Prostaglandin E2 secretion, cell maturation, and CD14 expression by monocyte-derived macrophages from localized juvenile periodontitis patients

Free eicosapentaenoic acid (EPA) was found to inhibit dose dependently the chemiluminescence of human neutrophil granulocytes phagocytosing zymosan and their chemotaxis induced by C5a-containing zymosan-activated serum (ZAS) and platelet-activating factor. Rigidification of plasma membranes in the ZAS-treated cells could be observed by measuring the fluorescence anisotropy. The cells were labeled by 3-[p-(6-phenyl-1,3,5-hexatrienoil) phenyl] propionic acid, reporting plasma membrane for determination of membrane fluidity. In resting, nonstimulated neutrophils, EPA dose dependently increased the fluidity of plasma membrane. In zymosan-activated cells, however, after a short fluidization, the basic effect of EPA was a rigidification compared to very low fluorescence anisotropy values of activated control cells. This diminished fluidity, increased membrane stability of plasma membranes can be one of the reasons for the decreased functions (phagocytosis and chemotaxis) of human EPA-treated neutrophils.     

Complementary roles for scavenger receptor A and CD36 of human monocyte-derived macrophages in adhesion to surfaces coated with oxidized low-density lipoproteins and in secretion of H2O2

Oxidized low-density lipoprotein (oxLDL) is considered one of the principal effectors of atherogenesis. To explore mechanisms by which oxLDL affects human mononuclear phagocytes, we incubated these cells in medium containing oxLDL, acetylated LDL (acLDL), or native LDL, or on surfaces coated with these native and modified lipoproteins. The presence of soluble oxLDL, acLDL, or native LDL in the medium did not stimulate H2O2 secretion by macrophages. In contrast, macrophages adherent to surfaces coated with oxLDL secreted three- to fourfold more H2O2 than macrophages adherent to surfaces coated with acLDL or native LDL. Freshly isolated blood monocytes secreted little H2O2 regardless of the substrate on which they were plated. H2O2 secretion was maximal in cells maintained for 4-6 d in culture before plating on oxLDL-coated surfaces. Fucoidan, a known ligand of class A macrophage scavenger receptors (MSR-A), significantly reduced macrophage adhesion to surfaces coated with oxLDL or acLDL. Monoclonal antibody SMO, which blocks oxLDL binding to CD36, did not inhibit adhesion of macrophages to oxLDL-coated surfaces but markedly reduced H2O2 secretion by these cells. These studies show that MSR-A is primarily responsible for adhesion of macrophages to oxLDL-coated surfaces, that CD36 signals H2O2 secretion by macrophages adherent to these surfaces, and that substrate-bound, but not soluble, oxLDL stimulates H2O2 secretion by macrophages.     

Human CD36 is a high affinity receptor for the native lipoproteins HDL, LDL, and VLDL

Mouse and hamster SR-BI glycoproteins and their putative human counterpart CLA-I are so far the only scavenger receptors known to bind both native and modified lipoproteins. CD36, a multigland glycoprotein structurally related to SR-BI and CLA-1, has been reported to bind oxidized low density lipoprotein (OxLDL) and acetylated LDL (AcLDL). In this report, we have studied the ability of CD36 to bind native lipoproteins. By transient expression of human CD36 in mammalian and insect cells, we demonstrate that CD36 is a high affinity receptor for the native lipoproteins HDL, LDL, VLDL, and, as previously reported, for OxLDL and AcLDL. The specificity of these interactions is supported by the dose-dependent inhibiton, effect of a monoclonal antibody against CD36. Furthermore, at least for HDL, binding to CD36 does not require the presence of apoE. These findings, together with preferential expression of CD36 in tissues performing very active fatty acid metabolism (skeletal muscle, heart, mammary epithelium, and adipose tissue) and its involvement in foam cell formation (macrophages), suggest that binding of lipoproteins to CD36 might contribute to the regulation of lipid metabolism, and to the pathogenesis of atherosclerosis.     

Binding of the 68-kilodalton protein of Mycobacterium avium to alpha(v)beta3 on human monocyte-derived macrophages enhances complement receptor type 3 expression

Attachment to and uptake by host cells are important early events in the pathogenesis of intracellular organisms such as Mycobacterium avium. Monocyte-derived macrophages (MDM) are known to express multiple surface receptors that play a role in binding to and uptake of M. avium. These include complement receptor type 3 (CR3), fibronectin receptor, mannose receptor, and transferrin receptor. In addition to these, we have previously reported that the integrin receptor alpha(v)beta3 also plays a role in binding to M. avium in a nonopsonic environment. Further, we have shown that a 68-kDa surface protein of M. avium binds to human monocytes and plays a role in attachment of M. avium to MDM. The present study provides direct evidence that this protein mediates attachment of M. avium to MDM by binding to alpha(v)beta3. Using the technique of cell surface enzyme-linked immunosorbent assay, we have shown that the M. avium 68-kDa protein inhibits the binding of monoclonal antibodies (MAb) against alpha(v)beta3 to MDM compared to control proteins such as ovalbumin and laminin (P < 0.05). Dual-labeling studies were performed to demonstrate that after phagocytosis, alpha(v)beta3 is present along with M. avium in phagosomes of M. avium-infected MDM. In addition, we have demonstrated that this interaction between alpha(v)beta3 and the M. avium 68-kDa protein resulted in enhancement of CR3 expression, which is known to play a role in complement-mediated uptake of M. avium. Attachment of MDM to wells coated with the M. avium 68-kDa protein resulted in a twofold increase in CR3 expression compared to attachment of MDM to wells coated with ovalbumin. This enhancement was completely inhibited by pretreatment of MDM with MAb against alpha(v)beta3. In summary, M. avium binds to MDM via alpha(v)beta3 with the help of the M. avium 68-kDa protein, and this ligation enhanced the expression of CR3 on MDM. Since CR3 has been known to play a role in M. avium uptake, enhanced expression of this receptor mediated by M. avium-alpha(v)beta3 interaction indicates a complex mechanism of communication among different receptors that participate in M. avium attachment and uptake. These findings add to current understanding of the roles played by multiple receptor-ligand systems in uptake and pathogenesis of intracellular pathogens such as M. avium.     

Differential human multiple myeloma cell line responsiveness to interferon-alpha. Analysis of transcription factor activation and interleukin 6 receptor expression

Although IFN-alpha is commonly used as maintenance treatment for multiple myeloma patients, its effectiveness is varied. In this study, we have used a panel of IL-6 responsive myeloma cell lines that vary remarkably in responsiveness to IFN-alpha. Three cell lines were growth arrested by IFN-alpha; however, IFN-alpha significantly stimulated growth of the fourth cell line, KAS-6/1. Our studies have focused on elucidating the mechanism of differential IFN-alpha responsiveness. First, we have shown that IFN-alpha-stimulated growth of the KAS-6/1 cells did not result from induction of autocrine IL-6 expression. Second, analysis of Stats 1, 2, and 3 and IFN regulatory factor-1 (IRF-1) and IRF-2 activation failed to reveal differences between the IFN-alpha growth-arrested or growth-stimulated cells. Third, although IFN-alpha treatment of the IFN-alpha growth-inhibited cell lines reduced IL-6 receptor (IL-6R) expression, IFN-alpha also reduced KAS-6/1 IL-6R expression. Finally, although IFN-alpha treatment reduced IL-6R numbers on each cell line, analysis of Stat protein activation revealed that the receptors were still functional. We conclude that myeloma cell responsiveness to IFN-alpha is heterogeneous and that mechanisms of IFN-alpha-mediated growth inhibition other than IL-6R downregulation must exist in myeloma. Identification of these mechanisms may allow development of agents that are more universally effective than IFN-alpha.     

Interferon gamma impairs the ability of monocyte-derived dendritic cells to present tumour-specific and allo-specific antigens and reduces their expression of CD1A, CD80 AND CD4

Eighteen linear antigenic sites were found in cytochrome P450 101 (P450cam) from Pseudomonas putida by the peptide scanning method. These sites accounted for about 30% of the protein sequence. We found no sequences that completely coincided with the antigenic sites of P450cam in cytochromes P450 from other sources. The linear B-epitopes of P540cam were mainly localized on the boundaries separating the elements of the secondary structure. Seventeen of eighteen antigenic sites were found to be on the protein surface and accessible to water molecules. Many functionally important sites or amino acid residues of the P450cam molecules coincided or were in close proximity to the linear B-epitopes found.     

Molecular cloning of a novel human receptor gene with homology to the rat adrenomedullin receptor and high expression in heart and immune system

Lipopolysaccharide was isolated from strain LMG 6999 of Burkholderia vietnamiensis. Degradative and NMR spectroscopic studies established the presence of two polymeric fractions based on the following trisaccharide repeating units: I:-->3)-alpha-D-Galp-(1-->3)-beta-D-Galp-(1-->3)-beta-D-GalpNAc- (1-->; II:-->3)-alpha-D-GalpNAc-(1-->3)-beta-D-GalpNAc-(1-->4)- alpha-L-Rhap-(1-->. The same polymers have previously been found together in lipopolysaccharide from the reference strain for Burkholderia cepacia serogroup O4 and, individually, in those from B. cepacia serogroups C (I) and A (II).     

Relationship between P-glycoprotein expression and cyclosporin A in kidney. An immunohistological and cell culture study

The role of assembled versus addressed phonology in reading was investigated by examining the size of the minimal phonological unit that is recovered in the reading process. Readers named words in unpointed Hebrew that had many or few missing vowels in their printed forms. Naming latencies were monotonically related to the number of missing vowels. Missing vowels had no effects on lexical decision latencies. These results support a strong phonological model of naming and suggest that even in deep orthographies, phonology is not retrieved from the mental lexicon as a holistic lexical unit but is initially computed by applying letter-to-phoneme computation rules. The partial phonological representation is shaped and completed through top-down activation.     

A novel strategy of cell targeting based on tissue-specific expression of the ecotropic retrovirus receptor gene

Gene transfer into specific tissues or cell types is a key technique in the development of gene therapy. Modification of vector particles such that they selectively bind to the target cells has been attempted, but the limitation of this approach is the low transduction efficiency. Here, we show that a two-step gene transfer system can be used for efficient cell targeting. With this strategy, and using a high-titer adenoviral vector containing a tissue-specific promoter, we have engineered a system in which only target cells become susceptible to retrovirus-mediated transduction. In a model experiment, we constructed an adenoviral vector (Ad.AFPEcoRec) containing the ecotropic retrovirus receptor (EcoRec) gene under the control of the alpha-fetoprotein (AFP) promoter. A binding assay showed that after transduction with AD.AFPEcoRec, EcoRec molecules were efficiently expressed in AFP+HepG2 cells, but not in AFP-HeLa and AFP-HLE cells. The EcoRec-expressing HepG2 cells could be stably transduced with ecotropic retroviral vectors, whereas HeLa and HLE cells remained highly resistant to retrovirus-mediated gene transfer. The apparent titer on HepG2 cells was greater than 2 x 10(5) CFU/ml. Because various tissue-specific promoter/enhancer elements are available, the two-step system could be used as a general strategy for both ex vivo and in vivo targeted gene transfer.     

Chemokine receptor CCR2 expression and monocyte chemoattractant protein-1-mediated chemotaxis in human monocytes. A regulatory role for plasma LDL

The subendothelial accumulation of macrophage-derived foam cells is one of the hallmarks of atherosclerosis. The recruitment of monocytes to the intima requires the interaction of locally produced chemokines with specific cell surface receptors, including the receptor (CCR2) for monocyte chemoattractant protein-1 (MCP-1). We have previously reported that monocyte CCR2 gene expression and function are effectively downregulated by proinflammatory cytokines. In this study we identified low density lipoprotein (LDL) as a positive regulator of CCR2 expression. Monocyte CCR2 expression was dramatically increased in hypercholesterolemic patients compared with normocholesterolemic controls. Similarly, incubation of human THP-1 monocytes with LDL induced a rapid increase in CCR2 mRNA and protein. By 24 hours the number of cell surface receptors was doubled, causing a 3-fold increase in the chemotactic response to MCP-1. The increase in CCR2 expression and chemotaxis was promoted by native LDL but not by oxidized LDL. Oxidized LDL rapidly downregulated CCR2 expression, whereas reductively methylated LDL, which does not bind to the LDL receptor, had only modest effects on CCR2 expression. A neutralizing anti-LDL receptor antibody prevented the effect of LDL, suggesting that binding and internalization of LDL were essential for CCR2 upregulation. The induction of CCR2 expression appeared to be mediated by LDL-derived cholesterol, because cells treated with free cholesterol also showed increased CCR2 expression. These data suggest that elevated plasma LDL levels in conditions such as hypercholesterolemia enhance monocyte CCR2 expression and chemotactic response and potentially contribute to increased monocyte recruitment to the vessel wall in chronic inflammation and atherogenesis.     

Low expression of CD40 and B7 on macrophages infiltrating UV-exposed human skin; role in IL-2Ralpha-T cell activation

After UV exposure of skin, epidermal Langerhans cells (LC) are depleted, whereas CD11b+CD36 CD1a- monocytes/macrophages (UV-Mphi) infiltrate. Different immunological outcomes in vivo are mediated by LC (sensitization) and UV-Mphi (tolerance) which may be related to the distinct T cell activation states that these antigen-presenting cells (APC) induce. We previously demonstrated that CD4+ T lymphocytes activated by UV-Mphi are, in contrast to LC-activated T cells, IL-2Ralpha deficient, and we hypothesize that this differential T cell activation is related to differences in co-stimulatory molecules between UV-Mphi and LC. Using four-color flow cytometry, we found a reduced capacity to up-regulate expression of the important co-stimulatory molecules CD40, B7-1 and B7-2 by UV-Mphi relative to LC. This alteration in co-stimulatory molecule expression was selective, because UV-Mphi express equal levels of ICAM-1 and ICAM-3, and increased levels of LFA-1, relative to LC. After bidirectional signaling with T cells during alloantigen presentation, UV-Mphi still exhibited less CD40 and B7-1 than LC. Addition of IFN-gamma induced CD40 and B7-1 expression on UV-Mphi and restored IL-2Ralpha expression on UV-Mphi-activated T cells but had no effect on IL-2Ralpha on resting or LC-activated T cells. The restoration of IL-2Ralpha expression on UV-Mphi-activated T cells by IFN-gamma was inhibited (67 %, p = 0.005) by addition of neutralizing anti-CD40. Therefore, differences in co-stimulatory molecule expression, in particular CD40, on UV-Mphi and LC are critical in determining the distinct T cell activation induced by these APC.     

Cloning and expression of PTP-PEST. A novel, human, nontransmembrane protein tyrosine phosphatase

The polymerase chain reaction was used to amplify protein tyrosine phosphatase (PTPase)-related cDNA from a template of total RNA isolated from human skeletal muscle. A novel PTPase, which we term PTP-PEST, was detected by this method. The polymerase chain reaction fragment was used to screen two different HeLa cell libraries to obtain full length cDNA clones. The cDNA predicts a protein of 510 amino acids, approximately 60 kDa, that does not contain an obvious signal sequence or transmembrane segment suggesting it is a nonreceptor type enzyme. The PTPase domain is located in the N-terminal portion of the molecule and displays approximately 35% identity to other members of this family of enzymes. The C-terminal segment is rich in Pro, Glu, Asp, Ser, and Thr residues, possessing features of PEST motifs which have previously been identified in proteins with very short intracellular half-lives. The protein was expressed in Escherichia coli as a fusion product with glutathione S-transferase. Intrinsic activity was demonstrated in vitro against a variety of phosphotyrosine-containing substrates including BIRK, the autophosphorylated cytoplasmic kinase domain of the insulin receptor beta subunit. It did not dephosphorylate phosphoseryl-phosphorylase a. PTP-PEST mRNA is broadly distributed in a variety of cell lines. Stimulation of human rhabdomyosarcoma A204 cells, a transformed muscle line, with insulin led to an approximately 4-fold induction of PTP-PEST mRNA within 36 h.     

A cell surface antigen that cross-reacts with My4, a monoclonal antibody to CD14, is expressed on human monoblastic cell line U937, B-lymphoma cells, and polymorphonuclear leukocytes

Occupational medicine in Poland has a long tradition, dating back to the establishment of the first health-care institutions for industrial workers at an early stage of Poland's industrialization. Legal foundations of the industrial health-care system, based on the Soviet model, were enacted in 1953. During its most dynamic period of development (1970s and 1980s) the industrial health-care system provided medical services to about 6 million workers. The process of the political and economic transition in Poland began in 1989, and since 1991 there have been numerous transformations in the structure and function of industrial health services. The present article describes the main stages of the transformation process, occupational health training, research, advisory bodies, and the system for setting of hygienic standards.     

Differential effects of mucosal pH on human (Caco-2) intestinal epithelial cell motility, proliferation, and differentiation

Mucosal pH abnormalities are associated with anastomotic dehiscence, ischemia, and malignancy. We postulated that intraluminal pH influences intestinal epithelial motility, proliferation, and differentiation and studied extracellular pHo (7.0-8.5) effects on human (Caco-2) intestinal epithelial motility, proliferation, and differentiation. Mucosal healing was modeled by sheet migration and differentiation by alkaline phosphatase and dipeptidyl dipeptidase specific activity. In parallel differentiation and motility studies, we inhibited proliferation with mitomycin to dissociate indirect mitogenic effects. Intracellular pHi was quantitated using BCECF/AM at varying extracellular pHo and in migrating cells. Motility was maximal at pHo 7.6 and proliferation at 7.2. Each decreased with acidity and alkalinity. By contrast, brush border enzyme activity was lowest at pHo 7.0 and highest at pHo 8.5. pHi was highest at pHo 8.5. Migrating cell pHi was higher than static cell pHi. Thus, extracellular pHo deviations perturb Caco-2 pHi homeostasis and motility. Alkalinity promotes differentiation while acidity induces proliferation and limits differentiation.     

A novel organ culture method to study the function of human odontoblasts in vitro: gelatinase expression by odontoblasts is differentially regulated by TGF-beta1

Odontoblasts cannot be cultured by traditional cell culture methods, thus restricting in vitro studies. Here we present an organ culture method for human odonto-blasts that utilizes the pulp chamber as a culture crucible. Crowns of human third molars were dissected, pulp was gently removed, and the odontoblasts attached to and in the walls of the pulp chambers were cultured in serum-free OPTI-MEM medium, or DMEM/Ham's F12 medium containing 10% serum. Pulp tissues were cultured separately. Cell content and morphology were analyzed by SEM, and the removed pulps were examined by light microscopy. Proteins secreted into the medium with or without TGF-beta1 supplementation were metabolically labeled with [35S]methionine, and the total protein content was assessed by TCA precipitation and SDS-PAGE/fluorography. To assess the role of gelatinolytic enzymes on dentin matrix remodeling, we used enzymography to analyze the effect of TGF-beta1 on gelatinase A and B expression. SEM revealed odontoblasts in pulp chambers after 5 days of culture, with only few or no fibroblasts, and no alterations in the odontoblast cell morphology or differences between the cells cultured in serum-free and serum-containing media. Rarely were any odontoblasts present in pulp tissue. Radiolabeling revealed protein synthesis and secretion until day 6 in both the odontoblast and pulp cultures, with no marked differences between TGF-beta1-treated and control cultures. The level of gelatinase A remained constant up to 7 days, while gelatinase B expression was always low and decreased with time in culture. However, gelatinase B levels were markedly increased upon TGF-beta1 treatment of cells and remained high to day 7. The results suggest that this method provides a novel technique for the study of human odontoblasts in vitro and that odontoblasts can be cultured even in serum-free conditions.     

Differential expression of human histone deacetylase mRNAs in response to immune cell apoptosis induction by trichostatin A and butyrate

Hepatocellular mitogen (HGF and EGF) inhibited lipopolysaccharide and cytokine mixture (referred as LPS/CM)-induced NO synthesis and cellular injury in hepatocytes. Mitogenic inhibitors such as hydroxyurea and Wortmannin could not reverse EGF or HGF-inhibited NO production, whereas both of them showed some inhibitory effect on hepatocyte NO synthesis. Although activation of protein kinase C (PKC) by phorbol 12-myristate 13-acetate (PMA) had no effect on hepatocyte NO synthesis, deletion of PKC activity by long-term treatment of hepatocytes with PMA abolished LPS/CM-induced NO production. In addition, pretreatment of hepatocytes with HGF and EGF also blocked LPS/CM-induced NO synthesis in the hepatocyte. These results suggest that proliferating signal is not directly involved in mitogen-inhibited NO synthesis in the hepatocyte, and LPS/CM-mediated NO synthesis is associated with the metabolic/redox state of hepatocytes.     

Response of four human ovarian carcinoma cell lines to all-trans retinoic acid: relationship with induction of differentiation and retinoic acid receptor expression

The response of 4 human ovarian carcinoma cell lines to retinoic acid was found to be related to the histological type and degree of differentiation of these tumor cells. The 2 serous cell lines NIHOVCAR3 and OVCCR1 were the most sensitive to the antiproliferative effect of RA. This inhibition was associated with morphological and biological changes that were indicative of differentiation. The undifferentiated IGROV1 cell line was not affected by RA. Since the effects of RA are thought to be mediated by nuclear retinoic acid receptors (RARs), the expression of RARs in human ovarian cancer cells was studied. RAR alpha was detected as mRNA species of 3.1 and 2.6 kb in all 4 cell lines. RAR beta was not detected in any of the cell lines, while RAR gamma (3 kb) was expressed in all of the ovarian cancer cells but at a very low level in the RA-resistant IGROV1 cells.