Proliferation in monocyte-derived dendritic cell cultures is caused by progenitor cells capable of myeloid differentiation

Dendritic cells (DC) can be generated by culture of adherent peripheral blood (PB) cells in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). There is controversy as to whether these DC arise from proliferating precursors or simply from differentiation of monocytes. DC were generated from myeloid-enriched PB non-T cells or sorted monocytes. DC generated from either population functioned as potent antigen-presenting cells. Uptake of [H-3]-thymidine was observed in DC cultured from myeloid-enriched non-T cells. Addition of lipopolysaccharide or tumor necrosis factor-alpha led to maturation of the DC, but did not inhibit proliferation. Ki67(+) cells were observed in cytospins of these DC, and by double staining were CD3(-)CD19(-)CD11c(-)CD40(-) and myeloperoxidase(+), suggesting that they were myeloid progenitor cells. Analysis of the starting population by flow cytometry demonstrated small numbers of CD34(+)CD33(-)CD14(-) progenitor cells, and numerous granulocyte-macrophage colony-forming units were generated in standard assays. Thus, production of DC in vitro from adherent PB cells also enriches for progenitor cells that are capable of proliferation after exposure to GM-CSF. Of clinical importance, the yield of DC derived in the presence of GM-CSF and IL-4 cannot be expanded beyond the number of starting monocytes. (C) 1998 by The American Society of Hematology.

H-ras activation is an early event in the ptaquiloside-induced carcinogenesis: Comparison of acute and chronic toxicity in rats

Bracken fern (Pteridium spp.) produces cancer of the urinary bladder and oesophagus in grazing animals and is a suspected human carcinogen, The carcinogenic principle ptaquiloside (PT), when activated to a dienone (APT), forms DNA adducts which eventually leads to tumor. Two groups of female Sprague-Dawley rats were given a chronic dose of 3 mg APT weekly for 10 weeks either by intravenous (iv) tail vein or by intragastric (ig) route, A third group was given a weekly dose of 6 mg of APT for 3 weeks by the ig route corresponding to acute dosing. Both chronic iv and ig dosed animals showed ischemic tubular necrosis in the kidney but only iv dosed animals developed adenocarcinomas of the mammary glands. Acutely dosed ig animals produced apoptotic bodies in the liver, necrosis of blood cell precursors in the bone marrow and ischemic tubular necrosis in the kidney but they did not develop tumors, No mutations were found in the H-ras and p53 genes in the mammary glands of either the ig rats or the tumor-bearing iv rats. However, the mammary glands of a fourth group of rats, which received APT by iv and killed before tumor development, carried Pu to Pu and Pu to Py double mutations in codons 58 and 59 of H-ras. This study indicates that the route of administration plays a role in the nature of the disease expression from ptaquiloside exposure. In addition to confirming the role of APT in the PT-induced carcinogenesis our finding suggests that activation of H-ras is an early event in the PT-carcinogenesis model. (C) 1998 Academic Press.

Proliferation in monocyte-derived dendritic cell cultures is due to cells capable of myeloid differentiation

Dendritic cells (DC) can be generated by culture of adherent peripheral blood (PB) cells in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). There is controversy as to whether these DC arise from proliferating precursors or simply from differentiation of monocytes. DC were generated from myeloid-enriched PB non-T cells or sorted monocytes. DC generated from either population functioned as potent antigen-presenting cells. Uptake of [H-3]-thymidine was observed in DC cultured from myeloid-enriched non-T cells. Addition of lipopolysaccharide or tumor necrosis factor-alpha led to maturation of the DC, but did not inhibit proliferation. Ki67(+) cells were observed in cytospins of these DC, and by double staining were CD3(-)CD19(-)CD11c(-)CD40(-) and myeloperoxidase(+), suggesting that they were myeloid progenitor cells. Analysis of the starting population by flow cytometry demonstrated small numbers of CD34(+)CD33(-)CD14(-) progenitor cells, and numerous granulocyte-macrophage colony-forming units were generated in standard assays. Thus, production of DC in vitro from adherent PB cells also enriches for progenitor cells that are capable of proliferation after exposure to GM-CSF. Of clinical importance, the yield of DC derived in the presence of GM-CSF and IL-4 cannot be expanded beyond the number of starting monocytes. (C) 1998 by The American Society of Hematology.

Thermal and mechanical properties of radiation crosslinked poly(tetrafluoroethylene-co-perfluoromethyl vinyl ether)

The gamma-radiolysis of poly(tetrafluoroethylene-co-perfluoromethyl vinyl ether) (TFE/PMVE) was investigated using chemical and mechanical analyses. The polymer was found to form an insoluble network with a dose of gelation of 15.8 kGy. Tensile and glass transition temperature measurements indicated the predominance of crosslinking, with optimal elastomeric properties reached in the dose range of 120 to 200 kGy. Photoacoustic FTIR spectroscopy CPAS) showed the formation of new carboxylic acid end groups on irradiation. These new end groups were shown to decrease the thermal oxidative stability of the crosslinked network as determined by thermal gravimetric analysis. Electron spin resonance (ESR) studies of the polymer at 77 K indicated the presence of radical precursors. A G-value of 1.1 was determined for radical production at 77 K. Comparison of radical concentrations for a copolymer with a different mole ratio of PMVE, indicated that the PMVE units contribute to scission reactions. (C) 1998 Elsevier Science Ltd. All rights reserved.

A novel two-chain proteinase inhibitor generated by circularization of a multidomain precursor protein

Female reproductive tissues of the ornamental tobacco amass high levels of serine proteinase inhibitors (PIs) for protection against pests and pathogens. These PIs are produced from a precursor protein composed of six repeats each with a protease reactive site. Here we show that proteolytic processing of the precursor generates five single-chain PIs and a remarkable two-chain inhibitor formed by disulfide-bond Linkage of Nand C-terminal peptide fragments. Surprisingly, PI precursors adopt this circular structure regardless of the number of inhibitor domains, suggesting this bracelet-like conformation is characteristic of the widespread potato inhibitor II (Pot II) protein family.

Novel composite membranes for gas separation: Preparation and performance

High performance composite membranes based on molecular sieving silica (MSS) were synthesized using sols containing silicon co-polymers (methyltriethoxysilane and tetraethylorthosilicate). Alpha alumina supports were treated with hydrochloric acid prior to sol deposition. Permselectivity of CO2 over CH4 as high as 16.68 was achieved whilst permeability of CO2 up to 36.7 GPU (10(-6) cm(3) (STP) cm(-2) . s(-1) . cm Hg-1) was measured. The best membrane's permeability was finger printed during various stages of the synthesis process showing an increase in CO2/CH4 permselectivity by over 25 times from initial support condition (no membrane film) to the completion of pore structure tailoring. Transport measurement results indicate that the membrane pretreated with HCl has highest permselectivity and permeation rate. In particular, there is a definite cut-off pore size between 3.3 and 3.4 angstroms which is just below the kinetic diameters of Ar and CH4. This demonstrates that the mechanism for the separation in the prepared composite membrane is molecular sieving (activated diffusion), rather than Knudsen diffusion.

Effect of temperature and a crosslinking promoter on the gamma-radiolysis of a perfluoroelastomer

Methods of promoting the radiation-induced cross linking of poly(tetrafluoro-ethylene-co-perfluoromethyl vinyl ether) (TFE/PMVE) have been investigated. Greater control of the crosslinking and chain-scission reactions was achieved by varying the radiolysis temperature. This was attributed to temperature affecting the mobilities of reactive species such as polymeric free radicals. These reactive species are precursors to radiation-induced cross links and chain-ends. Analysis of the sol/gel behaviour, tensile properties and FTIR indicated that the optimum temperature for the radiation crosslinking of TFE/PMVE, at a dose of 150 kGy, was 263 K. This temperature was 10 K below the glass transition temperature. Incorporation of 1 wt% triallyl isocyanurate (TAIC) greatly amplified the radiation crosslinking of TFE/PMVE, The dose for gelation was decreased by 70%, and the additive imparted superior mechanical properties compared to the neat irradiated TFE/PMVE. Electron spin resonance (ESR) measurements showed higher radical yields at 77 K with the 1 wt% TAIC, indicating that the crosslinking promoter was acting as a radical trap. (C) 1999 Society of Chemical Industry.

Differentiated dendritic cells expressing nuclear RelB are predominantly located in rheumatoid synovial tissue perivascular mononuclear cell aggregates

Objective. Differentiated dendritic cells (DC) and other antigen-presenting cells are characterized by the nuclear location of RelB, a member of the nuclear factor kappa B/Rel family. To characterize and enumerate differentiated DC in rheumatoid arthritis (RA) peripheral blood (PB), synovial fluid (SF), and synovial tissue (ST), the expression and location of RelB were examined. Methods. RelB protein expression and cellular location were determined in RA PB, SF, and ST by flow cytometry and immunohistochemical analysis of purified cells or formalin-fixed tissue. DNA-binding activity of RelB was determined by electrophoretic: mobility shift-Western immunoblotting assays. Results. Circulating RA PBDC resembled normal immature PBDC in that they did not express intracellular RelB protein. In RA ST serial sections, cells containing nuclear RelB (nRelB) were enriched in perivascular regions. A mean +/- SD of 84 +/- 10% of these cells were DC. The remaining nRelB+,HLA-DR+ cells comprised B cells and macrophages. Only 3% of sorted SFDC contained nRelB, However, RelB present in the nucleus of these SFDC was capable of binding DNA, and therefore capable of transcriptional activity. Conclusion. Circulating DC precursors differentiate and express RelB after entry into rheumatoid ST. Differentiated DC can thus be identified by immunohistochemistry in formalin-fixed ST. Signals for DC maturation may differ between RA ST and SF, resulting in nuclear location of RelB predominantly in ST. This is likely to have functional consequences for the DC in these sites.

Papillomavirus virus-like particles for the delivery of multiple cytotoxic T cell epitopes

Chimeric papillomavirus (PV) virus-like particles (VLPs) based on the bovine papillomavirus type 1 (BPV-1) L1 protein were constructed by replacing the 23-carboxyl-terminal amino acids of the BPV1 major protein L1 with an artificial polytope minigene, containing known CTL epitopes of human PV16 E7 protein, HIV IIIB gp120 P18, Nef, and reverse transcriptase (RT) proteins, and an HPV16 E7 linear B epitope. The CTL epitopes were restricted by three different MHC class 1 alleles (H-2(b), H-2(d), HLA-A*0201). The chimeric L1 protein assembled into VLPs when expressed in SF-9 cells by recombinant baculovirus. After immunization of mice with polytope VLPs in the absence of adjuvant, serum antibodies were detected which reacted with both polytope VLPs and wild-type BPV1L1 VLPs, in addition to the HPV16E7 linear B cell epitope. CTL precursors specific for the HPV16 E7, HIV P18, and RT CTL epitopes were also detected in the spleen of immunized mice. Polytope VLPs can thus deliver multiple B and T epitopes as immunogens to the MHC class I and class II pathways, extending the utility of VLPs as self-adjuvanting immunogen delivery systems. (C) 2000 Academic Press.

Characterization of the structural and surface properties of chemically modified MCM-41 material

Mesoporous Mobil catalytic materials of number 41 (MCM-41) silica was chemically modified using both inorganic and organic precursors and characterized using the techniques, XRD, XPS, MAS NMR, FTIR, W-Vis, and physical adsorption of nitrogen, hydrocarbons (hexane, benzene, acetone, and methanol) and water vapor. Modification using organic reagents was found to result in a significant loss in porosity and a shape change of surface properties (increased hydrophobicity and decreased acidity). With inorganic modifying reagents, the decrease in porosity was also observed while the surface properties were not significantly altered as reflected by the adsorption isotherms of organics and water vapors. Chemical modifications can greatly improve the hydrothermal stability of MCM-41 material because of the enhanced surface hydrophobicity (with organic modifiers) or increased pore wall thickness (with inorganic modifiers). (C) 2000 Elsevier Science B.V. All rights reserved.

MHC class II expression is regulated in dendritic cells independently of invariant chain degradation

We have investigated the mechanisms that control MHC class II (MHC II) expression in immature and activated dendritic cells (DC) grown from spleen and bone marrow precursors. Degradation of the MHC II chaperone invariant chain (li), acquisition of peptide cargo by MHC II, and delivery of MHC II-peptide complexes to the cell surface proceeded similarly in both immature and activated DC. However, immature DC reendocytosed and then degraded the MHC II-peptide complexes much faster than the activated DC. MHC II expression in DC is therefore not controlled by the activity of the protease(s) that degrade Ii, but by the rate of endocytosis of peptide-loaded MHC II. Late after activation, DC downregulated MHC II synthesis both in vitro and in vivo.

Mechanical loading modulates glutamate receptor subunit expression in bone

The cellular mechanisms coupling mechanical loading with bone remodeling remain unclear. In the CNS, the excitatory amino acid glutamate (Glu) serves as a potent neurotransmitter exerting its effects via various membrane Glu receptors (GluR). Nerves containing Glu exist close to bone cells expressing functional GluRs. Demonstration of a mechanically sensitive glutamate/aspartate transporter protein and the ability of glutamate to stimulate bone resorption in vitro suggest a role for glutamate linking mechanical load and bone remodeling. We used immunohistochemical techniques to identify the expression of N-methyl-D-aspartate acid (NMDA) and non-NMDA (AMPA or kainate) ionotropic GluR subunits on bone cells in vivo. In bone sections from young adult rats, osteoclasts expressed numerous GluR subunits including AMPA (GluR2/3 and GluR4), kainic acid (GluR567) and NMDA (NMDAR2A, NMDAR2B and NMDAR2C) receptor subtypes. Bone lining cells demonstrated immunoexpression for NMDAR2A, NMDAR2B, NMDAR2C, GluR567, GluR23, GuR2 and GluR4 subunits. Immunoexpression was not evident on osteocytes, chondrocytes or vascular channels. To investigate the effects of mechanical loading on GluR expression, we used a Materials Testing System (MTS) to apply 10 N sinusoidal axial compressive loads percutaneously to the right limbs (radius/ulna, tibia/fibula) of rats. Each limb underwent 300-load cycles/day (cycle rate, 1 Hz) for 4 consecutive days. Contralateral, non-loaded limbs served as controls. Mechanically loaded limbs revealed a load-induced loss of immunoexpression for GluR2/3, GluR4, GluR567 and NMDAR2A on osteoclasts and NMDAR2A, NMDAR2B, GluR2/3 and GluR4 on bone lining cells. Both neonatal rabbit and rat osteoclasts were cultured on bone slices to investigate the effect of the NMDA receptor antagonist, MK801, and the AMPA/kainic acid receptor antagonist, NBQX, on osteoclast resorptive activity in vitro. The inhibition of resorptive function seen suggested that both NMDAR and kainic acid receptor function are required for normal osteoclast function. While the exact role of ionotropic GluRs in skeletal tissue remains unclear, the modulation of GluR subunit expression by mechanical loading lends further support for participation of Glu as a mechanical loading effector. These ionotropic receptors appear to be functionally relevant to normal osteoclast resorptive activity. (C) 2005 Elsevier Inc. All rights reserved.

Subpopulations of corticotrophs in the sheep pituitary during late gestation: Effects of development and placental restriction

The prepartum surge in fetal plasma cortisol is essential for the normal timing of parturition in sheep and may result from an increase in the ratio of ACTH to proopiomelanocortin (POMC) in the fetal circulation. In fetuses subjected to experimental induction of placental restriction, the prepartum surge in fetal cortisol is exaggerated, whereas pituitary POMC mRNA levels are decreased, and in vitro, unstimulated ACTH secretion is elevated in corticotrophs nonresponsive to CRH. We therefore investigated the changes in the relative proportions of cells expressing POMC, ACTH, and the CRH type 1 receptor (CRHR1) shortly before birth and during chronic placental insufficiency. Placental restriction (PR) was induced by removal of the majority of placental attachment sites in five ewes before mating. Pituitaries were collected from control and PR fetal sheep at 140 d (control, n = 4; PR, n = 4) and 144 d (control, n = 6; PR, n = 4). Pituitary sections were labeled with specific antisera raised against POMC, ACTH, and CRHR1. Three major subpopulations of corticotrophs were identified that expressed POMC + ACTH + CRHR1, ACTH + CRHR1, or POMC only. The proportion of pituitary corticotrophs expressing POMC + ACTH + CRHR1 decreased (P < 0.05) between 140 (control, 60 +/- 1%; PR, 66 +/- 4%) and 144 (control, 45 +/- 2%; PR, 56 +/- 6%) d. A significantly higher (P < 0.05) proportion of corticotrophs expressed POMC + ACTH + CRHR1 in the pituitary of the PR group compared with controls. This study is the first to demonstrate subpopulations of corticotrophs in the fetal sheep pituitary that differentially express POMC, ACTH, and CRHR1 and the separate effects of gestational age and placental restriction on these subpopulations of corticotrophs.

Nuclear localization of RelB is associated with effective antigen-presenting cell function

Dendritic cells (DC) are potent APCs that enter resting tissues as precursors and, after Ag exposure, differentiate and migrate to draining lymph nodes. The phenotype of RelB knockout mice implicates this member of the NF kappa B/Rel family in DC differentiation. To further elucidate the role of RelB in DC differentiation, mRNA, intracellular protein expression, and DNA binding activity of RelB were examined in immature and differentiated human DC, as well as other PB mononuclear cell populations. RelB protein and mRNA were detected constitutively in lymphocytes and in activated monocytes, differentiated DC, and monocyte-derived DC. Immunohistochemical staining demonstrated RelB within the differentiated lymph node interdigitating DC and follicular DC, but not undifferentiated DC in normal skin. Active nuclear RelB was detected by supershift assay only in differentiated DC derived from either PB precursors or monocytes and in activated B cells. These RelB+ APC were potent stimulators of the MLR. The data indicate that RelB expression is regulated both transcriptionally and post-translationally in myeloid cells. Within the nucleus, RelB may specifically transactivate genes that are critical for APC function.

Synthesis of alpha-aspartyl-containing cyclic peptides

alpha-Aspartyl-containing cyclic pentapeptides were synthesised in high yields using a strategy that maintained fluorenylmethyl protection on the aspartic acid side chain during chain assembly, resin cleavage and cyclisation of the linear precursors. Tetra-n-butylammonium fluoride treatment of the fluorenylmethyl-protected cyclic peptides catalysed imide formation, whereas piperidine-induced deprotection resulted in good yields of the target cyclic peptides.