Regeneration of autotransplanted splenic tissue at different implantation sites.

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.

Immunoarchitecture and specific functions of splenic autotransplants at different implantation sites.

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-I+ 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 kappa-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-gamma 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.

[Autotransplantation of the spleen in rats: development, function and cytokine expression in intra-omental and subcutaneous regeneration]. / Autotransplantation der Milz an Ratten: Entwicklung, Funktion und Cytokin-Expression in intraomentalen und subkutanen Regeneraten.

The implantation of splenic tissue at different implantation sites (intraomental and subcutaneous) into one animal (Lewis rats) results in the development of splenic nodules at both sites. In a quantitative immunohistological analysis of splenic compartments such as red pulp (RP), periarteriolar lymphoid sheaths (PALS), marginal zone (MZ) and follicles (F) the T-cell reduction was related to the T(helper) cells in the MZ and T(supp/cyt) cells in the PALS. In contrast, the cell density of B cells and ED-1+ macrophages in the PALS and T(supp/cyt) cells in the MZ was increased. Significant differences between the implantation sites were restricted to CD5+ cells (thymocytes and T cells) in the MZ and OX-33+ cells (B cells with LCAB antigen) in the PALS. The reorganisation of the compartments of subcutaneous implants showed a delay of one week as compared with omental ones. Functional assays like haemolytic plaque assay, mitogen stimulation and mixed lymphocyte assay elicited an analogous delay of the functional maturation of IgM-positive B cells, a reduced proliferation of both transplant groups after pokeweed mitogen (PWM) stimulation, a decreased response after lipopolysaccharide (LPS) stimulation in solely subcutaneous replants and no differences concerning the mitogens concanavalin A (ConA) and phytohaemagglutinin (PHA). Both transplant groups showed a significantly reduced allogeneic response. The results of the functional analysis and the abnormal mRNA expression of Il-5, Il-6 (Interleukin 5 and 6), GMCSF (Granulocyte-Macrophage-Colony-Stimulation-Factor) and IFN-gamma (Interferon gamma) in subcutaneous replants indicate subtle molecular alterations (independent of a spleen-like immunoarchitecture) at this site.(ABSTRACT TRUNCATED AT 250 WORDS)

A modified primer extension procedure for specific detection of DNA-RNA hybrids on nylon membranes.

We have developed a modified primer extension procedure for specific detection of mRNA. Alkali-fragmented total cellular RNA or some RNA fraction is hybridized to single-stranded or double-stranded M13 DNA containing the insert of interest which is immobilized on nylon membranes. Hybridized RNA is then detected by incubation of membranes with Escherichia coli RNase H and DNA polymerase I. RNase H is used for nicking the RNA in the hybrids. The resulting 3'-OH groups can subsequently be used by DNA polymerase I to synthesize a labeled complementary strand. The method described is both relatively fast and sensitive and particularly useful for screening large numbers of DNA clones for their representation in RNA populations. Using total cellular RNA as hybridization probe and single-stranded M13 DNA as template as low as 0.25 ng of a specific mRNA was detected (2.5-fold background) when adding 1 microCi [3H]dCTP or 2.5 microCi [32P]d-CTP alternatively as radioactive precursor for the labeling reaction. The detection limit increased to 1 ng (2-fold background) with denatured replicative form double-stranded M13 DNA as template.