Heavy chain ferritin activates regulatory T cells by induction of changes in dendritic cells

Heavy chain ferritin (H-ferritin) Is a component of the Iron-binding protein, ferritin. We have previously shown that H-ferritin Inhibits anti-CD3-stimulated lymphocyte proliferation and that this was due to Increased production of Interleukin-10 (IL-10). In the present study we have shown that Induction of IL-10 production was due to effects of H-ferritin on adherent antigen-presenting cells (APCs) In blood and monocyte-derived dendritic cells (MoDCs). IL-10 was produced by a subpopulation of CD4 T cells, which expressed the CD25 component of the IL-2 receptor and the CTLA-4 receptor characteristic of regulatory T cells. The changes Induced In MoDCs were compared with those Induced by CD40L and their significance tested by Inhibition with monoclonal antibodies. These studies Indicated that H-ferritin Induced relatively greater expression of CD86 and B7-H1 on MoDCs and that monoclonal antibodies against their receptors, CTLA-4 and programmed death receptor-1 (PD-1), Inhibited IL-10 production from the regulatory T cells. H-ferritin did not appear to Induce direct production of the cytokines IL-2, IL-4, IL-6, IL-10, IL-12, or Interferon-gamma from the DCs. These results are consistent with the thesis that H-ferritin Induces B7-H1 and CD86 (B7-2) on APCs, which In turn Induce IL-10 production from regulatory T cells. This is possibly one mechanism by which melanoma cells may Induce changes In APCs In the vicinity of the tumor and result in suppression of Immune responses by induction of regulatory T cells. (C) 2002 by The American Society of Hematology.

Altered kinetics and benzodiazepine sensitivity of a GABA(A) receptor subunit mutation [gamma(2)(R43Q)] found in human epilepsy

The gamma-aminobutyric acid type A (GABA(A)) receptor mediates fast inhibitory synaptic transmission in the CNS. Dysfunction of the GABA(A) receptor would be expected to cause neuronal hyperexcitability, a phenomenon linked with epileptogenesis. We have investigated the functional consequences of an arginine-to-glutamine mutation at position 43 within the GABA(A) gamma(2)-subunit found in a family with childhood absence epilepsy and febrile seizures. Rapid-application experiments performed on receptors expressed in HEK-293 cells demonstrated that the mutation slows GABA(A) receptor deactivation and increases the rate of desensitization, resulting in an accumulation of desensitized receptors during repeated, short applications. In Xenopus laevis oocytes, two-electrode voltage-clamp analysis of steady-state currents obtained from alpha(1)beta(2)gamma(2) or alpha(1)beta(2)gamma(2)(R43Q) receptors did not reveal any differences in GABA sensitivity. However, differences in the benzodiazepine pharmacology of mutant receptors were apparent. Mutant receptors expressed in oocytes displayed reduced sensitivity to diazepam and flunitrazepam but not the imiclazopyricline zolpidem. These results provide evidence of impaired GABA(A) receptor function that could decrease the efficacy of transmission at inhibitory synapses, possibly generating a hyperexcitable neuronal state in thalamocortical networks of epileptic patients possessing the mutant subunit.

Effect of shear stress on expression of a recombinant protein by Chinese hamster ovary cells

A flow chamber was used to impart a steady laminar shear stress on a recombinant Chinese hamster ovary (CHO) cell line expressing human growth hormone (hGH). The cells were subjected to shear stress ranging from 0.005 to 0.80 N m(-2). The effect of shear stress on the cell specific glucose uptake, cell specific hGH, and lactate productivity rates were calculated. No morphological changes to the cells were observed over the range of shear stresses examined. When the cells were subjected to 0.10 N m(-2) shear in protein-free media without Pluronic F-68, recombinant protein production ceased with no change in cell morphology, whereas control cultures were expressing hGH at 0.35 mug/10(6) cells/h. Upon addition of the shear protectants, Pluronic F-68 (0.2% [w/v]) or fetal bovine serum (1.0% [v/v] FBS), the productivity of the cells was restored. The effect of increasing shear stress on the cells in protein-free medium containing Pluronic F-68 was also investigated. Cell specific metabolic rates were calculated for cells under shear stress and for no-shear control cultures performed in parallel, with shear stress rates expressed as a percentage of those obtained for control cultures. Upon increasing shear from 0.005 to 0.80 N m(-2), the cell specific hGH productivity decreased from 100% at 0.005 N m(-2) to 49% at 0.80 N m(-2) relative to the no-shear control. A concurrent increase in the glucose uptake rate from 115% at 0.01 N m(-2) to 142% at 0.80 N m(-2), and decreased lactate productivity from 92% to 50%, revealed a change in the yield of products from glucose compared with the static control. It was shown that shear stress, at sublytic levels in medium containing Pluronic F-68, could decrease hGH specific productivity. (C) 2002 Wiley Periodicals, Inc.

Effects of alcohol exposure during early life on neuron numbers in the rat hippocampus. I. Hilus neurons and granule cells

We previously showed that 16-day-old rats exposed to a relatively high dose of ethanol at 10-15 postnatal days of age have fewer neurons in the hilus region of the hippocampus compared with controls. Dentate gyrus granule cell numbers, however, showed no statistically significant changes attributable to the ethanol treatment. It is possible that some of the changes in brain morphology, brought about as a result of the exposure to ethanol during early life, may not be manifested until later in life. This question has been further addressed in an extension to our previous study. Wistar rats were exposed to a relatively high daily dose of ethanol on postnatal days 10-15 by placement in a chamber containing ethanol vapour, for 3 h/day. The blood ethanol concentration was found to be similar to430 mg/dl at the end of the period of exposure. Groups of ethanol-treated (ET), separation control (SC), and mother-reared control (MRC) rats were anaesthetised and killed either at 16 or 30 days of age by perfusion with phosphate-buffered 2.5% glutaraldehyde. The Cavalieri principle and the physical disector methods were used to estimate, respectively, the regional volumes and neuron cell numerical densities in the hilus and granule cell regions of the dentate gyrus. The total numbers of neurons in the hilus region and granule cell layer were computed from these estimates. It was found that 16-day-old animals had 398,000-441,000 granule cells, irrespective of group. The numbers of granule cells increased such that by 30 days of age, rats had 487,000-525,500 granule cells. However, there were no significant differences between ethanol-treated rats and their age-matched controls in granule cell numbers. In contrast, ethanol-treated rats had slightly but significantly fewer neurons in the hilus region than did control animals at 16 days of age, but not at 30 days of age. Therefore, it appears that a short period of ethanol exposure during early life can have effects on neuron numbers of some hippocampal neurons, but not others. The effects on hilar neuron numbers, observed as a result of such short periods of ethanol treatment, appeared to be transitory. (C) 2003 Wiley-Liss, Inc.

Blockade of vascular smooth muscle cell proliferation and intimal thickening after balloon injury by the sulfated oligosaccharide PI-88: Phosphomannopentaose sulfate directly binds FGF-2, blocks cellular signaling, and inhibits proliferation

Percutaneous transluminal coronary angioplasty is a frequently used interventional technique to reopen arteries that have narrowed because of atherosclerosis. Restenosis, or renarrowing of the artery shortly after angioplasty, is a major limitation to the success of the procedure and is due mainly to smooth muscle cell accumulation in the artery wall at the site of balloon injury. In the present study, we demonstrate that the antiangiogenic sulfated oligosaccharide, PI-88, inhibits primary vascular smooth muscle cell proliferation and reduces intimal thickening 14 days after balloon angioplasty of rat and rabbit arteries. PI-88 reduced heparan sulfate content in the injured artery wall and prevented change in smooth muscle phenotype. However, the mechanism of PI-88 inhibition was not merely confined to the antiheparanase activity of this compound. PI-88 blocked extracellular signal-regulated kinase-1/2 (ERK1/2) activity within minutes of smooth muscle cell injury. It facilitated FGF-2 release from uninjured smooth muscle cells in vitro, and super-released FGF-2 after injury while inhibiting ERK1/2 activation. PI-88 inhibited the decrease in levels of FGF-2 protein in the rat artery wall within 8 minutes of injury. PI-88 also blocked injury-inducible ERK phosphorylation, without altering the clotting time in these animals. Optical biosensor studies revealed that PI-88 potently inhibited (K-i 10.3 nmol/L) the interaction of FGF-2 with heparan sulfate. These findings show for the first time the capacity of this sulfated oligosaccharide to directly bind FGF-2, block cellular signaling and proliferation in vitro, and inhibit injury-induced smooth muscle cell hyperplasia in two animal models. As such, this study demonstrates a new role for PI-88 as an inhibitor of intimal thickening after balloon angioplasty. The full text of this article is available online at http://www.circresaha.org.

Antigen-specific suppression of inflammatory arthritis by dendritic cells

Purpose Antigen-specific suppression of a previously primed immune response is a major challenge for immunotherapy of autoimmune disease. We have shown that NF-κB inactivation in dendritic cells (modified DC) converts them into cells that tolerize rather than immunize to specific antigen [1]. Antigen-exposed modified DC prevent priming of immunity, and they suppress previously primed immune responses. Regulatory CD4+ T cells, which can transfer antigen-specific tolerance in an IL-10-dependent fashion, mediate the tolerance. We hypothesized that modified DC exposed to arthritogenic antigen would suppress clinical arthritis after disease onset. Methods Antigen-induced arthritis was induced in C57/Bl6 mice by priming to methylated bovine serum albumin (mBSA) antigen followed by challenge injection of mBSA to one knee. Knee swelling was apparent within 2 days, with peak clinical signs apparent at 5 days. Mice were treated with antigen-exposed modified DC between 2 and 6 days after mBSA challenge to the knee joint. Results Clinical arthritis was suppressed in each group receiving mBSA-exposed modified DC within 4 days compared with mice that received either no DC or keyhole limpet hemocyanin-exposed modified DC. Clinical improvement was associated with mBSA-specific tolerance in mice receiving mBSA-exposed modified DC. Tolerance induction was not impaired by concomitant administration of anti-tumor necrosis factor alpha monoclonal antibody. Subsequent rechallenge with intra-articular IL-1 induced flare of arthritis in all groups, which could be effectively suppressed by a second administration of mBSA-exposed modified DC. Conclusions The data indicate that modified DC induce antigen-specific immune suppression in this model of inflammatory arthritis, even after full clinical expression of the disease. These observations have important implications for antigen-specific therapy of autoimmunity.

Innate immune surveillance of spontaneous B cell lymphomas by natural killer cells and gamma delta T cells

Few studies have demonstrated that innate lymphocytes play a major role in preventing spontaneous tumor formation. We evaluated the development of spontaneous tumors in mice lacking beta-2 microglobulin (beta2m; and thus MHC class I, CD1d, and CD16) and/or perform, since these tumor cells would be expected to activate innate effector cells. Approximately half the cohort of perform gene-targeted mice succumbed to spontaneous disseminated B cell lymphomas and in mice that also lacked beta2m, the lymphomas developed earlier (by more than 100 d) and with greater incidence (84%). B cell lymphomas from perforin/beta2m gene-targeted mice effectively primed cell-mediated cytotoxicity and perform, but not IFN-gamma, IL-12, or IL-18, was absolutely essential for tumor rejection. Activated NK1.1(+) and gammadeltaTCR(+) T cells were abundant at the tumor site, and transplanted tumors were strongly rejected by either, or both, of these cell types. Blockade of a number of different known costimulatory pathways failed to prevent tumor rejection. These results reflect a critical role for NK cells and gammadeltaTCP(+) T cells in innate immune surveillance of B cell lymphomas, mediated by as yet undetermined pathway(s) of tumor recognition.

Gene-engineered T cells as a superior adjuvant therapy for metastatic cancer

The major limiting factor in the successful application of adjuvant therapy for metastatic disease is the lack of adjuvant specificity that leads to severe side effects. Reasoning that T cells of the immune system are highly specific, we generated tumor-specific T cells by genetic modification of mouse primary T cells with a chimeric receptor reactive with the human breast cancer-associated Ag erbB-2. These T cells killed breast cancer cells and secreted IFN-gamma in an Ag-specific manner in vitro. We investigated their use against metastatic breast cancer in mice in an adjuvant setting, and compared their effectiveness with the commonly applied adjuvants doxorubicin, 5-fluorouracil, and herceptin. Mice were inoculated orthotopically with the human erbB-2-expressing spontaneously metastatic mouse breast cancer 4T1.2 in mammary tissue, and the primary tumor was surgically removed 8 days later., Significant metastatic disease was demonstrated in lung and liver at the time of surgery on day 8 with increased tumor burden at later time points. T cell adjuvant treatment of day 8 metastatic disease resulted in dramatic increases in survival of mice, and this survival was significantly greater than that afforded by either doxorubicin, 5-fluorouracil, or herceptin.

A proteome analysis of conditioned media from human neonatal fibroblasts used in the maintenance of human embryonic stem cells

The pathways involved in the maintenance of human embryonic stem (hES) cells remain largely unknown, although some signaling pathways have been identified in mouse embryonic stem (mES) cells. Fibroblast feeder layers are used to maintain the undifferentiated growth of hES cells and an examination of the conditioned media (CM) of human neonatal fibroblasts (HNFs) could provide insights into the maintenance of hES cells. The neonatal foreskin fibroblast line (HNF02) used in this study was shown to have a normal 2n = 46, XY chromosomal complement and to support the undifferentiated growth of the Embryonic Stem Cell International Pte. Ltd.-hES3 cell line. The CM of HNF02 was examined using two-dimensional liquid chromatography-tandem mass spectrometry (2-D LCMS) and two-dimensional electrophoresis (2-DE) followed by matrix-assisted laser desorption/ionization-time of flight tandem mass spectrometry (2-DE/MALDI). A total of 102 proteins were identified, 19 by 2-DE/MALDI, 53 by 2-D LCMS and 30 by both techniques. These proteins were classified into 15 functional groups. Proteins identified in the extracellular matrix and differentiation and growth factor functional categories were considered most likely to be involved in the maintenance of hES cell growth, differentiation and pluripotency as these groups contained proteins involved in a variety of events including cell adhesion, cell proliferation and inhibition of cell proliferation, Writ signaling and inhibition of bone morphogenetic proteins.

Lipopolysaccharide and lipoteichoic acid induce different innate immune responses in bovine mammary epithelial cells

The objective of the present study was to characterize the innate immune responses induced by in vitro stimulation of bovine primary mammary epithelial cells (bMEC) using gram-negative lipopolysaccharide (LPS) and gram-positive lipoteichoic acid (LTA) bacterial cell wall components. Quantitative real-time PCR (qRT-PCR) was employed to examine the mRNA expression of a panel of 22 cytokines, chemokines, beta-defensins and components of the Toll-Like Receptor signaling pathway. Stimulation of bMEC with LPS for 24 h elicited a marked increase in mRNA expression for IL-1 beta, IL-8, TNF alpha, CXCL6 and beta-defensin while members of the Toll-Like Receptor pathway.. although present, were largely unaffected. Surprisingly, stimulation of these cells with LTA for 24 h did not significantly alter the expression of these genes. A time course of the expression of IL-1 beta, IL-8, TNF alpha, CXCL6 and beta-defensin was subsequently performed. The mRNA levels of all genes increased rapidly after stimulation for 2-4 h with both LPS and LTA but only the former treatment resulted in sustained responses. In contrast, the increased gene expression for LTA stimulated cells returned to resting levels after 8-16 h with the exception of beta-defensin, which remained up-regulated. The limited and unsustained cytokine response to LTA may explain why mastitis caused by gram-positive bacteria has greater potential for chronic intra-mammary infection than gram-negative infection. It was concluded that bovine mammary epithelial cells have a strong but differential capacity to mount innate immune responses to bacterial cell wall components. Crown Copyright (c) 2005 Published by Elsevier Ltd. All rights reserved.

Biological activity and metabolic clearance of recombinant human follicle stimulating hormone produced in Sp2/0 myeloma cells

Human follicle stimulating hormone is a pituitary glycoprotein that is essential for the maintenance of ovarian follicle development and testicular spermatogenesis. Like other members of the glycoprotein hormone family, it contains a common a subunit and a hormone specific beta subunit. Each subunit contains two glycosylation sites. The specific structures of the oligosaccharides of human follicle stimulating hormone have been shown to influence both the in vitro and in vivo bioactivity. Since the carbohydrate structure of a protein reflects the glycosylation apparatus of the host cells in which the protein is expressed, we examined the isoform profiles, in vitro bioactivity and metabolic clearance of a preparation of purified recombinant human follicle stimulating hormone derived from a stable, transfected Sp2/0 myeloma cell line, and pituitary human follicle stimulating hormone. Isoelectric focussing and chromatofocussing studies of human follicle stimulating hormone preparations both showed a more basic isoform profile for the recombinant human follicle stimulating hormone compared to that of pituitary human follicle stimulating hormone. The recombinant human follicle stimulating hormone had a significantly higher radioreceptor activity compared to that of pituitary human follicle stimulating hormone, consistent with a greater in vitro potency. Pharmacokinetic studies in rats indicated a similar terminal half life (124 min) to that of the pituitary human follicle stimulating hormone (119 min). Preliminary carbohydrate analysis showed recombinant human follicle stimulating hormone to contain high mannose and/or hybrid type, in addition to complex type carbohydrate chains, terminating with both alpha 2,3 and alpha 2,6 linked sialic acids. These results demonstrate that recombinant human follicle stimulating hormone made in the Sp2/0 myeloma cells is sialylated, has a more basic isoform profile, and has a greater in vitro biological potency compared to those of the pituitary human follicle stimulating hormone.

The intercalated disc of monkey myocardial cells and Purkinje fibers as revealed by scanning electron microscopy

The intercalated discs of working myocardium and Purkinje fibers of the monkey heart were examined by scanning and transmission electron microscopy. The NaOH/ultrasonication technique resulted in the digestion of connective tissue and a separation of the intercellular junctions of intercalated discs, such that these could be visualized three-dimensionally. The intercalated discs of ventricular myocytes, atrial myocytes and Purkinje fibers vary considerably in number and configuration, as do the intercalated discs of the three different layers of the ventricular myocardium. Myocytes in the subepicardial, middle and subendocardial layers of the ventricle have 1-3, 4-5 and 5-6 intercalated discs at the end of these cells, respectively, Those in the endocardial layer are characterized by the presence of small laterally-placed intercalated discs. Atrial myocytes and Purkinje fibers usually only have 1-2 intercalated discs, Individual intercalated discs in ventricular myocytes have complicated stairs with 10-30 steps and corresponding risers, while those of atrial myocytes and Purkinje fibers have simple stairs with 1-3 steps and risers, Steps equivalent to the plicate segments are characterized by densely-packed microplicae and finger-like microprojections which greatly increase surface area in vertricular myocytes, Microprojections in atrial myocytes and Purkinje fibers are sparse by comparison, Risers equivalent to the interplicate segments containing large gap junctional areas are most numerous in left ventricular myocytes, followed by right ventricular myocytes, Purkinje fibers and atrial myocytes in decreasing order. The geometric arrangement of the various types of myocytes may be related with impulse propagation. Large intercalated discs of cell trunks and series branches may participate in longitudinal propagation, while small laterally-placed ones may be the site of transverse propagation.

Chemical treatment of Escherichia coli .1. Extraction of intracellular protein from uninduced cells

Extraction of intracellular protein from Escherichia coli is traditionally achieved by mechanical disruption. A chemical treatment that destroys the integrity of the bacterial cell wall and could provide an alternative technique is examined in this study. Treatment with a combination of the chelating agent ethylenediaminetetraacetate (EDTA) (greater than 0.3 mM) and the chaotropic agent urea (6 M) is highly effective at releasing protein from uninduced E. coli. The 6 M urea in the presence of 3 mM EDTA can release cytoplasmic protein from both logarithmic-phase and stationary-phase E. coli cells at levels equivalent to mechanical disruption. The concentrations of the two chemical agents were the major variables affecting the maximum levels of protein release. Several minor variables and interactions were also identified. The kinetics of protein release is first order. For 2, 4, and 6 M urea with 3 mM EDTA, the time constant is approximately 2.5 min independent of urea concentration. Kinetics for 3 mM EDTA without urea is considerably slower, with a time constant of 12.3 min. (C) 1997 John Wiley & Sons, Inc.

Inhibition of natural killer cells by a cytomegalovirus MHC class I homologue in vivo

Herpesviruses, such as murine and human cytomegalovirus (MCMV and HCMV), can establish a persistent infection within the host and have diverse mechanisms as protection from host immune defences'. Several herpesvirus genes that are homologous to host immune modulators have been identified, and are implicated in viral evasion of the host immune response(2,3). The discovery of a viral major histocompatibility complex (MHC) class I homologue, encoded by HCMV(4), led to speculation that it might function as an immune modulator and disrupt presentation of peptides by MHC class I to cytotoxic T cells(5). However, there is no evidence concerning the biological significance of this gene during viral infection. Recent analysis of the MCMV genome has also demonstrated the presence of a MHC class I homologue(6). Here we show that a recombinant MCMV,in which. the gene encoding the class I homologue has been disrupted, has severely restricted replication during the acute stage of infection compared with wild-type MCMV, We demonstrate by in vivo depletion studies that natural killer (NK) cells are responsible for the attenuated phenotype of the mutant. Thus the viral MHC dass I homologue contributes to immune evasion through interference with NK cell-mediated clearance.

Effects of pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) on hormone secretion from sheep pituitary cells in vitro

Although vasoactive intestinal polypeptide (VIP) is thought to be a prolactin releasing factor, in vivo studies on sheep suggest that it is inactive in this species. Recent studies, based primarily on the rat, suggest that the related pituitary adenylate cyclase-activating polypeptide (PACAP) is also a hypophysiotrophic factor but again in sheep, this peptide has no in vivo effects on hormone secretion despite being a potent activator of adenylate cyclase in vitro. This lack of response to either peptide in vivo in sheep could be due to the low concentration of peptide that reaches the pituitary gland following peripheral injection. In the present study we therefore adopted an alternative approach of evaluating in vitro effects of these peptides on GH, FSH, LH or prolactin secretion from dispersed sheep pituitary cells. In a time-course study, PACAP (1 mu mol/l) increased GH concentrations in the culture medium between 1 and 4 h and again at 12 h but had no effect in the 6 and 24 h incubations. Prolactin, LH and FSH were not affected by PACAP. The response to various concentrations of PACAP (1 nmol/l-1 mu mol/l) were then evaluated using a 3 h incubation. Again prolactin and LH were not affected by PACAP and there was a small increase in GH concentrations but only at high concentrations of PACAP (0.1 and 1 mu mol/l; P<0.05), PACAP also stimulated FSH secretion in cells from some animals although this effect was small, The GH response to PACAP was inhibited by PACAP(6-38), a putative PACAP antagonist; but not by (N-Ac-Tyr(1), D-Arg(2))-GHRH(1-29)-NH2, a GH-releasing hormone (GHRH) antagonist. The cAMP antagonist Rp-cAMPS was unable to block the GH response to PACAP suggesting that cAMP does not mediate the secretory response to this peptide. At incubation times from 1-24 h, VIP (1 mu mol/l) had no effects on prolactin, LH or GH secretion and, in a further experiment based on a 3 h incubation, concentrations of VIP from 1 nmol/l-1 mu mol/l were again without effect on prolactin concentrations. Interactions between PACAP and gonadotrophin releasing hormone (GnRH), GHRH and dopamine were also investigated. PACAP (1 nmol/l-1 mu mol/l) did not affect the gonadotrophin or prolactin responses to GnRH or dopamine respectively. However, at a high concentration (1 mu mol/l), PACAP inhibited the GH response to GHRH. In summary, these results show that PACAP causes a modest increase in FSH and GH secretion from sheep pituitary cells but only at concentrations of PACAP that are unlikely to be in the physiological range. The present study confirms that VIP is not a prolactin releasing factor in sheep.