Inhibition Of Contact Research Articles

Analysis of apoptotic and antiapoptotic signalling pathways induced by Helicobacter pylori.

Although it is reported that Helicobacter pylori induces apoptosis on gastric epithelial cells, the mechanism remains unknown. Antiapoptotic effects generated by H pylori have not yet been evaluated. (1) H pylori strains (type 1 wild, TN2-DeltacagE, TN2-DeltavacA) were cocultured with MKN45, TMK1, and HeLa cells, and cell viability and apoptosis were assessed by trypan blue exclusion and DNA laddering, respectively. (2) Activation of caspases-3, 7, and 8, cytochrome c release from the mitochondria, and Fas, Fas associated death domain protein (FADD), Bax, Bak, and Bcl-X expression were evaluated by immunoblot analysis. (3) To investigate whether nuclear factor kappa B (NFkappaB) activation induced by cag pathogenicity island (PAI) positive H pylori affects antiapoptosis, MKN45 cells stably expressing super-repressor Ikappabetaalpha were cocultured with H pylori, and cell viability and caspase activation were evaluated. NFkappaB regulated gene expression was also evaluated by ribonuclease protection assay. (1) Wild-type and DeltavacA mutant H pylori induced apoptosis more potently than the DeltacagE mutant. Inhibition of cell contact between H pylori and cancer cells and heat killing H pylori diminished cell death. (2) Caspases-3, 7, and 8 were activated time dependently by H pylori as well as by the agonist anti-Fas. Cytochrome c release from mitochondria was observed and was not inhibited by caspase-8 inhibitor. Although protein expression of Fas, FADD, Bax, Bak, and Bcl-X in the whole cell lysates was not changed by H pylori, Bax was decreased from mitochondria free cytosol suggesting that Bax was translocated into mitochondria. (3) Cell death and the activities of caspases-3 and 8 were promoted in MKN45 cells stably expressing super-repressor Ikappabetaalpha that inhibits NFkappaB activation. Antiapoptotic proteins c-IAP1 and c-IAP2 were upregulated by the wild-type strains. cag PAI positive H pylori is capable of inducing apoptotic effects mainly through the mitochondrial pathway. Antiapoptotic effects mediated by NFkappaB activation were also observed.

Cortical bone ingrowth in growth hormone-loaded grooved implants with calcium phosphate coatings in goat femurs

Dental implants are succesfully used for tissue-integrated protheses, but the long-term survival in the maxilla is shorter than in the mandible [Cune MS, Thesis, University of Utrecht, 1993; Jaffin RA, Berman CL. J Periodontol 1991;62:2–4]. However, by adding growth hormone at implantation, increased bone apposition may be expected, since it is known that growth hormone has a stimulating effect on the differentiation and proliferation of osteoblastic cells [Ernst M, Froesch ER. Biochem Biophys Res Commun 1988;151:142–47; Scheven BAA et al. Growth Regul 1991;1:160–67; Stracke H et al. Acta Endocrinol 1984;107:16–24]. We studied bone ingrowth and bone contact of grooved implants impregnated with growth hormone in the cortex of femurs of female goats. We compared the effect of growth hormone on grooved implants with or without calcium phosphate coatings at the bottom of the grooves. The coatings used were hydroxyapatite, fluorapatite and heat-treated hydroxyapatite. The implants had both small and large grooves. The implants were positioned in the cortex of one femur and were treated with recombinant human growth hormone, while the implants on the opposite femur served as controls. After 6 weeks, the implants and surrounding tissues were dissected and evaluated histomorphologically and morphometrically by light microscopy. The bone ingrowth and the bone contact in the grooves were quantified by digital image analysis. Calcium phosphate coating at the bottom of the grooves resulted in a significant increase of bone ingrowth and bone contact. Small grooves had significantly more bone ingrowth and bone contact than the larger grooves. However, all implants impregnated with growth hormone showed inhibition of bone contact and bone ingrowth. We conclude that recombinant human growth hormone inhibits bone formation in the grooves coated with calcium phosphate. Without the addition of growth hormone, the calcium phosphate coatings improved bone ingrowth and bone contact in the grooves. Further studies are required to determine whether growth hormone could also possibly act as a bone growth promoting factor in these implants.

Synthesis of the Ca 2+-dependent cell adhesion molecule DdCAD-1 is regulated by multiple factors during Dictyostelium development

In Dictyostelium discoideum, the cadA gene encodes the cell adhesion molecule DdCAD-1, a protein of M r 24,000, which mediates Ca 2+-dependent cell-cell adhesion during development. We have examined the effects of cAMP, cell-cell contact, and growth conditions on cadA expression. cadA has a unique pattern of expression, which appears to be a combination of the expression patterns of early genes and aggregation-stage genes. Expression of the cadA gene in bacterially grown cells is activated at the beginning of the developmental cycle, followed by a period of rapid DdCAD-1 accumulation. The mRNA level reaches its maximum at 9 h of development and then declines to the basal level at ∼18 h, while the protein level remains constant after reaching its maximum at 12 h. Pulse-chase experiments have demonstrated that DdCAD-1 has a significantly longer half-life than the average cellular protein. Transcription of the cadA gene is stimulated by exogenous cAMP pulses, leading to a 3- to 5-fold increase in the transcription rate. In the fgdA mutant, which lacks a functional Gα2, cAMP fails to enhance cadA expression, suggesting that cAMP stimulates cadA transcription via a G protein-dependent pathway. However, inhibition of cell-cell contact has no effect on the synthesis of DdCAD-1. Growth conditions also have a major influence on cadA expression. Axenically grown cells produce a high level of cadA transcripts during vegetative growth. The mRNA level shows a steady decrease during development and is reduced to the basal level by 12 h. In contrast, the level of DdCAD-1 remains relatively high throughout development, suggesting that axenic growth affects the accumulation of cadA mRNA but not the stability of the protein. These results indicate that multiple mechanisms are involved to maintain a high level of DdCAD-1 during development.

Procoagulant Action of Aprotinin

In Response: In response to Dr. Alston's comment regarding our inability to see the procoagulant effects of aprotinin, our study was not designed to evaluate the procoagulant effects of aprotinin using ex vivo measurements but rather to determine whether in vitro or ex vivo aprotinin prolongs whole blood prothrombin time (PT) or activated partial thromboplastin time (APTT) measurements. This information could then be used by clinicians to avoid inappropriate administration of additional protamine or plasma in bleeding patients who have aprotinin-mediated prolongations of PT and APTT measurements. Accordingly, we demonstrated that prolonged APTT results are misleading in patients who are concurrently receiving aprotinin. As addressed in our manuscript, prolongation of coagulation measurements by aprotinin may be a reflection of this drug's anticoagulant properties [1,2] or in vitro inhibition of commonly used coagulation activators [3,4]. Nevertheless, we find Dr. Alston's evidence that aprotinin prolongs the modified PT assay intriguing. Although a previous study [5] indicated that protein C levels are unaffected by aprotinin, Dr. Alston's data confirm previous findings that have demonstrated inhibition of activated protein C by aprotinin [6]. We agree with Dr. Alston that aprotinin may have procoagulant properties and that PT and APTT assays may not detect these properties; however, we do not agree that these procoagulant properties necessarily account for reduced blood loss and transfusion requirements when this drug is used clinically. On the contrary, aprotinin may preserve hemostasis by enhancing anticoagulation via inhibition of contact [7,8] and potentially extrinsic-mediated [9] activation as reflected by reduced thrombin generation/activity [10], and these effects may be independent of its significant antifibrinolytic and antiinflammatory (e.g., neutrophil and complement inhibition) properties [7,11]. The concept of enhanced anticoagulation resulting in better preservation of hemostasis is supported by recent findings that demonstrate that maintenance of patient-specific heparin concentrations can reduce consumption of coagulation factors via better suppression of thrombin activity [12]. ATIII supplementation [13] has also been shown to suppress thrombin activity as demonstrated by reduced fibrinopeptide A levels in pediatric patients who received ATIII concentrates. Further studies are needed to characterize the pro- versus anticoagulant properties of aprotinin, which may vary significantly between individual patients. G. J. Despotis, MD A. Alsoufiev, MD L. T. Goodnough, MD D. G. Lappas, MD Departments of Anesthesiology, Internal Medicine, and Pathology Washington University School of Medicine St. Louis, MO 63110

Influence of cytokines (IL‐1α, IL‐3, IL‐11, GM‐CSF) on megakaryocyte‐fibroblast interactions in normal human bone marrow

The evolution of myelofibrosis accompanying chronic myeloproliferative disorders (CMPDs) is often linked with megakaryopoiesis. However, it is not known whether or to what extent megakaryocytes of normal human bone marrow are capable of stimulating fibroblast growth. For this reason, an in vitro study was performed to elucidate possible cytokine-dependent interactions between megakaryocytes and fibroblasts derived from healthy volunteers. Fibroblast growth was significantly promoted by the presence of megakaryocytes and modulated by additional application of various cytokines. While recombinant human (rh) interleukin (IL)-1 alpha had no obvious effect on fibroblast proliferation, a slight increase was detected on adding granulocyte-macrophage colony stimulating factor (rhGM-CSF). Application of rhIL-3 caused a significant increase in the number of fibroblasts. In contrast, administration of rhIL-11 suppressed the megakaryocyte-dependent growth-promoting effect and co-stimulation with rhIL-3 led to a significant decrease of fibroblast number in comparison to rhIL-3-stimulated co-cultures. Inhibition of cell-cell contact in unstimulated, as well as in rhIL-3-stimulated co-cultured led to a conspicuous impairment of fibroblast growth. A similar effect was observed when neutralizing antibodies directed against platelet-derived growth factor (PDGF) and transforming growth factor (TGF)beta 1 were added to rhIL-3-stimulated cultures. Our findings are in keeping with the assumption that interactions between megakaryocytes and fibroblasts involve in cytokine-mediated functional network regulated by factors such as spatial relationship, cytokine stimulation, and low concentrations of mediators, particularly PDGF and TGF beta. In this complex system rhIL-3 seems to play a crucial role in the promotion of these various interrelationships.

Apoptosis induced by inhibition of intercellular contact.

The LIM 1863 colon carcinoma cell line grows as structural organoids of goblet and columnar cells around a central lumen and provides a model for the development of stem cells in the normal colon. The organoid structure can be disrupted by removal of calcium from the medium, resulting in a suspension of single cells. Upon readdition of calcium, the cells reform the organoid structure over a period of 24 h, and ultrastructural examination of the reforming cells reveals that this involves a complex process that we have termed clutching. To determine the adhesion molecules involved in organoid formation we attempted to block this process by single cell suspensions of LIM 1863 reseeded in the presence of monoclonal antibodies. An anti-integrin antibody directed against a conformational epitope on the alpha v subunit totally inhibited organoid reformation. As a consequence of this inhibition of cell contact the colon carcinoma cells rapidly underwent apoptosis. Investigations of the apoptotic pathway involved suggested an induction mechanism since the onset of apoptosis in the contact-inhibited cells showed specific increased synthesis of 68- and 72-kD proteins. In addition, immunoblotting of cytosolic and nuclear extracts of the cells revealed the rapid translocation of the tumor suppressor gene product, p53 to the cell nucleus upon induction of apoptosis. These results suggest that cell-cell adhesion may be a vital regulator of colon development overcome in tumor cells by loss of adhesion molecules or of functional p53 protein.

Knowledge utilization: The role of new communication technologies

From the mid-1960s until the end of the 1970s, knowledge utilization was a framing concept for policy research on dissemination and social change in the U.S. The 1980s were a hiatus in the development of dissemination and social change strategies, but the present domestic refocusing of national policy brings knowledge utilization once again to the forefront. The communication technologies used in knowledge utilization programs of the 1960s and 1970s consisted of analog media such as printed materials and video. The technologies used in knowledge utilization programs of the 1990s will include several digital media such as ISDN, online search services, e-mail, facsimile, and CD-ROM. The sweeping claims made for digital media today are similar to those made for analog media 20 years ago, when in fact the analog media played only a secondary role to the prime movers of social networks and personal influence. Some properties of digital media such as asynchronicity and transformability will meet previously unmet needs in knowledge utilization. The challenge of matching specific communication technologies to phases and functions of knowledge utilization is renewed by the present mix of analog and digital media. Reasons why communication technologies succeed range from “meets an important need” to “avoids the technophobic pitfalls of deskilling, destatusing, undue technological literacy, and inhibition of human contact.” © 1993 John Wiley & Sons, Inc.

Human blood dendritic cells exhibit a distinct T-cell-stimulating mechanism and differentiation pattern.

In this study, the mechanisms underlying stimulation of T-cell proliferation by human blood dendritic cells (BDC) and their differentiation have been defined with a panel of monoclonal antibodies (MoAbs). It was found that the MoAbs against LFA-1 (CD11a), CD11c, LFA-3 (CD58), ICAM-1 (CD54) or HLA-DR could significantly suppress T-cell proliferation in an allogeneic mixed lymphocyte reaction (P < 0.05), while being unable to inhibit clustering of BDC with T cells. Addition of anti-CD18 or CD45 MoAbs increased the size of clusters after 18 h of culture, but had no effect on the proliferation of T cells (P < 0.05). The suppressive effect of the MoAbs may be viewed not as an inhibition of contact between BDC and T cells, but rather as a blocking of co-stimulatory signals for T-cell activation, which are mediated by interaction of the adhesion molecules. After depleting the BDC preparations of monocytes, we used a double staining in FACS analysis to demonstrate that BDC do not express specific T (CD3), B (CD20 and CD21) and myeloid cell markers (CD11b, CD13 and CD14), but abundant class II antigens. This pattern remained unaltered after 8 days of culture in the presence of 100 U/ml GM-CSF, although a threefold increase of HLA-DQ and ICAM-1 molecules on the cultured cells was observed.

Wind tunnel studies of sex pheromone-mediated behavior of the Hessian fly (Diptera: Cecidomyiidae)

In a wind-tunnel, male Hessian flies flying toward a source of the female-produced sex pheromone exhibited flight maneuvers very similar to those described for male moths. Upwind flight, consisting of zigzagging and straight flight upwind, was initiated within seconds after flies were placed in the odor plume. This upwind flight was sometimes interrupted by casting, which consisted of wide excursions in the horizontal plane ranging 10-35 cm across the central zone of the tunnel. Comparison of the flight maneuvers of males exposed to ten female equivalents of a hexane extract of female ovipositors and males exposed to 20 ng of (2S)-(E)10-tridecen-2-yl acetate (SE10-13:OAc), which has been identified as a component of the Hessian fly sex pheromone, indicated that the sex pheromone probably contains additional components. However, SE10-13: OAc elicited upwind flight and source location by a significant number of males, even at dosages as low as 2 ng on filter paper. At the highest dosage of SE10-13:OAc tested (200 ng on filter paper), there was a significant decrease in net flight velocity and a slight, but not significant, reduction in the number of males contacting the odor source. The addition of increasing amounts of the R enantiomer to the S enantiomer resulted in increased inhibition of upwind flight and source contact by males.

Formation of inclusion complexes between cyclodextrins and aromatic compounds under pressurized carbon dioxide

The effects of moisture content, pressure and temperature on the formation of inclusion complexes with cyclodextrins under pressurized carbon dioxide (CO 2) were studied to determine the optimal conditions for the recovery of aromatic compounds after extraction with supercritical or liquid CO 2. The presence of water in the cyclodextrin was essential for the formation of inclusion complex. However, an excess of water lowered the amount of inclusion complex due to inhibition of contact with CO 2. As the pressure increased under a constant temperature, the amount of inclusion complex increased until CO 2 became liquid, where the formation of inclusion complex started to decline. The amount of inclusion complex for modified cyclodextrins was very little, because the cyclodextrins became paste- or candy-like by the addition of a small amount of water.

Mechanical Properties of Aluminum Alloys Reinforced with Continuous Silicon-carbide Fibers and Whiskers or Particulates

Multifilament type silicon-carbide fiber-reinforced aluminum alloys and a new type of fiber composite, in which whiskers or particulates are distributed among continuous fibers, were prepared by squeeze casting. The mechanical properties and fracture behaviour of the composites were investigated.The hybrid composites were superior in the strength to the conventional fiber composites. The tensile strength of the composite with hybrid reinforcements of fibers and fine particulates coincided with the strength predicted from the simple rule of mixture. The effect of the addition of whiskers or particulates is to maintain fiber separation during composite fabrication, followed by inhibition of the direct fiber contact in the composites.

Purified human monocyte subsets as effector cells in antibody-dependent cellular cytotoxicity (ADCC).

Human peripheral blood monocytes are heterogeneous with respect to their size and function. Two monocyte subsets were isolated by countercurrent centrifugal elutriation and were studied with respect to their ability to effect antibody-dependent cellular cytotoxicity (ADCC) and for the presence of Fc receptors on their surface. Both monocyte subsets display Fc surface receptors and are effectors of ADCC against sensitized human erythrocyte target cells. The demonstration of ADCC by monocyte effectors is dependent on their concentration in the incubation mixture. Dilution of monocytes below 10% by unlabeled and unsensitized erythrocytes or lymphocytes significantly suppresses ADCC, presumably by steric inhibition of effector and target contact.

The effect of specific antibodies on the inhibition of leucocyte migration caused by staphylococcal peptidoglycan.

Specific antibodies to the various antigenic determinants of staphylococcal peptidoglycan are tested for neutralization of the inhibiting effect of peptidoglycan on leucocyte migration. Antibodies to the C-terminal D-Ala-D-Ala group of pentapeptides and to the C-terminal of the glycine bridge showed high neutralizing effect, whereas that of antibodies to the tetrapeptide and to the glycan chain was negligible. The observed neutralization of antibodies against the outermost parts of peptide chains may be due to the inhibition of contact between peptidoglycan and cells.