The effect of cyclosporin-A on the ultrastructure of gingival tissue in Beh?et's disease

Autoimmune processes are said to play an active role in aetiology of Beh?et's disease (BD), which is also known as a multisystem disease. In the treatment of this autoimmune disease, cyclosporin A (CyA) is used. Gingival hyperplasia (GH) is one of the important side effects that have been observed in some of the patients. We aimed to evaluate the CyA-induced gingival hyperplasia in BD patients. There were 3 study groups, each having 5 patients. In the first group displaying GH, mast cells were located between epithelial cells and in the connective tissue. Mast cell granules were in crystalline form and electron-dense cored form. Fibroblasts and plasma cells were present in the connective tissue. The second group did not display GH and the mast cells were similar to those in the first group. The third group, was the control group, in which the mast cells were located between the epithelial cells and connective tissue. Mast cell granules were in electron-dense cored form. We concluded that the development of CyA-induced gingival hyperplasia is determined mainly by individual sensitivity to CyA, because although both test groups which received CyA showed an increased number and activity of fibroblasts, only one group of patients developed GH.     

Morphometric analysis of cellular and vascular changes in gingival connective tissue in long-term insulin-dependent diabetes

This study examined cellular and vascular changes in gingival connective tissue samples by stereologic point-counting procedures and interactive digital analyzing systems in long-term insulin-dependent diabetes mellitus patients. Gingival connective tissue capillaries representing a clinically healthy sulcus with no evidence of periodontal disease at the site of biopsy were studied in 29 patients with diabetes. Based upon their long-term medical records, 19 were identified as having poorly controlled (PIDD) and 10 as controlled insulin-dependent diabetes mellitus (CIDD). Ten nondiabetic, age- and gender-matched individuals served as controls. Thirty-nine biopsies were processed for light microscopy, and the blood vessel area was analyzed using an interactive digital analyzing system; 9 gingival biopsies, 5 diabetic and 4 controls, were processed for morphometric electron microscopic analysis. For each individual, site-specific recordings were made for the plaque index, bleeding index, probing depth, loss of attachment, and radiographic loss of interproximal alveolar bone. No evident signs of periodontitis occurred at the biopsy sites. For each PIDD patient, respective volumetric and numeric densities of cellular components including fibroblasts, neutrophilic granulocytes, monocyte/macrophages, mast cells, lymphocytes, blast cells, and plasma cells were recorded in the inflamed connective tissue (ICT). Non-cellular components such as collagen fibers and blood vessels were also recorded. PIDD patients had elevated plasma cell levels relative to controls and they appeared also to have a decreased collagen fiber density. In addition, fibroblasts occupied less volume in the ICT of PIDD patients than in controls. PIDD patients had the largest mean area of cross-section of the blood vessels, but this difference was not statistically significant (P > or = 0.211; t-test). No specific characteristics of ICT or vascular changes were detectable in adult well-controlled long-term diabetics under similar plaque conditions. Swollen and proliferated endothelial cells were frequently found in PIDD patients and the mean distance from the lumen to the outer border of basement membrane was greater in the PIDD than in the controls (P < 0.001; t-test). Overall, our findings that cellular, vascular, and connective tissue changes indicative of increased catabolism rather than anabolism detected in gingiva are especially associated with poorly controlled long-term insulin-dependent diabetes.     

Discriminating PCR artifacts using directed heteroduplex analysis (DHDA)

A light microscopic, immunohistochemical and ultrastructural study was done on 2 patients with active pruritic keloids. The primary cell was the myofibroblast with prominent rough endoplasmic reticulum and bundles of myofilaments with focal densities in the cytoplasm. Enhanced secretory activity was reflected in the prominence of the Golgi apparatus and the frequent presence of intracellular collagen within the tubular membranes. The number of mast cells was increased and they were closely associated with myofibroblasts with frequent direct cell contacts. The filopodia of the mast cells were seen intimately applied along the cell membrane of the myofibroblasts. Degranulated mast cells contained few and electron-lucent granules. Discharge of the contents of granules into the interstitial matrix was encountered. Mast cell granules were also seen lying free in the interstitium. In the vicinity of the degranulated mast cells the interstitial matrix was oedematous containing granular material and the myofibroblasts showed considerable dilation of the rough endoplasmic reticulum and vacuole formation. The intimate relationship and interaction between mast cells and myofibroblasts support the important role of mast cells and their mediators in the pathogenesis of keloid.     

Evidence for an impaired ability to determine semantic relations in Alzheimer''s disease patients

Gingival and periodontal ligament (PDL) fibroblasts are the major cellular components of periodontal soft connective tissues, but the precise differences between these cells are not yet known. In the present study, we have therefore examined the phenotypic and functional features of the cells obtained from gingival and PDL biopsy samples. Spindle-shaped cells characteristic of fibroblasts were the main cell type observed in vitro, although epithelial cells were also present in primary gingival cell cultures. Flow cytometry was used to measure the size and granularity of the cultured cells, and showed that the gingival fibroblasts were smaller and less granular compared with the PDL cells. The expression of certain key extracellular matrix (ECM) proteins, fibronectin, collagen type I, and tenascin was measured by flow cytometry. Analysis of the fluorescence profiles of these cultures showed that the majority of cells expressed fibronectin and that the average fluorescence intensity of this antigen in the PDL cells was higher than that in the gingival fibroblasts. Moreover, the fibronectin-positive PDL cells apparently comprised two subpopulations which expressed fibronectin at different levels, suggesting that the cells in the PDL cultures were functionally heterogeneous. The level of collagen type I was also found to be up-regulated in the PDL compared with the gingival cells and, as with fibronectin, was expressed at two different levels by subsets of the PDL cells. In contrast, tenascin was expressed at very similar levels by both the gingival fibroblasts and PDL cells. In addition, measurement of alkaline phosphatase, a marker enzyme for mineralized tissue-forming cells, showed that the PDL cells had higher activity than the gingival fibroblasts and that the alkaline phosphatase activity in the PDL cells was far more markedly up-regulated by dexamethasone. Our findings demonstrate that, despite their similar spindle-shaped appearance, fibroblasts derived from gingival and PDL tissues appear to display distinct functional activities which are likely to play a vital part in the maintenance of tissue integrity and regenerative processes.     

Definitive characterization of rat hypothalamic mast cells

Mast cells have previously been identified in mammalian brain by histochemistry and histamine fluorescence, particularly in the rat thalamus and hypothalamus. However, the nature of brain mast cells has continued to be questioned, especially because the electron microscopic appearance often shows secretory granule morphology distinct from that of typical connective tissue mast cells. Here we report that mast cells in the rat hypothalamus, identified based on metachromatic staining with Toluidine Blue, fluoresced after staining with berberine sulfate, indicating the presence of heparin. These cells were also positive immunohistochemically for histamine, as well as for rat mast cell protease I, an enzyme characteristically present in rat connective tissue mast cells. In addition, these same cells showed a very strong signal with in situ hybridization for immunoglobulin E binding protein messenger RNA. However, use of antibodies directed towards immunoglobulin E or its binding protein did not label any cells, which may mean either the binding protein is below the level of detection of the techniques used or that it is not expressed except in pathological conditions when the blood-brain barrier becomes permeable. At the ultrastructural level, perivascular mast cells contained numerous, intact, electron-dense granules which were labeled by gold-labeled anti-rat mast cell protease I. These results clearly demonstrate the presence of perivascular mast cells in the rat hypothalamus, where they may participate in homeostatic processes.     

An increase in gingival volume induced by drugs (phenytoin, cyclosporine and calcium antagonists). A review of the literature

The aim of this review is to evaluate the side effects of some drug therapies on the gingival tissue in certain susceptible individuals. Phenytoin, cyclosporine-A and a variety of calcium channel blockers have been shown to produce gingival overgrowth. In this paper the pharmacodynamics and pharmacokinetics of these drugs, the pathogenesis, the clinical aspect of the enlargement and its treatment are examined. Several of the reviewed theories on pathogenesis are well documented in the literature, while others are controversial and less described. The old term gingival hyperplasia is not exact because histologically an increase in the number of fibroblasts has not been demonstrated, but an increase has been found out in the amount of collagen fibers and noncollagenous proteins in the connective tissue. The clinical findings have the same characteristics both in location and growth pattern while prevention is primarily directed at the removal of local irritant factors. The prevalence and severity of gingival enlargement increase in heart transplant patients who are often medicated with cyclosporin and channel blockers.     

Stress-induced sleep deprivation modifies corticotropin releasing factor (CRF) levels and CRF binding in rat brain and pituitary

OBJECTIVE: Mast cells are suggested to play an essential role during development of varices in the lower limbs. EXPERIMENTAL DESIGN: Investigation of the ultrastructure of mast cells in varicose lesions. SETTING: Saga Medical School, Yamamoto Surgical Hospital. PATIENTS: Eighteen varicose veins of 17 patients and 9 normal saphenous veins of 9 patients were examined. Patients who had undergone sclerotherapy for varices were excluded. INTERVENTIONS: Radical stripping surgery was performed on all varicose veins. MEASURES: Ultrastructural observations. RESULTS: In normal saphenous veins, mast cells usually singly embedded in dense collagen bundles as resident cells. They have characteristic crystalline granules of storage type. In varicose veins, mast cells show different features such as an increase of altered granules of discharging type, degranulation and intimate relationship with fibroblasts and lymphocytes. CONCLUSIONS: The observations suggest the presence of mast cell-mediated mechanism by releasing some mediators in the development of varices.     

Desmoplastic fibroblastoma

Previous reports of a distinctive, fibrous, soft-tissue tumor include eight patients with subcutaneous lesions and six patients with intramuscular lesions. We report a 48-year-old woman with a 2-cm cutaneous and subcutaneous nodule on the left arm with the same histologic features. An excisional biopsy showed a large, well circumscribed tumor replacing the reticular dermis and subcutaneous tissue. The tumor was relatively hypocellular and composed primarily of large, spindled, plump or stellate fibroblasts haphazardly dissecting between thickened fibrotic collagen bundles. The stroma contained a large amount of mucin which was positive with alcian blue at pH 2.5, and relatively numerous mast cells were present. The fibroblastic-like cells were positive with Vimentin and Factor XIIIA and negative with S-100, desmin, actin and keratin.     

Ultrastructural characteristics of fibroblasts, myofibroblasts, and fibroclasts in the process of wound healing (author's transl)

5 types of fibroblasts were characterized by histomorphometrical and ultrastructural examinations of the wound healing of rats, 3, 7, 14 and 21 days p. op. These cells were undifferentiated mesenchymal cells, immature fibroblasts, mature fibroblasts, myofibroblasts and fibroclasts. The functions of these cells were discussed with respect to cell proliferation, synthesis, and degradation of collagen.     

Scanning electron microscopy of cyclosporine-induced gingival overgrowth

Overgrown human gingival specimens were examined histologically and by scanning electron microscopy (SEM) to study structural changes caused by cyclosporine. The biopsy specimens were from organ transplant recipients receiving cyclosporine to suppress the rejection of the transplanted organ. The epithelium of the overgrown gingiva was thickened, acanthotic and parakeratotic. Retepegs were anastomosing and extending into connective tissue. The SEM examination of the outer surface of the attached gingival showed loss of cellular attachments and cells were exfoliating. The normal honeycomb structure formed by interconnecting microvilli surrounding the pits was distorted. Outer gingival cell surface showed numerous round, ovoid and dome-like structures instead of parallel, reticular or fingerprint-like microridges. It was concluded that cyclosporine not only caused hyperplasia but also changed the structure of the outer epithelial cell surface.     

Synthesis of proinflammatory cytokines by human gingival fibroblasts in response to lipopolysaccharides and interleukin-1 beta

We have examined the ability of gingival fibroblasts (GF) to participate in inflammatory response and function as accessory immune cells. The accessory immune function of GF cells was evaluated by their ability to elaborate proinflammatory cytokines following stimulation with lipopolysaccharides and interleukin-1 beta (IL-1 beta). Using three separate clonally derived and characterized human gingival fibroblast (GF) cell lines, we demonstrate that LPS from Actinobacillus actinomycetemcomitans (Aa) and Escherichia coli (Ec) induce mRNA and synthesis of proinflammatory cytokines, IL-1 beta, IL-6 and IL-8. IL-1 beta activation of GF cells showed that IL-1 beta non only induces the expression of IL-6, IL-8 and TNF-alpha, but also acts in an autocrine manner of GF cells and induces IL-1 beta expression. Furthermore, the continuous presence of IL-1 beta in GF cell cultures did not down regulate the response of GF cells to IL-1 beta. Pretreatment of GF cells with IL-1 beta resulted in the enhanced synthesis of TNF-alpha in response to additional IL-1 beta. These findings indicate that GF cells, in addition to providing structural support, may also function as accessory immune cells and play an important role in the initial inflammatory reaction as well as in the amplification of immune response.     

Macrophage-fibroblast interaction and its possible role in regulating collagen metabolism during wound healing

The role of macrophages in the resorption of denatured collagen in the wound tissue was demonstrated by transmission and scanning electron microscopy investigation. Tight cell contacts between the macrophages and fibroblasts in the granulation tissue preceded active collagen synthesis and fibrillogenesis. A hypothesis was suggested according to which these contacts played an important role in the transfer of specific signals for the initiation of collagen biosynthesis; collagen decomposition products were considered as the actual signals, thus connecting the collagen catabolism and biosynthesis. Such a feed-back mechanism probably served as an integral part of the general hemostasis regulating the growth of connective tissue.     

Fibroelastic hamartoma (fibroma) of the heart

Approximately 70 cardiac fibromas (fibroelastic hamartomas) have been reported in the literature and at least 15 have been successfully excised. However there is no well-documented ultrastructural study of these lesions. A successfully excised cardiac fibroma (FEH) was studied by light and elctron microscopy. The tumor was composed mainly of fibroblasts admixed with bundles of collagen and elastic fibers. No muscle fibers were demonstrated in the central region of the tumor either by light or electron microscopy. Mast cells were occasionally present. The lack of encapsulation enabled fibroblasts with collagen and elastic fibers to extend between groups of myocardial fibers at the periphery of the tumor. Within these myocardial fibers ultrastructural changes were limited to the mitochondria and myofibrillar structure.     

Field emission SEM, conventional TEM and HVTEM study of submandibular gland in prenatal and postnatal aging mouse

The development of acinar and ductal cells of the mouse submandibular gland was studied using field emission SEM, conventional TEM and HVTEM methods. The specimens, at 15 and 18 days of gestation and 1, 3, 7, 14, 21, 30, 90 and 180 postnatal days were fixed in 2.5% glutaraldehyde solution in 0.1 M sodium phosphate buffer (pH 7.3). At 15 and 18 days of gestation, the structure of mouse submandibular gland contains acinar and ductal cells in proliferation. The cytoplasmic organelles such as mitochondria, granular endoplasmic reticulum and Golgi apparatuses are scattered in the cytoplasm. At 18 prenatal days only several acinar cells present immature secretory granules in the apical portion. In this stage the acinar and ductal cells are enveloped by bundles of fine collagen fibrils disposed in several directions. There are also numerous capillaries located closely to the acinar cell membranes. In the aging stages of 1, 3, 7, 14, 21, and 30 postnatal days, the histo-differentiation of acinar, intercalated and ductal cell components are observed. At newborn day one the cytoplasmic organelles start to place themselves around the nucleus. Several immature secretory granules are observed at day one, however, they increase in the aging days. At postnatal day 30, the cytoplasms of acinar and ductal cells are filled with a large number of secretory granules of different sizes. The stacks of granular endoplasmic reticulum and Golgi apparatus and some vesicles and free ribosomes are noted. The intercellular membranes are attached by desmosomes and cytoplasmic interdigitations. The luminal surface shows several small projections of microvilli. An electron-dense line of basement membranes followed by fine collagen fibrils are recognized. Delicate capillaries are found in the outer surface of acinar cells. At postnatal day 90 and 180 the acinar, intercalated and striated ductal cells reveal numerous secretory granules in the apical portion. The acinar cells showed basal nuclei and the parallel arrangement of granular endoplasmic reticulum. The mitochondria are located at the base of ductal cells showing a typical pattern of cristae. In these stages the intercellular digitations of cytoplasmic protrusions and desmosomes are also noted. The cytoplasm of myoepithelial cells are seen along the cell membranes. The spongy-like structures constituting the basement membrane are followed by bundles of fine collagen fibers.     

Characterization of human orbital fat and connective tissue

This study was designed to evaluate the characteristics of human orbital fat and connective tissue. Two exenteration specimens were studied by light microscopy with special stains. Four distinct regions were identified on the basis of their connective tissue septa, which contained blood vessels and were composed of elastin and collagen types I, III, and IV. Transmission electron microscopy was performed on the opposite orbits. The fibroblasts and adipocytes appeared metabolically inactive and showed no regional differences. The fat was phase extracted from the connective tissue and subjected to biochemical analysis. No regional differences were found in the content of fatty acids and protein. The fatty acids included palmitic acid (22-24.6%), oleic acid (45-51.5%), and linoleic acid (15-18.6%). Despite demarcation of the orbital fat into distinct regions by the connective tissue septa, ultrastructural and biochemical analysis revealed no regional variations in the fat. The diagnostic and therapeutic implications of these findings are discussed.     

Induction of cell proliferation and collagen synthesis in human small intestinal lamina propria fibroblasts by lipopolysaccharide: possible involvement of nitric oxide

Recent studies suggest that tissue specific fibroblasts respond to inflammatory stimuli leading to the onset of inflammatory disorders. In the present study, we investigated cell kinetics, collagen synthesis, and nitric oxide (NO) level in cultured human small intestinal lamina propria fibroblasts (HSILPF, n = 45) in response to LPS of enteropathogenic E. coli. LPS treatment enhanced the 3[H] TdR uptake, increased the percentage of 'S' phase cells as early as 4 hrs, and decreased the population doubling time of HSILPF in a dose and time dependent manner. Collagen synthesis in HSILPF was also elevated by LPS. The LPS induced cell proliferation and collagen synthesis were inhibited by polymyxin B (10 micrograms/ml). LPS was found to suppress the NO production in these cells, whereas combination of LPS (10 micrograms/ml) and IFN gamma (100 U/ml) enhanced NO output and concurrently decreased the cell proliferation and collagen production in HSILPF. Inhibitors of NO, L-NG-monomethyl L-arginine, and aminoguanidine partially restored cell proliferation and collagen synthesis in cells exposed to LPS and IFN gamma. These findings suggest that LPS induces increased cell proliferation and collagen synthesis in HSILPF and these could be related to the suppression of NO production.     

Possible significance of cells within intraluminal collagen masses in equine oviducts

In addition to the unique feature of retention of unfertilized ova, the oviducts of mares frequently contain large intraluminal masses with a fibrillar component and some cells. The aim of this study was to identify the cells and examine their relationship to the extracellular components of these masses. Intraluminal masses were examined both in situ and flushed from the oviducts. The nature of the contained cells and their relationship to the fibrils were examined by light microscopy and by transmission and scanning electron microscopy. In some mares the large masses distended the oviduct, but neither loss of the oviductal epithelium nor damage to this epithelium was seen. Electron microscopy verified that the principal cellular component was fibroblasts, and that the fibrils were type I collagen. Collagen masses collected shortly after ovulation frequently contained viable fibroblasts with collagen fibrils associated with their cell surfaces and with surface clefts. Although such collagen masses were present in pregnant and nonpregnant mares, masses with viable fibroblasts were chronologically associated with recent ovulation. It was concluded that connective tissue drawn into the oviduct at ovulation is retained, and collagen synthesis continues at least for a few days. Although the fibroblasts eventually disintegrate, the collagen remains and may in some cases aggregate within the oviductal lumen to the extent that oviductal transport and embryonic viability could be affected.     

Permethrin-treated bed nets do not have a ''mass-killing effect'' on village populations of Anopheles gambiae s.l. in The Gambia

Juvenile hyaline fibromatosis is a multisystemic disorder characterized by a triad of cephalic fibrous outgrowths, gingival hyperplasia, and flexion contractures. The aim of this study was to find new ultrastructural features that could be useful for differentiating this entity from other types of fibromatosis. Mucosal lesions processed for light and electron microscopy by routine techniques showed hyperactive-appearing spindle-shaped fibroblasts and dysplastic mesenchymal cells. Dilated rough endoplasmic reticulum, prominent Golgi complexes, and multivesicular bodies as well as single membrane vesicles filled with fibrillogranular material were seen within the cytoplasm of dysplastic mesenchymal cells. Many fibrillogranular vesicles contained smaller vesicles. There were also invaginations of the cell membrane that contained fibrillogranular material similar to that seen in the single membrane vesicles, suggesting engulfment of an extracellular substance. The stroma contained both normal and serrated collagen fibrils, microfibrils, and two types of fibrillogranular material, one of them with a characteristic banding pattern. Our clinical and histopathologic findings resemble those previously described in cases of infantile systemic hyalinosis and juvenile hyaline fibromatosis. So many features of these two conditions overlap that it is difficult not to consider them as parts of a spectrum of the same disorder.     

Morphometric studies of collagen and fibrin lattices contracted by human gingival fibroblasts; comparison with dermal fibroblasts

Cell shape variations and substratum re-organization during contraction of floating collagen and fibrin lattices seeded with human gingival fibroblasts were determined by computerized image analysis of light and scanning electron microscopic images. Data were compared with those obtained with lattices populated with human dermal fibroblasts. The extent of collagen lattice contraction was similar with both cell types, resulting in a two-fold decrease in the area fractions occupied by collagen fibers. Fibroblasts exhibited a rounded shape (form factors equal to 0.8 and 0.7 for gingival and dermal cells, respectively) at day 1 of culture; they possessed a more elongated appearance (with form factors equal to 0.3 and 0.15 for gingival and dermal cells, respectively) at day 7. Continuous (gingival) and discontinuous (dermal) layers of cells were evidenced at the cortex of lattices. Contractions were associated with a significant reduction of the diameters of collagen fibers. Re-organization of substratum, as analyzed by the "Rose of Directions" technique, was evidenced only at the vicinity of filopodia where fibers ran parallel to these protrusions. Several lysed matrix cavities were observed when fibrin lattices were populated with gingival but not dermal fibroblasts at day 5 of culture. Although cells in fibrin lattices exhibited morphometric parameters comparable with those in collagen lattices, no fibroblast layers could be demonstrated at gel peripheries. Fibrin matrices consisted of an isotropic network of entangled fibrin filaments from the start of culture, and only a slight reduction of the diameters of fibrin fibers could be evidenced in dermal fibroblast-populated lattices. Fibrinolysis at the vicinity of gingival fibroblasts led to an entire re-organization of substratum toward the formation of larger fibers. The differential behavior of gingival vs. dermal fibroblasts inside fibrin but not collagen matrices could therefore partly explain the increased rate of remodeling of gingiva as compared with dermis.     

Ca2+-dependent exocytosis in mast cells is stimulated by the Ca2+ sensor, synaptotagmin I

Mast cells secrete a variety of biologically active substances that mediate inflammatory responses. Synaptotagmin(s) (Syts) are a gene family of proteins that are implicated in the control of Ca2+-dependent exocytosis. In the present study, we investigated the possible occurrence and functional involvement of Syt in the control of mast cell exocytosis. Here, we demonstrate that both connective tissue type and mucosal-like mast cells express Syt-immunoreactive proteins, and that these proteins are localized almost exclusively to their secretory granules. Furthermore, expression of Syt I, the neuronal Ca2+ sensor, in rat basophilic leukemia cells (RBL-2H3), a tumor analogue of mucosal mast cells, resulted in prominent potentiation and acceleration of Ca2+-dependent exocytosis. Therefore, these findings implicate Syt as a Ca2+ sensor that mediates regulated secretion in mast cells to calcium ionophore.