Prolonged ectopic calcification induced by BMP-2-derived synthetic peptide

Bone morphogenetic protein-2 (BMP-2) promotes the formation and regeneration of bone and cartilage, and therefore constitutes the most promising candidate for a bone repair material. However, it also has a wide range of functions, such as in organogenesis and apoptosis. Therefore, we investigated a novel synthetic peptide corresponding to residues 73-92 of BMP-2. This peptide bound to a BMP-2-specific receptor and elevated both alkaline phosphatase activity and osteocalcin mRNA in the murine cell line, C3H10T1/2. The 73-92 peptide also induced ectopic calcification when conjugated to a covalently crosslinked alginate gel. Here we report that the 73-92 peptide-conjugated alginate gel showed prolonged ectopic calcification for up to 7 weeks in rat calf muscle. In contrast, rhBMP-2-impregnated collagen gel showed maximum ectopic calcification at 3 weeks, and the calcified products that had formed disappeared after 5 weeks. Histological examination showed that the 73-92 peptide-conjugated alginate gel induced many osteoblast-like cells and few osteoclasts. In contrast, rhBMP-2-impregnated collagen gel induced many osteoclasts. These results suggest that the 73-92 peptide on alginate gel remains active at the implanted site, continuously induces differentiation of osteoblast precursor cells into osteoblasts, and activates osteoblasts to promote ectopic calcification.     

Roles of tumour necrosis factor-alpha (TNF-alpha), transforming growth factor-beta (TGF-beta), and IL-10 in the modulation of intercellular adhesion molecule-1 (ICAM-1) expression by macrophages during mycobacterial infection

Profiles of ICAM-1 expression on cultured murine peritoneal macrophages infected with Mycobacterium avium complex (MAC) were examined, with special reference to modulating roles of TNF-alpha, TGF-beta, and IL-10. When macrophages were infected with MAC, ICAM-1 expression, measured by microscopic counting of ICAM-1+ macrophages stained with anti-ICAM-1 antibody, ELISA, and flow cytometric analysis, was rapidly increased, peaking at day 3 (early-phase up-regulation) due to endogenous TNF-alpha, and thereafter gradually declined to the normal level within 1 week or more (late-phase down-regulation). The late-phase ICAM-1 down-regulation was also seen in macrophages phagocytosing heat-killed MAC and those stimulated with lipopolysaccharide but not in macrophages phagocytosing latex beads. ICAM-1 mRNA expression was augmented markedly at day 1 after MAC infection and thereafter decreased. While TNF-alpha and IL-10 production by MAC-infected macrophages was observed during the first 3 days, TGF-beta production was initiated from day 3 and continued until day 14. Exogenously added TGF-beta strongly inhibited the early-phase increase in ICAM-1 expression by infected macrophages, and the blockade of endogenous TGF-beta with anti-TGF-beta antibody markedly inhibited late-phase ICAM-1 down-regulation. Moderate blocking effect was also observed for anti-IL-10 antibody. On the other hand, late-phase ICAM-1 down-regulation was not prevented by the addition of exogenous TNF-alpha. Therefore, TGF-beta and IL-10, especially the former, appear to play active roles in the late-phase down-regulation of ICAM-1 in MAC-infected macrophages during long-term cultivation.     

Enhanced hydrogen peroxide release from macrophages stimulated with streptococcal preparation OK-432.

Wheat germ lectin was found to be a potent triggering agent for hydrogen peroxide release from mouse peritoneal macrophages. Macrophages stimulated by intraperitoneal injection of OK-432, a lyophilized attenuated streptococcal preparation, were highly responsive to wheat germ lectin.     

Characteristics of an anti-eosinophil monoclonal antibody that recognizes granulocytes from patients with blood eosinophilia but not from subjects without eosinophilia.

To investigate cell surface antigens of activated human eosinophils using monoclonal antibodies, we established a murine anti-human eosinophil monoclonal antibody AE500 by immunizing with blood eosinophils from patients with idiopathic hypereosinophilic syndrome (HES) and characterized the reactivity to a variety of human leucocytes by a fluorescence-activated cell sorter. AE500 reacted with blood eosinophils and neutrophils in nine out of 11 patients with marked eosinophilia (greater than or equal to 2500/microliters) (seven with idiopathic eosinophilia including HES and two with asthma), but not with those in asthmatic patients with mild eosinophilia (n = 10) or in healthy subjects (n = 8). AE500 did not react with blood lymphocytes, monocytes or platelets. AE500 did not react with human myeloid or lymphoid cell lines, including eosinophilic leukemia cell lines EOL-1 and EOL-3. The reactivity of AE500 to blood eosinophils and neutrophils in patients with marked eosinophilia changed in relation to blood eosinophil counts and prednisolone therapy. In addition, the reactivity of AE500 to blood eosinophils was increased in three out of four AE500-positive eosinophils by the incubation of the cells with granulocyte-macrophage colony-stimulating factor (GM-CSF) at 37 degrees C for 30 min, but not with interleukin 3 or interleukin-5. These results suggest that the anti-eosinophil antibody AE500 detects a cell surface antigen expressed on blood granulocytes in a hypereosinophilic state. This anti-eosinophil antibody would be useful for analysing the mechanism of eosinophilia.     

Immunohistochemical studies on oncogene products (EGF-R,c-erbB-2) and growth factors (EGF,TGF-α) in human breast cancer: their relationship to oestrogen receptor status,histological grade,mitotic index and nodal status

Summary In this investigation, 83 human mammary carcinomas were examined for the expression of oestrogen receptor (ER), epidermal growth factor receptor (EGF-R), epidermal growth factor (EGF), transforming growth factor alpha (TGF-), c-erbB-2, histological grade, mitotic index and nodal status, all of which are reportedly prognostically significant factors (Bloom and Richardson 1957; Baak et al. 1985; Wright et al. 1989). ER expression was biochemically recognized in 43.4% of mammary carcinomas, and EGF-R, EGF, TGF- and c-erbB-2 were histochemically recognized in 25.3, 14.5, 27.7 and 18.0% of mammary carcinomas examined respectively, using conventional sections of buffered formalin-fixed, paraffin-embedded tissue and monoclonal or polyclonal antibodies. There were significant relationships between negative ER and positive EGF-R or TGF-; positive EGF-R and TGF-; positive EGF-R and c-erbB-2; and positive c-erbB-2 and TGF-. The single changes which were the negative ER and the positive c-erbB-2 correlated with histological grade and mitotic index. Co-expression of EGF-R and TGF- correlated with positive nodal status. Therefore, the present investigation indicates that the negative ER, single expression of c-erbB-2 and co-expression of EGF-R and TGF- are important markers which contribute indirectly to prognosis, which reconfirms previous findings on the former two while adding the new finding that immuno-histochemical demonstration of expression of EGF-R and TGF- may provide useful information for selecting the appropriate treatment.     

MALLORY BODY-LIKE STRUCTURES IN THE LUNG

Mallory body-like structures (MBLs), which resemble Mallory body (MB) in the liver, were observed in lungs of 4 cases showing interstitial pneumonitis. MBLs were found in the cytoplasm of especially swollen alveolar epithelial cells. MBLs and MBs had almost similar characteristics morphologically and tinctorially under the light microscopy. MBLs were composed of intermediate type fine fibrils which were positive for anti-keratin antibody. These findings suggested that MBLs were a kind of pathological keratinization of alveolar epithelial cells. Although the origin of MBL containing cells could not be clarified, they were closely related to type 2 alveolar epithelial cells.     

Long term experiment of perfluorochemicals using rabbits

Summary Morphological changes six months to two years after the administration of Perfluorochemicals (PFC) in rabbits injected with a large amount of PFC (67 mg/kg) have already been reported by Ohnishi and Kitazawa (1979, 1980, and 1981). In this paper, the authors report on the persistance of large numbers of foamy cells in the liver, spleen and lymph nodes and many in the kidneys, lung and bone marrow, up to two years after administration. Quantitative analysis of PFC in various organs showed that it was retained. The liver revealed progressive fibrosis surrounding the foamy cells in or around the Glisson's sheath.     

Nerve growth factor enhances antigen and other secretagogue-induced histamine release from rat peritoneal mast cells in the absence of phosphatidylserine

The effects of 2.5S nerve growth factor (NGF) and epidermal growth factor (EGF), isolated from mouse submaxillary glands, on histamine release from rat peritoneal mast cells (PMCs) were studied. In the absence of phosphatidylserine, NGF (1 ng/ml to 1 microgram/ml) did not cause histamine release from PMCs isolated from normal rats and those infected with the nematode Nippostrongylus brasiliensis. However, when PMCs (greater than 97% pure) were preincubated with NGF and then challenged with worm antigen (Ag), there was a marked enhancement of histamine release (approximately twofold with a maximum effect at 10 ng/ml of NGF [3.8 X 10(-10) mol/L]) compared with the release induced by Ag alone. EGF (1 ng/ml to 1 microgram/ml) neither produced histamine release from PMCs in the presence of phosphatidylserine nor enhanced Ag-induced histamine release. This suggests that NGF acts directly on PMCs by activation of cell-surface receptors. The early kinetics of Ag-induced histamine release were altered by NGF that increased the initial rate at 15 seconds but did not prolong the overall duration of histamine release. Simultaneous addition of Ag and NGF did not cause enhanced histamine release; thus, some preincubation time with NGF (5 minutes or less) was required for the activation of PMCs. Moreover, after PMCs were activated by NGF, that state persisted for 1 hour, even when unbound NGF was removed by washing, and thereafter subsided gradually. Further studies revealed that NGF enhanced histamine release induced by concanavalin A, compound 48/80, and ionophore A23187. These results suggest that NGF might be an important molecule in inflammatory responses through the regulation of mediator release from mast cells.     

Association of HLA alleles with response (especially biochemical response) to interferon therapy in Japanese patients with chronic hepatitis C.

The response of chronic hepatitis C to interferon (IFN) treatment is classified as complete response (CR), biochemical response (BR), or no response (NR). Several studies have found no difference in prevention of hepatocellular carcinoma by IFN therapy between patients with CR and those with BR. We investigated whether specific human leukocyte antigen (HLA) alleles were associated with response to IFN, especially BR, in 138 patients with chronic hepatitis C. Comparing patients with and without CR, male, a low viral titer, genotype 2a or 2b, HLA-B55, and HLA-DRB1-0803 were more common in the group with CR. Multivariate analysis showed that age (adjusted odds ratio [OR], 0.95 by every year [95% confidence interval [CI] 0.90 - 0.99], p = 0.028), genotype 2a or 2b (5.21 [95% CI 1.63 - 16.6], p = 0.005), and low viral titer (8.58 (2.66 - 27.7), p < 0.001) were associated with CR. Comparing patients with BR and NR, the pretreatment alanine aminotransferase (ALT) level was lower in the BR group (p < 0.001). Both HLA-B7 and HLA-DRB1-0101 were more common in this group (p = 0.002). As the alleles HLA-B7 and HLA-DRB1-0101 were in linkage disequilibrium, the HLA-B7-DRB1-0101 haplotype may be associated with BR. Multivariate analysis indicated that a low ALT level (0.98 by every 1 IU/L [95% CI 0.98 - 0.99], p = 0.001) and HLA-B7-DRB1-0101 haplotype (32.3 [95% CI 1.50 - 693.1], p = 0.026) contributed significantly to BR. This study suggested that host HLA expression, but not viral factors, can influence BR.     

Mechanomyography of the human quadriceps muscle during incremental cycle ergometry

The mechanical activity of the human quadriceps muscle during maximal incremental cycle ergometry was investigated by mechanomyography (MMG). MMG and surface electromyography (EMG) recordings of vastus lateralis muscle activity were obtained from nine males. Cycle ergometry was performed at 60?rev/min and work load was incremented step wise by 20?W (3.2?Nm) every minute until volitional fatigue. The mean amplitudes of MMG (mMMG) and EMG (mEMG) during the contraction phase were calculated from the last six contractions in each load. The duration, load and work rate of exercise at exhaustion were 13.3 (1.6)?min, 44.1 (5.5)?Nm, 276.7 (34.7)?W, respectively. A linear relationship between mMMG and load was evident in each subject (r?=?0.868–0.995), while mEMG seemed to dissociate as the load became greater. In the grouped mean data, mMMG was linearly related to load whether aligned to the absolute (r?=?0.995) or maximal (r?=?0.995) load. Involvement of the noise component was further investigated by studying passive cycling by four subjects. Pedals were rotated passively for the first half of each stage (PAS) and the subject then pushed the pedals for the second half (ACT). In the lighter load region, the mMMG of ACT was as small as that of PAS. However, the change in the mMMG of PAS was very small compared with that of ACT. In conclusion, this study demonstrates a linear relationship between the mMMG of the quadriceps muscle and work load during maximal incremental cycle ergometry. The effect of movement noise was thought to be small and stable.