Magnitude and pattern of thymic lymphocyte migration in neonatal mice.

Neonatal mice were given a subcapsular, intrathymic injection of thymidine-(3)H using a modified microneedle technique, and the migration of labeled cells to spleen, lymph nodes, Peyer's patches, and bone marrow was followed radioautographically with time. Assuming that nonlabeled lymphocytes migrated in the same manner as labeled lymphocytes, it can be concluded that the majority of lymphocytes present within mesenteric lymph nodes (74%) and Peyer's patches (61%), and a large proportion of those located in popliteal lymph nodes (40%) and the spleen (26%), were of thymic origin. Evidence is presented indicating that these are minimum values. The difference in the magnitude of thymic cell migration to gut-associated lymphoid tissue on the one hand and to the spleen and popliteal lymph node on the other hand was tentatively attributed to antigenic stimulation from the intestinal flora which develops during the first days of life. Thymus-derived lymphocytes were scattered throughout the lymph node cortex and splenic follicles. No noticeable thymic cell migration to the bone marrow was found. Labeling indices in the peripheral lymphoid organs paralleled those of cortical thymic lymphocytes suggesting the thymic cortex as the major source of migrants. By 2 days postinjection, the mean grain counts of labeled lymphocytes in all peripheral lymphoid tissues were higher than the mean grain counts of labeled lymphocytes in the thymus. At 7 days postinjection heavily labeled cells constituted 11-16% of the labeled population in peripheral tissues while they were absent from the thymic cortex. These results indicate that a fraction of thymus-derived cells, upon settling in the periphery, remained in, or reentered, a nonproliferative phase for at least 7 days. Conversely, many thymus-derived lymphocytes underwent division in the periphery and/or penetrated the intestinal epithelium. Since the relative number of thymus-derived cells found in the mesenteric lymph nodes of 1- and 2-day old mice was considerably higher than the percentage of cells at this site having the theta (theta) alloantigen, as reported by other authors, the possibility exists that theta-antigen on thymus-derived lymphocytes may, at least in a fraction of these cells, no longer be detectable as they reach the peripheral organs.

Specific antibody within lymphoid germinal center cells of mice after primary immunization with horseradish peroxidase: a light and electron microscopic study.

The appearance in mice of specific antibody within newly formed germinal centers in lymph nodes was demonstrated by light and electron microscopy after regional primary antigenic stimulation with horseradish peroxidase (HRP). Lymphoid germinal center cells containing anti-HRP antibody in the perinuclear space and in the cytoplasm were detected from 17 to 26 days after antigenic stimulation. Extracellular anti-HRP antibody within germinal centers, localized between dendritic reticular cells and lymphoid elements, could not be found before the appearance of intracellular antibody. These findings strongly suggest antibody formation by lymphoid germinal center cells. Both antigen and corresponding antibody persisted in intercellular spaces up to 35 days after primary stimulation. The concomitant presence in a given lymph node of germinal centers which are positive or negative with regard to specific antibody provide evidence in favor of monospecificity of individual centers. The mechanisms of antigen-trapping within germinal centers are discussed in the light of the present observations.

Effects of single cyclophosphamide doses on the kinetics of thoracic duct lymph and blood leukocytes in calves.

After 5 or 20 mg cyclophosphamide per kg body weight, given once i.v., the output in thoracic duct lymph of small and large lymphoid cells, cells incorporating [3H]lymidine in vitro, mitotic cells, pyknotic cells, and/or the number of lymphocytes and neutrophils in peripheral blood were measured in six calves. The median grain count of labeled cells and the DNA content of pyknotic nucleic were determined. After both doses there was an exponential decrease and subsequent recovery of the median grain count. The larger dose caused a temporary cessation of lymphoid cell division, reduced the output of nondividing small lymphoid cells, and probably imparied proliferation of neutrophil precursors. The results suggest that increased cell production during recovery was due to changes in the growth fraction and that feedback mechanisms acting on G0-G1 cells controled the proliferation of lymphoid cells.

Chemotherapy-induced complete remission of a malignant fibrous histiocytoma of bone. An 11-year follow-up.

In a young woman with multiple malignant fibrous histiocytoma of bone and soft tissue with a rapidly progressive course, long-term chemotherapy with prednisone and cyclophosphamide resulted in a complete remission still lasting after an 11-year follow-up.

Heavy water (D2O)-induced shape changes, movements and F-actin redistribution in human neutrophil granulocytes.

Heavy water (D2O) induces characteristic shape changes and a distinct type of movement in human neutrophil granulocytes. In contrast to front-tail polarity as evoked by chemotactic peptides and microtubule-disassembling agents, D2O-based media produce non-polar neutrophils with many small or long surface projections. This phenotype is similar to that elicited by both phorbol myristate acetate and diacylglycerols, but the surface projections are smaller and more densely placed and are often associated with a single large projection. D2O-induced non-polar cells with surface projections perform continuous shape changes without front-tail polarity and without the unidirectional movement and cytoplasmic streaming seen in cells with front-tail polarity. Some of the cells show circus movements of a large projection indicating circular polarity. In neutrophils suspended in D2O, F-actin is shifted to the cell periphery, mainly into the surface projections of activated cells. The D2O-induced effects are reversed in H2O-based medium. D2O is dominant over the chemotactic peptide, N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP), colchicine and taxol in that the combined action of D2O with any of these agents results in the D2O-induced phenotype. In contrast, cytochalasin B alone and in combination with fMLP induces a considerable decrease of non-polar cells and an increase of spherical cells similar to non-stimulated cells in H2O-based medium. Earlier studies indicated that D2O acts on microtubules. Our results suggest that D2O may act on the microfilament system. Neutrophils suspended in D2O-based medium may represent a useful model to study the relationship between shapes, movements, and particular functions of these cells.

Physiology of chemotaxis and random motility.

The following aspects of random and directional migration of leukocytes have been considered: (1) basic cellular mechanisms involved in leukocyte locomotion; (2) the mechanisms of formation and the nature of various factors such as cytotaxins, cytotaxin-inactivators and inhibitors participating in the regulation of the chemotactic response, and (3) the relationship between chemotaxis and leukocyte emigration. The need for adequate techniques for measuring chemotaxis has been emphasized with special reference to clinical investigation.

The filter technique for measuring leucocyte locomotion in vitro. Comparison of three modifications.

Three forms of filter technique for measuring random and directional locomotion of leucocytes have been compared: (1) the conventional one filter technique of Boyden (lower surface count method); (2) the two filter system with a lower cell-impermeable filter designed to count the cells at the underside of the upper filter as well as those on the lower filter (two filter count method); and (3) two filter systems counting only cells associated with the lower filter (lower filter count method). In some instances all three methods produce qualitatively similar results. In others totally different results are reproducibly obtained with identical cell preparations, media and attractants. Compared to the two filter count method, the lower surface count method and the lower filter count method are not sufficiently reliable. The discrepancies are partly due to errors in measuring the response. They are caused by variable cell adhesion to the filters resulting in a varying distribution of cells between the upper and lower filter and/or detachment of neutrophils from the upper filter. Some of the discrepancies are not due to errors in assessing the response, but to differences in gradient formation and drift of chemokinetic and chemotactic materials from one compartment to the other.

Stimulation of proliferation of murine lymphocytes by the calcium ionophore A 23187 in the absence of serum: the requirement for thiols.

The proliferation of mouse spleen cells and T-lymphocytes, initiated by the calcium ionophore A 23187 was studied by a serum-free culture technique. In contrast to Con A, A 23187 was capable of stimulating cells only if 2-mercaptoethanol, cysteine and glutathione (reduced form), respectively, were present in the culture medium. In the absence of one of these compounds a stimulating activity of A 23187 was observed only with high concentrations of cells (i.e., 10(7)/ml). With glutathione present, the cells could be stimulated only at concentrations of A 23187 which were found to be suboptimal in cultures with 2-mercaptoethanol. Human serum, fetal calf serum and bovine serum albumin shifted the active and optimally stimulating concentrations of A 23187 to higher values. A similar effect was observed with sera- and Con A-treated cells. The effect of sera and albumin was paralleled by a protecting effect of cells against high concentrations of A 23187.

Interplay between extracellular -SH groups and Ca2+ in their mitogenic action on murine lymphocytes: the role of SiO2.

Murine spleen lymphocytes, cultured under serum-free conditions and stimulated with concanavalin A (Con A), showed an increased demand for supplemental concentrations of Ca2+ when 2-mercaptoethanol (2-ME) or L-cysteine were also present in the culture medium. The requirement for additional Ca2+ ions was particularly evident at high cell concentrations. The addition of SiO2 (Aerosil 200), in concentrations depending on the original level of Ca2+ in the culture medium, could substitute for the increased demand on calcium ions. This dependency of SiO2 effects on calcium levels in the medium was also evident when supraoptimal, i.e. inhibitory concentrations of silica were tested. Results of experiments with 45Ca suggest that silica particles may act as intercellular carriers of calcium. In contrast to spleen lymphocytes, Con A-stimulated thymus cells exhibited neither a need for an increase in the concentration of Ca2+ nor a dependency on the presence of 2-ME in the medium.

The role of 2-mercaptoethanol in the stimulation of spleen cell cultures: increased uptake of cystine into the TCA-soluble pool.

A sensitive system for the study of the mechanism of action 2-mercaptoethanol in stimulating of mouse spleen cell cultures is described. In this system the triggering of stimulation of cells by a mitogen or antigen was dependent on the presence of 2-mercaptoethanol (2-ME) in the medium. To obtain a similar stimulatory effect, 2-ME could be replaced by L-cystine or L-cysteine in the presence or absence of a mitogen. In the presence of a mitogen and below the saturation level, cystine was more effective than cysteine. By increasing in this system, the cystine concentration, to the double of that of the basal medium, a strong stimulation of cells was produced. Conversely, in the absence of mitogen, cysteine was significantly more effective than cystine. Addition of 2-ME led to an increased entry of [14C]L-cystine into the TCA-soluble pool of cells while the thiol had no influence on the uptake of a mixture of different amino acids. We conclude that in this system the role of 2-ME is to enable the utilization of otherwise ineffectively low concentrations of cystine by enhancing the entry of this amino acid into the amino acid pool of the cells.

Germinal center kinetics in lymph nodes of primed mice stimulated with complexed as opposed to free antigen.

Primed mice with low titers of circulating tetanus antitoxin (AB) were stimulated via the hind footpads with either fluid tetanus toxoid alone (AG) to create in vivo complexes in AG excess, or the same dose of toxoid complexed at equivalence with isologous antibody (AB-AG CPX), to have in vivo complexes in AB excess. All experimental animals reacted with three topically distinct consecutive waves of enhanced proliferative activity in popliteal lymph nodes, i.e., in the T-zone (peak on day 2), in the medullary area, the main site of plasmocytopoiesis (day 3), and in lymphoid follicles (day 5-6). Maximum serum AB titers following injection of AG-AB CPX were only about 25% of those found in animals boosted with AG alone. This suppressive effect was best reflected in a comparable reduction in plasmocytopoiesis, and to an lesser extent in the proliferative activity within the T-zone, and not at all in the overall magnitude of germinal center formation and/or expansion. However, the patterns of germinal center kinetics differed markedly between the two groups: a high sharp peak of development on day 5, followed by a marked drop on day 6 characterized the response in mice given AG alone, and a broad peak around day 6 that of those receiving AG-AB CPX. These differences could not adequately be accounted for by variations in centroblast/centrocyte proliferation rate vs. pycnotic indices, so that different patterns of lymphoid cell emigration from the centers may be considered. The results suggest that immune complexes, fixed on follicular dendritic cells, with different antigen-to-antibody ratios have divergent effects on the development and kinetics of germinal centers, the principal sites of memory B cell generation.

The post-natal development of lymph nodes in mice.

A total of ten major lymph node stations have been studied in ICR-strain mice at various time intervals between birth and the age of four weeks. Comparison of lymph node volumes and of volume changes during the observation period revealed that the mesenteric lymph node (MLN) complex was larger at birth and grew at a faster rate than all other nodes. A distinct numerical increase of cortical lymphocytes in MLN at the end of the first week of life was interpreted to be the result of both immigration of T cells and moderate local proliferation, while the quantitatively more important second phase of growth at the age of between two and three weeks might be the consequence of local lymphocyte production. The comparatively modest changes in lymph nodes removed from the gut (axillary and popliteal nodes) could be explained as being due in large measure to immigration of lymphocytes originating in gut-associated lymphoid tissues (GALT).

A proposal for a standardized system of reporting human lymph node morphology in relation to immunological function.

This memorandum proposes a standardized system of reporting the histology of human lymph nodes based on commonly used simple staining techniques. The purpose is to provide a uniform, internationally acceptable system by which the histological structure of lymph nodes can be correlated with other parameters of immunological status. The proposed protocols are intended to provide information that is not available in conventional written reports that use such terms as ;hyperplasia' or ;nonspecific lymphadenitis'.

Human lymph node morphology as a function of age and site.

Cervical, axillary, cubital, inguinal, popliteal, and mesenteric lymph nodes from subjects of various ages who had died a sudden death were examined histologically. Care was taken to establish by morphometry the proportional distribution in lymph node cross-sections of cortical, paracortical, and medullary areas. In addition, numbers and surface areas of cross-sectioned germinal centres were registered. Important differences related to age and anatomical site of lymph nodes were established by this survey. Germinal centre formation, particularly evident in infants and children, less so in young adults, and often absent in ageing individuals, was most impressive in lymph nodes normally exposed to antigenic stimulation (mesenteric and cervical lymph nodes). Paracortical and medullary areas exhibited a slight but gradual reduction with advancing age. Replacement of lymphatic parenchyma by fat tissue (lipomatous atrophy) was a characteristic of more peripheral lymph nodes usually subjected to little antigenic stimulation, that is, cubital, axillary, and popliteal nodes. It should be emphasised that both age-related and regional differences have to be taken into account in a meaningful functional interpretation of lymph node morphology.

Thymic lymphopoiesis: protected from, or influenced by, external stimulation?

The mechanisms regulating thymic lymphopoiesis are still a matter of debate. Intracortical proliferation and differentiation of thymocytes are thought to be controlled by locally produced humoral factors and close contact with epithelial, possibly also phagocytic, cells, and restricted by products of the major histocompatibility complex. The observation of a translocation of intraabdominally introduced PVP-coated silica particles (Percoll) via parathymic lymph vessels and through the thymic capsule into the cortical parenchyma demonstrates that the thymic cortex is accessible to materials carried with the transcapsular flux of interstitial fluid, and that this barrier is less effective than the blood-thymus barrier. The proliferative activity of cortical thymocytes following an intraabdominal injection of particulate tetanus toxoid was compared in sites adjacent to, and distant from, parathymic lymph nodes. Absolute numbers of DNA-synthesizing thymocytes were found to be much higher in cortical areas close to the lymph nodes, where lymphatic vessels are most numerous, than on the opposite sides of the thymic lobes. Taken together, these findings indicate that--in addition to intrinsic control mechanisms--cortical thymocyte production may be influenced by peripheral stimulation to some extent, and that materials from sites which are drained by parathymic lymph nodes may be important in this respect.

Mitotic activity and nuclear DNA damage of large cells in Hodgkin's disease: comparison with the expression of p53 and bcl-2 proteins and the presence of Epstein-Barr virus.

The roles of the bcl-2 and p53 proteins in Hodgkin's disease (HD) are poorly understood. We therefore compared their detected presence in Hodgkin and Reed-Sternberg/large atypical (H-RS/LA) cells immunohistochemically with the percentages of these cells double-labeled for CD30 and DNA strand breaks (DNA fragmentation index, DFI); mitotic indices (MI); and the EBV infection status. We found a highly significant inverse correlation between the fractions per case of H-RS/LA cells expressing bcl-2/p53 proteins and the DFI of CD30+ elements. No marked effect of these two oncoproteins on MI was noticed, although these parameters and DFI of CD30+ cells were linearly related. EBV infection of H-RS/LA cells exerted only a limited effect on the parameters tested. The results of this study suggest that overexpressed bcl-2 and, to some extent, p53 proteins in H-RS/LA cells of HD primarily counteract deletion of these cells.

Cell kinetics, morphology, and molecular IgVH gene rearrangements in Hodgkin's disease.

The present study dealt with the question of whether any cellular kinetic patterns correlate with clonal rearrangement of the IgVH gene as revealed by polymerase chain reaction on DNA extracted from lymph nodes with classical Hodgkin's disease (HD) and/or from single CD30+ cells (Hodgkin [H] and Reed-Sternberg [RS] cells). In 15/4 cases with H-RS cells of B or Null phenotype, signs of such monoclonality could be detected (group I) but not in the others (group II). CD30+/H-RS cells in group I differed slightly but significantly from those in group II in that they a) exhibited a larger fraction of cells attaining the anaphase/telophase stage of mitosis, and b) produced relatively more mononucleated cells (H) at the expense of multinucleated (RS) cells. In addition, reactive lymphoid cell (CD30-) infiltrates were considerably less dense in group I that in group II. These findings suggest that the cytokinesis of H-RS cells in group I was moderately more efficient than in group II. However, signs of monoclonality were not associated with the normalization of the mitotic process, which also proved to be disturbed in group I.

Abortive mitoses and nuclear DNA fragmentation in CD30+ large cells of Hodgkin's disease.

This study was undertaken to better comprehend the reasons for the scarcity of Hodgkin and Reed-Sternberg (H-RS) cells in Hodgkin's disease (HD) despite their expression of "proliferation-associated antigens". To this end, we assessed the relative frequency of mitotic phases and nuclear damage (detected by in situ end-labeling of DNA strand breaks) in CD30+ large cells of nodular sclerosis and mixed cellularity HD. Our results show that a) most CD30+ cells in HD exhibit abortive mitoses, with a highly significant arrest at the metaphase-ana/telophase transition, and b) many of these elements, i.e. mainly H-RS cells, show fragmentation of nuclear DNA, suggesting imminent or actual death. Percentages of CD30+ cells that entered mitosis and those with DNA strand breaks were of a similar order of magnitude and correlated significantly in a linear fashion. These findings are consistent with the concept that cell deletion is the major cause of the paucity of H-RS cells in HD.

Oncocytic (Hürthle cell) tumors of the thyroid: distinct growth patterns comparedwith clinicopathological features.

Neoplastic growth results from cell production that exceeds cell loss. We registered mitotic and apoptotic indices (MI and AI) in 97 immunohistochemically verified oncocytic (Hürthle cell) tumors of the thyroid (OT; 50 adenomas [OA], 20 atypical adenomas [aOA], and 27 carcinomas [OC]) and compared these kinetic data with histological diagnoses and other parameters. MI, although very low in all, was significantly higher in carcinomas than in adenomas. Conversely, AI did not differ as much among the 3 groups. This indicates that the magnitude of cell deletion did not play a prominent role in determining the disparate growth of the 3 types of oncocytic tumors. Cluster analysis with MI and AI per case as variables revealed the existence of 3 groups of neoplasms with highly distinct growth characteristics: (1) near-steady state (n = 78, all diagnostic categories represented); (2) progressive (n = 9, mostly carcinomas); and (3) regressive (n = 10, mostly adenomas). MI distinguished between histologically benign and malignant with the greatest discriminant power of the variables tested. Proliferative indices should thus be included in the differential diagnostic evaluation of oncocytic thyroid tumors. Our study also suggests that invasiveness and growth are 2 diverging properties of carcinomas.

Fixation of bone fragments with BIOCEM. First observations on humans.

BIOCEM is a recently developed material that consists of bisphenol-a-glycidyl methacrylate ("epoxide methacrylate") as the organic matrix and pentacalcium hydroxide triphosphate ("tricalcium phosphate") with or without bioceramic A2 as the filling particles. Previous animal experimentation has demonstrated that BIOCEM can establish and maintain direct contact with bone without compromising tissue vitality. Rather, it favors with time the ingrowth of, and coverage by, newly formed bone, thus creating interdigitations and strong fixation of the implant. This novel technique has now, for the first time, successfully been applied in humans, ie, for the fixation of frontal sinuses. Clinical, radiological, and histological findings are briefly reported, and it is also shown that the frontal sinus mucosa had recovered at the inside of the lesions filled with BIOCEM.