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Emission of atmospheric ammonia (NH 3 ) is an environmental challenge because of its harmful effects on humans and animals including birds. Among all organisms, NH 3 is highly sensitive to birds. Autophagy plays a critical role in Bursa of fabricius (BF)-mediated immune responses against various hazardous substances. Therefore, we designed our work to demonstrate whether NH 3 can induce autophagy in broiler chicken BF. In this study, the downregulated levels of mammalian target of rapamycin and light chain-3 (LC-Ⅰ), as well as the upregulated levels of phosphate and tensin homology (PTEN), protein kinase B (AKT), autophagy related-5, light chain-3 (LC3-Ⅱ), Becline-1, and Dynein, were found. Our results of transmission electron microscopy displayed signs of autophagosomes/autophagic lysosomes, and immunofluorescence assay displayed that NH 3 exposure reduced the relative amount of CD8 + B-lymphocyte in chicken BF. Exposure of NH 3 led to energy metabolism disturbance by decreasing mRNA levels of glucose metabolism factors aconitase-2, hexokinase-1, hexokinase-2, lactate dehydrogenase-A, lactate dehydrogenase-B, pyruvate kinase, phosphofructokinase and succinate dehydrogenase complex unit-B, and adenosine triphosphates (ATPase) activities (Na + /K + ATPase, Ca 2+ ATPase, Mg 2+ ATPase, and Ca/Mg 2+ ATPase). Moreover, phosphate and tensin homology was found as target gene of microRNA-99a-3p which confirmed that high concentration of NH 3 caused autophagy in chicken BF. In summary, these findings suggested that ammonia induced autophagy via miR-99a-3p, the reduction of ATPase activity, and the alteration of autophagy-related factors, and energy metabolism mediation in BF. Our findings provide information to assess the harmful effects of NH 3 on chicken and clues for human health pathophysiology., (Copyright © 2020. Published by Elsevier Inc.)

The bursa of Fabricius is a primary lymphoid organ for B-cell development and gut-associated lymphoid tissue. After hatching, IgG-containing cells with reticular branches are found in the medulla of bursal follicles on frozen sections stained with anti-Cγ antibody, and IgM(+)IgG(+) B cells are detected in single-cell suspension of the bursa. IgG-containing cells in the medulla do not biosynthesize IgG and are composed of aggregated maternal IgG and environmental antigens. Then, those cells in the medulla are acknowledged as follicular dendritic cells retaining immune complexes. Also, it is presumed that IgM(+)IgG(+) B cells are generated by the attachment of immune complexes to IgM(+) bursal B cells because IgM(+)IgG(+) B cells are induced by antigen-dependent attachment of maternal IgG. Therefore, it is reasonable to suppose that immune complexes exert further B-cell differentiation in the medulla., (© 2014 Elsevier Inc. All rights reserved.)

We evaluated the immunomodulatory effects of a proanthocyanidin-rich extract (PAE) from Pinus radiata bark in Korean native chickens. In experiment 1, proliferation of peripheral blood mononuclear cells was significantly enhanced in chickens administered 1.25, 2.5, 5, and 10 mg/kg of PAE for 2 wk once daily by oral gavage compared with no administration of PAE in vitro. Proliferation of splenocyte and thymocyte cells was also significantly enhanced in chickens administered 1.25, 2.5, 5, and 10 mg/kg of PAE for 5 wk. In experiment 2, proliferation of peripheral blood mononuclear cells, splenocytes, and thymocytes in chickens administered PAE for 6 wk was continuously higher than that in other groups. Additionally, increasing cell populations for which CD4(+)CD8(-)(Th cells) and Bu-1(+) (B cells) protein was expressed on the cell surface were significantly different after 4 wk of PAE administration in chickens. Thus, the immunomodulatory effects of a PAE from P. radiata bark differed by dosing period in chickens.

Antibiotics are commonly used to prevent and treat poultry microbial infections, but certain antibiotic families depress humoral immunity, such as antibody production. Poultry humoral immunity depends on the normal functioning of the bursa of Fabricius and the B lymphocytes that mature in that gland. In this study, recommended therapeutic doses of enrofloxacin, florfenicol, or ceftiofur were administered to 2-d-old chicks. On d 7 post-hatch, bursae were sampled for histological, immunohistochemical, and flow cytometric determination of Bu-1-positive (Bu-1+) cell number, percentage, and distribution. The bursa of Fabricius from all treatment and control groups had normal morphology. The administration of antibiotics significantly decreased the number of Bu-1+ cells in the bursal medulla, with a simultaneous increase of these cells in the cortex. Flow cytometry revealed a significant decrease in the percentage of bursal Bu-1+ cells from all of the studied antibiotics: enrofloxacin (93.91 ± 3.27), florfenicol (87.84 ± 7.14), and ceftiofur (89.16 ± 5.68) compared with that of the control (96.48 ± 2.60). The combination of reduced percentages of Bu-1+ cells and a decrease in these cells in the medullary region suggests lower B cell maturation.

The immunoregulatory actions of conjugated linoleic acid (CLA) of relevance to viral disease pathogenesis and immune responses were investigated. To test the hypothesis that CLA ameliorates immunosuppression, we developed a viral challenge model by infecting chickens with infectious bursal disease virus (IBDV). After 14 d of dietary supplementation with either soybean oil or CLA, half of the chickens in each group were challenged with IBDV. We examined the effect of CLA on the development of lesions (i.e., lymphoid depletion and necrosis) and observed the immune responses against IBDV. The IBDV infection depleted lymphocytes in the medullary area and significantly stimulated interferon (IFN)-γ and IL-6 mRNA relative expression of bursa (P < 0.05) compared with the uninfected bursa. Compared with the CLA diet, lymphocytes depletion was more accentuated in chickens fed the control diet, whereas IFN-γ and IL-6 mRNA relative expression were upregulated (P < 0.05). Additionally, histopathological examination of the bursa revealed that the pathological changes tended to be more severe in infected chickens fed the control diet, which also significantly decreased (P < 0.05) on lymphocyte proliferation. Significant interactions were found between infection and diets for lymphocyte proliferation, antibody titers, and IFN-γ mRNA relative expression (P < 0.05). The results of this study indicate that dietary CLA enhanced immune function in chickens, particularly those of the IBDV-immunosuppressive status. Furthermore, at the molecular level, the immunoregulatory functions of CLA on chickens are attributable mainly to the antiinflammatory properties of CLA and are mediated, at least in part, through suppressing IBDV-specific proinflammatory cytokines mRNA relative expression.

Proanthocyanidins are naturally occurring compounds that are widely found in fruits, vegetables, nuts, seeds, flowers, and bark. We evaluated the immunomodulatory effects of proanthocyanidin-rich extract (PAE) from Pinus radiata bark in specific-pathogen-free White Leghorn chickens. Proliferation of peripheral blood mononuclear cells was significantly enhanced in chickens treated for 2 wk with 20 mg/kg of PAE. Proliferation of splenocytes and bursal cells was significantly enhanced in chickens treated for 5 wk with 5, 10, and 20 mg/kg of PAE. Thymocyte proliferation was significantly enhanced in chickens treated for 5 wk with 5 and 10 mg/kg of PAE. These effects were markedly enhanced by the presence of lipopolysaccharide, which acted on B cells responsible for humoral immunity, and concanavalin A, which acted directly on T cells involved in cell-mediated immunity. The PAE significantly promoted the expression of T helper 1 cytokine (interferon-γ) and decreased the expression of T helper 2 cytokine (IL-6). Thus, P. radiata PAE has immunomodulatory effects in specific-pathogen-free White Leghorn chickens.

Chemokine receptors guide immune cells to specific organs during health and disease. The mRNA content of the chemokine receptors CCR2, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CXCR4, CXCR5, and CX3CR1 in CD4(+) cells (T-helper cells) isolated from blood, bursa, cecal tonsil, spleen, and thymus and in CD8(+) cells (T-cytotoxic cells) isolated from blood, cecal tonsil, spleen, and thymus were investigated. The CD4(+) cells isolated from thymus had the highest amount of CCR7 and CCR8 mRNA. The CD4(+) cells isolated from bursa, cecal tonsil, and thymus had the highest amount of CCR5 mRNA. The CD4(+) cells isolated from cecal tonsils had the highest amount of CCR9 mRNA. The CD4(+) cells isolated from bursa and thymus had the highest amount of CXCR5 mRNA. The CD8(+) cells isolated from cecal tonsil had the highest mRNA amount of all receptors studied except CCR9 and CX3CR1. The CD4(+) cells treated with concanavalin A had increased CCR2, CCR4, CCR7, CCR8, and CXCR5 mRNA amounts at 24 h of stimulation. The CD8(+) cells treated with concanavalin A had increased CCR4 mRNA at 72 h, increased CCR6 mRNA at 24 h, and decreased CCR8 and CXCR4 mRNA at 24 h of stimulation.

Bursa of Fabricius is a primary lymphoid organ in the birds and its microenvironment is responsible for the B cell maturation. The epithelial anlage develops through epithelial-mesenchymal interaction into which -as a third party - hemopoietic cells immigrate. Chemical bursectomy can be made by dipping or injecting eggs by testosterone solution. Both methods of treatments compromise the development of the whole embryo. Heparin beads of 70-150 microm in diameter were soaked in 5% testosterone solution and implanted into the tail bud of 4 day old chicken embryos before the bursal anlage began to develop at embryonic day 5. The locally applied testosterone inhibited the differentiation of the bursal secretory dendritic cell (BSDC) precursors, which were the inducer of the follicular epithelial bud formation. Testosterone treatment did not blocked the entering of B cells and other hemopoietic cells into the bursal mesenchyme, but in the absence of BSDC precursor cells the dendroepithelial microenvironment is not established, subsequently B cell precursors are lodged only in the bursal mesenchyme. This method is suitable for locally introducing other regulatory molecules, involved in bursal development.

Early B lymphopoiesis in mammals is induced within the bone marrow (BM) microenvironment, but which cells constitute this niche is not known. Previous studies had shown that osteoblasts (OBs) support hematopoietic stem cell (HSC) proliferation and myeloid differentiation. We now find that purified primary murine OBs also support the differentiation of primitive hematopoietic stem cells through lymphoid commitment and subsequent differentiation to all stages of B-cell precursors and mature B cells. Lin(-)Sca-1(+)Rag-2(-) BM cell differentiation to B cells requires their attachment to OBs in vitro, and this developmental process is mediated via VCAM-1, SDF-1, and IL-7 signaling induced by parathyroid hormone (PTH). Addition of cytokines produced by nonosteoblastic stromal cells (c-Kit ligand, IL-6, and IL-3) shifted the cultures toward myelopoiesis. Confirming the role of OBs in B lymphopoiesis, we found that selective elimination of osteoblasts in Col2.3Delta-TK transgenic mice severely depleted pre-pro-B and pro-B cells from BM, preceding any decline in HSCs. Taken together, these results demonstrate that osteoblasts are both necessary and sufficient for murine B-cell commitment and maturation, and thereby constitute the cellular homolog of the avian bursa of Fabricius.

Adequate energy status in lymphocytes is vital for their development. The ability of developing chicken lymphocytes to acquire and metabolize energy substrates was determined during embryonic days (e) and neonatal days (d) of life when primary-energy substrate metabolism is altered at the whole-animal level. In 3 experiments, bursacytes and thymocytes were isolated on e17, e20, d1, d3, d7, or d14 to analyze markers associated with glucose, glutamine, and lipid metabolism. Bursacyte glucose transporter-3 (Glut-3) mRNA abundance increased from d1 to d14 and hexokinase-1 (HK-1) mRNA abundance was maximum on e20 (P<0.05). Thymocyte Glut-1, Glut-3, and HK-1 mRNA abundance increased from e17 to d14 (P<0.05). HK enzyme activity increased from e20 to d3 in bursacytes and d3 to d7 in thymocytes (P<0.05). Glucose uptake by bursacytes and thymocytes was greater on d14 compared to d1 and d7 (P<0.05). Bursacyte and thymocyte sodium coupled neutral amino acid transporter-2 and glutaminase (GA) mRNA abundance increased from e20 to d7 (P<0.05). GA enzyme activity increased from e20 to d7 in bursacytes (P<0.05) and did not change in thymocytes. Carnitine palmitoyl transferase enzyme activity did not change over time in either cell type. These studies suggest that developing B and T lymphocytes adapt their metabolism during the first 2 wk after hatch. Developing lymphocytes increase glucose metabolism with no change in fatty acid metabolism and bursacytes, but not thymocytes, increase glutamine metabolism. Understanding the factors that regulate lymphocyte development in neonatal chicks may help promote their adaptive immune responses to pathogens in early life.

During the last decades of the 20th century, studies on the vertebrate hematopoietic and immune systems have largely been performed, on mammalian models. The mouse has been the preferred material for several cogent reasons: (i) numerous well defined genetic strains are available; (ii) this species has been and still is instrumental in the study of gene activity through transgenesis; and (iii) in vitro culture techniques and in vivo assays for blood cells together with a wide array of antibodies and nucleic acid probes have been developed to investigate the cellular interactions occurring during hematopoiesis and immune reactivity. However, important and fundamental notions have emerged from using another higher vertebrate model, the avian embryo. The distinction among small lymphocytes of two populations, the T and B lymphocytes, endowed with different roles in adaptive immunity and dependant on different environments for their specification, has relied on experiments carried out in birds. The avian model has been critical for the analysis of the origin and traffic of hematopoietic precursor cells. It allowed the demonstration that both hematopoietic and angioblastic lineages arise from a common precursor, a cell whose existence had been proposed but never undoubtedly proven, the hemangioblast. Finally a form of thymus-dependant 'dominant' tolerance was demonstrated on the basis of experiments in the avian embryo, which initiated a large current of studies on 'regulatory T-cells'. Work in this model during the last decades has relied strongly on the construction of chimeras between quail and chick embryos that allowed a refined analysis of cell behaviour during embryogenesis. The novel perception of developmental neuropoiesis and immunopoiesis that followed proved to be largely applicable to lower and higher vertebrates, notably mammals.

Apoptosis, or programmed cell death, is believed to be the mechanism for depletion of lymphocytes recognizing self-antigens following clonal expansion in the bursa of Fabricius. Although bursal apoptosis has previously been shown to increase following in vivo exposure to glucocorticoids, the microanatomical site of induced or normal apoptosis has not been unequivocally established. Presently, we adapted the existing terminal deoxynucleotidal transferase-mediated dUTP nick-end labeling (TUNEL) assay for use with neonatal bursae. Similar to previous reports, TUNEL revealed that normal apoptosis is preferentially, but not exclusively, ongoing in bursal follicular cortical cells. Administration of a single dose of a synthetic glucocorticoid (dexamethasone) or androgen (19-nortestosterone) did not significantly (P < 0.05) alter follicular lymphocyte numbers or apoptosis per unit of area at the time points evaluated post-administration (6 or 24 h). However, administration of 19-Nortestosterone increased the interfollicular epithelial thickness, a change usually associated with edema, within 6 h following treatment. Additionally, administration of the androgen 19-nortestosterone significantly decreased the number of proliferating cells as detected using mouse anti-proliferating cell nuclear antigen (PCNA) as a primary immunohistochemical antibody. In normal (control) bursal sections, occasional follicles consisting of predominantly apoptotic cells were observed (0.26% of follicles). Such follicles were consistently one-tenth the area of normal follicles. This incidental finding may suggest occasional occurrence of a common signal for deletion, such as a common integral or clonal mistake, viral infection, or an aberrant paracrine signal.

Density gradient-separated embryonic spleen and bone marrow cells were enriched for chB6(+) cells with positive and negative selection procedures and magnetic cell sorting. The majority of chB6(+) cells, consisting of small, dense, strongly chB6(+) cells, were prone to apoptosis, which was further accentuated after exposure to monoclonal antibodies directed against chB6 alloantigen, but was largely inhibited by PDBU, leading to maintenance and frequently numerical increases of chB6(+) cells after an initial decline. sIgM(+) cells within that population followed a very similar pattern, suggesting a PDBU-induced upregulation of sIgM expression on a proportion of chB6(+) cells. The protective effect of PDBU on anti-chB6-exposed cells was confirmed with bursal lymphocytes and shown to be entirely PDBU concentration dependent. It was calculated that each 14-day embryonic spleen contained a minimum of 250,000 chB6(+) and 3000-4000 sIgM(+) cells, respectively. Endogenous apoptosis appeared to be increased with embryonic age, reaching a peak with bursal cells in the posthatching period. A second population of larger, less dense, and weak chB6(+) cells, often with vacuoles in a more abundant cytoplasm, differed functionally, expanding numerically in unstimulated cultures and being inhibited by PDBU. No sIgM(+) cells developed within this population. It is proposed that this chB6(+) fraction may represent progenitors of a previously suggested chB6(+) subset of macrophages, in contrast to the dense chB6(+), small cells, viewed as B cell progenitors. chB6(-) cells, consisting predominantly of granulocytes, proliferated vigorously in unstimulated cultures, but were consistently inhibited by PDBU. Coculture of age-matched embryonic bursal stroma with positively and negatively enriched chB6(+) cells revealed enhanced protection from apoptosis for chB6(+) cells and a PDBU-induced upregulation of sIgM expressing cells. Bursal stroma also had a pronounced positive effect on the proliferation of chB6(-) cells., (Copyright 2000 Academic Press.)

The bursa of Fabricius represents the primary immune organ where immature B cells undergo maturational changes in avian species. Isolation of bursal lymphocytes for analysis in cell culture results in the rapid endogenous activation of apoptosis. After 2 h of incubation, over 45% of the lymphocytes were shown to be undergoing apoptosis and by 6 h 80% were undergoing apoptosis as demonstrated by a terminal deoxynucleotidyltransferase-mediated dUTP-fluorescein isothiocynate nick end-labeling flow-cytometric analysis. These results were corroborated by a propidium iodide-staining flow-cytometric assay and by an agarose gel electrophoresis DNA fragmentation assay that demonstrated internucleosomal DNA cleavage of genomic DNA in apoptotic bursal lymphocytes. Endogenous activation of apoptosis in bursal lymphocytes could be inhibited in a dose-dependent fashion with the phorbol ester, phorbol 12-myristate 13-acetate, but not the phorbol ester antagonist 4 alpha-phorbol 12,13-didecanoate. In addition, apoptosis could be inhibited in a dose-dependent fashion with inhibitors of protein translation, cycloheximide, and puromycin, as well as the transcriptional inhibitor actinomycin D. These results suggest that endogenous activation of bursal lymphocyte apoptosis may be mediated by the protein kinase C signal transduction pathway and activation of this process appears to be dependent upon de novo protein biosynthesis.

The objective of this study was to examine possible interactions between drinking water contaminants and suboptimal nutritional status for performance and immune function in male broiler chickens. Experimental drinking water contained a mixture of arsenic, benzene, cadmium, lead, and trichloroethylene (TCE) at low concentrations (0.80, 1.3, 5.0, 6.7, and 0.65 ppm) and high concentrations (8.6, 13, 50, 67, and 6.5 ppm). These chemicals were selected because they are among the most common contaminants found in ground water near hazardous waste sites. The experimental diets included feed containing 50% added vitamins and minerals (V&M) and feed without added V&M. Increasing levels of drinking water contaminants and decreasing levels of V&M in diet resulted in significantly (P < or = 0.05) decreased water and feed intake, decreased weight gain, and suppression of natural, humoral, and cell-mediated immune response. In a paired-water study, feed consumption, body weight, and immune function were decreased in chickens provided low and high concentrations of the chemical mixture in drinking water compared with chickens given control drinking water equal to the volumes consumed by the chickens given the low and high concentration of mixture, respectively. A deficiency of dietary V&M caused increased sensitivity to adverse effects of drinking water contaminants.

The tetrapeptide AcSer-Asp-Lys-Pro (AcSDKP) is a physiological negative regulator of hematopoiesis in mammals. It acts by blocking the cell cycle entry of quiescent stem cells and progenitors. In the present study we report that AcSDKP blocks the proliferation of human as well as chicken lymphocytes. It inhibits by 25 to 40% the polyclonal mitogen- (phytohemagglutinin (PHA), pokeweed mitogen (PWM), or concanavalin A) or mixed lymphocyte reaction-induced proliferation of chicken lymphocytes. A comparable degree of inhibition was observed in human whole blood cultures stimulated by "T" (PHA) or "T and B" (PWM) mitogens. Our results obtained on two phylogenetically distant species show that AcSDKP reduces the lymphocyte proliferation probably by blocking or retarding entry into the cell cycle as previously demonstrated for hematopoietic progenitors and hepatocytes. Therefore, this endogenous, non-species-specific tetrapeptide may be involved in the regulation of immune response.

The bursa of Fabricius is an ideal model system to answer the plethora of questions related to the origin of B cells and microenvironmental issues leading to the education of the stem cell. Prior to the 1960s, lymphocytes were thought to be derived from epithelial or mesenchymal cells. Later work demonstrated the bloodborne nature of the stem cell contributing to B cells. Stem cells entered the bursa of quail and chicken between 7 and 11 and 7.5 and 14 d of embryogenesis, respectively. Interspecific chimeric studies, quail and chick, emphasized the intraembryonic origin and sites of the stem cell. The bloodborne and stromal cells that contribute to the microenvironment of the bursa orchestrate the events leading to B cell differentiation. The separation of the endodermal and mesodermal components of the bursa revealed a singular role for the endoderm in the genesis of the bursa but did not exclude a role for the mesoderm. A dark mesenchymal cell was shown to play a role in bud formation. This cell gave rise to the bursal secretory dendritic cell (BSDC), unique in its membrane association with IgG. A receptor-paracrine thesis has been proposed to explain the interaction between in-frame B cells and Ig-positive BSDC in the expansion of in-frame B cells and the subsequent development of the B cell repertoire. Cell adhesion molecules have been integrated into this thesis.

The bursa of Fabricius is the site of primary B cell generation in the chicken. Analysis of the rate of bursal cell emigration demonstrated that about 1% of the blood B cell pool was replaced per hour by bursal emigrants. Surgical bursectomy distinguished three populations of blood B cells. About 60% of blood B cells in 3-wk-old chickens were short lived, with a lifespan of 2 to 3 d, and included most bursal emigrants. These cells migrate directly from the bursal follicular cortex to the periphery, express the LT2 antigen, and are proposed to represent a diversified repertoire of B cell specificities that have emigrated from the bursa in the absence of interaction with environmentally derived antigens in the follicular medulla. About 35% of blood B cells were much longer lived cells, having a lifespan exceeding 2 wk. These cells represent about 10% of bursal emigrants, which do not express the LT2 antigen and do not divide in the peripheral blood following emigration from the bursa. This population may represent cells that have undergone positive selection in the bursa by interacting with environmentally derived antigens and should, therefore, contain a restricted repertoire of B cell specificities. The third population, about 5% of blood B cells, were short-lived cells that represent the progeny of postbursal B cell production. The frequency of these cells progressively increases with time, taking over from the short-lived bursal emigrants as the bursa involutes, likely as a diversified repertoire of B cell specificities.

A central feature of the vertebrate humoral immune system is that an organism must have a vast repertoire of antibodies to protect it against foreign pathogens. Chickens create a diverse immunological repertoire by intrachromosomal gene conversion of the single variable gene segments of the Ig heavy and light chain genes. This diversification process has been shown to require the bursa of Fabricius. Immature cells commit to the B cell lineage by rearranging their Ig genes prior to migration to the bursa. Recent work has suggested that the ability of a developing B cell to migrate to the bursa may depend on the expression of the carbohydrate structure sialyl Lewis x. Developing B cells in the spleen with the ability to migrate to the bursa have been shown to express sialyl Lewis x. Cells expressing sialyl Lewis x begin appearing in the bursa anlage between embryonic Days 10 and 12. These sialyl Lewis x-positive cells appear to form the nascent bursal follicles and are induced to proliferate. Coincident with the time that B cells initiate the gene conversion process, cells cease expressing sialyl Lewis x and begin expressing the related surface epitope Lewis x. As cells mature further, they undergo another phenotypic change and switch from expressing high levels of Lewis x to become Lewis x-low. At the same time that Lewis x-low cells accumulate in the bursa, cells with this phenotype begin to appear in the spleen. These phenotypic markers may be useful in identifying chicken B cells at different developmental stages.

The immune system's repertoire is generated in two stages: Stage I results in a small size high copy number repertoire that is diversified by "mutation" to result in a large size low copy number repertoire referred to as Stage II. The Stage I or high copy number repertoire is derived from information stored directly in the genome by two mechanisms. (a) The copy-cassette mechanism: the Ig-locus has one rearrangeable V gene segment which acts as recipient for controlled gene conversion in cis from a set of donor V gene segments that results in a family of subunits, L and H. This is illustrated by the avian systems. (b) The cassette-exchange mechanism: the Ig-locus has many rearrangeable V gene segments which are fused into transcription units, the products of which are a family of L and H subunits identical in function to those resulting from the copy-cassette mechanism. This is illustrated by the murine or human systems. It is possible for a species to use both mechanisms, copy-cassette at one Ig locus and cassette-exchange at the other Ig locus. This seems to obtain in the rabbit system. Further, it is possible to encode the high copy number repertoire directly in the genome as tandemly repeated rearranged transcription units as one sees in shark (a genomic analogue of the cassette-exchange mechanism). We have discussed here and elsewhere (Cohn and Langman, 1990) the consequences of these mechanisms for haplotype exclusion and functional responsiveness to antigen. The Stage I or high copy number repertoire generated by any of the above mechanisms is now a substrate for "mutation" which generates the low copy number or Stage II repertoire. These three species are compared in table V. The high copy number repertoire is small but the response to any antigen that it recognizes is rapid. The low copy number repertoire is large but responsiveness to any antigen it recognizes is slow. Cooperativity between the two repertoires optimizes the overall responsiveness with respect to rapidity of response and range of responsiveness. The use of a copy-cassette mechanism requires that the phi B cell undergoing gene conversion have a single rearranged L- and H-chain haplotype (L+/oH+/o). The reason is that conversion can correct an aberrantly rearranged transcription unit and generate an unacceptable level of doubles. In order to have one chromosome functionally rearranged and the homologue in the germline configuration, a selection mechanism is required.(ABSTRACT TRUNCATED AT 400 WORDS)

In 1978, Olah and Glick identified in the medulla of the bursa of Fabricius a dendritic cell, the bursal secretory dendritic-like cell (BSDC). The morphology of BSDC, the functional relationship of BSDC to bursal follicle development and the spleen, and the identification in the embryo of the BSDC precursor was reported. The precursor BSDC appeared in the bursa before B cells. A monoclonal antibody to vimentin, an intermediate filament, will identify BSDC and not B cells in posthatch chickens. The vimentin-positive cells, but not B cells, in hatched chicks express complete IgG chain. Taken together, the present data may contribute to the events occurring during the bursal-dependent stages of B cell development. Immunoglobulin gene rearrangement, within the bursa or at extra-bursal sites, and clonal expansion at extra-bursal sites are antigen-independent and dependent, respectively. The bursal-dependent stage includes the expansion of the Ig rearranged cells and gene conversion, which contributes to antibody diversity. The IgG and granular products produced by BSDC may act as ligand and signals for B cell expansion and gene conversion.

Recent studies have demonstrated that the action of nerve growth factor (NGF) is not restricted to neuronal cells but also affects cells of the immune system. In a previous work on the effect of NGF on the chick embryo bursa of Fabricius both in vivo and in vitro, we observed that NGF prolongs bursal cell survival in vitro. In the present study we report that the increase of viable cells in NGF-treated cultures is not due to a proliferative effect of NGF on bursal cells but to a reduction of cell mortality. The morphological analysis revealed that bursal cells in cultures die by apoptosis, which was also shown by the typical pattern of DNA fragmentation, a hallmark of this cell death process. It is concluded that NGF, with an action similar to that described in sympathetic neurons and PC12, could retard bursal cell death by influencing apoptosis.

The purpose of the study was to determine what type of non-lymphoid cells develop in chicken embryos during ontogeny, and whether these cells are functional. To detect these cells, we used monoclonal antibodies specific to two groups of non-lymphoid cells: CVI-ChNL-68.1, specific for mononuclear phagocytes, and CVI-ChNL-68.2, specific for a subpopulation of reticulum cells in spleen, liver and bursa. Monoclonal antibodies HIS-C7, HIS-C1, and HIS-C12, which are specific to leukocytes, B lymphocytes, and IgM respectively, were used to correlate the ontogeny of non-lymphoid cells and lymphoid cells. Mononuclear phagocytes and reticulum cells were detected in the liver, spleen, yolk sac, bursa, gut, and thymus at about the same time as leukocytes, but earlier than B lymphocytes. To determine whether mononuclear phagocytes and reticulum cells in spleen and liver absorb antigen, we injected embryos intravenously with colloidal carbon and the antigen FITC-Ficoll. In addition, acid phosphatase was used as a marker for phagocytic activity. Reticulum cells in the liver and spleen were functional from the first point of detection, whereas mononuclear phagocytes in the liver and spleen started to absorb antigen a few days after their development.

The Bursa Fabricii, restricted to birds, specifically provides the microenvironment for B-cell maturation. The presence of nerve fibers containing immunopotent neuropeptides in immune organs opens interesting perspectives on the understanding of neuroimmune communication. As an organ for the development of only B-lymphocytes is not known in mammals, the contribution of a peptidergic innervation to the microenvironment of B-cells is not known. Therefore, we studied the peptidergic innervation of the Bursa Fabricii as an organ of B-cell maturation. Four different neuropeptides were found in nerve fibers of the Bursa Fabricii: tachykinins (TK), vasoactive intestinal peptide (VIP), galanin (GAL), and calcitonin gene-related peptide (CGRP). All these neuropeptides were distributed throughout the different bursal compartments, with the exception of the medulla of the bursal follicles, where the early stages of lymphocyte maturation are found. Double immunofluorescent studies revealed three different fiber populations: One containing coexisting VIP and GAL, the second TK and CGRP, and the last TK and no CGRP. Some of the TK-ir fibers may contain coexisting VIP. As demonstrated by double labeling for neuropeptides and lymphocytes, these heterologous fiber populations were found adjacent to B-cells in the follicle cortex. In addition, VIP-ir fibers were seen in association with macrophages. The origins of the fiber populations and the possible functional implications of our findings are discussed.

Treatment of animals with exogenous adrenal steroids or elevation of endogenous glucocorticoids results in a profound involution of lymphoid tissue. In rodent species, this lymphoinvolution is accompanied by lymphocyte cell death and extensive degradation of the genome prior to lymphocytolysis. In the present study, this process was investigated in the bursa of Fabricius of domestic fowl. Four-wk-old chicks were treated with a single injection of dexamethasone, and bursal regression and cell viability were monitored over a 72-h period. Following hormone treatment, DNA was extracted from bursal lymphocytes and analyzed by agarose gel electrophoresis. Dexamethasone treatment resulted in a rapid regression of bursal tissue that could be detected as soon as 6 h posttreatment, but lymphocyte viability was not altered until 24 h afterward. The DNA isolated from bursal lymphocytes of glucocorticoid-treated birds appeared to be degraded at internucleosomal sites and generated a "ladder" of discrete DNA fragments when analyzed by agarose gel electrophoresis. This form of hormone-induced cell death, referred to as programmed cell death, may play a key role in glucocorticoid-mediated immunosuppression.

The ontogeny of chicken lymphocytes expressing J chain (LEJ) was investigated in the embryonic bursa of Fabricius, the spleen, and the thymus. Simultaneous appearance of LEJ was detected in the bursa and spleen on Day 14 of incubation. These cells were detected later in the thymus. The LEJ were found to increase rapidly in the spleen from the 19th to 20th incubation day. In adult chickens, the highest percentage of LEJ was also found in the spleen. These cells were seen in the thymus at a lower frequency. Intermediate numbers were found in bursal and peripheral blood lymphocytes. The frequencies of the LEJ were similar to those of lymphocytes positive for cytoplasmic immunoglobulins (Ig) IgA and IgM, but were not related to the number of lymphocytes expressing surface Ig. It is possible to consider that the suitable site for LEJ is the spleen, on the basis of the rapid increase in the number of LEJ just before hatching and from the fact that the highest value is found in adult chickens. Furthermore, LEJ may participate in secretion of IgA or IgM but not be associated with the expression of surface Ig.

In this report, we identify in the medulla of the starling's bursa, a cell that resembles the secretory cell previously revealed in the bursa of the chicken. The secretory cell possessed prominent cytoplasm, internal granules, a ruffled surface, and a medium dense substance on the cell surface.

Analysis in a cell flow cytometer (Ortho Spectrum III, Ortho Inst.) of single cell suspensions from the bursa and thymus of 20-day embryos revealed two distinct cell clusters. The two clusters were less apparent after fixation of the cells in paraformaldehyde and assumed a comet-like appearance at 30 min fixation in ethyl alcohol (EA). The G (postmitotic), S [deoxyribonucleic acid (DNA) synthesis], and G2/M (premitotic and mitotic) phases of the life cycle were visualized in two cell flow cytometers (Ortho Spectrum III and FACS IV, Bectin Dickinson) after treating the cells with EA, ribonuclease (RNase), and propidium iodide (PI, a fluorescent dye). Bursal cell suspensions exposed to the EA-RNase-PI protocol and stored for 3 weeks in phosphate-buffered saline showed minor changes in the G1 coefficient of variation, G1, and S percentages, but marked changes in these parameters occurred after 6 weeks of storage. Thymic cells treated in a similar fashion could not be maintained for 3 weeks. Bursal and thymic cells may remain in the EA for one day and possibly as long as 7 days prior to preparing them for DNA life cycle analysis. Paraformaldehyde was not a satisfactory cell fixative for assessing a cell's DNA life cycle.

Cell flow cytometry was used to assess the life cycle of embryonic lymphocytes. Cells were prepared for flow cytometry by fixation for 30 min in a final concentration of 70% ethyl alcohol, treated with ribonuclease (50 to 70 Kunitz/ml) and incubated for 30 min. in propidium iodide. Our data demonstrated that bursal lymphocytes from 20-day embryos (DE) exhibited significantly fewer cells in pre-DNA (deoxyribonucleic acid) and more cells in DNA synthesis (S) than thymic cells from 20 DE, and thymic lymphocytes from 16 DE expressed a more active S than bursal cells. Lymphocytes were separated from granulocytes by a Ficoll-hypaque double density gradient. The DNA cycle of bursal lymphocytes was more easily disrupted with cyclophosphamide (Cy) than the DNA cycle of thymic lymphocytes. However, unfractionated splenic cells from embryos treated with Cy revealed a greater percentage of cells in S and post-DNA synthesis and mitosis than splenic cells from control embryos. Because the splenic cells of Cy-treated embryos were predominantly immature granulocytes, it was concluded that Cy stimulated myelopoiesis in the spleen.

Lys-His-Gly-NH2 has been claimed to selectively induce B cell precursors to differentiate into mature B lymphocytes. In the present study, the effects of this tripeptide and a control compound having the reverse sequence (Gly-His-Lys-NH2) on growth and differentiation of chicken and mouse B cell precursors were investigated. When chicken bone marrow (BM) cells from 15-day-old embryos were treated for 18 hr with either of the tripeptides, the frequency of Bu-1 antigen-bearing cells increased. Moreover, when embryonic bursa cells were stimulated in vitro with phorbol myristate acetate, which induces them to proliferate and undergo terminal differentiation into immunoglobulin (Ig)-secreting cells, these compounds caused a 10-fold increase in the number of Ig-secreting cells but did not increase cell proliferation. They had no effect on neonatal or adult bursa cells. Embryonic bursa cells were cultured in the presence of either of the tripeptides and metabolically labeled with [35S]methionine. When immunoprecipitated Ig was analyzed by two-dimensional gel electrophoresis, no differences in mu heavy or lambda light chain diversity patterns could be detected, indicating that neither of these compounds enhances Ig diversification. The effect of these tripeptides on murine B cell precursors was assayed in cultures of BM cells depleted of mature B cells by 5-fluorouracil. When precursor cells were incubated without adherent BM stromal cells, they did not respond to the tripeptides. However, after incubation of precursors with adherent stromal BM cells for 2 days, followed by treatment with either of the two tripeptides, differentiation into lipopolysaccharide-reactive mature B cells took place. Incubation of precursors with adherent stromal BM cells in the absence of tripeptides was not sufficient to allow the precursors to complete differentiation. In addition, both tripeptides acted synergistically with interleukin 1 or interleukin 3. In conclusion, these tripeptides seem to enhance precursor B cell differentiation in a lineage-nonspecific manner rather than to function as lineage-specific differentiation hormones.

In the chicken, and perhaps in all birds, the development of the cells which form antibodies, the B-cells, differs substantially from that in mammals. In birds, committed B-cells colonize a specialized organ, the bursa of Fabricius, which consists of some 10(4) follicles. Diversification, i.e., the development of the antibody repertoire, takes place in bursal follicles by a process termed "gene conversion." The avian bursa is easily accessible experimentally, and in the chicken, it has been the subject of extensive research. As an aid to experimentation in this field, we present here a formal mathematical model of bursal development. Formulae are derived which allow one to estimate the number and sizes of B-cell clones in bursal follicles, and hence the size of the overall antibody repertoire. Particular attention is paid to the problem of estimating experimental errors.

Using the quail-chick marker technique, cells have been traced in the haemopoietic organs of 149 developing chimaeras composed of a quail embryo with a chick yolk sac. The existence of intraembryonic stem cell relaying yolk sac stem cells, previously demonstrated with this system, is confirmed. Thymus, bursa of Fabricius and bone marrow are preferentially populated by intraembryonic stem cells. The spleen shows a transitory phase of colonization by chick yolk sac stem cells at 11--12 days of incubation. From 7 days onwards the yolk sac receives quail stem cell emigrating from the embryo.

The binding of membrane immunoglobulin (mIg) by anti-Ig antibodies is known to initiate a mitogenic signal in B lymphocytes. Because in many instances growth control appears to be correlated with phosphokinase activity, as well as with alterations in cytoskeletal architecture, we asked the question whether antibodies binding to mIg would also lead to the specific phosphorylation of lymphocyte actin-associated proteins. Utilizing a myosin affinity technique, we directly examined phosphoproteins that were associated with actin in the chicken B cell. We found that in a few instances the level of phosphorylation was indeed modulated by mIg:anti-Ig interactions. These actin-binding phosphoproteins may be important control elements in the lymphocyte cytoskeleton.