Two morphotypes, fusiform and oval, were isolated from a single clone of the diatom Phaeodactylum tricornutum Bohlin and maintained as subclones by culturing in liquid and solid substrates, respectively. Salinity of the medium, from brackish to marine, had no effect on expression of the phenotypes. The oval cell is generated endogenously within a “transformed”fusiform cell upon transfer from liquid medium to agar plates. With the light microscope, normal and “transformed”fusiform cells, prior to giving rise to oval cells, can be discriminated by means of their staining response to toluidine blue. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis of protein extracts from lysed cells revealed slight differences in polypeptide composition between fusiform and oval types. A phenotype‐restoration experiment from oval to fusiform demonstrated that the oval type readily reestablished not only fusiform morphology but also the protein pattern characteristic for the fusiform type. Immunochemical analyses (western blots) using antisera raised against whole and lysed cells of both morphotypes revealed antigenic alterations of the oval morphotype. Several antigenic determinants restricted mainly to the surface of oval cells were detected. Results indicate that environmentally induced phenotypes of Phaeodactylum may be not only the consequence of specific gene expression but also the result of significant, general post‐translational modifications.
Inbred animals (Lewis rats) were used to investigate the regeneration of autologously implanted splenic tissue at intra-omental and subcutaneous sites. Quantitative immunohistology with monoclonal antibodies against lymphocytes and macrophages was performed to analyse the cell density of red pulp (RP), periarteriolar lymphoid sheath (PALS), marginal zone (MZ) and follicle, 7-180 days after transplantation. Antigenic, allogeneic and mitogenic stimulation and Northern blotting were also performed. Transplant groups differed from spleen only in the reduced size of PALS; however, quantitative analysis demonstrated subtle differences between spleen and transplants. The cell density of B-cells and ED-1+ macrophages was reduced in the RP, Tsupp/cyt-cells were decreased and B-cells increased in PALS, and B-cells and Thelper-cells reduced in the MZ. No differences could be detected between the transplant groups. Flow-cytometric analysis of cell suspensions from spleen and transplants revealed a reduction of T-cells (OX-19+), MHC-I and transferrin-receptor-bearing cells in both transplant groups, and a decrease in the number of Thelper-cells and ED-3+ macrophages in subcutaneous transplants. Both transplant groups were defective regarding the allogeneic and pokeweed mitogen response. Aberration of the lipopolysaccharide response was restricted to subcutaneous transplants, which additionally showed abnormal expression of interferon-gamma, interleukin-5 and interleukin-6 mRNA. Thus, subtle alterations of the newly developed microenvironment and/or lymphocyte-homing may influence the regeneration of splenic tissue; the implantation site may represent an important parameter in functional reorganisation.
The present study deals with the morphological and functional development of intraomentally and subcutaneously implanted splenic tissue. Spleens and splenic transplants from 138 Lewis rats were investigated with immunohistological, immunological and molecular biological methods at different times after operation (up to 200 days postoperatively). The analysis of the development revealed a nonsignificant reduction concerning the weight of subcutaneous replants and a nonsignificant decrease of the weight of female transplants of both groups at different phases after operation. The cell composition of cell suspensions from spleen and both transplant types showed a deficiency of T, B, MHC-L cells and a certain macrophage subset (ED-3+ cells) in transplants. In a quantitative immunohistological analysis of compartments (red pulp, periarteriolar lymphoid sheaths, marginal zone and follicles) the T cell reduction was related to the Tsupp/cyt cells and T cell receptor bearing cells in the periarteriolar lymphoid sheaths, whereas the density of T helper cells was normal. In addition, a different homing of K-light chain positive and leukocyte common antigen (B cell type)-positive B cells in follicles and marginal zone was detected. The amount of two macrophage subsets (ED-1+ and ED-2+ cells) was increased in the red pulp. Only minor differences in the immunoarchitecture of transplants at different implantation sites were measured. A functional analysis of spleen compared to both transplant groups elicited a B cell defect after LPS stimulation in subcutaneous transplants and a reduced allogeneic response of both transplant types but a normal proliferation of T cells after ConA stimulation and a correct IgM antibody response against sheep red blood cells. The in vivo mRNA expression and the expression kinetics of interferon-γ and granulocyte-macrophage colony-stimulating factor after antigen stimulation differed in both transplant groups with a remarkable permanent expression of both mediators in subcutaneous transplants. It can be summarized that the results clearly indicate a development of spleen-like immunoarchitecture of intraomental replants with subtle cellular, functional and molecular alterations. In contrast, despite a comparable development, some severe functional defects occurred in subcutaneous implants pointing out the important role of interactions between the regenerating splenic tissue and the target tissue on a functional and molecular level.
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