Increase in gap junctional intercellular communication by high molecular weight hyaluronic acid associated with fibroblast growth factor 2 and keratinocyte growth factor production in normal human dermal fibroblasts

The effects of different molecular weights of hyaluronic acid (HA), a major component of extracellular matrix, on gap junctional intercellular communication (GJIC) in normal human dermal fibroblasts (NHDF cells) were investigated. NHDF cells were cultured for 4 days with different molecular weights of HA and then the extent of GJIC was assessed by the scrape-loading dye transfer method, using Lucifer yellow. The area of dye transfer was greater in the dishes coated with HA than in those to which HA was added. Thus, NHDF cells cultured on surfaces coated with high molecular weight (HMW) HA (MW, 800 kDa) showed greatly enhanced GJIC. Furthermore, another aim of this study was to evaluate the effects of different molecular weights of HA on the production of FGF-2 and KGF, because both are important cytokines produced by NHDF cells. When FGF-2 and KGF cultured levels of cell extracts and media were determined by ELISA, both levels were significantly enhanced when cells were grown on plates coated with HMW HA. This finding indicated that the function of gap junction channels in NHDF cells grown on plates coated with HMW HA may promote the biosynthesis of growth factors such as FGF-2 and KGF.     

Comparison of deltaFVC between patients with allergic rhinitis with airway hypersensitivity and patients with mild asthma.

BACKGROUND: In asthmatic individuals, airway sensitivity and maximal airway response are increased. Airway sensitivity is usually evaluated by measuring the provocation concentration of inhaled methacholine or histamine that causes a decrease in forced expiratory volume in 1 second of 20% (PC20). The percentage decrease in forced vital capacity at the PC20 (deltaFVC) has been proposed as a surrogate marker for maximal airway response. Individuals with allergic rhinitis and no clinical evidence of asthma frequently exhibit airway hypersensitivity. OBJECTIVE: To compare the deltaFVC between patients with allergic rhinitis and mild asthmatic patients with a similar degree of airway hypersensitivity. METHODS: A retrospective analysis of methacholine challenge test data from 72 children with allergic rhinitis and airway hypersensitivity (methacholine PC20 < 16 mg/mL) (rhinitis group) and from 72 children with mild atopic asthma matched to the rhinitis group regarding the methacholine PC20 (asthma group). The deltaFVC was calculated on the concentration-response curve to methacholine. RESULTS: The mean +/- SD deltaFVC was significantly lower in the rhinitis group (15.0% +/- 3.6%) vs the asthma group (17.4% +/- 5.3%) (P = .002). There was no significant correlation between the deltaFVC and PC20 in the rhinitis (r = -0.101; P = .41) and asthma (r = -0.023; P = .85) groups when 2 patients with PC20 less than 1 mg/mL were excluded from each group. CONCLUSIONS: Patients with allergic rhinitis and airway hypersensitivity had a significantly lower deltaFVC than methacholine PC20-matched mild asthmatic patients, suggesting that the level of maximal airway response in patients with allergic rhinitis is lower than that in mild asthmatic patients with a similar degree of airway hypersensitivity.     

Activation dependence of isotonic transient in response to step tension reduction in cardiac muscle segment during barium contracture

Summary To clarify the activation-dependence of dynamic mechanical characteristics of contracting cardiac muscle, we analysed the healthy central segment length (SL) response to step decrease in tension at two different levels of barium contracture (0.2 mM and 0.5 mM Ba²⁺) in rat papillary muscles with a fixed initial SL. The time course of this response is thought to reflect the kinetics of actin-myosin interaction. The muscle was released stepwise from the steady contracture tension (T_c) to new steady tension levels (T_r) of varying magnitudes at 22° C. The SL responses consisted of four phases at T_r/T_c > 0.3. The amplitude of shortening in the second phase, after the initial rapid and minute shortening in the first phase, increased with an increase in amplitude of step tension reduction, and was greater at the higher activation level when compared at an identical amount of T_r/T_c. The fourth phase, after the remarkable lengthening in the third phase, was an extremely slow and minute shortening toward a new steady SL under the new tension. The duration of the second and third phase was quite insensitive to activation level at T_r/T_c > 0.85, but became longer at the higher activation level with larger amounts of tension reduction. The velocity measured from the initial quasi-steady SL shortening in the second phase increased significantly with the increase in activation level. These results are discussed in terms of cross-bridge kinetics underlying the isotonic SL transients at two different activation levels.     

Down-regulation of transforming growth factor-beta gene expression by antisense oligodeoxynucleotides increases recombinant interferon-gamma-induced nitric oxide synthesis in murine peritoneal macrophages.

Increasing evidence indicates that the production of nitric oxide (NO) by inducible NO synthase (iNOS) is tightly regulated. Transforming growth factor-beta (TGF-beta) is a family of multifunctional peptides secreted during macrophage activation, but several lines of evidence suggest that TGF-beta is selectively suppressive for macrophage NO production. We therefore reasoned that a strategy employing oligodeoxynucleotides (ODN) complementary to TGF-beta mRNA (antisense ODN) might increase NO production in interferon-gamma (IFN-gamma) treated murine peritoneal macrophages. To evaluate this concept, we tested the effects of antisense ODN targeted to TGF-beta mRNA (25-mer ODN complementary to TGF-beta mRNA sequences) by introducing them into the medium of cultured macrophages. Phosphorothioation of ODN was employed to retard their degradation. Antisense ODN had no effect on NO production by itself, whereas IFN-gamma alone had a modest effect. When antisense ODN were used in combination with IFN-gamma, there was a marked cooperative induction of NO production. These effects of antisense ODN were associated with decreased TGF-beta expression in activated macrophages. However, sense ODN had no effect. Adding anti-TGF-beta antibodies to the IFN-gamma-treated macrophages mimicked the positive effect of antisense ODN on NO production. In addition, the effects of either antisense ODN or anti-TGF-beta antibodies were blocked by adding exogenous TGF-beta in cultured macrophages. These results indicate that the generation of TGF-beta by activated macrophages provides a self-regulating mechanism by which the temporal and perhaps spatial production of NO, a reactive and potentially toxic mediator, can be finely regulated.     

Effects of altered body fluid balance and high blood pressure on the plasma brain natriuretic peptide in rats.

The present study was aimed to investigate the regulatory mechanisms of BNP release. Effects of acute and chronic perturbations in body fluid balance, changes in BP, and regulatory roles of NO and endothelin systems on BNP release were examined in rats. Although acute extracellular volume expansion did not have significant effects on plasma BNP, prolonged high-salt intake increased plasma BNP levels. Plasma BNP levels were also higher in 2K1C rats compared with the control. Although infusion of L-NAME increased the plasma BNP in control, it did not further affect the plasma BNP in rats with high-salt intake. Although L-arginine (20 mg.kg-1 per min) per se did not have significant effects on plasma BNP, it blocked the stimulatory effect of L-NAME (200 micrograms.kg-1 per min). Plasma BNP was severalfold increased following a single injection of endothelin (0.3 micrograms/kg) in normal and high-salt intake groups, the magnitude of which was not significantly affected by the high-salt intake. Although indomethacin did not have significant effects on plasma BNP in normal rats, it blocked the stimulatory effect of 2K1C hypertension. It is concluded that BNP is regulated by chronic changes in body fluid balance and blood pressure. It is also suggested that endothelin and NO systems may directly regulate the secretion of BNP in vivo. An endogenous prostaglandin synthesis may be involved in the stimulated release of BNP in hypertension.     

Pharaoh ant (Monomorium pharaonis): newly identified important inhalant allergens in bronchial asthma

The nonstinging house ant, Monomorium pharaonis (pharaoh ant), was recently identified as a cause of respiratory allergy. This study was performed to evaluate the extent of sensitization to pharaoh ant, and its clinical significance in asthmatic patients. We carried out skin prick tests in 318 patients with asthma. Specific IgE (sIgE) to pharaoh ant was measured by ELISA, and cross-reactivity was evaluated by ELISA inhibition tests. Bronchial provocation testing was performed using pharaoh ant extracts. Fifty-eight (18.2%) of 318 patients showed positive skin responses to pharaoh ant, and 25 (7.9%) had an isolated response to pharaoh ant. Positive skin responses to pharaoh ant were significantly higher among patients with non-atopic asthma than among those with atopic asthma (26.0% vs. 14.9%, p<0.05). There was significant correlation between sIgE level and skin responses to pharaoh ant (rho=0.552, p<0.001). The ELISA inhibition tests indicated that pharaoh ant allergens had various pattern of cross-reactivity to house dust mites and cockroaches. Bronchial provocation tests to pharaoh ant were conducted for 9 patients, and eight showed typical asthmatic reactions. In conclusion, pharaoh ant is an important source of aeroallergens, and it should be included in the skin test battery for screening the causative allergens in patients with asthma.     

Molecular Epidemiology of Oral Treponemes Associated with Periodontal Disease

Periodontitis, a disease responsible for tooth loss worldwide, is characterized by chronic inflammation of the periodontium, eventually leading to destruction of periodontal ligaments and supporting alveolar bone. Spirochetes, identified by dark-field microscopy as being the most predominant bacteria in advanced lesions, are thought to play a causative role. Various spirochetal morphotypes were observed, but most of these morphotypes are as yet uncultivable. To assess the role of these organisms we designed oligonucleotide probes for the identification of both cultivable and so far uncultivable spirochetes in periodontitis patients. Subgingival plaque specimens taken from diseased sites (n = 200) and healthy control sites (n = 44) from 53 patients with rapidly progressive periodontitis (RPP) were submitted to direct in situ hybridization or dot blot hybridization after prior amplification with eubacterial primers. Spirochetes were found in all patients, but their distributions varied considerably. Parallel use of oligonucleotide probes specific for cultivable or so far uncultivable treponemes suggested the presence of novel yet unknown organisms at a high frequency. These uncultivable treponemes were visualized by fluorescence in situ hybridization, and their morphologies, sizes, and numbers could be estimated. All RPP patients included in this study harbored oral treponemes that represent either novel species, e.g., Treponema maltophilum, or uncultivable phylotypes. Therefore, it is necessary to include these organisms in etiologic considerations and to strengthen efforts to cultivate these as yet uncultivable treponemes.     

Transient expression of osteopontin mRNA and protein in amoeboid microglia in developing rat brain

To investigate a potential role of osteopontin (OPN) in developing rat brain, the expression of OPN mRNA and protein in the developing rat brain relative to the distribution of brain macrophages was investigated using in situ hybridization and immunohistochemistry, and the phagocytic capability of OPN-expressing cells was accessed using rhodamine isothiocyanate (RhIc) as a tracer. OPN-expressing cells appeared from embryonic day 16. During the first week of postnatal life, OPN-labeled cells increased markedly, and peaked around P7, then declined and had completely disappeared by the end of the second postnatal week. The spatiotemporal distribution pattern of OPN mRNA closely matched that of OPN protein. Their morphology and localization were compared with those of cells expressing the established microglial marker OX-42 in adjacent sections, and double-labeling studies demonstrated that OPN was localized to the amoeboid microglia which stain with the lectin GSI-B₄, another marker for microglia. Furthermore, OPN-labeled cells were confirmed to be active phagocytes emitting RhIc fluorescence indicating that the tracer into the brain tissues was engulfed by phagocytosis. Therefore, these results provide the first evidence that OPN is transiently expressed in active brain macrophages in the embryonic and early postnatal brain, and suggest that OPN may contribute to the migration and phagocytic function of brain macrophages in the developing brain.This work was supported by a Korea Research Foundation grant (KRF-2002-015-EP0106)     

Evaluation of an Immunoglobulin M Capture Enzyme-Linked Immunosorbent Assay for Diagnosis of Orientia tsutsugamushi Infection

To differentiate scrub typhus from other acute febrile diseases, a rapid and reliable serological diagnosis is important. We developed an immunoglobulin M (IgM) capture enzyme-linked immunosorbent assay (ELISA) for diagnosis of recent Orientia tsutsugamushi infections in humans. The 56-kDa major outer membrane protein of O. tsutsugamushi is well known as the most immunodominant antigen in scrub typhus. The test is based on the use of the biotinylated recombinant 56-kDa protein of O. tsutsugamushi Boryong, Bor56, which was expressed as a fusion protein with a maltose-binding protein in Escherichia coli. In the test, the serum IgM antibodies were captured by anti-human IgM antibodies coated onto a microtiter plate. The captured IgM antibodies were revealed through sequential addition of biotinylated Bor56 antigen and peroxidase-conjugated streptavidin to the plate. The IgM capture ELISA was compared with the immunofluorescence antibody assay (IFA) by testing 176 serum samples from patients with diagnosed cases of rickettsial disease and patients with other acute febrile diseases. Of the 81 IgG IFA-positive samples, 78 tested positive (sensitivity, 96.3%) and all 31 IgM IFA-positive samples tested positive (sensitivity, 100%) by the IgM capture ELISA. The specificity of the IgM capture ELISA was 99%, and 1 of the 95 IFA-negative samples was positive in the assay. These results strongly suggest that IgM capture ELISA using the recombinant Bor56 antigen is a reliable and detailed method for the detection of early O. tsutsugamushi infection.     

Characterization of iridium film as a stimulating neural electrode

Iridium films having near-bulk properties were formed by electron-beam evaporation with simultaneous bombardment of Ar ion beam. The charge-injection capabilities of Ir film were investigated, and the detrusor pressure induced by S2 stimulation with Ir-coated Pt electrode was measured and compared with the uncoated Pt electrode. The charge densities of Ir film were continuously increased with increase in the number of cycles in 0.1 M H2SO4 due to the accumulation of the iridium oxide phase. The iridium oxide formed contained nano-pores, and oxides had different dielectric properties. The Ir film could inject various amounts of charge in physiological solution under the identical stimulating condition depending on the degree of activation in 0.1 M H2SO4. S2 stimulation by Ir-coated Pt electrode caused more efficient bladder contraction of the male dog than the uncoated Pt electrode under the identical stimulus condition.