Locally applied testosterone is a novel method to influence the development of the avian bursa of Fabricius

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 µm 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.     

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

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

The influence of embryonic testosterone treatment on bursal epithelial pinocytotic activity

Developing White Leghorn chicks were treated with testosterone propionate $\text{in}$$\text{ovo}$ to effect a partial chemical bursectomy. After hatching, birds were administered carbon particles or horseradish peroxidase via the cloaca, and the transport of these tracers by the bursal epithelium was examined. Uptake of both tracers was inhibited in the treated when compared to untreated birds. The degree of development of interaction between bursal epithelium and underlying lymphoid tissue appeared to play a major role in determining ability of the epithelium to transport these tracers. Epithelium associated with follicles possessing medullary and cortical divisions was able to transport, while intraepithelial follicles or regions of the epithelium overlying diffuse lymphoid tissue did not transport either carbon or horseradish peroxidase. Bursal function in regional defense of the gut may depend on a local interaction of bursal epithelium with lymphoid, and perhaps other cell types.     

Effect of surgical bursectomy on the ellipsoid, ellipsoid-associated cells, and periellipsoid region of the chicken's spleen

Surgical bursectomy resulted in cellular depletion of the periellipsoid white pulp, confirming its bursa dependency. Also, in bursectomized birds, the ellipsoid could not be identified, although a small number of abnormal ellipsoid-associated cells (EAC) were observed in the periellipsoid region. The most characteristic finding was the degeneration of the EAC. Degeneration of EAC indicated that the intact bursa was mandatory for normal differentiation of cells of the periellipsoid white pulp into EAC. The promoting effect of the bursa might take place by a bursal hormone. The histological impairment of the EAC was followed by reduced carbon binding and migrating capabilities. Bursectomy resulted in a shift in bacterial phagocytosis in that many cells of the periellipsoid phagocytosed Salmonella. The reduced heterophil infiltration of the ellipsoid in bursectomized birds might be explained by the impaired granular content of the EAC. The impaired migration capability of the EAC might contribute to the low number of germinal centers in bursectomized birds.     

The behavior of bursal lymphoid follicle-associated cells after treatment with testosterone

Administration of androgens produces damage in lymphoid tissue and in the bursa of Fabricius. After IM administration of 5 mg of testosterone propionate (TP) beginning at hatching and continued during the following 4 days, a significant reduction in the bursal weights is observed. Histologically, an increase in the connective tissue is observed and cystic formations are also found. In all cysts examined, there is continuity of the cystic lumen with the free surface. The follicle-associated epithelial (FAE) cells are on the bottom of the pseudocysts and form a separation between the pseudocystic cavity and the lymphoid tissue which is still further inwards. These cells do not lose their esterase activity, even though they are often flattened. Furthermore, they disappear in the pseudocysts deprived of lymphoid tissue. A new hypothesis is advanced that the FAE cells originate from the mesenchyme with differentiation in the histocytic line.     

Inhibition of membrane bone formation by vitamin A in the embryonic chick mandible

Embryonic chick mandibles (Hamburger and Hamilton [HH] stages 17-25) were cultured in the presence of various concentrations of vitamin A to determine the effect of hypervitaminosis A on membrane bone formation. In normal development, the mandible differentiates a centrally located Meckel's cartilage surrounded by membrane bones. Mandibles cultured without added vitamin A differentiated normally, though the timing of differentiation was retarded from that in ovo. Treatment with vitamin A interfered with skeletogenesis to varying degrees depending upon the initial age of the explant and the concentration of vitamin A. At low concentrations of vitamin A (1 microgram/ml), neither cartilage nor membrane bone formed in young explants (HH stage 17), whereas cartilage formed in 78% and membrane bone in 11% of older explants (HH stage 25). Higher concentrations of vitamin A (2-4 micrograms/ml) inhibited membrane bone formation in all explants, and 4 micrograms/ml of vitamin A inhibited chondrogenesis in most (88%) of the older explants. To determine whether tissue interactions influence this effect of vitamin A on skeletogenesis, mandibular mesenchyme was separated from its epithelium and treated with vitamin A. Under normal culture conditions, isolated mesenchyme (HH stage 25) differentiated both cartilage and membrane bone. Hypervitaminosis A inhibited membrane bone formation in the isolated mesenchyme at all levels tested (1-4 micrograms/ml) and inhibited chondrogenesis at levels 2-4 micrograms/ml. Hence, vitamin A can act directly upon the mesenchyme to inhibit both membrane bone formation and chondrogenesis, but its action is mitigated by the presence of the epithelium.     

The Inability of Surgical Bursectomy in the Newly Patched Pepiod to Affect Antibody Formation in the Pigeon (Columba Livia)

Surgical bursectomy was performed in newly hatched pigeons within 24 hours of hatching. The effect of bursectomy on the immune responsiveness was evaluated by studying antibody formation to sheep red blood cells (SRBC) and Brucella abortus organisms and by the presence of serum immunoglobulins after immunoelectrophoresis. Surgically bursectomized pigeons responded as well to SRBC and Brucella as did the non-bursectomized controls. There was no statistically significant difference in the geometric mean antibody titers between the bursectomized and non-bursectomized groups of pigeons. Treatment of the serum with 2-mercaptoethanol (2-ME) indicated that the major amount of antibody produced was resistant to 2-ME and there was no significant difference in the amount of 2-ME-resistant antibody produced in the bursectomized and the control pigeons. Immunoelectrophoretic analysis of the sera from the bursectomized pigeons showed both IgG and IgM present in similar quantities to that of the non-bursectomized controls. Histological sections of the spleens showed germinal centers in both groups of pigeons. Sections of the bursal area from the bursectomized birds indicated that none of the pigeons in the bursectomized group had any remaining identifiable bursal tissue, follicular structures or other organized lymphoid tissue.     

Significance of bursa of Fabricius as target organ in infectious bursal disease of chickens.

The highly virulent strain Cu-1 of infectious bursal disease virus caused 100% mortality in 4-week-old specific pathogen-free chickens. In contrast, chickens infected after bursectomy did not become sick and only showed some discrete and transient necrosis in lymphatic tissues. However, these chickens contained infectious virus and, subsequently, produced specific antibodies. The virus concentrations in the organs studied reached their maximum 2 days postinfection, but were about 1,000 times lower in non-bursectomized animals. It may be assumed that in bursectomized chickens the early events of infection are the same as in non-bursectomized ones. Virus is spread in varius organs, but due to the absence of a sufficient number of susceptible cells, virus multiplication is moderate and can be kept in check by the host defense mechanism. With the occurrence of circulating specific antibodies the virus can be rapidly eliminated. The studies particularly stress that the availability of a large number of highly susceptible cells is a crucial point in acute viral infections.     

Alkaline Phosphatase in the Developing Bursa of Fabricius

The ontogeny of alkaline phosphatase in the bursa of Fabricius was studied by histochemical and biochemical methods. According to the quantitative determinations, the activity of alkaline phosphatase increased from the 11th to 17th day of incubation--that is, during the time of the lymphoid follicle formation in the developing bursa. The activity was localized in the mesenchymal tissue surrounding the lymphoid follicles. Testosterone given in ovo prevented the appearance of alkaline phosphatase in the bursal mesenchyme but had no effect on the activity of the embryonic liver. In contrast, in ovo treatment with cyclophosphamide had no effect on the alkaline phosphatase in the bursa. By using transplantation of embryonic bursal stem cells, it was further shown that, in contrast to cyclophosphamide, testosterone destroys the capacity of the bursa to serve as a differentiation site for the B-cell lineage. The results indicate that testosterone affects the stromal cells of the bursa, whereas cyclophosphamide destroys only the lymphoid population undergoing differentiation and leaves the bursal stroma intact.     

Chemical bursectomy of chickens with colchicine applied to the anal lips.

To induce chemical bursectomy, 30 microliter colchicine dissolved in saline solution (1 mg/ml) was applied on the anal lips of White Leghorn chickens once daily for four consecutive days after hatching. Histologic characteristics of the bursa of Fabricius, spleen, thymus, cecal tonsils, and rectal wall were studied 1-7 days after hatching. Total necrosis of the lymphoid cells and the follicle-associated epithelium in the bursa was observed during the four days of colchicine application. The bursal stroma remained unchanged, and only minor changes were found in the interfollicular surface epithelium. After colchicine application ceased, some regeneration of the epithelium, as evidenced by small epithelial buds, was found. At the end of the observation period the epithelial buds were often covered by the follicle-associated epithelium, which was capable of phagocytizing carbon. However, practically no lymphoid repopulation was seen in the buds. Since this method of colchicine application had no direct effect on other lymphoid organs or on the survival or weight of the chickens, this bursectomy model seems to be a new tool for use in studies of bursal function.     

Scanning electron microscopy of the bursa of fabricius from normal and testosterone-treated embryos

Scanning electron microscopy (SEM) has revealed the presence of projecting follicles (PF) and button-like follicles (BLF) in the bursa of Fabricius. This study was designed to examine the embryonic bursa with SEM to ascertain which type of follicle appears first and to compare the SEM of the bursa from normal embryos with those having received testosterone propionate (TP) on the 11th day of incubation. The bursa of the latter embryos exhibits an arrested lymphoid development. PF appeared in normal embryos by 16 days and were well developed by 18 days of embryonic development. Inter-follicular epithelium was apparent by 21 days of embryonic development in normal embryos. On the other hand, bursal follicles and interfollicular epithelium failed to form in TP birds. The TP-birds exhibited a characteristic pebble-like epithelium which may attest to the regressive influence of TP on bursal epithelium or to an arrested stage of epithelial development. The PF may lead to the development of BLF or the BLF may be derived independently of PF.     

Cellular composition of the bone marrow in the chicken: II. The effect of age and the influence of the bursa of fabricius on the size of cellular compartments

The blood and bone marrow of New Hampshire chicks were analyzed quantitatively from the time of hatch to 8 weeks of age. Hormonal bursectomy was performed by treating embryonating eggs on the 11th day of incubation with testosterone propionate (TP) which resulted in severe hypogammaglobulinemia and complete atrophy of the bursa of Fabricius. TP-treated birds exhibited some lymphocytopenia, reduced splenic weight, and lack of plasma cells in their bone marrow. The number of cells per milligram bone marrow was comparable in normal and TP-treated birds, falling in the range reported for laboratory rodents. The chick medullary hemopoiesis is characterized by the predominance of erythroblasts throughout early development; granulocytes and lymphocytes represent much smaller cellular compartments than in rodents. In the chick granulocytes tend to decrease after hatch, whereas in rodents they tend to increase. The normal chick shows a temporary increase in marrow lymphocytes after hatch, similar to that observed in some young rodents, but on a much smaller scale. Hormonal bursectomy did not prevent the development of a lymphocyte population in the bone marrow. These cells were fewer in TP-treated birds at hatch and at 4 weeks than in normal birds, but at 8 weeks of age normal and bursectomized chicks had comparable numbers of lymphocytes in their marrow. Although some lymphocytes in avain bone marrow may depend on the bursa of Fabricius for their development, a substantial proportion of bone marrow lymphocytes in the chick are bursa independent. The cell surface phenotype and site of origin of these cells remains to be investigated.     

Study on the Effect of Calf Thymic Peptides Preparation (Tp) on Human B' Lymphocytes

Extensive experimental evidence has shown that thymic hormones (or factors) affect and regulate the differentiation and function of T lymphocytes. However, little is known about the action, if any, of thymic hormones on B lymphocytes. This paper reports the results of an investigation of the effect of a calf thymic peptide preparation (TP) prepared in our laboratory, on the proliferation and differentiation of human B lymphocytes.

TP at concentrations higher than 1 μg (protein)/ml inhibited the proliferative responses of human B lymphocytes to the stimulation by Staphylococcus aureus Cowen strain I (SAC) and F(ab')₂ fragments of goat anti-human IgM μ chain specific antibody (anti-μ). TP itself had neither toxic nor mitogenic effect on B cells. TP at concentrations of 60 and 100 μg/ml did not affect the differentiation of B cells driven by SAC and PWM in a reverse PFC assay, but appeared to suppress the production in some individuals of total IgG and IgM in culture supernatants in a PWM system. Preculture of B cells with 60 μg/ml of TP for 40 hrs showed a suppression of the proliferative response to SAC and anti-μ stimulation, suggesting that TP might act on cells directly and persistently for some time. When TP was added to the culture on day 0 or on day 1, a similar decrease of inhibition of B cell proliferation was observed. A decrease in monocytes from 15-17% to 5% did not appreciably influence the suppression of SAC-or anti-μ-induced proliferation of B cells by TP. These preliminary results suggest that a calf thymic peptides preparation might have some direct effect on B lymphocytes.     

Ontogenic restriction of colonization of the bursa of Fabricius

The capacity of hemopoietic precursor cells (HPC) to home to embryonic bursal and thymic grafts was investigated in embryonic and newly hatched chickens. Whereas thymic grafts developed normal histogenesis in both types of recipients, the bursal rudiment was colonized and developed in embryonic, but not in newly hatched hosts. In the latter, noncolonized bursal grafts developed neither lymphoid follicles nor granulopoiesis in the mesenchyme. These results are interpreted in terms of ontogenic “maturation” of the HPC which lose their homing potential towards the bursa while they preserve their thymic seeding capacity. This hypothesis is consistent with previously reported data which indicated cyclic continuous recruitment of the thymic lymphoid population, but restricted bursal colonization to a relatively brief period of embryonic life.     

Immunofluorescent detection of surface antigens specific to T and B lymphocytes in the chicken

Rabbit antisera specific for chicken T and B cells as judged by surface immunofluorescent staining have been raised. Specificity was established by the staining of thymus and bursal cell suspension and by the effects of thymectomy and bursectomy on the staining of peripheralized lymphocytes. Furthermore, double labeling experiments showed that anti-T and anti-B sera reacted with different populations of blood lymphocytes. Comparable numbers of cells in blood and spleen stained for B and light chain determinants. No evidence for “null cells” was obtained. There was little change in the percentage of cells staining in the various lymphoid organs from 4 days to 12 months of age. The thymus contained approximately 7 % B cells, although no T cells were demonstrable in the bursa. One antiserum showed only thymocyte specific antibodies not reacting with peripheral T cells. The specific B and T markers seem to be acquired during differentiation within the appropriate central lymphoid organ. Demonstrable surface immunoglobulins appear later in ontogeny than the B antigens. The majority of cells bearing the B marker in bone marrow were large cells lacking surface light chain determinants.     

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

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

Bursa-dependent subpopulations of peripheral B lymphocytes in chicken blood

By selective labeling of juvenile chicken bursal cells with colloidal fluorescein isothiocyanate in situ, the emigration rate of bursal lymphocytes to the periphery was estimated at approximately 0.84% and 0.96% of the peripheral blood lymphocyte (PBL) and splenic B cell pool per hour, respectively. Emigrant bursal cells were found primarily in blood and spleen, with very small numbers migrating to thymus, bone marrow, and gut-associated lymphoid tissues. Emigrant bursal cells expressed high levels of both major histocompatibility complex class II antigen and the Ov alloantigen, a phenotype found on a population comprising approximately 4% of bursal cells from which the bursal emigrants may be derived. Surgical bursectomy at 3 weeks of age revealed that peripheral blood B cells could be divided into three distinct populations. Specifically, 60% of the peripheral blood B cells were short lived with a half-life of about 30 h in the blood. These cells accounted for the great majority of emigrants from the bursa to the peripheral blood. Approximately 35 % of PBL B cells had a half-life of 12 days following bursectomy and comprised cells which did not divide in the periphery. Consequently, we propose that physiological differences between this population and the majority of bursal emigrants are established intrabursally. The remaining PBL B cells, whose relative proportion increases with age from about 5 % of PBL B cells at 2-3 weeks of age, are short lived and are being continually produced from (a) post-bursal site(s) of B cell production.     

Development of intrahepatic bile ducts in humans. Immunohistochemical study using monoclonal cytokeratin antibodies

Cytokeratins (CKs) are major structural proteins of intermediate filaments of epithelia. Recent availability of monoclonal antibodies (MoAbs) against various CK polypeptides has made it possible to study their development during cellular differentiation. We analyzed the expression of CKs in the human liver during development. Twenty-four liver specimens were tested by the avidin-biotin complex immunohistochemical method by using three MoAbs against different CK polypeptides (CAM 5.2 against CKs 50, 43, and 39 kd; AE1 against acidic CKs 56.5, 50/50', 48, and 40 kd; and 34 beta E12 against CKs 58, 56.5, and 56 kd). Liver parenchymal cells in fetuses as early as 4 weeks of gestational age reacted with MoAbs CAM 5.2 and AE1, but the expression of AE1-positive CK polypeptides in hepatocytes disappeared by 24 weeks of gestational age. Small cells, presumably ductal plate cells, in direct contact with mesenchyme around the portal vein and along the branches of portal veins, showed strong staining with MoAbs CAM 5.2, AE1, and 34 beta E12, identical to that of bile ducts. In neonates, children, and even in adults, residual MoAbs CAM 5.2-,Ae1, and 34 beta E 12-positive cells were present around the branches of portal veins. These findings suggested that the CK profile of liver parenchymal cells changes during their differentiation into hepatocytes, whereas that of ductal plate cells and bile ducts remains unaltered with respect to the polypeptides tested here. Some ductal plate cells may persist in neonates, children, and even in adults.     

Slit and robo expression in the developing mouse lung.

Mammalian lung development is mediated through complex interactions between foregut endoderm and surrounding mesenchyme. As airway branching progresses, the mesenchyme undergoes dramatic remodeling and differentiation. Little is understood about the mechanisms that direct mesenchymal organization during lung development. A screen for candidate genes mediating this process identified Slit, a ligand for the Roundabout (Robo) receptor previously associated with guidance of axonal projections during central nervous system development. Here, we demonstrate by in situ hybridization that two Slit genes (Slit-2 and Slit-3) and two Robo genes (Robo-1 and Robo-2) are expressed in fetal lung mesenchyme. Slit-2 and Robo-1 expression is present throughout mesenchyme at midgestation and is not detectable by newborn day 1. Slit-3 and Robo-2 expression is restricted to specific, complementary subsets of mesenchyme. Robo-2 is expressed in mesenchymal cells immediately adjacent to large airways, whereas Slit-3 expression predominates in mesenchyme remote from airway epithelium. The temporal and spatial distribution of Slit and Robo mRNAs indicate that these genes may direct the functional organization and differentiation of fetal lung mesenchyme.     

Questioning the role of adenosine deaminase in the development of B lymphocytes in chicken bursa

Adenosine deaminase, a purine metabolic enzyme, was studied in lymphoid tissues of the developing chicken in order to evaluate whether enzyme activity is related to development of the immune system in birds in the same way as for mammals, in which adenosine deaminase is essential for lymphocyte differentiation, especially for the T-cell lineage. Enzyme activity was assayed in thymocytes and bursal lymphocytes at different times during chicken development ranging from the 17th day of embryonic life up to the 50th day after hatching. Adenosine deaminase activity was significantly higher in the bursa than in the thymus, regardless of whether such an activity was expressed per mg protein or per 10(8) cells; moreover, no substantial difference in the relative levels of adenosine deaminase was observed in thymocytes at the various stages of thymus development studied. Significant changes in enzyme activity, however, were found in bursal lymphocytes in which different amounts of adenosine deaminase appeared to be related to definite stages of bursal development and to specific immunological responsiveness of B lymphocytes to intravenously injected antigens. Therefore, if adenosine deaminase does play a role in the functional maturation of the immune system in birds, such a role appears to be related to the differentiation of the B- rather than the T-cell lineage.