Electron microscopy of bone.

Mineralized tissues like bone, dentin and mineralized cartilage are difficult to prepare for ultrastructural analysis. In general, the higher the level of mineralization is, the more difficult it is to obtain ultrathin sections of these tissue. Tissues with a low level of mineral, e.g. from young animals, are rather simple to prepare and sectioning is not that much of a problem. In the present chapter we describe step-by-step how to prepare mineralized tissues for ultrastructural examination.

Treatment and complications in elderly stage III colon cancer patients in the Netherlands.

BACKGROUND: We evaluated which patient factors were associated with treatment tolerance and outcome in elderly colon cancer patients. DESIGN: Population-based data from five regions included in the Netherlands Cancer Registry were used. Patients with resected stage III colon cancer aged ≥75 years diagnosed in 1997-2004 who received adjuvant chemotherapy (N = 216) were included as well as a random sample (N = 341) of patients who only underwent surgery. RESULTS: The most common motives for withholding adjuvant chemotherapy were a combination of high age, co-morbidity and poor performance status (PS, 43%) or refusal by the patient or family (17%). In 57% of patients receiving chemotherapy, adaptations were made in treatment regimens. Patients who received adjuvant chemotherapy developed more complications (52%) than those with surgery alone (41%). For the selection of patients who had survived the first year after surgery, receiving adjuvant chemotherapy resulted in better 5-year overall survival (52% versus 34%), even after adjustment for differences in age, co-morbidity and PS. CONCLUSION: Despite high toxicity rates and adjustments in treatment regimens, elderly patients who received chemotherapy seemed to have a better survival. Prospective studies are needed for evaluating which patient characteristics predict the risks and benefits of adjuvant chemotherapy in elderly colon cancer patients.

[Spontaneous dental abscesses in familial hypophosphatemic rickets]. / Multipele spontane odontogene abcessen bij familiaire hypofosfatemische rachitis.

Symptoms of familial hypophosphatemic rickets are growth retardation, the formation of O- or X-legs, pain of the joints, spontaneous dental abscesses, and delayed tooth eruption. The dental symptoms are predominantly attributable to the demineralization of dentin. In absence of adequate preventive measurements,familial hypophosphatemic rickets may lead to spontaneous pulpal necrosis. The prophylactic application of occlusal sealants might be effective in preventing abscess formation.

Rapid combined light and electron microscopy on large frozen biological samples.

The use of large unfixed frozen tissue samples (10 x 10 x 5 mm(3)) for combined light microscopy (LM) and electron microscopy (EM) is described. First, cryostat sections are applied for various LM histochemical approaches including in situ hybridization, immunohistochemistry and metabolic mapping (enzyme histochemistry). When EM inspection is needed, the tissue blocks that were used for cryostat sectioning and are stored at -80 degrees C, are then fixed at 4 degrees C with glutaraldehyde/paraformaldehyde and prepared for EM according to standard procedures. Ultrastructurally, most morphological aspects of normal and pathological tissue are retained whereas cryostat sectioning at -25 degrees C does not have serious damaging effects on the ultrastructure. This approach allows simple and rapid combined LM and EM of relatively large tissue specimens with acceptable ultrastructure. Its use is demonstrated with the elucidation of transdifferentiated mouse stromal elements in human pancreatic adenocarcinoma explants grown subcutaneously in nude mice. Combined LM and EM analysis revealed that these elements resemble cartilage showing enchondral mineralization and aberrant muscle fibres with characteristics of skeletal muscle cells.

Involvement of V-ATPases in the digestion of soft connective tissue collagen.

The contribution of vacuolar H+-ATPases (V-ATPases) to collagen degradation was investigated in soft connective tissue explants (periosteum). Immunolocalisation showed faint to intense staining of cells throughout the periosteum. The V-ATPase inhibitors, bafilomycin A1 and folimycin, decreased overall collagen degradation by 40 and 50% after 24 and 48 h, respectively. The participation of V-ATPases in intracellular degradation of collagen was demonstrated by the decrease of the amount of phagocytosed collagen in fibroblasts upon inhibition of pump activity. The inhibition of degradation was not due to a reduction in activity of gelatinase A, an enzyme previously found to mediate collagen degradation, as assessed by zymographic analysis of tissue and conditioned medium. Bafilomycin A1 even induced an increase of gelatinase A and B levels in both fractions. In conclusion, acidification by V-ATPases may represent an important mechanism in extracellular and intracellular collagen degradation in soft connective tissue.

Participation of intracellular cysteine proteinases, in particular cathepsin B, in degradation of collagen in periosteal tissue explants.

The involvement of cysteine proteinases in the degradation of soft connective tissue collagen was studied in cultured periosteal explants. Using cysteine proteinase inhibitors that were active intracellularly or extracellularly (Ep453 and Ep475, respectively), it was shown that over-all collagen degradation, as measured by the release of hydroxyproline, decreased significantly on inhibition of the intracellular pool of cysteine proteinases by Ep453. This inhibitor also induced an accumulation of intracellular fibrillar collagen in fibroblasts, indicating a decreased degradation of phagocytosed collagen. The extracellular inhibitor, Ep475, had minor or no effects. Histochemical analysis using a substrate for the cysteine proteinases cathepsins B and L revealed a high level of enzyme activity, which was completely blocked in explants preincubated with a selective intracellular inhibitor of cathepsin B, Ca074-Me. Moreover, the cathepsin B inhibitor strongly affected collagen degradation, decreasing the release of hydroxyproline and increasing the accumulation of phagocytosed collagen. These effects were comparable or slightly stronger than those found with the general intracellular inhibitor (Ep453). Taken together, these data strongly suggest that intracellular cysteine proteinases, in particular cathepsin B, play an important role in the digestion of soft connective tissue collagen.

EGF and IL-1 alpha modulate the release of collagenase, gelatinase and TIMP-1 as well as the release of calcium by rabbit calvarial bone explants.

Matrix metalloproteinases (MMPs), among which is collagenase (MMP-1), are likely to be involved in various steps of the bone resorption process. As both production of these enzymes and bone resorption appear to be mediated by cytokines, we investigated the effects of two cytokines, IL-1 alpha and EGF, on the release of collagenase, gelatinase A (MMP-2), gelatinase B (MMP-9), TIMP-1 and calcium by rabbit calvariae. It was found that all these parameters increased under the influence of these cytokines. The release of calcium--used as a parameter of bone resorption--was highest in the combined presence of the cytokines. Although the absolute and relative enhancement by a combination of IL-1 alpha and EGF was most pronounced for collagenase (7-fold), both gelatinase A (5-fold) and gelatinase B (1.5-fold) had increased simultaneously. Calvariae produced a high level of MMP inhibitor (TIMP-1), especially under the influence of the cytokines; periosteum released little inhibitor. It is concluded that IL-1 alpha and EGF are likely to play a modulating role in the process of bone resorption.

Cytokines modulate phagocytosis and intracellular digestion of collagen fibrils by fibroblasts in rabbit periosteal explants. Inverse effects on procollagenase production and collagen phagocytosis.

Degradation of fibrillar collagen may occur in the extracellular space by enzymes, such as the metalloproteinase collagenase, or in the lysosomal apparatus of fibroblasts following phagocytosis. As the mechanisms involved in the regulation of the latter process are unknown, we investigated possible modulating effects of the cytokines epidermal growth factor (EGF), platelet-derived growth factor (PDGF), interleukin-1 alpha (IL-1 alpha) and transforming growth factor-beta (TGF-beta) on both collagen phagocytosis and the release of collagenase in an in vitro model employing periosteal tissue explants. The data demonstrated that the level of intracellular collagen digestion could be influenced by cytokines: IL-1 alpha inhibited and TGF-beta enhanced phagocytosis of fibrillar collagen by periosteal fibroblasts, whereas the cytokines had an opposite effect on the release of procollagenase. In combination, IL-1 alpha and TGF-beta proved to have an antagonizing effect on either parameter. PDGF and EGF had no effect on phagocytosis or collagenase release. The level of phagocytosed collagen correlated positively with the actual breakdown of collagen as assessed by the release of hydroxyproline but negatively with the level of released procollagenase. Our findings demonstrated that cytokines are able to modulate both the phagocytosis of collagen fibrils by fibroblasts and their subsequent intracellular breakdown, as well as the release of procollagenase, an enzyme considered crucial for extracellular collagenolysis. Moreover, our data show a negative correlation between these two parameters. It is concluded that IL-1 alpha, EGF and TGF-beta may be important in modulating the contribution of the intracellular and extracellular route of collagen breakdown.

Ectopic mineralized cartilage formation in human undifferentiated pancreatic adenocarcinoma explants grown in nude mice.

Mineralized as well as nonmineralized cartilage-like structures enclosing cells resembling chondrocytes were found in human-derived undifferentiated but not in poorly differentiated pancreatic adenocarcinoma explants grown in nude mice. The structures reacted with anti-mouse IgG but not with antibodies against human cytokeratin 19, indicating that the newly formed tissue was of mouse origin. High activity of alkaline phosphatase was found in cell layers surrounding the structures and in cells embedded in the matrix. The extracellular matrix was strongly positive after Sirius red staining, reacted with anti-collagen type II antibodies, and the presence of proteoglycans was demonstrated with Alcian blue staining and by metachromasia after Giemsa staining. Electron microscopic inspection revealed the presence of bundles of both thick collagenous fibrils with low levels of fine filamentous material and thin collagenous fibrils with high concentrations of filamentous components. The majority of both types of matrices was found to be partially or completely calcified. The mean area density of the cartilage-like structures in the undifferentiated tumors was 0.31%. The frequent formation of the cartilage-like structures in the rapidly growing undifferentiated explants and its absence in the slowly growing, more differentiated explants suggest that low oxygen tensions in combination with altered levels of growth factors, such as members of the transforming growth factor beta superfamily, create conditions that induce differentiation of fibroblasts to chondrocytes. It is concluded that these human tumors grown in nude mice can be used as an in vivo model to study ectopic formation of mineralized cartilage.

Immunolocalisation of collagenase in rabbit periosteal tissue explants and extraction of the enzyme. The effect of the cytokines IL-1 alpha and EGF.

The effect of interleukin-1 alpha (IL-1 alpha) and murine epidermal growth factor (EGF) on incorporation of endogenously produced collagenase in the extracellular matrix of soft connective tissue was studied in an in vitro model system using periosteal explants obtained from rabbit calvariae. Immunohistochemical analysis indicated the highest level of collagenase in explants cultured for 72 hours with IL-1 alpha in combination with EGF. Most enzyme appeared to be associated with the extracellular matrix, but labeling was also found in numerous fibroblast-like cells. Explants cultured in the presence of IL-1 alpha alone contained less enzyme and in periostea treated without cytokines, or with EGF alone, only a faint label, if any, was seen. Freshly isolated, non-cultured periostea contained no detectable enzyme. Extraction of collagenase from periostea revealed that: (1) non-cultured periosteum did not contain detectable levels of enzyme. (2) The amount of total activatable enzyme synergistically increased (10-fold) under the influence of IL-1 alpha and EGF, whereas IL-1 alpha alone showed a 4-fold enhancement compared to control or EGF-incubated explants. (3) The latent fraction of the enzyme was synergistically increased (up to 100-fold or more) in periostea cultured in the presence of IL-1 alpha + EGF (21.17 mU/explant versus 0.05 mU/explant in controls). (4) Active collagenase, on the other hand, appeared to be present in a relatively high concentration in explants cultured without cytokines (2.45 mU/explant versus 0.36 mU/explant in IL-1 alpha + EGF-treated explants).(ABSTRACT TRUNCATED AT 250 WORDS)

The role of sialic acid in the localization of lymphocytes in the spleen.

The role of carbohydrate structures in the interaction of lymphocytes and endothelial cells is well established. Here the influence of sialic acid in the entrance and localization of lymphocytes in the lymphoid white pulp area of the spleen was studied by injecting sialidase in vivo. A role for sialic acid molecules on stromal elements of the spleen was determined. Although the identity of the cells that bear sialidase sensitive receptors could not be established, a role for marginal zone macrophages could be ruled out by macrophage depletion studies.

Use of frozen biologic material for combined light and electron microscopy.

A simple and rapid method that enables the use of unfixed frozen material for light and electron microscopic purposes is described. At the light microscopic (LM) level, unfixed cryostat sections were used for enzyme histochemistry. When electron microscopic (EM) inspection was needed, tissue blocks, which were stored at -80 degrees C, were fixed at 4 degrees C and prepared for EM according to standard procedures. Ultrastructural analysis of this material demonstrated that most morphologic aspects of normal (human pancreas and rat liver) and pathologic (human pancreatic adenocarcinoma and rat colon carcinoma metastases in liver) tissue were rather well retained. Cryostat sectioning at -25 degrees C did not appear to have damaging effects on the morphology. The method was applied to correlate enzyme histochemical (LM) data with ultrastructural (EM) aspects of mineralization of stroma in explants of human pancreatic adenocarcinoma grown in nude mice and of nonparenchymal cells around metastases of colon carcinoma in rat liver.

Interleukin-1 alpha and epidermal growth factor synergistically enhance the release of collagenase by periosteal connective tissue in vitro.

The effects of recombinant human interleukin-1 alpha (IL-1 alpha) and murine epidermal growth factor (EGF) on the release of collagenase were studied in an in vitro model system using periosteal explants from rabbit calvariae. Following an incubation period of 72 h it was shown that IL-1 alpha in combination with EGF (IL-1 alpha + EGF) induced a synergistic increase in the amount of collagenase released by periosteal explants. This increase appeared to be at least 10-fold. Most of the enzyme was present in a latent form since the increase in enzyme activity was only detectable after activation by APMA and the molecular weight as determined in immunoblots corresponded to the latent form of this enzyme. Incubations carried out with IL-1 alpha alone resulted in a 2- to 4-fold increase of total enzyme activity, whereas the amount of collagenase in media of EGF-treated periosteal did not surpass control values. A neutralizing anti-IL-1 alpha antibody completely blocked the enhanced release of collagenase as induced both by IL-1 alpha and by IL-1 alpha + EGF. Indomethacin partially prevented the IL-1 alpha + EGF-induced increase in enzyme release, suggesting the involvement of prostaglandins. The amount of tissue inhibitor of metalloproteinases (TIMP) as determined by ELISA was slightly elevated in culture media obtained from all cytokine-treated explants. Comparable results were obtained by Western blot analysis as well as by a functional bioassay. It is suggested that the concomitant presence of the cytokines IL-1 alpha and EGF may play an important role in collagenase-mediated degradation of collagen.

The release of tissue inhibitor of metalloproteinases by calvarial bone explants and its immunolocalization.

Tissue inhibitors of metalloproteinases (TIMPs) play an important role in the regulation of the activity of matrix metalloproteinases (MMPs) such as collagenase, stromelysin and gelatinase. Although it has been shown that upon culturing bone tissue releases relatively large amounts of TIMP, little is known as to the source of the inhibitor. In an attempt to investigate this in more detail calvarial bone explants from young rabbits were cultured in serum-free medium. The explants were cultured with or without adhering periosteum. In some experiments solitary periosteal fragments were maintained in the absence of bone. Media were analyzed for the presence of TIMP by immunoblotting and ELISA as well as for their capacity to inhibit the activity of collagenase. In addition, TIMP was immunolocalized in cryosections of the explants. The data demonstrated that bone-conditioned medium contained significantly more (2-10 times) collagenase inhibitor than periosteum-conditioned medium. Removal of the (convex and/or concave) periosteum from the calvariae did not significantly affect the amount of inhibitor released. Immunoblots and ELISA showed the presence of TIMP in the media, being more in bone- than in periosteum-conditioned medium. In immunolabeled cryosections TIMP appeared to be present in osteoblast-like cells lining both the outer bone surface as well as the endosteal spaces. Label was also found in a number of osteocyte lacunae. The periosteum was almost negative. It is suggested that TIMP contributes to the regulation of MMP-activity involved in the remodeling and turnover of bone.

Migration of alveolar macrophages from alveolar space to paracortical T cell area of the draining lymph node.

In this report we studied the translocation of fluorescent particulate antigens to the draining lymph node, and the migration of fluorescent labeled alveolar macrophages (AM) and peritoneal macrophages (PM) in mice. The results show that intratracheally (IT) instilled particulate antigens translocate to the paracortical T cell area of the draining lymph node. When labeled AM were injected IT, they were found to migrate from the alveolar space into the paracortical T cell area of the draining lymph node. An identical localisation was found after IT injection of labeled PM. When either labeled AM, or PM were injected into the peritoneal cavity, a different migration pattern was observed. Via this route the labeled macrophages migrated to the subcapsular sinus and medulla of the draining lymph nodes. It is shown that the migrated cells are not dendritic cells (DC) present in the cell preparations. A possible role for the micro-environment of the injection site, and the significance of the specific migration pattern of AM is discussed.

The influence of dietary fat on the interaction of lymphocytes with high endothelial venules.

The specific adherence of lymphocytes to high endothelial venules (HEV) represents the first step in the lymphocyte emigration from blood into most lymphoid tissues. The interaction of lymphocytes with HEV exhibits a remarkable organ specificity, which appears to be mediated by complementary receptors on both recirculating lymphocytes (homing receptors) and tissue-specific HEV (vascular addressins). The expression of homing receptors varies and depends on factors such as lymphocyte subtype, stage of activation and maturation. As these receptors are glycoproteins, which are anchored in the cell membrane, it can be envisaged that their position and function are determined by the overall composition of the cell membrane itself. In this study we investigated the significance of dietary fat concentration and saturation for the interaction between lymphocytes and HEV. In addition to these functional studies, the expression of homing receptors in combination with T and B cell markers were analyzed. Using immunohistochemistry the effect on the presence and characteristics of lymphocytes and HEV in situ was studied. Changes in the dietary fatty acid composition resulted in an altered ability of T and B cells to adhere to HEV, without affecting their binding preference. The changed adhesion patterns seemed to be associated with alterations in the expression of adhesion molecules, that are essential for the lymphocyte migration. The latter observation might in turn be explained by the observed modifications in the fatty acid composition of the lymphocytes. These results suggest a role for the fatty acid composition of the nutrition in the process of lymphocyte recirculation.

Alveolar macrophages down-regulate local pulmonary immune responses against intratracheally administered T-cell-dependent, but not T-cell-independent antigens.

The role of alveolar macrophages in the pulmonary immune response against various antigens was studied after elimination of alveolar macrophages by intratracheal administration of liposome-encapsulated dichloromethylene diphosphanate. When the responses against T-cell-independent type 1 and type 2 antigens were compared, it was found that elimination of alveolar macrophages had no effect on T-cell-independent antigens. Intratracheal antigen administration resulted in low lung associated, local responses, although some response was observed in the spleen. In contrast, elimination of alveolar macrophages resulted in an increase in local pulmonary immune response against T-cell-dependent antigens. We conclude from these experiments that alveolar macrophages play an important role in controlling the local pulmonary immune response against T-cell-dependent antigens by down-regulation of local T-cell populations. The alveolar macrophages do not down-regulate the response against intratracheally administered T-cell-independent antigens, although they are important in the protection against inflammatory damage caused by bacterial endotoxins.

Different epitopes on the dendritic cell-associated NLDC-145 molecule during ontogeny.

A monoclonal antibody, 6D2, is described that recognizes a different epitope on the NLDC-145 dendritic cell associated molecule in the mouse. During ontogeny the epitope appears on interdigitating cells in lymphoid organs only around birth, whereas the NLDC-145 antigen can be detected as early as day 16 of gestation. No differences can be observed in the expression of the two antigenic determinants on the epithelial cells of the thymus during ontogeny. Evidence is presented that the two antibodies recognize different epitopes on the same molecule.

Lymphocyte migration in the spleen: the effect of macrophage elimination.

To study the influence of macrophages on the migration and distribution of lymphocytes in the spleen, macrophages were eliminated from the spleen of mice by injection of liposomes in which DMDP was encapsulated. This leads to an elimination of macrophages in both the red pulp and marginal zone of the spleen within 1-2 days. In these animals the distribution of lymphocytes was determined by transfer of either syngeneic fluoresceinated or Ly 5 congeneic cells. It was found that after elimination of the macrophages the number of lymphocytes immigrating into the spleen had decreased, although a comparable mode of compartimentalization was found with an initial localization in the marginal zone and a subsequent distribution into the white pulp. After this elimination spleen macrophage subsets return with different kinetics, and in this way the influence of the red pulp macrophages, the marginal zone macrophages and the marginal metallophilic macrophages on lymphocyte immigration and redistribution could be investigated. A quantitative decrease of immigration was still found when red pulp and marginal metallophilic macrophages had repopulated their compartments, but was only fully restored when the last population to repopulate the spleen after treatment with DMDP-liposomes, the marginal zone macrophages, had returned. Experiments with isolated T and B cells showed that the elimination of macrophages had a profound effect on the localization of B cells in the white pulp, whereas it hardly affected T cells.

Lymphocyte depletion in thymic nurse cells: a tool to identify in situ lympho-epithelial complexes having thymic nurse cell characteristics.

In situ pre-existing complexes of epithelial cells and thymocytes having thymic nurse cell characteristics were visualized in the murine thymus cortex using dexamethasone as a potent killer of cortisone-sensitive thymocytes. The degradation and subsequent depletion of cortisone-sensitive thymocytes enclosed within cortical epithelial cells appeared to be paralleled by thymocyte degradation and depletion in thymic nurse cells isolated from thymic tissue fragments from dexamethasone-treated animals. This suggests that thymic nurse cells are derived from pre-existing sealed complexes of cortical epithelial cells and thymocytes. Not all thymocytes situated within in situ epithelial or thymic nurse cells complexes appear to be cortisone-sensitive: a minority of 1-2 thymocytes per complex survives the dexamethasone-treatment, thus constituting a minor subset of cortical cortisone-resistant thymocytes predominantly localized within cortical epithelial cells in situ and within thymic nurse cells derived from such structures. Cortisone resistance in thymocytes thus seems to be acquired within the cortical epithelial cell microenvironment. Cortisone-resistant thymocytes in thymic nurse cells express the phenotype of mature precursors of the T helper lineage, indicating that the in situ correlates of thymic nurse cells may play an important role in T cell maturation and selection.