Paavo Toivanen

A solid-phase radioimmunoassay for IgG and IgM antibodies against measles virus.

A solid-phase radioimmunoassay (RIA) was used to determine the presence of IgG and IgM antibodies to measles virus in human serum and cerebrospinal fluid (CSF). Purified measles virus was adsorbed on to polystyrene balls, which were then exposed to serial dilutions of test serum or CSF. The presence of antibody was measured by its capacity to bind 125I-labelled specific anti-human IgG or IgM. Serum from a variety of patients as well as measles-immune clinically healthy persons were tested; binding ratios (using negative human serum controls) were usually between 10 and 30, but with subacute sclerosing panencephalitis (SSPE) ratios were as high as 50. Of ten CSF specimens tested, all but one, which was taken early in the convalescent phase of measles infection, had detectable IgG antibody. In six patients with acute measles, IgM antibodies were found in all serum specimens taken one or more days after the onset of rash. Maximal titers of 1:10000 to 1:40000 were found about 7 days later. Thereafter, IgM titres decreased rapidly but were still detectable at 40 days. A purified ribonucleoprotein of measles virus was also used successfully as an antigen in this RIA method.

Effect of in ovo treatment with cyclophosphamide on lymphoid system in chicken

Abstract To develop a model for studies on the embryonic lymphoid cell maturation in chicken, cyclophosphamide (CY) was administered intravenously to chick embryos on three consecutive days, starting either on Day 12, 14, or 16 of incubation. To evaluate the effects of CY, survival and growth patterns, antibody-forming capacity, lymphocyte responses to phytohemagglutinin (PHA) and concanavalin A (Con A), and weight and microscopic morphology of bursa of Fabricius, thymus, and spleen were assessed. Like CY treatment in the newly hatched period, CY given in ovo caused a long-lasting and deep humoral immunodeficiency, when administered on Days 14–16 or on Days 16–18 of incubation. In microscopic examination at the age of 44 days, only rudimentary follicles and increased interfollicular connective tissue were found in the bursa. Thymus weights and lymphocyte responses to PHA and Con A were significantly reduced following in ovo treatment with CY. In reconstitution studies, this model of embryonic immunodeficiency will provide a possibility for a match according to the maturity of transplanted cells and of the microenvironment they require for further development.

B‐Cell Differentiation in the Chicken: Expression of Immunoglobulin Genes in the Bursal and Peripheral Lymphocytes

We have studied the expression of immunoglobulin genes in the chicken B‐cell precursors, and of a B‐cell surface marker (Bu‐1) on the bursal and peripheral B‐cells during normal ontogeny. Since there is no way of distinguishing the precursor cells from the more mature bursal lymphocytes on the basis of surface markers, we chose to study the total bursal lymphocyte population at ages when the numbers of the various precursor cells (bursal, early post‐bursal, and post‐bursal stem cells) in the bursa are estimated to be at their highest. Thereafter, comparisons with the more mature lymphocytes in the peripheral organs were made. As a result, levels of the γ and μ transcripts and expression of Bu‐I antigen in the chicken B‐cell precursors were found to be unchanged during the post‐hatching period. In the light of these experiments, the later events of B‐cell differentiation, i.e. the development from the bursal to post‐bursal B lymphocytes, occurs without the γ, μ, and Bu‐1 gene loci involved. On the other hand, the higher level of γ and μ expression in the splenic B lymphocytes indicates that the post‐bursal stem cells mature into highly active plasma cells after seeding to the peripheral organs.

IgG Fc Receptor-bearing cells during early lymphoid cell development in the chicken

Abstract Cells from intraembryonic mesenchyme, yolk sac, bursa of Fabricius, and thymus from chicken embryos at different stages of development were studied for the presence of IgG Fc receptors by EA-rosette formation and binding of heat-aggregated chicken IgG (agg IgG). Cells with Fc receptors were found in high frequency in the intraembryonic mesenchyme as early as on the third day of incubation, in the yolk sac on the 7th day, in the bursa on the 10th day, and in the thymus on the 16th day of embryonic development. In the bursa the number of agg IgG binding cells increased with the age of the embryo and remained high after hatching, whereas in the thymus the peak value (76%) was observed on the 16th embryonic day, and after hatching only about 10% of the cells expressed the agg IgG receptors. The results also suggest that the appearance of IgG Fc receptors precedes the expression of B-L (Ia-like) antigens and of cytoplasmic and surface immunoglobulins on early lymphoid cells of the chicken embryo.

γδ and αβ T Cells are Equally Susceptible to Apoptosis

Little is known about the role of apoptosis in the regulation of gamma delta T cell development and function. We have used chicken as a model to study apoptosis of gamma delta T cells at different stages of their development. Apoptosis was measured with electrophoretic analysis of DNA fragmentation and flow cytometric determination of DNA content combined with immunofluorescence staining of cell surface molecules. In vitro culture, dexamethasone, and gamma-irradiation induced apoptosis of both gamma delta TCR+ thymocytes and peripheral gamma delta T cells. Apoptosis could be induced even in the earliest thymic gamma delta thymocytes on embryonic day 13. Resting peripheral blood gamma delta T cells were more resistant to apoptosis than thymocytes and spleen cells. Following polyclonal activation of splenic gamma delta T cells by Con A, the proportion of the CD8+ gamma delta T cell blasts decreased significantly when recultured without further stimulation. These results indicate that gamma delta T cells are susceptible to apoptosis in a manner similar to alpha beta T cells, and suggest that apoptosis plays an important role in the regulation of the development and function of both thymic and peripheral gamma delta T cells.Little is known about the role of apoptosis in the regulation of γδ T cell development and function. We have used chicken as a model to study apoptosis of γδ T cells at different stages of their development. Apoptosis was measured with electrophoretic analysis of DNA fragmentation and flow cytometric determination of DNA content combined with immunofluorescence staining of cell surface molecules. In vitro culture, dexamethasone, and γ‐irradiation induced apoptosis of both γδ TCR thymocytes and peripheral γδ T cells. Apoptosis could be induced even in the earliest thymic γδ thymocytes on embryonic day 13. Resting peripheral blood γδ T cells were more resistant to apoptosis than thymocytes and spleen cells. Following polyclonal activation of splenic γδ T cells by Con A, the proportion of the CD8+γδ T cell blasts decreased significantly when recultured without further stimulation. These results indicate that γδ T cells are susceptible to apoptosis in a manner similar o γδ T cells, and suggest that apoptosis plays an important role in the regulation of the development and function of both thymic and peripheral γδ T cells

Susceptibility to tolerance induction of bursal and peripheral B cells.

We investigated cellular aspects of immunological tolerance to protein antigens in chickens by examining the immune responses of bursal and splenic cells from tolerant or normal chickens after transfer into cyclophosphamide (CP)‐treated recipients. Newly‐hatched chicks were made tolerant to bovine serum albumin (BSA) by injection of 100 mg of the antigen. When bursa cells from 4‐day‐old BSA‐unresponsive chicks were transferred into CP‐treated recipients, the reconstituted birds were able to respond to a subsequent injection of BSA almost as well as normal birds, and as well as CP‐treated birds that had been reconstituted with normal bursa cells. To investigate whether the presence of the BSA antigen might affect recovery from tolerance, we injected CP‐treated recipients with BSA at the time of transfer of bursal cells. The presence of the antigen prevented the recovery of the anti‐BSA response in reconstituted birds. When spleen cells from 6.5‐week‐old unresponsive chicks were transferred into CP‐treated recipients, no recovery of responsiveness to BSA could be demonstrated. A likely reason for the failure of splenic B cells to recover responsiveness on transfer is their inability to generate somatic variants of Ig genes in the same way as bursal stem cells. Thus, when the bursa involutes, the chickens antibody repertoire may be frozen in a less adaptable state than that of a mammal.

Cell Transplantation into Immunodeficient Chicken Embryos Reconstituting Capacity of Different Embryonic Cells

The findings to be described in this paper form a step in our studies on the B cell development in the chicken. In these studies we have largely used the cyclophosphamide model of immune deficiency first described by Lerman and Weidanz (1). Based on a series of experiments we have characterized the postbursal and bursal stages of B cell development (2-6). As a postbursal cell we have defined a cell which is capable of further function and maturation without any influence by the bursa of Fabricius, i.e., this cell can be transferred into cyclophosphamide-treated surgically bur- sectomized newly-hatched recipients and a full functional restoration of immune functions occurs in the recipients. Postbursal cells appear in significant numbers in the bursa around the third and fourth week after hatching, and shortly thereafter in the spleen, bone marrow and also in the thymus. In contrast, a cell type defined in functional terms as a bursal stem cell needs the bursal microenvironment for further maturation. This cell is capable of inducing a full functional as well as morphological re- constitution only when the bursal rudiment is available. No effect occurs if this cell is transferred into cyclophosphamide- treated surgically bursectomized recipients. The bursal stem cell is present in the bursa during the late embryonic development and shortly after the hatching.

Mother/child mixed lymphocyte reaction: is it depressed?

Several alternative reasons have been offered for the immunological inertia between the pregnant mother and her fetus. None of these alone has proved correct and sufficient. In this review Paavo Toivanen and Christer Granberg look at one interesting mechanism - the inhibition of maternal lymphocyte reactivity by fetal suppressor cells observedin vitro when untreated populations of maternal and neonatal lymphocytes are cultured together.

Usage of beta 1 integrin ligands by B cells is developmentally regulated in avian bursa.

Chicken B cell development takes place in a separate organ, the bursa of Fabricius, which provides the blood‐borne stem cells, a microenvironment specialized for B cell maturation. Therefore, chicken can be used as a model to study specifically the molecules and interactions which control the development of the B cell compartment. In this work, we studied expression, localization and function of β1 integrins on maturing B cells and bursal stroma. The expression of β1 integrins on B cells increases during the embryonic development and β1 integrin‐positive cells can be found both in the medulla and the cortex throughout the bursal development. The binding assays show that the attachment of B cells to stroma is mediated by β1 integrins. Binding to 10‐day‐old embryonic stroma is fibronectin‐independent, whereas fibronectin‐mediated binding takes place in an increasing manner during further embryonic maturation. After hatching fibronectin appears to be the main binding site for B cells. However, the epitope of β1 integrin which takes part in the adhesion of B cells to stroma and to fibronectin is not crucial for the homing of cells into the bursa in a cell transfer model. Our results indicate that the interaction of β1 integrins with their ligands is developmentally regulated in the bursa and suggest that B cell maturation may be partially controlled by this interaction.