine doses of 20, 40 and 80 micrograms/kg body wt i.m. Atipamezole was injected (i.m.) 20 min later at dose rates two, four, six and ten times higher (in micrograms/kg) than the preceding medetomidine dose. Placebo treatment was included in the study. The deeply sedated dogs showed signs of arousal in 3-7 min and took their first steps 4-12 min after atipamezole injection. The dose-related reversal effect of atipamezole proved to be optimal with doses which were four, six or ten times higher than the preceding medetomidine dose. Drowsiness was found 0.5-1 h after atipamezole injection in 41% of the cases. No adverse effects nor cases of over-alertness or excitement were found.

An in vitro method for organ culture of embryonic bursa of Fabricius is presented. It is shown that bursa cells proliferate in in vitro culture as evidenced by [3H]-thymidine incorporation. We assessed the expression of B-cell alloantigen (Bu-la and Bu-lb), class I (B-F) and class II (B-L) antigens of chicken major histocompatibility complex (MHC), and surface immunoglobulin (sIg) on cultured bursa cells using specific monoclonal antibodies (mAb). Cells from 13-day and 14-day embryonic bursae incubated in organ culture for 1 to 2 weeks developed characteristic patterns of surface phenotype observed in adult chicken except for B-F antigen, whose expression was much lower than in vivo. These results indicate that the maturation of bursa cells in organ culture follows the in vivo development, except for the expression of MHC class I antigens. Furthermore, we demonstrate the in vitro repopulation of bursae from cyclophosphamide(cy)-treated chickens by cells from Bu-l antigen disparate normal bursae.

We have performed immunoperoxidase staining on cryostat tissue sections and immunofluorescence analysis on cell suspensions to identify cells expressing the alpha/beta T cell antigen receptor during ontogeny and adult life in chickens. We used the mouse monoclonal antibody, TCR2, which was previously shown to recognize the alpha/beta TCR in chickens. TCR2+ cells were observed in thymic cortex and medulla and in T-dependent areas of spleen, intestine, and cecal tonsils of young adult chickens. Some TCR2+ cells were found in the cortex of bursal follicles and in liver. The first TCR2+ cells appear in thymus on Day 13 of the embryonic life and it is only after hatching that TCR2+ cells begin to migrate to the periphery.

Treatment of neonatal chickens with cyclophosphamide depletes bursal lymphocytes while maintaining the bursal epithelium intact. The bursae of normal young chickens contain "bursal stem cells" which can reconstitute the lymphoid compartment in the bursa of the cyclophosphamide-treated recipient. Using bursal stem cells from IgM allotype-heterozygous donors we show that most bursal follicles in the reconstituted host are colonized by single stem cells which are committed to the expression of one or other IgM allotype. In addition we show that the reconstituting bursal stem cells express allelically excluded surface IgM at the time of transfer. Our results suggest that B lymphocyte numbers in hatched chickens are maintained by self-renewal of committed precursors rather than by de novo production from multipotential stem cells.

Germinal center formation was studied in the spleen of young chickens immunized in ovo and at the time of hatching. When immunization was performed on day 18 in ovo and on the day of hatching, the first germinal centers were observed at 4 days. This is markedly earlier than in unimmunized chickens, where the first germinal centers appear at the age of 10 days or later. Germinal center formation preceded significant antibody production. The possible role of germinal centers in the generation of immunological memory is discussed in the light of these and earlier observations.

Plasma cells of line 151(5) chickens have been shown to express elevated levels of endogenous retroviral envelope glycoprotein (VEG), measured relative to levels expressed by both immature B cells and resting peripheral B lymphocytes. In this study we analyzed the relationship between peripheral blood lymphocyte (PBL) maturation and the level of VEG expression. A culture system was developed that would support maturation of pokeweed mitogen-activated peripheral B lymphocytes. As analyzed by cytofluorometry, both Ig+ and Ig- lymphoblasts present in the pokeweed mitogen-stimulated cultures expressed detectable levels of VEG in contrast to bursacytes and PBL. Similarly, Ig- blasts, which were present in concanavalin A-stimulated cultures of PBL and presumed to represent activated T cells, were also positive for the expression of VEG. Immature T cells, i.e., thymocytes, although negative by immunofluorescence analysis, expressed VEG at levels that were detectable by radioimmunochemical techniques. These results indicate that T cells as well as B cells constitutively express VEG, and that mitogenic activation of the resting lymphocyte induces an increase in VEG expression.

We will briefly outline mammalian B cell ontogeny to provide a comparison with the avian model. The earliest defined stage of mammalian B cell development is the pre-B cell (itself derived from a multipotent stem cell) which expresses heavy chains within the cytoplasm and is a large, rapidly dividing cell. This cell drops out of division, reduces in size and, over about 24 hours, rearranges Ig light chain V region gene. As a consequence of light chain rearrangement and expression, intact IgM is expressed on the cell surface and the cell leaves the bone marrow as a small virgin B lymphocyte with the capacity to respond to antigen (Opstelten and Osmond 1983). This pathway occurs throughout the life of the animal and so there is constant production of B cells from the bone marrow which are recently derived from sIg- precursors, which have, in turn, recently rearranged their Ig V region genes. Thus there is a constant influx of new V region gene combinations into the peripheral mammalian B cell pool. Furthermore, the mammalian B cell repertoire is very large with estimated germ line diversity approaching 5 X 10(7) as a consequence of various germ-line V region recombinations (Honjo 1983). The pre-bursal stem cell is present in the periphery of the embryo from before day 8 to about day 16 - 17 of embryonation. Most colonisation of bursal follicles probably occurs prior to day 13. Individual bursal follicles are populated by a low number (2-3) of precursor cells. These rapidly become committed for the expression of particular V region genes, probably as a consequence of productive V region recombination. This recombination is restricted to the embryo and may not require the bursal microenvironment for its induction. The available germ-line repertoire of heavy and light chain V region in the chicken is very limited and little diversity can be generated from this initial recombination event (see Weill et al 1986). By day 12 of embryonation, sIg+ cells are present in the bursa and from this time sIg+ cells rapidly divide within the medulla of the bursal follicle. By about day 18 of embryonation, there are no pre-bursal cells in the periphery and sIg+ cells begin to seed from the bursa. It is from about this time that the cortex appears within the bursal follicle and it is tempting to suggest that cells migrate from the medulla to the cortex where further cell division occurs prior to export into the periphery.(ABSTRACT TRUNCATED AT 400 WORDS)

The disease induced by the avian myeloblastosis associated virus MAV-2-O in the susceptible chicken strains Brown Leghorn (BLH) and Prague CB (CB) was compared with that induced in the resistant G-B1 strain. Osteopetrosis, stunting and lymphoid organ atrophy were more severe in BLH than in CB chickens. G-B1 animals remained superficially normal until the end of the experiment. In contrast to the other two strains, the histopathological changes were very mild and there was no sign of immunosuppression. After 4 months, however, nephroblastomas could be detected in more than 50 per cent of the infected G-B1 chickens. Similar tumors were also found in CB birds kept for up to 5 months. Antibodies against MAV-2-O specific viral proteins were detected in plasma from infected G-B1 chickens but the titers were less than in plasma of convalescent birds. Virus could be demonstrated in peripheral blood until the end of the experiment (at 8 weeks). Therefore the resistance of the G-B1 strain is due neither to a restriction at the receptor level nor the result of a humoral immune reaction, but represents a new type of relative resistance at the cellular level. From (CC X G-B1)F1 and (CC X G-B1)F2 crosses the resistant phenotype is determined by a single genetic factor. This gene is not linked to the major histocompatibility complex. There is also a sex-dependent factor, possibly hormonal, involved in the resistant phenotype.

MHC class-I antigens on peripheral blood macrophages and bursa cells were analysed after adoptive bursa cell transfer to cyclophosphamide-treated immunodeficient chickens. We studied the expression of B-F locus-encoded antigens (B-F is homologous to mouse H-2K, D) on macrophages and B cells and found that macrophage B-F antigens are of host origin, whereas bursa cells express only donor-type B-F antigens. Both syngeneic and allogeneic bursa cells restored IgM antibody production to a T-cell-independent antigen, Brucella, but only syngeneic bursa cells could restore the IgG antibody response to a thymus-dependent antigen, sheep erythrocytes. The results indicate that B-cell maturation and macrophage-B-cell interaction are not MHC-restricted but that a restriction exists in T-cell-B-cell collaboration.

Maturation of bursal stem cells in an allogeneic environment was studied. The occurrence of surface IgM, surface IgG, and Ia-like antigens on B cells after differentiatoin within an allogeneic bursa was studied. Furthermore, the immediate capacity of B cells to form germinal centres after differentiation in an allogeneic bursa was studied by injecting them with histocompatible T cells into "test-tube" birds. The results indicate that the switch to surface-IgG-bearing cells occurs in an allogeneic bursa in a similar manner as in a syngeneic bursa. Studies of the occurrence of Ia-like antigens on bursa cells demonstrate that they retain their original Ia-like antigens, even during maturation in an allogeneic host. Third, the results demonstrate that after differentiation within an allogeneic environment bursa cells have acquired the ijmediate capacity to cooperate with histocompatible T cells as measured by germinal centre formation in "test-tube" birds.

Chicken peripheral blood leukocytes (PBL) proliferate in vitro in response to a wide range of pokeweed mitogen (PWM) concentrations. The PWM response was not influenced by the major histocompatibility complex (MHC) haplotype nor by the intrinsic responsiveness of the PBL to concanavalin A (Con A). The results indicate that PWM under our conditions stimulates B-lineage cells, although a T-cell subset is also clearly induced to division. The PBL from B-cell-depleted animals gave substantially lower responses than those from normal controls. Pokeweed mitogen stimulation of PBL was adherent cell dependent. Thus the low PWM response of adherent cell-depleted PBL was reconstituted by the addition of irradiated unseparated PBL. Furthermore, purified irradiated adherent cells containing greater than 90% macrophages were also able to reconstitute PWM responses. Finally, we have shown that PWM was able to induce large numbers of B cells to produce cytoplasmic immunoglobulin. However, only a minor proportion of such cells were induced to immunoglobulin secretion.

To determine the presence of precursor B cells in chick embryos surgically bursectomized at 72 h of incubation (E-Bx) we studied chick chimeras that were produced by establishing parabiotic connections between blood vessels of chorioallantoic membranes of normal and surgically bursectomized chick embryos. Using sex chromosomes and a B cell alloantigen (Bu-1a) as markers we showed that chick embryos bursectomized at 72 h of incubation contain B cell precursors capable of colonizing the bursa of Fabricius and developing into B lymphocytes. The repopulation capacity of 14-day-old embryonic spleen cells from E-Bx recipients was tested by transferring them into age-matched X-irradiated Bu-1-disparate embryos. The results show that B cell precursors are present in 14-day spleen of chick embryos bursectomized at 72 h of incubation. These precursors carry the Bu-1 B cell alloantigen, suggesting that commitment to the B cell lineage can take place in the absence of bursa.

For transplantation of semiallogeneic bursal stem cells into cyclophosphamide-treated 3-day old chicks, two lines of chickens homozygous at the major histocompatibility locus and their F(1) hybrids were used in reciprocal combinations. The semiallogeneic transplantations resulted in a complete restoration of antibody formation to sheep red blood cells (SRBC) and Brucella, of microscopic morphology of bursa fabricii, and of germinal center formation in the spleen. In contrast, allogeneic bursal stem cells were not effective in restoring secondary response to SRBC and germinal center formation, while they were able to reconstitute anti-Brucella responses and bursal morphology. These findings indicate an effective cooperation of donor and host cells leading to a complete restoration of the bursa-dependent lymphoid system, when the donor and recipient share at least one haplotype determining the major histocompatibility antigen complex.

Supernatants from concanavalin A (Con A)-stimulated chicken spleen cells were used to generate a long-term cultured cell line from antigen-primed chicken peripheral blood leukocytes. This line has been kept in continuous proliferation in vitro for more than 25 weeks. Morphologically these cells were lymphoblastoid and expressed class I and class II antigens of the major histocompatibility complex as well as T-cell (but not B-cell or macrophage) antigens. In addition they contained no peroxidase or non-specific esterase activity, neither were they phagocytic. Proliferation of the line was totally dependent on exogenous T-cell growth factor (TCGF) activity provided by the Con-A-stimulated spleen cell supernatant, comparable with the proliferation of Con-A-induced T-cell blasts. TCGF activity from the supernatant was absorbed both by the long-term cultured T cells and by Con A blasts, demonstrating the presence of receptors for the same TCGF species on the two populations. We have used the long-term cultured cell line to characterize chicken TCGF further. The molecular weight of the biologically active fractions found by gel filtration on Sephadex G-100 was approximately 13,000 and isoelectric focusing showed chicken TCGF to have a pI of pH 5.9. We propose that the TCGF described here is the chicken analogue to the mammalian interleukin 2.

The first event in the initiation of an immune response is the capture and presentation of antigen to T cells. Such presentation involves two distinct steps: (1) display of the antigen, which requires uptake, processing and re-expression of the antigen in association with MHC molecules on the presenting cell surface; and (2) triggering, in which the presenting cell provides signals leading to the activation of the responding T cell. Two sorts of cells can capture antigens, the 'professional' antigen-presenting cells (APCs) such as dendritic cells and macrophages, and the B cells. Both types of cells can display antigens and the APCs are known to be able to trigger resting T cells. But despite in vitro evidence that certain B-cell types can reactivate previously-activated T cells, it is not yet clear whether a B cell can initiate an immune response by providing the signals necessary to activate a resting T cell. We reasoned that resting B cells should not have this capacity because of the problems this would present with tolerance to self idiotypes. By exploiting the unique properties of the avian haematopoietic system, we have examined the presenting capacity of B cells in vivo and found that resting B cells are indeed unable to activate resting T cells.

The detailed study of the genetic control of T-B cell interactions in the chicken has been hampered by the lack of defined major histocompatibility complex (MHC) recombinant chicken lines. In the present study we have used some recently described MHC recombinant chicken lines separating regions encoding antigens that are homologous to class I and class II antigens of mammals in adoptive bursa cell transfer experiments, in which bursa cells from newly hatched chicks were transplanted into cyclophosphamide (Cy)-treated chicks. Subsequent immunizations of the recipients with a thymus-dependent antigen (SRBC) and a thymus-independent antigen (Brucella abortus) showed that the generation of germinal centers in the spleen and the production of antibodies to SRBC required identity between donor and recipient class II antigens (B-L antigens), whereas response to Brucella antigen did not require identity at any of the known MHC loci of the chicken. The results thus reveal that also in the chicken class II (B-L) region genes encode cell-surface glycoproteins that serve as restriction elements in T-B cell cooperation.

A total of 10 laboratory beagles was used to determine the cardiovascular and respiratory effects of medetomidine. The effects of atropine sulphate and glycopyrrolate on heart rate were also observed. Xylazine was included as a positive control. Medetomidine induced initial hypertension followed by a longer lasting hypotensive period. Evident bradycardia with second degree atrioventricular blocks and decrease in respiratory frequency was observed. Atropine sulphate and glycopyrrolate transiently abolished the bradycardic effect of medetomidine. Xylazine exhibited a similar cardiovascular and respiratory pattern to medetomidine.

In chickens rendered neonatally tolerant to BSA the germinal center formation was significantly decreased after stimulation with BSA at the age of 3 weeks. At the breakdown of tolerance after the age of 6 weeks the IgG antibody formations recovered before the IgM production. Stimulation of tolerant birds with unrelated antigens resulted in slightly decreased antibody response but the germinal center formation was on the same level as in normal controls.

The avian bursa of Fabricius contains about 1 X 10(4) discrete follicles, each of which is colonized by a small number of lymphoid progenitor cells during embryonic life. We have previously shown (J.R.L. Pink et al., Eur. J. Immunol. 1985. 15:617) that all, or almost all B cell progenitors in the bursae of 4-day-old chicks express cell surface IgM. In this report, we have analyzed the distribution of cell surface (s)IgM-1 allotypes within individual follicles of (M-1a/M-1b) allotype heterozygous birds. Although the majority of follicles contained a mixture of sIgM-1a+ and sIgM-1b+ cells, a significant proportion of isolated follicles contained exclusively sIgM-1a+ or sIgM-1b+ cells. Statistical analysis of the frequency of such "M-1a" and "M-1b" follicles demonstrated that the sIg+ B cells in the bursae of 4-8-week-old birds are derived from 2-4 allotypically committed precursor cells per follicle. Since we have previously shown that each bursal follicle is colonized by 2-5 pre-bursal stem cells, these cells must be committed to the eventual expression of one or other allotypic haplotype before they have undergone extensive proliferation within the bursa. In addition, we show that almost all B progenitor cells from the bursae of chicks which had been allotype suppressed as embryos were committed to synthesis of the nonsuppressed allotype, showing that this commitment was essentially complete at the time of suppression (i.e. before 19 days of incubation). Finally the bone marrow of 16-day embryos was used to reconstitute the bursal lymphocytes of cyclophosphamide-treated host embryos. Reconstitution was inhibited by anti-Ig antiserum indicating that most 16-day embryonic BM-derived bursal cell precursors also express sIgM. These results raise the possibility that expression of sIgM may be controlled by a "biological clock" rather than by any inductive capacity of the bursal microenvironment. Furthermore, these results provide further evidence that in normal birds a self-renewing sIg+ B cell population in the hatched chicken is the sole source of B cells in the adult.

Transplantation of bursa cells into surgically thymectomized (STx), cyclophosphamide(CY)-treated and X-irradiated (X) newly hatched chicks resulted in a morphological and functional reconstitution of the bursa-dependent immune system only when the transplanted bursa cells were supplemented with thymus cells. Without supplementation with thymus cells, morphological restoration of the bursa, antibody formation to sheep red blood cells and Brucella abortus, and germinal center formation remained deeply deficient. Bursa cells alone induced a full reconstitution of the bursa-dependent immunity in non-thymectomized. CY-treated and X-irradiated cell recipients. The incomplete restoration of STx-CY-X chickens after transplantation of bursa cells was not due only to lack of T helper cells, as indicated by the incomplete restoration of the bursal morphology and of antibody formation against Brucella abortus, a thymus-independent antigen. It is concluded that T cells are necessary for the B cell differentiation in the bursa of Fabricius. The mechanism behind this phenomenon remains unclear but our results indicate that a complete T cell function is not a prerequisite for this, since STx-CY-X birds reconstituted with bursa and thymus cells together did not have their mitogen (PHA, Con A) responses normalized.