Electron microscopic study on the early histogenesis of thymus in the toad,Xenopus laevis

Summary Sequential electron microscopic observations of thymic histogenesis in the toad, Xenopus laevis, reveal that the thymus arises as epithelial buddings of the visceral pouches at Nieuwkoop-Faber stage 40, and acquires its basic histological features at stages 48–49. In the rudiments and the surrounding mesenchyme at stages 43–45, there are non-epithelial cells with pseudopodia, abundant ribosomes, and marginated heterochromatin. These cells, possible precursor cells of thymic lymphocytes, are frequently observed to attach and pass through the basal lamina which coats the thymic rudiment. The proliferation and differentiation of large lymphocytes are evident at stage 47. During stages 48–49 the small lymphocytes, lymphoid cortex and epithelial medulla including the thymic cysts, differentiate, and vascularization occurs.The results provide an ultrastructural basis for recent experimental evidence that the thymus exerts its essential function at stages 47–48. The possibility of non-epithelial derivation of thymic lymphocytes is discussed.The author wishes to express his thanks to Asst. Prof. Ch. Katagiri for his helpful advice during the course of this study     

Implantation of cultured thymic fragments in congenitally athymic (nude) rats

Summary Cultured thymic fragments correspond to the thymic microenvironment depleted of lymphocytes and dendritic cells. When these fragments are implanted under the kidney capsule of congenitally athymic rats, lymphocytes and dendritic cells of host origin enter the graft and induce thymus-dependent immunity in the recipient. This paper describes the ultrastructure of the fragments and the changes that occur during the restoration of normal thymic architecture. At the end of the culture period of 6–9 days and in the early stages after implantation, the grafts consist of keratin-containing epithelial cells of unusual morphology that can be labelled with antibodies raised against the epithelium of the mid/deep cortex and the subcapsule/medulla. Normal thymic architecture develops, including nerves and blood vessels, as lymphocytes populate the environment, and by 4–6 weeks the epithelial cells are the same phenotypically and ultrastructurally as those found in normal rat thymus. However, some areas without lymphocytes still contain the atypical epithelial cells seen before implantation. Large multinucleated giant cells are also present with a few associated epithelial cells of subcapsular/medullary phenotype. In conclusion, the cultured thymic fragments contain a hitherto unknown precursor epithelial cell with an atypical ultrastructure and phenotype that is not seen in normal development.     

Morphogenesis of rat lingual filiform papillae.

The morphogenesis of filiform papillae on rat tongue was investigated with the electron microscope. Tongue rudiments were first seen on the 12th day of gestation. At 15-17 days, dermal papillae had formed and were arranged in hexagonal array on the dorsal lingual surface. Capping each dermal papilla was a two-layered epithelium that protruded slightly above the lingual surface, thus forming the early filiform papilla. In the next stage of development, at 18-19 days of gestation, the epithelium lining the papilla had differentiated into two cell populations, one producing hard keratin, the other producing soft keratin. Some of the keratinized epithelial cells assumed a position at an acute angle to the tongue surface and extended deep into the epithelium. In the next stage, 20-21 days, a cleft appeared within these angularly oriented cells. This resulted in the division of the epithelium into keraatin-lined individual filiform papillae. Finally, the individual papillae increased in size to the adult form.     

An ADAM family member with expression in thymic epithelial cells and related tissues

We have analyzed the tissue-specific expression, mRNA isoforms, and genomic structure of murine ADAM28, an ADAM family member recently discovered in human and mouse. While human ADAM28 is expressed in lymphocytes (J. Biol. Chem. 274 (1999) 29251), we observe expression of murine ADAM28 in thymic epithelial cells and developmentally related tissues including the trachea, thyroid, stomach, and lung, but not in lymphocytes. The expression patterns in adult and day 15.5 embryos are similar. We have detected multiple mRNA isoforms varying in the cytoplasmic domain coding sequence and 3prime prime or minute untranslated region due to alternative polyadenylation and splicing events that occur in the final four exons and three introns. The entire ADAM28 gene spans 55 kb and contains 23 exons. The protein sequence contains all conserved residues required for metalloprotease activity, indicative of a role in ectodomain shedding and extracellular matrix modeling. Given its unique expression pattern and potential functions, murine ADAM28 may play a role in organogenesis and organ-specific functions such as thymic T cell development.     

Post-hatching development of the thymic epithelial cells in the rainbow trout Salmo gairdneri: an ultrastructural study

This study reports the ultrastructure of subpopulations of epithelial cells of the thymic parenchyma during the post-hatching development of the rainbow trout, Salmo gairdner, kept at 14 degrees C. At hatching, the thymus contained a small number of medium and large thymocytes interspersed among three different types of epithelial cells: (1) epithelial cells adjacent to the connective tissue capsule; (2) ramified dark epithelial cells with electron-dense cytoplasm; and (3) pale electron-lucent epithelial cells displaying secretory-like features. All these cells types were anchored to one another by desmosomes and had apparently differentiated from the pharyngeal epithelium. At 4 days after hatching, the thymus enlarged, and numerous gaps occurred between the cell processes of contiguous epithelial cells adjacent to the capsular connective tissue. In 21-day-old trout, thymic trabeculae developed carrying blood vessels, and a subcapsular zone became evident containing lymphoblasts and large subcapsular epithelial cells. In 30-day-old trout, an outer thymic zone developed consisting of spindle-shaped epithelial cells which formed a dense network. At this stage, scattered cystic cells, which apparently differentiated from the pale epithelial cells, were present.     

Implants of quail thymic epithelium generate permanent tolerance in embryonically constructed quail/chick chimeras

In situ implantation of a quail wing bud into a chick embryo at 4 days of incubation (E4) regularly results in the normal development of the implant followed by its acute rejection starting within two weeks post-hatching. If the epithelial thymic rudiments of the quail donor are implanted into the branchial arch area of the chick recipient after partial removal of its own thymic primordia, a chimeric thymus develops in the chick host and this induces tolerance to the quail wing by the chick recipient. The species identity of cells in chimeric thymuses was mapped using Feulgen-Rossenbeck' staining and immunolabelling with monoclonal antibodies directed against quail or chick B-L antigens. Certain lobes contained only chick cells both at the stromal and hemopoietic cell levels. Others had a quail epithelial stroma containing host hemopoietically derived cells. Only chimeras in which at least one third of the thymic lobes were chimeric showed permanent tolerance to the grafted wing. Since the two species exhibit distinct developmental rates, we decided to study the kinetics of thymic involution after birth. Although the changes in thymus weight and histological structure are fundamentally similar in quail and chick, those in the quail start about 7-8 weeks earlier. In the chimeric thymuses, the lobes whose epithelial cells were quail involuted at the rate of control quail showing no influence of the hemopoietic thymic compartment in this process. Tolerance induced by the thymic epithelium during embryogenesis and in early postnatal life was maintained after a profound involution of the quail thymic graft had occurred.     

Mouse thymic virus infection: ultrastructural and immunocytochemical studies of infected thymus cells.

Only limited attention has been paid to the cell population that is affected in the course of Mouse Thymic Virus (MTV) infection. In the present study, thymic cells of newborn mice infected with MTV were examined for general ultrastructural and immunocytochemical characteristics. The earliest sign of infection was detected 5 days after inoculation. Lymphocytes, epithelial reticular cells, macrophages, and lymphoepithelial cell complexes (thymic nurse cells) were affected. Viral particles and filamentous structures were present in both the nucleus and the cytoplasm of these cells. At more advanced stages of cellular necrosis, 6 to 7 days post-infection, cytoplasmic granulation and loss in definition of cytoplasmic organelles became apparent. This was followed by nuclear degradation and cellular aggregation. The selective effect of MTV on lymphocyte subpopulations was also observed. Two populations of infected lymphocytes were identified by single and double immunogold labelling employing monoclonal antibodies and different sizes of gold particles. CD4+8+ and CD4+8- lymphocytes were found to be selectively lysed by MTV.     

Neoplastic response of turkey liver to MC29 leukosis virus.

Twelve to fourteen days after intravenous inoculation of MC29 virus (1 x 10(5)LD50) into 1-day-old turkeys, liver tumour developed in 100% of the infected animals and led to death of birds. The tumour proved to be hepatomas histologically and electron microscopically. Numerous C-type particles were detectable among the tumour cells, as well as budding from the cell membrane. C-type particles were also observed in the tumour-free liver tissue in the spaces between the cells, budding was not detectable. Large number of virus particles were found in spleen extracellularly and intracellular vacuoles. Kidney tumours did not develop, but a few extracellular virus particles were located in the tissue. The MC29 virus-induced primary liver tumour in the turkey seems to be a suitable model for the morphological study of the relationship between oncogenic viruses and eukaryotic cells.     

Relationships between terminal transferase expression, stem cell colonization, and thymic maturation in the avian embryo: studies in thymic chimeras resulting from homospecific and heterospecific grafts

Terminal deoxynucleotidyl transferase (TdT) can be detected in 11- to 12-day-old embryonic chick thymuses 5 to 6 days after the first influx of lymphoid stem cells into the thymic rudiment. To identify the main factors of TdT induction, grafting experiments were devised in such a way that the age of the grafted thymus and that of the host were different. Uncolonized embryonic chick thymuses were grafted into chick hosts of different ages. Under these conditions, lymphoid differentiation arose from host lymphoid stem cells (LSC) invading the thymic rudiment. TdT immunofluorescent detection in the first wave of thymocytes showed that the percentages of TdT+ cells were related to the total age of the explant and not to the age of the host (11 to 17 days). Similar results were obtained when the chick thymic rudiment was transplanted into quail embryos, showing that quail LSC have TdT inducibility similar to that of chick LSC while developing in a chick thymic environment. Colonized chick thymuses were also grafted into quail embryos to compare the TdT inducibility of the first lymphoid generation (of chick type) and of the second (of quail origin), taking advantage of the different chromatin structure of quail and chick cells. In these experiments, the majority of chick cells remained TdT negative for as long as 10 days, whereas most lymphocytes of the second generation became TdT+ soon after their arrival in the grafted thymus. Therefore, during embryonic life, most TdT+ cells were derived from the second wave of stem cells, but some early stem cells were also able to acquire the enzyme. In a final series of experiments, early thymic rudiments were cultured in vitro with 14- to 16-day-old bone marrow and then grafted into 3-day-old host embryos. Under these conditions, bone marrow LSC contributed to a variable proportion of the first generation of thymocytes. The percentage of TdT+ cells among the progeny of these bone marrow stem cells was found to be two times higher than that of thymocytes derived from host LSC. These results suggest that, in addition to intrathymic environmental factors, the origin of LSC influences the frequency of TdT expression in their progeny.     

Ontogeny and tissue distribution of leukocyte-common antigen bearing cells during early development of Xenopus laevis

To analyze the ontogenic emergence of leukocytes during early development, a mouse monoclonal antibody (IgG1), designated as XL-1, was produced against the peritoneal macrophages of adult Xenopus laevis. The XL-1 determinant was expressed on all types of leukocytes, including lymphocytes, granulocytes, thrombocytes and macrophages, but not on erythrocytes of either larvae or adults. Immunohistochemical observations of the hemopoietic organs revealed that the XL-1+ cells with granulocyte and/or macrophage morphology appeared at st.36-37 in the liver, at st.44-45 in the mesonephric and the thymus rudiments, and at st.47 in the spleen. The XL-1 determinant was expressed on the precursor cells of T lymphocytes in the thymus rudiments at st.46-47, on the pre-B cells in the liver rudiments at st.47, and on lymphocytes in the spleen at st.48-49. A few XL-1+ cells were present in the ventral blood island of the st.35/36 embryos, where differentiating erythrocytes had predominated since st.28. XL-1+ cells with a macrophage-like morphology were found in several locations of the mesenchyme in the st.32 embryos, before the establishment of vascularization at st.33/34 and far earlier than the emergence of lymphocytes.     

Thymic Dendritic Cells Are Primary Targets for the Oncogenic Virus SL3-3

The murine retrovirus SL3-3 causes malignant transformation of thymocytes and thymic lymphoma in mice of the AKR and NFS strains when they are inoculated neonatally. The objective of the present study was to identify the primary target cells for the virus in the thymuses of these mice. Immunohistochemical studies of the thymus after neonatal inoculation of the SL3-3 virus showed that cells expressing the viral envelope glycoprotein (gp70⁺ cells) were first seen at 2 weeks of age. These virus-expressing cells were found in the cortex and at the corticomedullary junction in both mouse strains. The gp70⁺ cells had the morphology and immunophenotype of dendritic cells. They lacked macrophage-specific antigens. Cell separation studies showed that bright gp70⁺ cells were detected in a fraction enriched for dendritic cells. At 3 weeks of age, macrophages also expressed gp70. At that time, both gp70⁺ dendritic cells and macrophages were found at the corticomedullary junction and in foci in the thymic cortex. At no time during this 3-week period was the virus expressed in cortical and medullary epithelial cells or in thymic lymphoid cells. Infectious cell center assays indicated that cells expressing infectious virus were present in small numbers at 2 weeks after inoculation but increased at 5 weeks of age by several orders of magnitude, indicating virus spread to the thymic lymphoid cells. Thus, at 2 weeks after neonatal inoculation of SL3-3, thymic dendritic cells are the first cells to express the virus. At 3 weeks of age, macrophages also express the virus. In subsequent weeks, the virus spreads to the thymocytes. This pathway of virus expression in the thymus allows the inevitable provirus integration in a thymocyte that results in a clonal lymphoma.     

Differential participation of ventral and dorsolateral mesoderms in the hemopoiesis of Xenopus, as revealed in diploid-triploid or interspecific chimeras

The thymocytes in the early larvae of Xenopus laevis have been shown to be derived from precursor cells immigrating interstitially through the mesenchyme into the organ rudiments at 3-4 days of age (Nieuwkoop and Faber stages 42-45). Orthotopic grafting of diploid tissues onto triploid stage 22 embryos followed by ploidy analyses of their hemopoietic cells revealed that both thymocytes and erythrocytes in early larvae are derived from the ventral blood islands (VBI), whereas those in late larvae and adults come mainly from the dorsolateral plate (DLP). To study how the VBI cells of embryos at stage 22 participate in hemopoiesis, a number of interspecific chimeras were produced in X. laevis and X. borealis embryos. Sections of the chimeras at various developmental stages were examined by employing the unique stainability of X. borealis nuclei to quinacrine as a marker; the results show that the VBI-derived cells enter into the circulation around stage 35/36, and that some of them leave the blood vessels to migrate interstitially through the mesenchyme toward the thymic rudiment during stages 43-45. A minor population of the VBI-derived cells was also found extravascularly in the mesonephric primordia. In contrast to the VBI, the DLP-derived cells contributed to the hemopoietic cell population not in early larvae, but in late ones as a major constituent in the mesonephros, thymus, liver, and peripheral blood.     

Lymphopoiesis in the nude fetal thymus following sympathectomy

This study was carried out to examine the innervation of the nude fetal thymus during ontogeny and to see if lymphopoietic activity would occur within these thymic lobes in the absence of sympathetic neuronal input. Fetal thymic rudiments from nu/nu mice were removed and examined for galoxylic acid-induced histofluorescence to detect the catecholaminergic nerves. Some of these lobes were organ cultured for 5 to 7 days in the presence of deoxyguanosine to eliminate any existing lymphoid cells within the rudiments. Such "nonlymphoid" thymic rudiments were implanted into the anterior eye chambers of syngenic BALB/c mice (heterozygous) from which cervical sympathetic ganglia and part of the sympathetic chain had been surgically removed (right side) one week earlier. The left side was only sham operated. The thymic implants were allowed to grow for up to 21 days on both sides; they were then removed and examined by histofluorescence, immunofluorescence, and light microscopy. The results indicate for the first time that the nude fetal thymus is innervated by sympathetic nerves and that following sympathectomy the nude thymus is able to sustain lymphopoietic activity and generate lymphoid cells which have characteristics present on thymocytes during in vivo development in normal mice, such as binding to peanut agglutinin and expression of Thy-1 antigen. The relationship between the presence of sympathetic inhibitory influence and the thymic atrophy seen in the nude mice during ontogeny, is being investigated.     

Measles Virus Infection Induces Terminal Differentiation of Human Thymic Epithelial Cells

Measles virus infection induces a profound immunosuppression that may lead to serious secondary infections and mortality. In this report, we show that the human cortical thymic epithelial cell line is highly susceptible to measles virus infection in vitro, resulting in infectious viral particle production and syncytium formation. Measles virus inhibits thymic epithelial cell growth and induces an arrest in the G₀/G₁ phases of the cell cycle. Moreover, we show that measles virus induces a progressive thymic epithelial cell differentiation process: attached measles virus-infected epithelial cells correspond to an intermediate state of differentiation while floating cells, recovered from cell culture supernatants, are fully differentiated. Measles virus-induced thymic epithelial cell differentiation is characterized by morphological and phenotypic changes. Measles virus-infected attached cells present fusiform and stellate shapes followed by a loss of cell-cell contacts and a shift from low- to high-molecular-weight keratin expression. Measles virus infection induces thymic epithelial cell apoptosis in terminally differentiated cells, revealed by the condensation and degradation of DNA in measles virus-infected floating thymic epithelial cells. Because thymic epithelial cells are required for the generation of immunocompetent T lymphocytes, our results suggest that measles virus-induced terminal differentiation of thymic epithelial cells may contribute to immunosuppression, particularly in children, in whom the thymic microenvironment is of critical importance for the development and maturation of a functional immune system.     

Electron Microscopy of Bursa of Fabricius of Chicks Infected with a Field Strain of Infectious Bursal Disease Virus

Chicks were infected in the bursa with a field strain of infectious bursal disease virus. Inter- and intracellular edema, condensation and margination of nuclear chromatin, increased number of lysosomes in macrophages, and lymphocytolytic changes appeared earliest by 8 hours post infection. Inclusions containing spheroid to hexagonal virus particles were seen in the cytoplasm of the macrophages. Multiplying virus particles in crystalline arrays arranged either in single or in multiple clusters were seen in the cytoplasm of macrophages, lymphocytes and light stained reticular epithelial cells.     

Cellular and metabolic requirements for induction of macrophage procoagulant activity by murine hepatitis virus strain 3 in vitro.

The cellular basis for the variation in induction of monocyte procoagulant activity (PCA) by murine hepatitis virus strain 3 (MHV-3) was examined using a set of recombinant inbred strains of mice derived from the resistant (A/J) and susceptible C57B1/6J (B) progenitors. Induction of PCA by MHV-3 required live virus and host protein and RNA synthesis. Absolute restriction for induction of PCA was observed at the level of the macrophage. Peritoneal macrophages from resistant parental A/J and RI strains (AXB5) could not be induced to express PCA when stimulated by MHV-3 alone or in the presence of lymphocytes from susceptible and H-2 compatible RI mice (AXB3) although they did respond to endotoxin (LPS). In contrast, macrophages from both susceptible (AXB3) and semisusceptible (AXB1) RI strains of mice expressed a similar increase in PCA after stimulation with MHV-3 in the absence of lymphocytes. The levels of PCA expressed by macrophages in the presence of Thy-1.2+ lymphocytes correlated with susceptibility to disease. Thy-1.2+ lymphocytes from susceptible RI AXB3 mice could induce levels of PCA in macrophages from semisusceptible RI AXB1 mice equivalent to that seen in cultures of macrophages and lymphocytes from susceptible mice. Further subfractionation of Thy-1.2+ cells demonstrated that L3T4+ cells instructed macrophages to produce PCA. Thy-1.2+ cells from MHV-3 immunized resistant AXB5 mice, but not from non-immunized mice, were able to suppress induction of PCA. This suppressor cell activity could be detected 4 days after immunization, reaching maximal activity at day 7 with significant suppression even at 28 days. The PCA was shown to have direct prothrombin cleaving activity (prothrombinase) by ELISA and immunofluorescence staining using the mAb 3D4.3. These results demonstrate that induction of a unique PCA (prothrombinase) is restricted at the level of the macrophage and define a regulatory role for T lymphocytes in its induction.     

Ontogeny of reticular cells in the ileal Peyer's patch of sheep and goats.

The ontogeny of reticular cells in the ileal Peyer's patch of sheep from 70 days gestational age was studied by light and electron microscopy and by enzyme histochemistry. Small to medium-sized lymphocytes were seen in the lamina propria at 97 days, when the stroma was essentially still mesenchymal. By 110 days, the stromal cells in the dome/follicle primordia had differentiated into reticular fibroblasts, whose processes and fibers were seen to surround groups of lymphocytes. With advancing age the number and size of primordia increased, and proliferation was obvious among the lymphocytes. Processes of reticular cells increased in number and penetrated between individual lymphocytes of the groups. Coarser desmosome-like contacts were seen between the reticular cells from 115 days onwards. A central light area in the follicle was apparent from 130 days onwards. The fine structure of the stromal cells in this light follicle center developed towards but never became similar to that of follicular dendritic cells in a typical germinal center. The fine interdigitating end branches of the stromal cells were less numerous, and the dense homogeneous material present in between the end branches was not observed in the ileal Peyer's patch follicle. Instead, small particles and vesicles were seen between the various cell types of the light center and were not restricted to the intercellular spaces between the stromal cells. In the dark peripheral zone of the follicle, the stromal cells retained more immature features. The follicle became bordered by a capsule at an early stage. This capsule was formed by multiple layers of flattened fibroblasts separated by small amounts of intercellular material only. The alkaline phosphatase, Mg(2+)-dependent adenosine triphosphatase and 5' nucleotidase reactivities of the follicular dendritic cells in the ileal Peyer's patch were similar to those of early prenatal primary follicles of sheep lymph nodes. This study indicates that the stromal cells of the ileal Peyer's patch are mesenchymal in nature and different from those of germinal centers and the epithelial stromal cells of bursa Fabricii of birds.     

A decay of gap junctions in association with cell differentiation of neural retina in chick embryonic development.

Ultrastructural studies of thin-sectioned and freeze-cleaved materials were performed on developing retinal tissues of 3- to 9-day-old chick embryos to clarify the junctional structures between neural retinal cells and between neural retinal cells and cells of the pigmented epithelium. Frequency, size and position of gap junctions in developing neural retina are different at each stage of development. In 3-day-old embryos, some cells adhere to each other by gap junctions immediately below the outer limiting membrane of neural retinae. The size and number of gap junctions increase remarkably during 5-6 days of incubation. In this period of development, well developed gap junctions consisting of subcompartments of intramembrane particles are found between cell surfaces at both the outer limiting membrane region and the deeper portion of the neural retina. Gap junctions disappear thereafter, and at 7-5 days of incubation, small gap junctions are predominant between cell surfaces at the outer limiting membrane region, while the frequency of gap junctions in the deeper portion is very low. At 9 days of incubation, gap junctions are rarely found. Typical gap junctions are always found between neural retinal cells and those of the pigmented epithelium in embryos up to 7-5 days of incubation. Tight junctions are not found in the neural retina or between neural retina and pigmented epithelium throughout the stages examined.     

In-vivo expression of a C-type RNA virus in rat ventral prostate epithelial cells.

During evaluation of a procedure for separating rat ventral prostate epithelial from connective tissue cells, isolated fractions were examined by transmission electron microscopy. Characteristic C-type RNA viruses were seen budding from or in close proximity to the plasma membranes of isolated epithelial cells. Dissociation of rat prostate cells and concentration of the epithelial fraction facilitated detection of this virus.     

Ontogeny and organization of the stationary non-lymphoid cells in the human thymus

Summary Ontogenetic differentiation of the human thymus was investigated in 50 embryos by means of light and electron microscopic methods in an attempt to clarify the morphogenesis of the complicated microecology of thymic tissue. At the 8th gestational week (g.w.), the primordium of the thymus contains almost exclusively undifferentiated epithelial cells. At the 10th g.w., the epithelial cells in the central part are spindle-shaped. During the subsequent weeks the cortical region of the thymus becomes separated into lobes by mesenchymal septa containing hemopoietic precursor cells and large electronlucent cells with irregularly shaped nuclei. The latter cells are also found in the deeper presumptive medullary regions of the thymus; they differentiate into interdigitating reticulum cells (IDC). The permeation of the medulla of the thymus by non-epithelial IDC occurs concurrently with the formation of cortical and medullary epithelial cells. Between the 12th and 14th g.w. the cortical and medullary differentiation is completed. At this time-stage cortical small lymphocytes differ in morphological shape from medullary lymphocytes, the latter acquiring the appearance of immunocompetent T cells and establishing intimate contact with the IDC.These findings indicate that the thymic cortex and medulla contain different epithelial cells. In addition, the thymic medulla displays cells characterized by the morphology of typical interdigitating reticulum cells of peripheral lymphoid tissue. The structural pattern of the thymus is correlated to morphologically differing lymphoid cell populations in the cortical and medullary regions.This investigation was supported by grants from the Deutsche Forschungsgemeinschaft and by the Sonderforschungsbereich 111The authors dedicate this paper to Professor Helmut Leonhardt on the occasion of his 60th birthday. The authors also appreciate the excellent technical assistance of Mrs. I. Knauer, Mrs. H. Waluk and Mrs. H. Siebke