Induction of alloimmune tolerance in heart transplantation through gene silencing of TLR adaptors.

Toll-like receptors (TLRs) activate biochemical pathways that evoke activation of innate immunity, which leads to dendritic cell (DC) maturation and initiation of adaptive immune responses that provoke allograft rejection. We aimed to prolong allograft survival by selectively inhibiting expression of the common adaptors of TLR signaling, namely MyD88 and TRIF, using siRNA. In vitro we demonstrated that blocking expression of MyD88 and TRIF led to reduced DC maturation. In vivo treatment of recipients with MyD88 and TRIF siRNA significantly prolonged allograft survival in the BALB/c > C57BL6 cardiac transplant model. Moreover, the combination of MyD88 and TRIF siRNA along with a low dose of rapamycin further extended the allograft survival (88.8 ± 7.1 days). Tissue histopathology demonstrated an overall reduction in lymphocyte interstitium infiltration, vascular obstruction and hemorrhage in mice treated with MyD88 and TRIF siRNA vector plus rapamycin. Furthermore, treatment was associated with an increase in the numbers of CD4(+) CD25(+) FoxP3(+) regulatory T cells and Th2 deviation. To our knowledge, this study is the first demonstration of prolonging the survival of allogeneic heart grafts through gene silencing of TLR signaling adaptors, highlighting the therapeutic potential of siRNA in clinical transplantation.

Mobile phones, heat shock proteins and cancer.

There are several reports which indicate that electromagnetic radiation (such as from mobile phones) at non-thermal levels may elicit a biological effect in target cells or tissues. Whether or not these biological effects lead to adverse health effects, including cancer, is unclear. To date there is limited scientific evidence of health issues, and no mechanism by which mobile phone radiation could influence cancer development. In this paper, we develop a theoretical mechanism by which radiofrequency radiation from mobile phones could induce cancer, via the chronic activation of the heat shock response. Upregulation of heat shock proteins (Hsps) is a normal defence response to a cellular stress. However, chronic expression of Hsps is known to induce or promote oncogenesis, metastasis and/or resistance to anticancer drugs. We propose that repeated exposure to mobile phone radiation acts as a repetitive stress leading to continuous expression of Hsps in exposed cells and tissues, which in turn affects their normal regulation, and cancer results. This hypothesis provides the possibility of a direct association between mobile phone use and cancer, and thus provides an important focus for future experimentation.

Biological effects of electromagnetic fields--mechanisms for the effects of pulsed microwave radiation on protein conformation.

Microwave exposure under "athermal" conditions occurs when no temperature rise can be measured by conventional thermometry. The existence of biological effects arising from the athermal exposure is still controversial, partly because of a lack of the linear dose response relation. We propose a model in which pulsed microwave radiation causes a triggering of the heat shock or stress response by altering the conformation of proteins through a transient heating of the protein and its close environment. We support this by modelling using the heat diffusion equation and show that pulsed exposure even when athermal can lead to transient temperature excursions outside the normal range. We propose that the power window phenomenon in which biological effects are observed at low power levels may be caused by an incomplete triggering of the heat shock response.

Effects on protein kinase C and gene expression in a human mast cell line, HMC-1, following microwave exposure.

We used a resonant cavity which delivered a continuous wave exposure at 864.3 MHz at an average specific absorption rate (SAR) of 7 W/kg to determine non-thermal biological effects of microwave exposure. A human mast cell line, HMC-1, was used as the biological target. Cells were given three exposures each of 20-min duration daily for 7 days. The temperature of the cell culture medium during the exposure fell to 26.5 degrees C. Effects were seen on localization of protein kinase C, and expression of three genes of 588 screened. The affected genes included the proto-oncogene c-kit, the transcription factor Nucleoside diphosphate kinase B and the apoptosis-associated gene DAD-1. Stress response genes were variably upregulated. No significant effect on morphology or on F-actin distribution was detected. We conclude that low-power microwave exposure may act on HMC-1 cells by altering gene expression via a mechanism involving activation of protein kinase C, and at temperatures well below those known to induce a heat shock response.

Effects of exposure to electromagnetic radiation at 835 MHz on growth, morphology and secretory characteristics of a mast cell analogue, RBL-2H3.

A mast cell line, RBL-2H3, was exposed to 835 MHz for 20 minutes, three times per day for 7 days at a power density of 8.1 +/- 3 mW/cm2. From day 4 onwards, it was observed that the rate of DNA synthesis and cell replication increased, that actin distribution and cell morphology became altered, and the amount of beta-hexosaminidase (a marker of granule secretion) released in response to a calcium ionophore was significantly enhanced, in comparison to unexposed cultures. There were no effects seen on levels of cytoskeletal protein synthesis or of beta-actin mRNA. Morphological changes persisted following subculture for at least 7 days in the absence of further exposure. It is hypothesized that effects of exposure to an electromagnetic field at 835 MHz may be mediated via a signal transduction pathway.

Human macrophages contain a stretch-sensitive potassium channel that is activated by adherence and cytokines.

A variety of stimuli, including cytokines and adhesion to surfaces and matrix proteins, can regulate macrophage function, in part through changes in Ca(2+)- dependent second messengers. While fluctuation in intracellular Ca2+ is an important modulator of cellular activation, little attention has been paid to the roles of other ions whose cytoplasmic concentrations can be rapidly regulated by ion channels. To examine the role of ion channels in macrophage function, we undertook patch clamp studies of human culture-derived macrophages grown under serum-free conditions. The major ionic current in these cells was carried by an outwardly rectifying K+ channel, which had a single-channel conductance of 229 pS in symmetrical K(+)-rich solution and macroscopic whole-cell conductance of 9.8 nS. These channels opened infrequently in resting cells but were activated immediately by (i) adhesion of mobile cells onto a substrate, (ii) stretch applied to isolated membrane patches in Ca(2+)-free buffers, (iii) intracellular Ca2+ (EC50 of 0.4 microM), and (iv) the cytokine IL-2. Furthermore, barium and 4-aminopyridine, blockers of this channel, altered the organization and structure of the cytoskeletal proteins actin, tubulin and vimentin. These cytoskeletal changes were associated with reversible alteration to the morphology of the cells. Thus, we have identified an outwardly rectifying K+ channel that appeared to be involved in cytokine and adherence-mediated macrophage activation, and in the maintenance of cytoskeletal integrity and cell shape.

The unique extracellular disulfide loop of the glycine receptor is a principal ligand binding element.

A loop structure, formed by the putative disulfide bridging of Cys198 and Cys209, is a principal element of the ligand binding site in the glycine receptor (GlyR). Disruption of the loop's tertiary structure by Ser mutations of these Cys residues either prevented receptor assembly on the cell surface, or created receptors unable to be activated by agonists or to bind the competitive antagonist, strychnine. Mutation of residues Lys200, Tyr202 and Thr204 within this loop reduced agonist binding and channel activation sensitivities by up to 55-, 520- and 190-fold, respectively, without altering maximal current sizes, and mutations of Lys200 and Tyr202 abolished strychnine binding to the receptor. Removal of the hydroxyl moiety from Tyr202 by mutation to Phe profoundly reduced agonist sensitivity, whilst removal of the benzene ring abolished strychnine binding, thus demonstrating that Tyr202 is crucial for both agonist and antagonist binding to the GlyR. Tyr202 also influences receptor assembly on the cell surface, with only large chain substitutions (Phe, Leu and Arg, but not Thr, Ser and Ala) forming functional receptors. Our data demonstrate the presence of a second ligand binding site in the GlyR, consistent with the three-loop model of ligand binding to the ligand-gated ion channel superfamily.

Focal adhesion formation is associated with secretion of allergic mediators.

Adherence of cells to the extracellular matrix via focal adhesions is known to modulate many cellular functions. However, the role of focal adhesions in the regulation of secretion is unclear. To examine this we have used the RBL-2H3 rat mast cell line, in which we and others have observed cytoskeletal rearrangements and increased cell spreading during secretion. All activators of secretion examined, whether acting specifically through or bypassing the IgE-receptor, induced the assembly of focal adhesions, as defined by the localization of vinculin and talin. The extent of focal adhesion formation correlated with the extent of secretion and the time course of secretion also correlated with that of the assembly of focal adhesions. To examine the mechanism by which focal adhesion formation occurred, the protein kinase C inhibitor bisindolylmaleimide was used. Bisindolylmaleimide caused complete inhibition of both secretion and focal adhesion formation induced by antigen or the calcium ionophore A23187. Although PMA did not induce secretion, it induced focal adhesion assembly which was inhibited by bisindolylmaleimide. The inhibitor had no effect on secretion or focal adhesion formation induced by the ATP analogue, ATP gamma S in permeabilized cells, indicating ATP gamma S acts after the activation of protein kinase C in the secretory pathway. These data provide novel evidence that the formation of focal adhesions may have a role in the process of secretion from mast cells.

ATP gamma S induces actin and myosin rearrangement during histamine secretion in a rat basophilic leukemia cell line (RBL-2H3).

Rat basophilic leukemia cells (RBL-2H3) undergo morphological and cytoskeletal changes during antigen (DNP-BSA) or calcium ionophore-induced secretion of allergic mediators from intact or permeabilized cells. We describe the novel finding that the phosphatase-resistant ATP analogue, ATP gamma S, mimics antigen-induced serotonin secretion and cytoskeletal rearrangements in permeabilized cells. Confocal microscopy of unstimulated cells shows that myosin and F-actin are concentrated at the plasma membrane. Upon addition of ATP gamma S, F-actin becomes rearranged into membrane ruffles and also associates with myosin in a cytoplasmic meshwork, concentrated perinuclearly. F-actin and myosin ultimately become colocalized into parallel microfilament bundles located on the basolateral membrane. During this period the cell height decreases whilst the cell area increases more than twofold. Gel electrophoresis shows that the cytoskeletal proportion of actin remains unchanged, indicating that the rearrangements occur within the total F-actin pool. The distribution of microtubules and intermediate filaments is unchanged in the presence of ATP gamma S. These results suggest that overcoming a phosphatase may be sufficient to induce secretion in RBL-2H3 cells, and that this secretion may be regulated by F-actin and myosin rearrangements.

PMA and calcium ionophore induce myosin and F-actin rearrangement during histamine secretion from RBL-2H3 cells.

Rat basophilic leukemia (RBL-2H3) cells undergo morphological and cytoskeletal changes during antigen-induced secretion of allergic mediators. The exact role these changes play in the process of secretion is unclear. Using confocal microscopy we now show that PMA+A23187 causes extensive F-actin rearrangements during secretion of [3H] 5-HT. We also describe for the first time the association of myosin with F-actin during this secretory process. In unstimulated cells, myosin and F-actin are concentrated at the plasma membrane with no evidence of stress fibres. Upon addition of PMA or A23187, both F-actin and myosin are rearranged into membrane ruffles and discrete aggregations (foci), followed by the formation of parallel stress fibres located on the ventral membrane. This is in contrast to reports in other cell types in which PMA has been described as causing the disruption of F-actin stress fibres. The time course of secretion coincides with the formation of the foci and ruffles whilst the stress fibres form after the majority of secretion has occurred. These changes are accompanied by a 40% decrease in cell height and a two-fold increase in cell spreading and they occur in the absence of extracellular calcium but are inhibited by the protein kinase C inhibitor, Bisindolylmaleimide, which also inhibits secretion. The formation of myosin-decorated stress fibres, foci, and ruffles is not sufficient to cause secretion, as PMA alone induces these changes without any secretion. The relevance of actin and myosin rearrangements for the regulation of secretion is discussed.

A confocal microscopy study of anticytoskeletal antibody activity in patients with connective tissue disease.

The significance of the presence of antibodies to cytoskeleton proteins in patients with connective tissue diseases is not clear, as there is a high level of these antibodies in healthy controls. In an attempt to improve the visualization of the immunofluorescence binding pattern of autoantibodies to cytoskeletal structures in cultured fibroblasts, we have used confocal microscopy. Of the 256 serum samples tested, 155 (61%) WERE reactive with cytoplasmic structures. These reactive samples could be divided into seven patterns of binding, as determined by double-blind examination of single-section confocal images. While confirming the results of previous immunofluorescence studies which have shown that autoantibodies that bind to filamentous structures in the cytoplasm of cultured cells are common in patients with connective tissue diseases, we were able to identify three patterns of cytoskeletal binding which may be useful as an adjunct to other tests for the diagnosis of some connective tissue diseases, in particular systemic sclerosis (scleroderma) and rheumatoid arthritis/Sjogren's syndrome. None of the seven patterns was exclusive to a particular disease. We conclude that confocal microscopy may be of limited use as an adjunct to other serological assays in the diagnosis of some forms of connective tissue disease.

Antibodies to tubulin and actin bind to the surface of a human monocytic cell line, U937.

Intermittent reports of cytoskeleton proteins (actin and tubulin) on the cell surface have appeared over the last 13 years. Whereas most have concentrated on lymphocytes, this study provides evidence for the presence of these proteins on the surface of a human cultured monocyte-like cell line, U937. Both actin and tubulin were detected on the surface of U937 cells by flow cytometry, using an indirect staining procedure based on biotin-streptavidin-phycoerythrin, chosen for greater sensitivity. By use of this procedure, the majority of viable unstimulated U937 cells stained positively for actin and tubulin, although the level of fluorescence intensity was low. With an antibody specific for tyrosine-tubulin, most of the surface tubulin was also found to be tyrosinylated. For vimentin, an intermediate filament protein abundantly present in the cytoplasm of U937 cells, no staining could be detected. Confirmation of the flow cytometry data for surface actin and tubulin on unstimulated U937 cells was achieved by direct vesualization using a confocal laser scanning microscope. When U937 cells were activated with PMA and LPS, a marked reduction in the level of cell surface actin and tubulin occurred. The role of cell surface actin and tubulin on unstimulated U937 cells, in terms of monocyte function, remains to be elucidated.

Neural membrane glycoproteins associated with chicken Thy-1: an anti-idiotypic antibody study.

In order to investigate the possible binding of Thy-1 to other neuronal cell surface proteins, anti-idiotypic antibodies were raised using a panel of anti-Thy-1 monoclonal antibodies. Anti-idiotypic antibodies were selected for their ability to bind to day-old chick brain membrane components in enzyme-linked immunosorbent assays (ELISA), and to bind to membrane glycoproteins as determined by Western transfer immunoblotting assays. The 5 monoclonal anti-idiotypic antibodies bind to a membrane glycoprotein component of 70 kDa, and one of the antibodies also binds to 3 higher molecular weight components of 160 kDa, 120 kDa and 90 kDa. These antibodies bind to areas of the chicken cerebellum known to be rich in Thy-1. It is postulated that these molecules are associated with Thy-1, and that the role of Thy-1 on the neuronal cell surface, may be to form complexes with, and/or to stabilise these higher molecular weight glycoproteins during synaptic development.

Effects of sperm protamine on human cervical epithelial cells and BHK 21 cells in vitro.

The addition of protamine, of both human and animal origin, to cultures of epithelial and fibroblastic cells has demonstrated that this component of sperm proteins may be capable of bringing about neoplastic transformation in vitro.

A population of Thy-1 molecules associated with the cytoskeleton.

Using a monoclonal antibody, SB1 38.456, raised against chicken neuronal Thy-1, we have shown that a significant amount of Thy-1 is associated with the non-ionic detergent-insoluble fraction of chicken brain membranes. Furthermore, this population of Thy-1 is not released by any biochemical method which disrupts microfilaments, microtubules or intermediate filaments, but is released by raising the temperature to 40 degrees C in high ionic strength, cation-containing buffer. We propose, therefore, that a population of Thy-1 molecules is associated with the cytoskeleton either via a linker protein or via other cell surface glycoproteins which are directly associated with the cytoskeleton.

Localization of low-sulfur keratin proteins in the wool follicle using monoclonal antibodies.

Monoclonal antibodies that recognize components of the low-sulfur keratin proteins extracted from Merino wool have been used to locate these components within the wool follicle. Immunoblotting procedures showed that all of the monoclonal antibodies bound more than one of the eight low-sulfur protein components, indicating that these proteins have antigenic determinants in common. Immunofluorescence studies showed that those antibodies specific for the component 7 family of the low-sulfur proteins bound to the developing wool fiber, whereas those antibodies recognizing the component 8 family bound to areas throughout the wool follicle, particularly the inner and outer root sheaths, but also to the fiber, the cuticle, and the epidermis. One of the monoclonal antibodies also bound to intermediate filament networks of cultured human epithelial cells.

Partial characterization of chicken Thy-1 glycoprotein by monoclonal antibodies.

A panel of monoclonal antibodies has been raised to Thy-1 purified from chicken brain. They were produced by immunization of Balb/c mice with a Thy-1-enriched (Sephacryl B) fraction of a lentil lectin-positive fraction from solubilized brain membrane proteins. Antibody-secreting clones were found to be specific for Thy-1 by enzyme-linked immunosorbent assay (ELISA) and Western transfer and immunoblotting. Four of the monoclonal antibodies were of the IgG2b isotype, one was IgG1, and one was IgM. In additivity and competition ELISA, it was found that three of the antibodies bound to the same (or similar) epitopes, and three had different epitope specificities. These monoclonal antibodies have been used to examine the development and distribution of Thy-1 in the central nervous system of chicken.

Monoclonal antibodies to a subfraction of merino wool high-tyrosine proteins.

Monoclonal antibodies were prepared which react with members of the high-tyrosine type proteins from Merino wool. Specificity was confirmed by the use of Western transfer immunoassays and by enzyme-linked immunosorbent assay on purified fractions. Immunofluorescent staining of sections of wool follicles using the antibodies showed that the proteins were present in the developing wool shaft but that staining was asymmetric, indicating specific location of the proteins in the orthocortex of the fibres. Immunogold-electron microscopy confirmed that one of the antibodies bound to the keratin microfibril bundles.

Sperm basic proteins in cervical carcinogenesis: Correlation with socioeconomic class.

Two types of basic protein, a histone and a protamine, were separated from single ejaculates of human sperm and a ratio between their contents in a given number of sperm established. The ratio varied widely in different males and correlated with ranking by social class: the lower the social class, the greater the proportion of protamine. Statistically similar correlations link social class with the incidence of venereal disease and the incidence of in-situ carcinoma of the cervix, both of which are epidemiological variables in squamous cervical cancer. The basic proteins of the sperm head, especially the protamines, may thus also have a role in the aetiology of squamous cervical cancer.

PIP: 2 types of basic protein a histone and a protamine, were separated from single ejaculates of human sperm and a ratio between their contents in a given number of sperm established. The ratio varied widely in different males and correlated with ranking by social class: the lower the social class, the greater the proportion of protamine. Statistically similar correlations link social class with the incidence of venereal disease and incidence of carcinoma in situ of the cervix, both of which are epidemiological variables in squamous cervical cancer. The basic proteins of the sperm head, especially the protamines, may thus also have a role in the etiology of squamous cervical cancer.