Ultrastructural study of the FVB/N mouse thymus: presence of an immature epithelial cell in the medulla and premature involution.

FVB/N inbred mice have been widely used to generate a variety of transgenic lines, but their physiology and especially their immunological characteristics are poorly documented. We therefore studied the ultrastructure of the thymus and the distribution of thymocyte subpopulations in FVB/N mice at several ages. In young FVB/N mice the stromal microenvironment exhibits the three types of epithelial cells and the two types of bone-marrow derived cells (macrophages and interdigitated cells) previously described in other strains of mice. Moreover, in the thymic medulla of young FVB/N mice, a fourth cell type with the morphological characteristics of an immature epithelial cell was present in relatively high number. Furthermore, thymocyte subpopulations distribution shows an earlier thymocyte maturation than in other strains. Finally, changes associated with thymic involution were observed about 5 months earlier than in many other mouse strains. Our results demonstrated that the FVB/N strain has a specific immunological status.

Apoptosis in parathyroid hyperplasia of patients with primary or secondary uremic hyperparathyroidism.

BACKGROUND: Chronic oversecretion of parathyroid hormone (PTH) is associated with parathyroid hyperplasia, reflecting a disturbed balance between cell proliferation and apoptosis. This study addressed the unsolved issue of apoptosis in hyperparathyroidism. METHODS: Parathyroid glands from 19 patients with primary (1 degrees ) and 11 patients with secondary (2 degrees ) uremic hyperparathyroidism, as well as 13 normal parathyroid glands, were examined. Apoptosis was evaluated by terminal deoxynucleotidyl transferase (Tdt)-mediated dUTP nick end-labeling assay (TUNEL). Because the apoptotic process is regulated by several oncoproteins, the expression of Bcl-2 and Bax was analyzed by immunohistochemistry. RESULTS: The numbers of apoptotic cells in 1 degrees parathyroid adenoma (0.99 +/- 0.03 per 1000 cells, mean +/- SE, P < 0.009) and 2 degrees parathyroid hyperplasia (1.20 +/- 0.54 per 1000 cells, P < 0.005) were significantly higher than in normal parathyroid tissue (0.13 +/- 0. 06 per 1000 cells). Light microscopy examination of hyperplastic parathyroid tissue from a uremic patient showed the presence of nuclei with dense chromatin characteristic of apoptosis. Bcl-2 staining was strong in normal tissues but weak or negative in several sections of 1 degrees and 2 degrees hyperparathyroid tissues, mostly in nodular areas. Bax staining was homogeneous in normal tissue but patchy in several hyperplastic tissues. CONCLUSION: These results suggest that hyperparathyroidism is associated with a compensatory increase in apoptosis, possibly favored by a diminished Bcl-2/Bax ratio. This renders highly improbable the hypothesis that parathyroid hyperplasia is due to a decreased rate of apoptosis.

Persistence of Ca2+-sensing receptor expression in functionally active, long-term human parathyroid cell cultures.

An original human parathyroid cell culture model from uremic patients with IIo hyperparathyroidism has been developed, with its main feature being long-term functionally active viability up to 5 months, as assessed by persistent responsiveness to changes of extracellular Ca2+ concentrations ([Ca2+]e). In addition to the inhibitory effect of increasing [Ca2+]e, increasing extracellular phosphate exerted a biphasic effect on parathyroid hormone (PTH) secretion. The presence of the Ca2+-sensing receptor (CaR), on which depends the response to [Ca2+]e and its persistence, has been demonstrated in our culture system both by direct detection and by inhibition of its activity. CaR protein was detected by Western blot analysis with a specific anti-CaR antibody. CaR gene transcripts have been identified by reverse transcription-polymerase chain reaction analysis. mRNA (by in situ hybridization) and protein (by immunocytochemistry) expression were detected for both CaR and PTH. Adding a specific anti-CaR antibody to the medium induced a marked reduction of low [Ca2+]e-stimulated PTH release, which decreased to levels equivalent to those obtained in high [Ca2+]e medium. The described long-term functionality could be due to several factors, including the clustered cell type of culture yielded by our preparation procedure, the growth characteristics of hyperplastic uremic tissue, and the use of a phosphate-rich medium. The present model, because of its long-term functionality, is a unique tool for the exploration of PTH synthesis and secretion and for studies of parathyroid cell growth in vitro.

Massive mammary gland infection and pregnancy-dependent mammary tumor development in mice infected neonatally with mouse mammary tumor virus (SW) but not in mice infected as adults, despite a dramatic local response.

Mouse mammary tumor virus (MMTV) (SW) caused a high incidence (65%) of pregnancy-dependent adenocarcinomas in BALB/c(SW) mice infected as newborns by suckling their mothers' milk. These tumors were type B adenocarcinomas which developed early, at about 1 year of age. Uninfected breeding females and those infected at an age of 8 weeks by injection of virus had the same low incidence of malignant tumors (13%), and the tumors developed later (at approx. 23-24 months). The low incidence of tumors in adult-infected mice was correlated with partial infection of the mammary glands, and delayed transmission of MMTV(SW) to the offspring. Although the virus was rapidly disseminated in both types of infection, the responses of neonatally infected and adult-infected mice to MMTV(SW) infection and viral superantigen (vSAG) presentation were different. Activation by and presentation of the vSAG was impaired in mice infected neonatally, and tolerance induction by clonal deletion was delayed. Local activation was dramatic in mice infected as adults and clonal deletion followed rapidly. Although interaction between B and T cells is needed for completion of the virus life cycle and viral amplification, the strong local immune response to MMTV(SW) in adult-infected mice limits mammary gland infection, and protects them against mammary tumor development.

Premature thymic involution, observed at the ultrastructural level, in two lineages of human-SOD-1 transgenic mice.

The human Cu/Zn superoxide dismutase (hSOD-1) gene, catalyses the dismutation of O2 to H2O2 and O2. It is located on chromosome 21 in q22.1 and is overexpressed in Down's syndrome (DS) patients. These patients present various abnormalities including mental retardation, congenital heart disease, immunological deficits and premature aging. In order to explore the potential role of SOD-1 overexpression in DS, we have generated two lineages of transgenic mice for the hSOD-1 gene and studied, at the ultrastructural level, the effect of hSOD-1 overexpression on the thymic microenvironment. Modification of the cellular architecture and morphology associated with a lipidic invasion, signs of a premature involution of the thymus, were observed in both lineages. A rupture of the filamentous network in the extracellular and probably also in the intracellular matrix was first observed. These results correlate the thymic alterations visualized in light microscopy, on the thymus from DS patients, and raise the question of the relationship between the SOD-1 overexpression and the different morphological alterations associated with the premature thymic involution observed in SOD-1 transgenic mice. They suggest that thymic and immunological impairments present in DS patients may be related to the SOD-1 gene dosage effect.

Mouse mammary tumor virus production by thymic epithelial cells in vivo.

Exogenous mouse mammary tumor viruses (MMTV) replicate in the mammary glands of infected females, and so infect the suckling pups. We have previously shown that the virus is rapidly disseminated to all the lymphoid organs, including the thymus. The present electron microscope immunohistochemical study describes the viral production site in the thymus. Viral buds and viral proteins were restricted to the thymus medullary epithelial cells. MMTV-encoded proteins were identified on the free viral particles and on the budding ones, the ribosomes, the membrane of the endoplasmic reticulum, and on the membrane of the medullary type II epithelial cell vacuolar network. The thymus medullary epithelial cells can thus integrate the virus and allow viral replication. The results support earlier results indicating that in some experimental conditions, epithelial cells may be involved in MMTV-induced negative selection by showing that thymic epithelial cells do express MMTV-encoded proteins.

Ultrastructural study of thymic microenvironment involution in aging mice.

Aging involves morphological alterations of the thymus and deregulation of various immune response parameters. Altogether, these phenomena have been termed thymic involution. Using electron microscopy, we studied the morphological ultrastructure of the thymic microenvironment in aged mice. We observed cellular damages which progressively affected all the thymic stroma. At later stages (i.e., about 18-20 months old), a disappearance of the organ architecture with a drastic decrease in lymphocyte number was observed. The loss of cellular integrity of the microenvironment with lysis of cellular membranes and formation of a large and clear cytoplasmic layer engulfing a few remaining lymphocytes was noted. Extensive lipidic invasion surrounding the remaining epithelial cells grouped in nest formations and/or bordering cytics cavities was also present in these thymus from aged mice. Because the thymic microenvironment plays an important role in the "education" and functional maintenance of T cells and because the alteration of this cellular entity precedes a decline in certain immune functions, it can be suggested that membrane alterations, lack of cellular microenvironment integrity, and T cell dysfunction are correlated.

Transgenic mice overexpressing the human Cu/Zn-SOD gene: ultrastructural studies of a premature thymic involution model of Down's syndrome (trisomy 21).

It has been suggested that the overexpression of copper-zinc superoxide dismutase (SOD-1) in Down's syndrome (DS) patients may be involved in expression of some of the phenotypic characteristics observed in these patients. To explore the possible role of SOD-1 overexpression in the premature thymic involution and immunologic disorders observed in DS patients, transgenic mice overexpressing the human SOD-1 gene have been generated and their thymuses have been studied at the ultrastructural level. Our observations show premature involution of the thymus in SOD-1 transgenic mice, with a strong modification of the thymic microenvironment starting at approximately 3-4 months of age. The thymic microenvironment in 7-month-old transgenic mice is similar to that observed in 20-month-old control mice. We suggest that these results are consistent with the role of SOD-1 overexpression in the early thymic involution observed in DS patients. These transgenic mice provide an interesting model to investigate the deleterious effect of increased dosage of some chromosome 21 genes such as SOD-1 in the pathogenesis of DS.

Thymic reticulum of mice. III. The connective compartment (innervation, vascularisation, fibrous tissues and myoid cells).

T lymphocytes interact at various levels of differentiation, with cells of the thymic reticulum, forming a peculiar and complex microenvironment. Following earlier descriptions by electron microscopy of three types of epithelial cells and two types of non-epithelial cells (macrophages and interdigitated cells) forming the thymic microenvironment, we report a study on a third compartment, the connective tissue, whose elements occur throughout the organ. The components of the capsule and trabeculae, the vascularisation and the innervation of the thymus and the presence of a few myoid cells are described. This is very rarely studied in ultrastructure. All these cells are completely imbricated and form a network trapping the lymphocytes, playing an essential role in the differentiation, maturation and selection of T cells.

The ER-TR4 monoclonal antibody recognizes murine thymic epithelial cells (type 1) and inhibits their capacity to interact with immature thymocytes: immuno-electron microscopic and functional studies.

The thymic stroma is heterogeneous with regard to cellular morphology and cellular function. In this study, we employed the monoclonal antibody ER-TR4 to characterize stromal cells at the ultrastructural level. To identify the labelled cell type, we used two techniques: immunogold labelling on ultrathin frozen sections and immunoperoxidase staining on thick "vibratome" sections. ER-TR4 reacted with thymic Type 1 epithelial cells (according to our classification). A dense labelling appears in the cytoplasm of cortical cells using the two techniques. Immunogold labelling identified small cytoplasmic vesicles whereas the cytoplasm and the cell membrane seem to be labelled with the immunoperoxidase technique. ER-TR4 also identified isolated thymic nurse cells (TNC), and was observed in vitro to inhibit the capacity of some type 1 epithelial cells to establish interactions with immature thymocytes. This finding supports the hypothesis that the factor is involved in the formation of lymphoepithelial interactions within thymic nurse cells, and thus in the relations that immature thymocytes establish with the thymic microenvironment.

Stimulation of ileal calcium absorption by sorbitol, L-xylose, or creatine via a decrease in luminal sodium concentration: relation with concomitant changes in enterocyte energy metabolism.

Ligated ileal loops, 30 cm in length, of 4-month-old male Wistar rats were instilled with 3 ml of a 10 mM CaCl2 solution (added with 0.25 muCi 45Ca) in the absence (control) or presence of 100mM sorbitol, L-xylose, or creatine. Ileal calcium (Ca) transport, measured by plasma 45Ca appearance, was found to be similar 30 minutes after fluid instillation in all four instances. However, thereafter, 45Ca appearance in plasma did not increase further in control animals whereas it increased twice as much during the subsequent 30 minutes in the presence of sorbitol, L-xylose, or creatine. However, when loops of similar length were instilled with only 1.0 ml of such solutions, the sorbitol effect was already observed during the first 30 minutes. The stimulation of ileal Ca absorption induced by the presence of sorbitol appeared to be due to a cellular effect, associated with a decreased flux across the paracellular pathway, as indicated by 3H-mannitol absorption. The presence of sorbitol in instilled ileal solution induced a significant decrease in luminal Na, K, bicarbonate, and Cl concentrations at each time point studied (30, 60, 120, or 240 minutes after instillation). Thirty minutes after instillation, no difference in soluble Ca concentration was observed between control and experimental rats. After 60 minutes, Ca concentration was dramatically decreased in control rats but it remained nearly constant in experimental animals. Thus, the presence of substances enhancing ileal Ca transport favored the maintenance of soluble Ca in ileal solution during longer time periods than their absence. In the ileal enterocyte, these substances induced a twofold increase of ATP content compared with controls.(ABSTRACT TRUNCATED AT 250 WORDS)

[In vitro effect of citrate and phosphates on calcium transport in the rat ileum]. / Effet du citrate et des phosphates sur le transport du calcium dans l'iléon de rat in vitro.

OBJECTIVES AND METHODS: The effect of the presence of phosphates or citrate on ileal calcium (Ca) absorption was studied by using rat ileum mounted in a Ussing chamber. RESULTS: Mucosa-to-serosa and serosa-to-mucosa undirectional fluxes were not modified by mucosal addition of 2.8 mM Na phosphate or 2.0 mM Na citrate to Krebs-Ringer-bicarbonate buffer containing 1.25 mM CaCl2. However, 50 mM Na citrate induced a significant and similar increase of both unidirectional Ca fluxes, leading to no modification of net Ca flux. This high citrate concentration was associated with a significant increase of tissue conductance and no alteration of zona adherens and tight junction as observed by electron microscopy. CONCLUSIONS: Thus in the absence of any Ca gradient along the epithelium, even at high concentration, citrate did not induced modification of ileal Ca absorption.

Mixed phenotype murine leukemias.

Cell lines were derived from eight individual leukemias induced by X-rays in NFS mice. First typed as null cells (surface immunoglobulin negative, Thy-1 negative), they turned out to have a mixed phenotype with myeloid cytochemical markers, pre-B surface antigens and molecular markers of pro-B lymphocytes. They represent murine models for mixed phenotype (pro-pre-B-myeloid) leukemias.

Increased brush-border membrane calcium transport in the intestine, but not the kidney tubule, of spontaneously hypertensive rats.

Since we and others have found a decrease in intestinal Ca2+ absorption and renal Ca2+ reabsorption in the mature spontaneously hypertensive rat (SHR) at the tissue and cell level, we asked whether the transport defect was located at the luminal or the basolateral side of the epithelial cell. We studied intestinal and renal Ca2+ transport using isolated epithelial brush-border membrane vesicles (BBMVs) in order to examine the luminal side of this transport. For technical reasons, the preparation of intestinal BBMVs was performed using a centrifugation technique, but for renal BBMVs a precipitation method was used. The vesicles obtained with these two different techniques had markedly different aspects by electron microscopy analysis. However, no morphological difference was apparent between the two rat strains for BBMVs of either preparation. SHR and normotensive control Wistar-Kyoto (WKY) rats were studied at the age of 5 and between 12 and 14 weeks, receiving a normal Ca (1%) and P (0.46%) diet. In 5 week old SHR, duodenal BBMV Ca2+ uptake kinetics were similar to that of WKY of same age. However, in 12 week old rats mean (+/- SD) Vmax of duodenal Ca2+ uptake was significantly enhanced in SHR compared with WKY (0.59 +/- 0.21 v 0.38 +/- 0.09 nmol/mg protein and 10 s, P < .01), whereas Km was similar in the two strains. By contrast, no difference was found for Vmax or Km of Ca2+ uptake in renal BBMVs in 12 week old rats. In conclusion, Ca2+ uptake was either enhanced (duodenum) or normal (kidney tubule) in the mature SHR, compared with the WKY of same age.(ABSTRACT TRUNCATED AT 250 WORDS)

Mast cells and their committed precursors are not required for interleukin-3-induced histamine synthesis in murine bone marrow: characteristics of histamine-producing cells.

In the present study we investigate the nature of the murine bone marrow cell subset responsible for the marked increase in histamine synthesis induced by interleukin-3 (IL-3). Because mast cells, and eventually their committed precursors, represent a potential source of histamine in this context, we examined their possible participation in this biologic activity with particular attention. We provide evidence that neither of these populations respond to IL-3 in terms of histamine synthesis and that other differentiated end cells or stromal components of the bone marrow are also not involved in this phenomenon. Starting from these findings, we further characterized the immature hematopoietic compartment responsible for IL-3-induced histamine synthesis using fluorescence-activated cell sorter (FACS) sorting based on rhodamine retention or wheat germ agglutinin (WGA) affinity. These procedures have allowed us to ascribe the following features to histamine-producing cells: (1) They belong to a low-density, progenitor-enriched bone marrow subset containing cells of relatively important size and internal structure. (2) The highest histamine levels are generated by the rhodamine-bright fraction of this population, while the most primitive rhodamine-dull cells do not express this biologic activity. (3) Histamine-producing cells do not copurify with colony-forming units in spleen day 7 and day 12 in WGA-bright fractions. (4) Their enrichment is associated with increased frequencies of cells forming colonies in methylcellulose (CFU-C), suggesting the involvement of several progenitors with partially limited differentiation potential in this biologic activity.

[Study of histidine decarboxylase activity (HDC) in murine fetal hematopoietic organs. Description of basophil-enriched splenic cell population during fetal life]. / Etude de l'activité histidine décarboxylase (HDC) dans les organes hématopoïétiques foetaux murins. Description d'une population de cellules spléniques riche en basophiles au cours de la vie foetale.

The role of histamine in vitro during hematopoiesis has been described by several authors. This work was carried out to determine whether histamine could be available in hemopoietic organs by measuring the HDC activity and the histamine content of developing hemopoietic tissues from C 57 BL/6 mice during fetal life (liver from the 12th day of gestation, spleen from the 14th day, and bone-marrow from the 17th day) and postnatal life. High values were found in the liver, the bone marrow, and especially the spleen between the 17th and the 19th days of gestation. A rapid fall in these values was seen near birth. Interestingly, electron microscopy analysis of day 18 fetal spleen cells, provides evidence for a basophil-rich cell population (25%).

Thymus reticulum of autoimmune mice. 3. Ultrastructural study of NOD (non-obese diabetic) mouse thymus.

The non-obese diabetic (NOD) mouse develops spontaneous insulin-dependent diabetes mellitus. Converging lines of evidence indicate that the disease is of autoimmune origin and is primarily mediated by T cells. It thus appeared interesting to study the morphology of the thymic microenvironment in order to determine whether the architecture and/or the cellular components of the organ are altered. In the NOD mouse, significant aspects of involution were observed as early as the first month of life, forming a heterogeneous pattern with non-involuted areas. With time, these involuted aspects increased in surface and severity. In non-involuted zones vacuolization of epithelial cells was noted, as well as infiltration by plasma cells and the presence of numerous macrophages with high phagocytic activity. Involuted areas, forming a cellular layer as if cells had lost their limiting membranes, were crossed by a great number of cystic cavities bordered by epithelial cells and cells containing granulations. Their lumens contained lymphocytes and a few macrophages. These observations, which are reminiscent of similar reports made in other autoimmune mouse strains, may be related to the functional thymic abnormality thought to participate in the pathogenesis of autoimmune disease.

Effect of cytokines on thymic hematopoietic precursors. Phenotypic and electron-microscopic study.

We have previously shown that the interaction of thymocytes with thymic accessory cells (macrophages and/or interdigitating cells) is one of the factors required for thymocyte activation. Precursors of both thymic accessory cells and thymocytes are included in the CD4- CD8- Mac-1- Ia- subpopulation, and their respective maturation and/or activation may be modulated by granulocyte-macrophage colony-stimulating factor, interleukin 1 and interleukin 2. When CD4- CD8- thymic cells are activated with granulocyte-macrophage colony-stimulating factor plus interleukin 2, both macrophages and interdigitating-like cells are present, as shown by electron microscopy. When activated with interleukin 1 plus interleukin 2, the interdigitating-like cell is the only accessory cell present. In both culture conditions, large clusters are formed between interdigitating cells and lymphoid cells. These results have led us to propose two-step signals for thymocyte proliferation: first, the maturation of macrophages under granulocyte-macrophage colony-stimulating factor control and the production of interleukin 1, and secondly, the maturation of interdigitating cells under interleukin 1 control, their clustering with thymocytes which are then activated.

Ultrastructural studies of mouse thymic reticulum. II. Non-epithelial component.

In this study we describe two types of non-epithelial cells forming a part of the thymic reticulum: macrophages with high phagocytic function, present in the cortex and medulla of the organ, and interdigitated cells present at the corticomedullary junction and in the medulla. These cells, in relation with epithelial cells, form a meshwork, a thymic microenvironment which influences the differentiation and maturation of T lymphocytes. These non-epithelial cells were probably mobile and their precursors exist in bone marrow. It has not yet been determined whether they are both of the same lineage and whether there is or is not common lineage between macrophages and interdigitated cells. Their role as accessory cells in the immune response seems evident. We will compare our observations with those of other authors. We will also discuss several issues concerning these two cell types; their nomenclature, their interrelationship in the thymic reticulum, their function, and their relationship to other similar cells in situ and to cells isolated in vitro, which perhaps are similar.