Basement membrane formation and lung cell differentiation in vitro.

In high density cultures of mouse fetal lung cells, so-called "mass cultures", development of organoid structures, formation of a basement membrane (BM), and differentiation of pneumocytes type II occur accompanied by synthesis and secretion of lamellar bodies. The relationship between the formation of a BM, on the one hand, and morphogenesis as well as differentiation of pneumocytes type II, on the other hand, has been investigated by use of antibodies against BM components in the lung mass culture. It is shown here that anti-laminin antibodies prevented BM formation, but morphogenesis and pneumocyte differentiation occurred as in untreated cultures. Short-term treatment with the antibody revealed that the BM is formed only during the first 2 to 3 days in vitro. Already formed BM could not be removed by anti-laminin. Anti-collagen type IV antibodies showed no effect in the lung mass culture except for a stronger staining of the BM. Anti-BM-1 antibodies caused no changes in morphogenesis, cell differentiation and BM formation either, but the mesenchymal intercellular space exhibited a dark staining, which is probably due to antigen-antibody complexes. The results obtained with anti-laminin antibodies indicate that a BM is not necessary for lung cell differentiation in vitro.

Connective tissue of the livers of newborn and adult marmosets (Callithrix jacchus).

Immunofluorescence microscopic and electron microscopic investigations revealed components of the matrix and of the basal lamina (collagen type I, III, IV and V, BL-heparan sulfate and fibronectin) in the sinus wall (Disse's space) of the livers of newborn and adult marmosets (Callithrix jacchus). Collagen type I was missing in both the two age groups. Small amounts of laminin were present in the livers of newborn and absent in those of adult animals, whereas collagen type III occurred in the form of delicate fibres. Light microscopic inspection showed a continuous distribution of all other components in the sinus wall. The amount of collagen type III and V increased depending on the age. Electron microscopic investigations revealed single or bundled fibrils (20-30 nm) and filaments (10-12 nm). After addition of tannic acid, plaques of a fine-filamentous network and incorporated granules were observed. After addition of resting Ruthenium Red, electron-dense granules (20-60 nm) were irregularly distributed in the structureless space, resting on collagenous fibrils and cell membranes. The fibrils were allocated to collagen type III, the filaments to collagen type V. The plaques were supposed to contain heparan sulfate, collagen type IV and fibronectin. The absence of a Lamina densa of the basal lamina was attributed to the absence of laminin which probably plays an important role in the formation of this layer. Differences in the distribution pattern of the matrix components and thus a functional mosaic of the permeability of Disse's space were assumed. The complete absence of collagen type I and laminin in the lobules makes the adult marmoset liver especially suited for studies on the importance of this collagen type under pathological conditions, since both components are expressed in this way.

Ultrastructural changes induced by alpha-sarcin in a human pulmonary tumor grown in naked mice.

alpha-Sarcin is a cytotoxic polypeptide produced by Aspergillus giganteus. It suppresses protein synthesis in yeast and wheat germ extracts and has a purine-specific RNase activity. The substance has been tested for its antitumor properties in a series of induced tumor systems in mice such as sarcoma and carcinoma among others. Although some of the in vitro effects of alpha-Sarcin on certain cellular components have been elucidated, the biological effects leading to cellular damage are still obscure. In this work we analysed the morphological changes in tumor cells derived from human pulmonary adenocarcinoma heterotransplanted and grown in naked mice, induced shortly (24 hours) after a single intratumoral injection of alpha-Sarcin (0.4 mg/tumor). The results obtained were: 1) swelling of mitochondria; 2) cell necrosis with partial removal of necrotic cells by phagocytosis; 3) thickening of interlobular connective tissue; 4) hyperplasia of goblet-cell-like clear cells. The mode of action concerning these cellular changes is presently uncertain. In view of the severity of these structural alterations it seems conceivable that alpha-Sarcin may enter the cell undergoing interactions with different intracellular structures. This would require a selective membrane permeabilization, perhaps induced upon formation of complexes with negatively-charged membrane phospholipids.

Down-regulation of adhesion receptors on cells of primate embryos as a probable mechanism of the teratogenic action of thalidomide.

In spite of ongoing speculation, there has been no evidence that adhesion receptors are expressed on the cells of mammalian embryos. In this report, we provide the first proof that a variety of such receptor (beta 1-, beta 2-, and beta 3-integrins and selectin) are indeed expressed on cells of essentially all primordia of marmoset embryos at early organogenesis (developmental stages 11 to 13, or even earlier). Treatment with low doses (20 or as little as 1 mg/kg body weight) of a highly teratogenic derivative (EM12) of thalidomide, the most notorious human teratogen, triggers a dramatic and statistically highly significant down-regulation of several surface adhesion receptors (e.g. CD11a/CD18, CD49d/CD29, CD61, etc.) on early limb bud cells and on cells of some other primordia during early organogenesis of embryos of a primate (marmoset, Callithrix jacchus). Some of these receptors almost disappear, or they are expressed at a lower epitope density in the exposed embryos. These down-regulations of surface adhesion receptors may be expected to alter cell-cell- and cell-extracellular matrix interactions, and they are suggested to be a long-sought primary mechanism of the teratogenic action of thalidomide-type substances.

Effects of ofloxacin on chondrogenesis in murine cartilage organoid culture.

The fluoroquinolone, ofloxacin, a widely used antimicrobial agent, has been shown to cause athropathogenic syndromes in juvenile animals. In the present study, the effect of ofloxacin on chondrogenesis in cartilage organoid cultures of limb-bud mesenchymal cells obtained from day-12 mouse embryos was investigated. Cultures treated with increasing concentrations of ofloxacin (10, 30 and 100 mug/ml medium) for 6 days showed no significant changes in overall protein content and dry weight. Collagen type II, as a specific marker for cartilage, and collagen type I, as a marker protein in the internodular loose connective tissue in this culture system, were estimated by an inhibition-ELISA after cyanogen-bromide digestion. The collagen type I content of treated cultures remained constant, but the collagen type II level decreased in a dose-dependent manner to about 40% of that of the controls. There was no detectable increase in the concentration of collagen type II in the medium suggesting that ofloxacin inhibits synthesis rather than stimulate degradation of collagen type II. Cultures treated with 100 mug ofloxacin/ml were further investigated by indirect immunofluorescence and electron microscopy. Anticollagen type II antibodies demonstrated irregularities, several defects in the cartilage nodules, and much weaker staining in the treated cultures compared with controls. Similar results were obtained with antibodies directed against the core protein of large chondroitin sulphate proteoglycan monomers. Using monoclonal antibodies specific for unsulphated, 4-sulphated and 6-sulphated disaccharide "stubs" that remain attached to the core protein after chondroitinase ABC digestion of proteoglycans, different changes in these glycosaminoglycans were observed. While unsulphated chondroitin seemed to disappear nearly completely from the cartilage matrix, the level of chondroitin 4-sulphate remained unchanged and in the case of chondroitin 6-sulphate a slight increase in staining intensity was observable in the ofloxacin-treated cultures. Ultrastructurally, there was a reduction in the number of collagenous fibrils in the cartilage matrix of treated cultures and necrotic chondroblasts could be demonstrated. The present results resemble, in some aspects, observations that have been made in vivo after ofloxacin treatment, indicating that this in vitro model may provide a suitable system for examining the mechanism of quinolone-induced athropathia.

[The distribution of different collagen types and laminin in the gingiva of the common marmoset (Callithrix jacchus)]. / Die Verteilung verschiedener Kollagentypen und des Laminins in der Gingiva von Marmoset-Affen (Callithrix Jacchus).

The distribution of the collagen types I, III, IV, V, VI and of the glycoprotein laminin have been examined in the healthy gingiva of the marmoset with the indirect immunofluorescence method. It has been shown that collagen type I formed thick fibrils in a wave-like pattern, while collagen type III formed thinner fibrils, especially under the epithelial basal membrane. Collagen type IV and laminin showed a strong fluorescence in the basal membrane (epithelium, vessels, nerves). Collagen type V has been demonstrated as a fine fibrillar pattern in the lamina propria and occasionally close to the basal membrane. Collagen type VI has been represented as a microfibrillar network in the connective tissue matrix with a brilliant pericellular fluorescence around vessels and nerves. The study showed the structural model of the connective tissue matrix of the marmoset gingiva and allows a later comparison between inflamed and healthy gingiva.

In vitro system for toxicological studies on the development of mammalian limb buds in a chemically defined medium.

In organ culture systems using the Trowell setup, morphogenetic differentiation (which largely mimics the development reached in vivo within 2--3 days) can be obtained in limb buds of mouse embryos during a culture period of 6 days. We succeeded in improving the technique and in achieving good differentiation of the limb buds in a chemically defined culture medium from which all additions of the heterologous serum used in previous studies were omitted. With this technique of using a chemically defined medium, the following results were obtained: (1) A high degree of reproducibility can be obtained in the grade of differentiation if the experimental conditions are standardized and limb buds of the same developmental stage (somite stage) are used. (2) The technique is applicable to species other than mice, such as rats and rabbits, however, the results obtained so far are not so satisfactory as those acquired with mouse limb buds. (3) In an attempt to offer some colloid osmotic pressure, macromolecules like polyvinylpyrrolidone (Periston), polydextrans (Macrodex) or polypeptides (Haemaccel) were added to the culture medium. None of these macromolecules had a beneficial effect on the differentiation of the limb buds in vitro; in the case of Macrodex the differentiation was even impaired. (4) In comparison with the development in a serum-containing medium, the results attained with a chemically defined medium are just as good when judged from microscopical and some biochemical studies (total content of DNA, RNA, protein, and protein-bound hydroxyproline in the explants). The applicability of the test system for the evaluation of embryotoxic effects is discussed.

Production and specificity of antibodies against the central region of type II collagen.

Collagen type-specific antibodies as well as antibodies to particular portions of the molecule are extremely useful tools especially for the quantification of collagens and for immunohistochemical examinations in developing embryos. Quantification of collagens in CNBr-solubilized tissue samples presupposes the production of antibodies against CNBr-derived collagen fragments. For the first time, as antigens for the immunization of rabbits, cyanogen-bromide derived fragments of collagen type II were used, obtained by direct digestion of tissue (Swarm chondrosarcoma from rat) and separation by gel filtration chromatography. Antisera were applied to affinity chromatography and the eluted antibodies were characterized by ELISA, immunoblotting, inhibition studies and immunohistochemistry. The antibodies from five different rabbits show high specificity for type II collagen and are directed against sequential determinants in the central portion of the type II collagen molecule. The easy way of obtaining the fragments directly from tissue, combined with their immune response in rabbits, gives the possibility of producing type II collagen-specific, fragment-directed antibodies in a convenient and rapid way.

Distribution of fibronectin in healthy, inflamed and drug-induced gingival hyperplasia.

The distribution of fibronectin (FN) in the healthy, inflamed and hyperplastic human gingiva was investigated by indirect immunofluorescence. FN appeared as a fibrillar structure in the lamina propria of the healthy gingivae. In the inflamed specimens, FN demonstrated parallel fibres, especially in the coronal areas of the tissue. In the phenytoin gingival overgrowth, tissue FN was observed as thin fibres with variable length. The thin fibres gave the appearance of penetrating the basement membrane of the epithelium. Cyclosporin A gingival enlargement could be differentiated by phenytoin lesions because of the higher length and the parallel distribution of the FN. Finally, FN was observed in the nifedipine gingival overgrowth, where a microfibrillar delicate network gave the appearance of a "cloud"-pattern of distribution. In all of the specimens, blood vessels and nerves could not be stained. These findings show that FN distribution could differentiate the structure of the gingival lesions.

Immunohistochemical localization of collagenous components in healthy periodontal tissues of the rat and marmoset (Callithrix jacchus). I. Distribution of collagen types I and III.

The distribution of collagen types I and III was demonstrated in healthy periodontal tissues of the rat and marmoset using immunofluorescent localization after decalcification of the maxillae and mandiblae in 0.2 N HCl. An intense fluorescence in the alveolar bone and cementum matrix, as well as in the soft periodontal tissue, was demonstrated with anti-collagen type I antibodies. In the gingival connective tissue and in the periodontal ligament thick fibers of collagen type I could be observed. The fluorescent reaction in the rat periodontal ligament was not strong in comparison to the marmoset periodontal ligament. Sharpey's fibers, inserting into the cementum and alveolar bone, were also stained. On the other hand, collagen type III could not be demonstrated in the hard periodontal tissues, but could be in the bone marrow stroma and the incremental lines as well as around the Sharpey's fibers of the cementum, in accordance to previous studies. In the gingival connective tissue a strong staining was evident, especially near the basement membrane. The periodontal ligament showed an intense fluorescence that was, in some areas, continuous with Sharpey's fibers inserting into the cementum. The distribution of collagen types I and III was demonstrated with immunohistochemical techniques in the rat and marmoset periodontium. These results provide necessary information on healthy tissues that will be required for future studies on the effects of pathological, reparative and regenerative processes.

The extracellular matrix in cartilage organoid culture: biochemical, immunomorphological and electron microscopic studies.

Limb bud mesenchymal cells obtained from day-12 mouse embryos were grown at high density on a membrane filter (pore size 0.2 micron) at the medium/air interphase. Chondrogenesis in this so-called cartilage organoid culture was monitored quantitatively by immunological estimation of type I and type II collagen and qualitatively by indirect immunofluorescence and electron microscopy in the course of a 36 days culture period. Three stages of cartilage development could be substantiated: 1. Formation of cartilage between days 2 and 7; 2. maturation of cartilage between days 9 and 13; 3. degeneration of cartilage beginning at day 20. Differentiation in cell aggregates and a loose mesenchymal tissue occurred during the first two days of the culture period. Type II collagen synthesis started in cell aggregates two days after plating and after 6 days in culture distinct cartilage nodules had developed which were embedded in loose connective tissue that contained type I collagen. During this period the type II collagen content increased progressively from 2.3 micrograms (day 3) to nearly 40 micrograms (day 7) per mg dry weight, whereas the type I collagen level increased more linearly from 2.7 to 21.3 micrograms/mg dry weight. The second period was characterized by enlargement and fusion of cartilage nodules and a diminished increase in type II collagen content from 45 to 60 micrograms/mg dry weight. Enlargement and fusion occurred by matrix production as well as by transformation of perichondrial cells into chondroblasts. Type I collagen synthesis enhanced from 29 to 54 micrograms/mg. Hypertrophic chondrocytes could be demonstrated ultrastructurally. At the third stage a nearly continuous layer of cartilage on the membrane filter covered by noncartilagenous tissue had developed. To some extent chondrocytes lost their matrix capsule and changed into fibroblast-like cells accompanied by a switch of collagen synthesis from type II to type I collagen. Quantitative studies yielded a constant level of about 60 micrograms/mg type II collagen and a further increase in type I collagen from 77 to 116 micrograms/mg dry weight. This study reveals an in vitro model of a prolonged, but almost identical image of chondrogenesis in vivo prior to endochondral mineralization which may be useful for investigations on cartilage differentiation, maturation and degeneration.

Immunohistochemical localization of collagenous components in healthy periodontal tissues of the rat and marmoset (Callithrix jacchus). II. Distribution of collagen types IV, V and VI.

The immunohistochemical distribution of collagen types IV, V and VI has been demonstrated in healthy periodontal tissues of rats and marmosets following decalcification of the maxillae and mandibulae in 0.2 N HCl. An intense fluorescence with anti-collagen type IV antibodies was demonstrated in the basement membranes of the epithelium and of the blood vessels and nerves. In the alveolar bone stroma and in the periodontal ligament (PL) collagen type IV was present only in the basal membranes of the blood vessels and nerves. In comparison, collagen type V was observed in a fibrillar pattern in the gingival connective tissue, as well as the PL. In the PL, type V collagenous fibers demonstrated a parallel distribution with stronger fluorescence near the cementum surface. Collagen type VI could be demonstrated in fine fibers present in the gingival connective tissue and the PL. Blood vessels and nerves were not stained in the marmoset, but were in the rat, where a localization of collagen type VI was demonstrated in these areas. Alveolar bone and cementum, as well as the Sharpey's fibers embedded in these tissues, were not stained with antibodies against collagen type V and type VI, but a pericellular localization of these collagenous components could be observed. Collectively, these results provide basic information on the relative distribution of different collagen types in normal tissues of rats and marmosets that will be required for future studies on the effects of pathological, reparative and regenerative processes.

Immunohistochemical distribution of the collagen types IV, V, VI and glycoprotein laminin in the healthy rat, marmoset (Callithrix jacchus) and human gingivae.

The purpose of this study was to demonstrate the localization of collagen types IV, V and VI as well as the glycoprotein laminin in biopsies of healthy rat, marmoset (Callithrix jacchus) and human gingivae. The slices, after the use of indirect immunofluorescence (incubation with antibodies against these extracellular matrix components), showed the same distribution with the anti-type IV and laminin antibodies on the basement membranes of the epithelium, blood vessels and nerves. Collagen type V, as a filamentous, and collagen type VI, as microfibrillar components, were localized in a similar pattern in the different species. In contrast to the other species, collagen type VI could not be found near the basement membranes of vessels and nerves of the marmoset gingiva. This result shows differences between human and monkey tissues, but not between rat and human gingivae, and conforms the heterogeneity of collagen type VI in the various cell and tissue types.

Immunofluorescence-microscopic localization of collagen type IV and VI, laminin and nidogen in the livers of Callithrix jacchus during pre- and postnatal development.

The distribution of collagen type IV and VI, laminin and nidogen was investigated by immunofluorescence microscopy in the livers of marmosets (Callithrix jacchus) at various stages of development, i.e. on days 93, 111, 116 and 134 of gestation, 1 day postpartum and at the mature stage. Large amounts of collagen type IV could in all cases be demonstrated in the sinus wall and in all basal laminae outside the lobule. After the application of antibodies against collagen type VI the sinus wall showed a weak fluorescence reaction at the early stages and a strong binding towards the end of gestation which persisted up to the adult stage. In the periportal field it was mainly localized at the border between lobule and connective tissue. Laminin also increased gradually but could be demonstrated only until birth. In contrast, nidogen was present during the total prenatal and postnatal period. Therefore, collagen type IV and VI were not very suitable for the demonstration of an increase in matrix components under pathological conditions, because they occur already in large amounts in normal livers. However, the occurrence of laminin that was missing in the adult liver must be interpreted as pathological indication. The different occurrence of laminin and nidogen showed that these two substances were expressed and regulated independently of each other.

Extracellular matrix analysis of nifedipine-induced gingival overgrowth: immunohistochemical distribution of different collagen types as well as the glycoprotein fibronectin.

The purpose of this study was to demonstrate the localization of collagen types I, III, IV, V, VI and VII as well as the glycoprotein fibronectin in nifedipine-induced gingival overgrowth. The slices, after the use of indirect immunofluorescence (incubation with antibodies against these extracellular matrix components), showed a diffuse distribution with the anti-types I and III in the stroma and fluorescent staining of the basement membranes of the epithelium, blood vessels and nerves with collagen type IV antibodies. The increased number of vessels was localized near the surface of the lesion. Collagen type V - seen as a filamentous - and collagen type VI - as microfibrillar - components were also localized in the tissue, showing completely different patterns of distribution. Collagen type V appeared "crater"-like and type VI displayed a "honeycomb"-shaped structural model. The blood vessels were not stained but the area around their walls demonstrated an intense fluorescence with these antibodies. Collagen type VII showed a characteristic linear staining near to the epithelial basement membrane. In contrast to this, fibronectin localized with a varied intensity in the different areas of the tissues and presented a "cloud"-like structure. This shows differences between the matrix components in nifedipine-induced hyperplasia and confirms the heterogeneity of the matrix in health and in gingival alterations.

Ofloxacin in juvenile non-human primates and rats. Arthropathia and drug plasma concentrations.

Arthropathia in juvenile animals is the most important toxic effect induced by quinolones. We conducted pharmacokinetic and morphological studies with ofloxacin on non-human primates (Callithrix jacchus, Marmosets) and rats. In the marmoset, electron microscopy and the application of immuno-morphological methods proved to be suitable for the detection of specific alterations in cartilage (e.g. loss of proteoglycans and altered chondrocytes). Subsequently performed electron microscopic examinations in rats showed similar specific alterations of the femur cartilage surface after multiple oral applications of 600 mg ofloxacin/kg body wt. These results were correlated with pharmacokinetic data obtained for the same species. After single oral application of 100, 300 or 600 mg ofloxacin/kg body wt to 5 week-old rats peak plasma levels were achieved 15-45 min after administration indicating a rapid absorption of the drug. The following peak concentrations were measured for the three doses applied (mean +/- SD): 8.9 +/- 2.1, 22.6 +/- 7.5 mg/l and 33.5 +/- 9.8 mg/l, respectively. After 360 min the concentrations were 1.1 +/- 0.4, 5.9 +/- 2.5 and 15.9 +/- 5.1 mg/l, respectively. After subcutaneous injection of 100 mg ofloxacin/kg body wt the mean peak concentration was 27.7 +/- 2.6 mg/l after 45 min (0.5 +/- 0.2 mg/l after 360 min). In the marmoset higher plasma concentrations were measured with comparable doses. One, 3, and 6 h after the last of nine administrations of 200 mg ofloxacin/kg body wt, the mean (+/- SD) plasma concentrations were: 42.7 +/- 16.7, 40.6 +/- 9.5, and 26.5 +/- 3.6 mg ofloxacin/l plasma. Typical alterations of the joint cartilage of juvenile rats (e.g. opened chondrocyte cavities, swelling of rough endoplasmic reticulum and mitochondrial swelling in the chondrocytes) were induced by oral administration of ofloxacin at doses that were approximately 100 times higher than therapeutic ones, but led to peak plasma concentrations which were only approximately 10 times above the therapeutic level.