Effect of busui shengxue granule (see text) on chronic aplastic anemia patients' hematopoietic adhesion molecule VLA-6/CD49f and its ligand laminin.

OBJECTIVE: To observe the effect of Busui Shengxue Granule ((see text) Herbal granule for replenishing marrow to produce blood) on chronic aplastic anemia (CAA) patients' integrin alpha 6 (VLA-6/CD49f) and laminin (Ln). METHODS: Sixty-five patients were divided into experimental group and control group through random number table. There were 34 patients, 17 were male and 17 female, aged 2-67, with a medianage of 30.2 +/- 8.6, in the experimental group, including 17 patients of kidney-yin deficiency and 17 of kidney-yang deficiency, treated by Busui Shengxue Granule. There were 31 patients in the control group, 16 were male and 15 female, aged 4-65, with a medianage of 31.2 +/- 8.0; administered Zaizhang Shengxue Tablet (see text) Herbal tablet for chronic aplastic anemia). Both groups were treated for six months and compared with 10 normal persons after the treatment. Flow cytometry was adopted to detect the change in the expression of VLA-6/CD49f, receptor in mononuclear cells of CAA patients and normal persons. Enzyme-linked immunosorbent assay was applied to detect the expression of peripheral serum Ln. RESULTS: CAA patients' VLA-6/CD49f was in the state of low expression and Ln in the state of high expression. After the treatment, both VLA-6/CD49f and Ln were regulated to some extent and the change in the experimental group was better than that of the control group. Compared with the kidney-yin deficiency patients, those indices of kidney-yang deficiency patients were easier to correct. CONCLUSION: The VLA-6/CD49f and Ln expressions of CAA patients are abnormal. The treatment with Busui Shengxue Granule makes both of them improved.

The effects of selective COX-2 inhibitor/celecoxib and omega-3 fatty acid on matrix metalloproteinases, TIMP-1, and laminin-5gamma2-chain immunolocalization in experimental periodontitis.

BACKGROUND: Matrix metalloproteinases (MMPs) play important roles in tissue-destruction mechanisms-associated periodontitis. MMP-8 and -13 are the predominant collagenases that are important in the extracellular matrix degradation in periodontal tissues. MMP-14 is a membrane-type MMP, whereas laminin-5 indicates basal membrane modification and epithelial induction. The purpose of the present study was to evaluate the effects of celecoxib and omega-3 fatty acid administration on the gingival tissue expression of MMP-8, -13, and -14, tissue inhibitor of MMP (TIMP)-1, and laminin (Ln)-5gamma2-chain in rat experimental periodontitis induced by Escherichia coli endotoxin (lipopolysaccharide [LPS]). METHODS: Experimental periodontitis was induced in rats by repeated LPS injection. Fifty-one adult male Sprague-Dawley rats were divided into six study groups: saline control, LPS, LPS + celecoxib, LPS + therapeutic omega-3 (TO3), prophylactic omega-3 + LPS + omega-3 (P+TO3), and LPS + celecoxib + omega-3 fatty acid. Celecoxib and omega-3 fatty acid were given as a single agent or as combination therapy for 14 days. On day 15, all rats were sacrificed, and gingival tissues were analyzed immunohistochemically for the expression of MMP-8, -13, and -14, TIMP-1, and Ln-5gamma2-chain. Alveolar bone loss was evaluated morphometrically under a stereomicroscope. Data were tested statistically by Kruskal-Wallis and Mann-Whitney tests and Spearman correlation analysis. RESULTS: Alveolar bone loss was significantly higher in all study groups compared to the saline control group (all P <0.01). MMP-8 expression was significantly higher in the LPS group than in the saline group (P = 0.001). Very low expression of MMP-8 was found in the celecoxib, P+TO3, and combination groups. TO3 increased TIMP-1 expression significantly compared to the LPS group (P <0.05). Individual celecoxib and P+TO3 administration increased MMP-14 significantly compared to saline control and LPS groups (P <0.05). No significant differences were found among the study groups with regard to Ln-5gamma2-chain and MMP-13 expressions (P >0.05). CONCLUSIONS: Selective cyclooxygenase-2 inhibitor, prophylactic omega-3 fatty acid, and a combination of these two agents can inhibit gingival tissue MMP-8 expression. Moreover, the individual administration of therapeutic omega-3 may increase gingival TIMP-1 expression in contrast to no effect on MMP-8, -13, and -14 expressions in experimental periodontitis. These experimental findings in a rat model of LPS-induced periodontitis need to be verified by clinical human studies.

Effects of aluminium sulphate in the mouse liver: similarities to the aging process.

Aluminium (Al) is a ubiquitous metal that is potentially toxic to the brain. Its effects on other fundamental organs are not completely understood. This morphological in vivo study sought to compare sublethal hepatotoxic changes and Al deposition in adult mice that orally ingested Al sulphate daily for 10 months, in age matched control mice that drank tap water and in senescent mice (24 months old). Livers were examined for collagen deposition using Sirius red and Masson, for iron accumulation using Perls' stain. Light, electron microscopy and morphometry were used to assess fibrosis and vascular changes. Scanning transmission electron microscopy and EDX microanalysis were used to detect in situ elemental Al. Iron deposition, transferrin receptor expression were significantly altered following Al exposure and in the aged liver but were unaffected in age matched control mice. In Al treated mice as in senescent mice, endothelial thickness was increased and porosity was decreased like perisinusoidal actin. Furthermore, Al stimulated the deposition of collagen and laminin, mainly in acinar zones 1 and 3. Pseudocapillarization and periportal laminin in senescent mice were similar to Al treated adult liver. In conclusion, prolonged Al sulphate intake accelerates features of senescence in the adult mice liver.

Fractones and other basal laminae in the hypothalamus.

The physiological role of basal laminae (BL) and connective tissue (meninges and their projections) in the adult brain is unknown. We recently described novel forms of BL, termed fractones, in the most neurogenic zone of the adult brain, the subependymal layer (SEL) of the lateral ventricle. Here, we investigated the organization of BL throughout the hypothalamus, using confocal and electron microscopy. New types of BL were identified. First, fractones, similar to those found in the lateral ventricle wall, were regularly arranged along the walls of the third ventricle. Fractones consisted of labyrinthine BL projecting from SEL blood vessels to terminate immediately beneath the ependyma. Numerous processes of astrocytes and of microglial cells directly contacted fractones. Second, another form of BL projection, termed anastomotic BL, was found between capillaries in dense capillary beds. The anastomotic BL enclosed extraparenchymal cells that networked with the perivascular cells coursing in the sheaths of adjacent blood vessels. Vimentin immunoreactivity was often detected in the anastomotic BL. In addition, the anastomotic BL overlying macrophages contained numerous fibrils of collagen. We also found that the BL located at the pial surface formed labyrinthine tube-like structures enclosing numerous fibroblast and astrocyte endfeet, with pouches of collagen fibrils at the interface between the two cell types. We suggest that cytokines and growth factors produced by connective tissue cells might concentrate in BL, where their interactions with extracellular matrix proteins might contribute to their effects on the overlying neural tissue, promoting cytogenesis and morphological changes and participating in neuroendocrine regulation.

Upregulation of adhesion complex proteins and fibronectin by human keratinocytes treated with an aqueous extract from the leaves of Chromolaena odorata (Eupolin).

The fresh leaves and extract of the plant Chromolaena odorata are a traditional herbal treatment in developing countries for burns, soft tissue wounds and skin infections. We have previously shown that the extract had an effect on the growth and proliferation of keratinocytes and fibroblasts in culture. This study has demonstrated that Eupolin extract increased expression of several components of the adhesion complex and fibronectin by human keratinocytes. Using indirect immunofluorescence we found increased expression (dose-dependent) of laminin 5, laminin 1, collagen IV, and fibronectin. The expression of the b1 and b4 integrins was upregulated by the extract at low concentrations (0.1 and 1 microg/ml), but the expression was decreased at higher doses of Eupolin (10 microg-150 microg/ml). A number of clinical studies carried out by Vietnamese and international medical investigators have demonstrated the efficacy of this extract on the wound healing process. In this study we have shown that Eupolin stimulated the expression of many proteins of the adhesion complex and fibronectin by human keratinocytes. The adhesion complex proteins are essential to stabilise epithelium and this effect could contribute to the clinical efficacy of Eupolin in healing.

Extracellular matrix proteins increase the expression of pro-TRH and pro-protein convertase PC1 in fetal hypothalamic neurons in vitro.

External clues for neuron development include extracellular matrix (ECM) molecules. To explore ECM influence on the early development of peptide phenotype in the CNS, we have compared pro-TRH levels in primary cultures of rat hypothalamic cells plated either on poly-lysine (PL) (control) or on PL plus one of various ECM molecules at 10 microgram/ml. Fetal day 17 cells plated at a density of 1250/mm(2) were grown in a serum free medium made of Neurobasal medium supplemented with B27 (GIBCO). Cultures, consisting mainly of neurons, were analyzed at DIV 2. ECM proteins induced morphological effects in agreement with previously published studies. The amount of pro-TRH per dish, quantified by Western blotting, was increased to 275% for laminin, 191% for fibronectin and 173% for tenascin-C (control=100%); there was no effect of vitronectin. Laminin or fibronectin did not change pro-TRH mRNA or TRH levels but enhanced levels of the pro-protein convertase PC1 suggesting that the ECM molecules did regulate the translational status of pro-TRH. In conclusion, we have shown that some ECM proteins increased pro-TRH level in vitro; this may contribute to the enhancement of pro-TRH levels observed early in vivo in the hypothalamus.

Heparan sulphate and HB-GAM (heparin-binding growth-associated molecule) in the development of the thalamocortical pathway of rat brain.

Extracellular matrix (ECM) molecules, such as laminin, tenascin, chondroitin sulphate proteoglycans and heparan sulphate proteoglycans have been suggested to have 'signpost' and directing roles in the formation of axonal projections in cortical development. We show here that the expression of the neurite outgrowth-promoting protein heparin-binding growth-associated molecule (HB-GAM) and N-syndecan, a transmembrane heparan sulphate proteoglycan previously isolated as a receptor for HB-GAM, is spatiotemporally associated with the developing thalamocortical pathway in the rat brain. Using in situ hybridization, thalamic neurons were shown to express mRNA for N-syndecan, and in vitro, thalamic neurons grew more neurites on HB-GAM than on laminin. The HB-GAM-induced neurite outgrowth in thalamic neurons was inhibited by heparitinase, heparin, soluble N-syndecan and by an excess of soluble HB-GAM in the culture medium. In a pathway assay, thalamic neurons selectively preferred attaching and growing neurites on matrices containing HB-GAM than on those containing fibronectin or laminin alone, suggesting that HB-GAM may modulate the effect of other ECM proteins. On an unfixed brain slice preparation, thalamic neurons repeatedly showed a typical neurite outgrowth and attachment pattern resembling the expression pattern of HB-GAM. On the brain slices, the neurite outgrowth was significantly inhibited by heparitinase, heparin and soluble HB-GAM, thus displaying features of neurite outgrowth on matrix-bound HB-GAM. Our results suggest that HB-GAM is important for the neurite outgrowth of thalamic neurons and it may function as an ECM-bound guidance cue for thalamic neurons that possess HB-GAM-binding heparan sulphates on their cell membrane.

Assessment of bone formation during osteoneogenesis: a canine model.

Distraction osteoneogenesis, callotasis, has been demonstrated to be an effective means of lengthening long bones. A variation of Ilizarov's technique produces a transport disk from one cut surface of bone within a defect and advances the disk to the opposite surface to close the defect. This process, previously described by Costantino et al. (Arch Otolaryngol Head Neck Surg 1990; 116:535-45), demonstrated bone formation within the distraction site. The precise mechanism of bone formation has not yet been described for the mandible. Four conditioned beagles were studied, with one control dog maintained in neutral fixation and three dogs distracted at 0.25 mm every 8 hours. A two-cm defect was closed, and dogs were kept in fixation for 1 week after closure, after which they were killed. Three sites were evaluated: (1) the distraction seam, (2) the interface of the cortical and distracted bone, and (3) the cortexes at the closed defect. Each site was bisected, and one half was decalcified for immunohistochemical and hematoxylin and eosin pathologic evaluation. The vascular basement membrane was labeled for laminin and type IV collagen. Both of these substances demonstrate the differentiation of the vascular matrix component predisposing primary bone formation. Labels were intense at the distraction seam where intense angiogenesis occurred. No hyalin cartilage was observed at the distraction site, which indicates that the fixation was stable and that ossification occurred primarily without intermediate callous formation. This model demonstrated that osteoclasts within the canine model produce bone through primary bone formation within an angiogenic matrix rich in basement membrane laminin and type IV collagen. Likewise, bone is species specific in mineral composition for dog mandible. Understanding the formation and composition of distracted bone is essential for understanding application of this technique within the clinical setting.

A unique rat model of bile ductular hyperplasia in which liver is almost totally replaced with well-differentiated bile ductules.

A novel rat model was developed in which furan combined in a unique synergistic manner with bile duct ligation to induce replacement of most of liver with well-differentiated hyperplastic bile ductules. Multiple tissue sections of liver from Fischer 344 male rats first subjected to a bile duct ligation and 1 week later given furan by gavage at 45 mg/kg body weight, once a day, five times weekly for 5 to 6 weeks, exhibited a mean percent of bile ductule tissue per total liver section area of 72.6 +/- 16.3% compared to control values of 20.0 +/- 4.2% for bile duct-ligated rats that received corn oil by gavage instead of furan and 11.9 +/- 3.1% for rats that were given a sham operation followed by furan. This dramatic difference was also reflected by the very high mean gamma-glutamyl transpeptidase specific activity of liver homogenates from the bile duct-ligated/furan-treated rats, which was approximately 8 x 10(3) nmoles p-nitroaniline/mg protein/hour versus values of approximately 2 x 10(3) for bile duct-ligated/corn oil control, approximately 1 x 10(3) for sham-operated/furan-treated control, and 44.9 for untreated rat. The data presented support a potentially powerful experimental model for investigating bile ductular cell functions, differentiation, and proliferation.