Comparison of infrared thermography and laser speckle contrast imaging for the dynamic assessment of digital microvascular function.

OBJECTIVES: Laser speckle contrast imaging (LSCI) is a novel non-invasive microvascular imaging modality. The present study evaluates the validity and reliability of LSCI by comparison with infrared thermography (IRT) for the dynamic assessment of digital microvascular function in healthy volunteers. METHODS: Subjects attended on 3 occasions. Simultaneous assessment of cutaneous perfusion at 3 distinct regions of interest (ROI) within the hands was undertaken using LSCI and infrared thermography (IRT) at baseline, and at 13s intervals over 15 min following a standardised local cold challenge. Endpoints for evaluation included absolute measurements at baseline and following cold stress, in addition to the characteristics of the re-warming curves (maximum % recovery and maximum gradient). Visits 1 and 2 were undertaken in identical conditions (ambient temperature 23°C) to assess reproducibility, whereas visit 3 was undertaken at a lower ambient room temperature of 18°C to evaluate responsiveness to reduction in ambient room temperature. RESULTS: Fourteen healthy participants completed the study. There was greater variability in the data generated using LSCI compared with the highly damped IRT, reflecting greater sensitivity of LSCI to physiological variation and movement artefact. LSCI and IRT correlated well at baseline and following cold challenge for all endpoints (r(s) for pooled data between 0.5 and 0.65, p<0.00005). Reproducibility of both IRT and LSCI was excellent (ICCs>0.75) for absolute assessments but lower for re-warming curve characteristics. LSCI provides greater spatial resolution than IRT identifying variation in cutaneous perfusion within the hands most likely associated with the presence of arteriovenous anastamoses. Both techniques were responsive to reduction in ambient room temperature. Effect sizes were greatest for IRT than LSCI (e.g. -1.17 vs. -0.85 at ROI 1 at baseline) although this may represent heat transfer rather than altered vascular perfusion. DISCUSSION: In the dynamic assessment of digital vascular perfusion, LSCI correlates well with IRT, is reproducible and responsive to reduction in ambient room temperature. Absolute measurements appear preferable to parameters derived from re-warming curve characteristics when assessing digital perfusion following cold challenge. The greater temporal and spatial resolution of LSCI compared with IRT may facilitate the development of novel assessment tools of autonomic function and digital cutaneous perfusion.

Des-gamma-carboxy prothrombin stimulates human vascular endothelial cell growth and migration.

Des-gamma-carboxy prothrombin (DCP) is an aberrant prothrombin produced by hepatocellular carcinoma (HCC) cells. Serum and tissue DCP expressions are thought to reflect the biological malignant potential of HCC. However, the role of DCP in the development of angiogenesis is not well understood. Herein, we report the effects of DCP on growth and migration of human vascular endothelial cells. DCP significantly stimulated the proliferation of HUVEC (ECV304) cells in a dose and time dependent manner, as measured by the MTT assay. A continuous rapid migration of ECV304 cells was observed in the presence of DCP measured by the scratch wound assay. The continuous rapid invasive activity, measured by transwell chamber assay also showed that DCP increased endothelial cells migration through the reconstituted extracellular matrix (Matrigel). Further, the tube formation of vascular endothelial cells on 3-D Matrigel showed an increased number of branch points of ECV304 cells induced by DCP in a dose dependent manner. The levels of vascular endothelial cell growth-related angiogenic factors and matrix metalloproteinase were also examined. DCP significantly stimulated the expression levels of epidermal growth factor receptor (EGFR), vascular endothelial growth factor (VEGF), and matrix metalloproteinase (MMP)-2 (latent and active). Together, these data suggest that DCP is a novel type of vascular endothelial growth factor that possesses potent mitogenic and migrative activities in angiogenesis of HCC.

Des-gamma-carboxy prothrombin increases the expression of angiogenic factors in human hepatocellular carcinoma cells.

AIMS: Des-gamma-carboxyl prothrombin (DCP) is a serum protein produced by hepatocellular carcinoma (HCC) cells. The aim of this study was to evaluate the angiogenic activity of DCP in HCC cells. MAIN METHODS: The proliferation of HCC cells was measured by 3-[4, 5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay. The growth of HCC cells was also evaluated in vivo by using the xenografts in nude mice. The enzyme-linked immunosorbent assay (ELISA) was employed to measure the levels of angiogenic factors in supernatant of cell culture. The expression of angiogenic factors was examined by Western blot analysis and immunohistochemical staining. KEY FINDINGS: DCP displayed the stimulation of HCC cell growth in a dose (5-80 ng/ml) and time (24-96 h) dependent manner. The increase of cell growth was also observed in nude mice bearing well-established, palpable HepG2 and SMMC-7721 xenografts after 2 weeks administration of DCP. HCC cell growth was accompanied by the elevated levels of angiogenic factors. The levels of vascular endothelial growth factor (VEGF), transforming growth factor-alpha (TGF-alpha) and basic fibroblast growth factor (bFGF) in supernatant of SMMC-7721 cells were increased from 47, 126, and 60 pg/10(6) cells/24 h to 400, 208, and 298 pg/10(6) cells/24 h, respectively, after 72 h incubation with 80 ng/ml of DCP. The results of Western blot analysis and immunohistochemical staining of HCC xenografts also showed the significant increase of VEGF, TGF-alpha, and bFGF in HCC cells. SIGNIFICANCE: These results provide the information that DCP is a type of growth factor in progression of HCC.

Inhibition activity of sulfated polysaccharide of Sepiella maindroni ink on matrix metalloproteinase (MMP)-2.

SIP-SII is the sulfated S. maindroni ink polysaccharide (SIP) isolated from cuttlefish Sepiella maindroni. SIP-SII weakly inhibited tumor cell growth without cytotoxicity in vitro assay. Herein, we examined the effects of SIP-SII on the expression of matrix metalloproteinase MMP-2 and MMP-9 as well as tumor cell invasion and migration. SIP-SII (0.8-500 microg/ml) significantly decreased the expression of MMP-2 activity in human ovarian carcinoma cells SKOV3 as evidenced by the gelatin zymography analysis. No significant decrease of MMP-9 was detected in the cell line after SIP-SII treatment. The expression of MMP-2 was also evaluated using Western blot analysis. The results showed that SIP-SII inhibited the expression of MMP-2 in SKOV3 and human umbilical vein vascular endothelial cells ECV304 after 24 h incubation. Furthermore, the activity of invasion and migration of SKOV3 and ECV304 cells were measured. SIP-SII displayed an inhibitory effect on the penetration of SKOV3 cells through Matrigel-coated membrane in transwell chamber. A significant inhibition of ECV304 cell migration was observed in the presence of SIP-SII. These results suggest that SIP-SII might suppress invasion and migration of carcinoma cells via inhibition of MMP-2 proteolytic activity.

New drug targets in inflammation: efforts to expand the anti-inflammatory armoury.

Inflammation is a beneficial host response to challenge by foreign bodies or to tissue injury. When this normal physiological process (which is designed to restore normal tissue structure and function), becomes dysregulated, it can become harmful and destructive leading to inflammatory diseases that are a major burden on humanity. Despite some notable successes, there are still major unmet medical needs in the treatment of inflammatory diseases and the development of new anti-inflammatory drugs features prominently in the research portfolios of most pharmaceutical and biotech companies. New insights into inflammatory processes and new anti-inflammatory drug targets were the subjects of a Focus Topic organized for the Life Sciences 2007 meeting in Glasgow (July 2007). The speakers from this meeting were invited to generate reviews on the basis of their presentations and these reviews contribute to this themed issue and are summarized in this short article.

T-lymphocyte navigation and migration: beyond the PI3K paradigm.

The co-ordinated and directional trafficking of T-lymphocytes in lymphoid and peripheral tissues is an important process in lymphoid development, immunosurveillance and immune responses. Members of the chemokine superfamily play a key role in providing navigational cues for T-cells and chemokine receptors couple with a wide range of biochemical signals including phosphoinositide lipid metabolism, elevation of intracellular calcium levels, activation of a wide array of protein kinases as well as small GTPases. One of the most robust biochemical signals elicited by chemokines in T-lymphocytes is the activation of several members of the PI3K (phosphoinositide 3-kinase) family. In many cell systems, PI3Ks are known to contribute to several aspects of the migratory machinery, although their role in T-cell migration has been unclear and will be considered in the present paper.

Phosphoinositide 3-kinases in the gut: a link between inflammation and cancer?

Carcinoma of the gastrointestinal tract is the most common internal malignancy affecting men and women in Western countries. Chronic intestinal inflammation, especially of the colon, is also a Western disease and correlates with a significantly increased risk of developing cancer. This has suggested that the immune processes involved in both conditions might share some common pathways. Indeed, there is increasing evidence that phosphatidylinositol 3-kinases (PI 3-kinases) are involved in both the pathogenesis of colorectal carcinoma and intestinal inflammation. Here, we discuss this rapidly progressing area of research, presenting evidence for a pivotal role of PI 3-kinase(s) in intestinal pathophysiology.

The sheepshead minnow as an in vivo model for endocrine disruption in marine teleosts: a partial life-cycle test with 17alpha-ethynylestradiol.

The sheepshead minnow (Cyprinodon variegatus Lacépède), an estuarine fish species, was exposed to 17alpha-ethynylestradiol (EE2) at nominal test concentrations of 0.2, 2, 20, 200, 400, 800, 1,600, and 3,200 ng/L. Fish were exposed for up to 59 d, from subadult stages to sexual maturity, under flow-through conditions. The exposure period was followed by an evaluation of reproductive success and survival of progeny. The reproductive success of exposed sheepshead minnows, as determined from data on egg production from two subsequent spawning trials, was reduced in fish exposed to 200 ng/L EE2 and, in one spawning trial, in the 20-ng/L treatment. Hatching success was reduced in the progeny of fish exposed to 200 ng/L EE2, but survival was good among fry that successfully hatched. Histological examination indicated generalized edema, damage to gill epithelia, hepatic toxicity, fibrosis of the testis, and evidence of sex reversal, including testes-ova and spermatagonia-like cells in ovaries. The maximum acceptable toxicant concentration (MATC) for gonadal development in males was within the normal range of EE2 concentrations in sewage treatment plant effluents. The exposure regimen and choice of test organism, combined with histological examination, allowed independent evaluation of ecologically significant acute, reproductive and estrogenic endpoints. Estrogen receptor-mediated effects occurred at concentrations where reproductive effects were measurable under standard reproduction assays. The sheepshead minnow appears to be a sensitive in vivo model for partial life-cycle testing of compounds that have the potential to disrupt the endocrine system as well as reproduction in estuarine and coastal marine fish species.

PI 3-K and T-cell activation: limitations of T-leukemic cell lines as signaling models.

Phosphoinositide 3-kinases (PI 3-Ks) phosphorylate the d3-hydroxyl position of phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P(2)] resulting in the generation of the 3'-phosphoinositide lipid PtdIns(3,4,5)P(3). PI 3-Ks are activated by a diverse set of receptors that play a role in determining T-cell function. It now seems that leukemic T cells, which are widely used as models for T-cell biology, show constitutive activation of PI 3-K-mediated signal-transduction pathways. Hence, studies of the role of PI 3-K in T-cell biology using leukemic cell lines might have misinterpreted the importance of this pathway for T-cell signal transduction.

Phosphoinositide 3-kinases in T lymphocyte activation.

Biochemical experiments have established that the metabolism of inositol phospholipids by phosphoinositide 3-kinases (PI3Ks) and lipid-phosphatases is triggered by many receptors that control T lymphocyte function, including antigen-receptors, costimulatory molecules, cytokines and chemokines. Novel effectors of PI3K have been identified in the immune system and shown to be important in the control of lymphocyte activation. Moreover, key lipid-phosphatases have been identified that act to terminate or modulate PI3K signalling in cells of the immune system.

Regulatory role of phosphatidylinositol 3-kinase on TNF-alpha-induced cyclooxygenase 2 expression in colonic epithelial cells.

BACKGROUND AND AIMS: Cyclooxygenase (COX)-2 is up-regulated in most colonic cancers and in inflammatory bowel disease in which tumor necrosis factor (TNF)-alpha is believed to play a central role. There has been recent speculation on the activation of phosphatidylinositol 3-kinase (PI 3-kinase) by TNF-alpha and its role in the regulation of genes controlled by NF-kappaB. We investigated the regulatory role of PI 3-kinase on COX-2 expression in colonic epithelial cells. METHODS: In HT-29 and Caco-2 colonic epithelial cells, COX-2 expression was induced by either TNF-alpha or interleukin (IL)-1alpha as observed by Northern and Western analyses. COX-2 activity was assessed by measuring prostaglandin E(2) (PGE2) production by enzyme-linked immunosorbent assay. NF-kappaB binding activity was assessed by electrophoretic mobility shift assay. PI 3-kinase activity was measured by quantifying the accumulation of PI 3-kinase-dependent D-3 lipid products by high-performance liquid chromatography. RESULTS: The PI 3-kinase inhibitor wortmannin up-regulated induced COX-2 expression in a concentration-dependent manner in both HT-29 and Caco-2 cells. An alternative PI 3-kinase inhibitor, LY294002, caused up-regulation of induced COX-2 messenger RNA (mRNA) in HT-29 cells at concentrations of < or =1 micromol/L. IL-4 and IL-13, which are known to activate PI 3-kinase, down-regulated HT-29 COX-2 mRNA, protein, and PGE2 production. NF-kappaB binding activity was unaltered by PI 3-kinase inhibition in HT-29 cells, in which TNF-alpha was shown to activate PI 3-kinase directly. CONCLUSIONS: COX-2 is negatively regulated by PI 3-kinase; we propose that the inhibitory effect of IL-4 and IL-13 is mediated via a PI 3-kinase-dependent pathway. This mechanism does not appear to involve NF-kappaB because PI 3-kinase inhibition did not alter NF-kappaB binding activity. TNF-alpha can activate PI 3-kinase directly in addition to inducing COX-2.

Collection, separation, and activation of human T lymphocytes.

There is increasing evidence that T lymphocytes play a central role in regulating both the initial and chronic inflammatory cascades of allergic asthma (1,2) and can also regulate baseline airway responsiveness in mouse models of asthma (3). In particular, activation of antigen-specific CD4(+)T cells of the Th2 subset in the lungs, resulting in interleukin (IL)-5 secretion, plays a major role in allergic inflammation of the airways (4). T-cell activation requires two signals, one provided by interaction between the TCR and specific antigen in association with major histocompatability antigens (MHC) class II molecules, the other provided by costimulatory molecules (5,6). CD28 is the best-characterized costimulatory molecule and is constitutively expressed on the surface of both CD4(+)and CD8(+)T lymphocytes (5,6). CD28 and its homolog CTLA-4 bind the natural ligands B7.1 and B7.2 (also known as CD80 and CD86, respectively), which are present on antigen presenting cells (APCs) (5,6). CD28 ligation provides cyclosporin A-resistant biochemical signals to T cells, which are an absolute requirement to drive proliferation and IL-2 production from CD3-stimulated T cells, as well as enhance cell survival (5,6). In contrast, CTLA-4 negatively regulates the immune response (7,8). Costimulation through B7.2 has been demonstrated to be required for the induction of lung mucosal Th2 immune response and altered airway responsiveness (4), whereas B7.1 costimulation has been reported to be essential for the maintenance or amplification of lung inflammatory responses (9). Hence, better understanding of the biochemical and functional effects of CD28 and CTLA-4 is essential to formulating better strategies aimed at inhibition of CD28 interaction with its ligands as these may represent a possible therapeutic target for the treatment of lung mucosal allergic inflammation.

Disparate effects of phorbol esters, CD3 and the costimulatory receptors CD2 and CD28 on RANTES secretion by human T lymphocytes.

This study has examined the stimuli required for secretion of regulated upon activation, normal T-cell expressed, presumed secreted (RANTES) from T lymphocytes and found that stimuli such as phorbol 12-myristate 13-acetate (PMA), which are unable to support T-cell proliferation and interleukin-2 (IL-2) production, are nevertheless able to elicit strong secretion of RANTES. Conversely, stimuli such as CD2 and CD28 ligation, which are able to support T-cell proliferation, are unable to elicit RANTES secretion. Coligation of CD3 and CD28 drives T-cell proliferation to a similar degree as CD2 and CD28 coligation, yet also supports modest RANTES secretion. Furthermore, CD28 ligation enhances the secretion of RANTES stimulated by PMA and this costimulatory effect is abrogated by the phosphoinositide 3-kinase inhibitor wortmannin. Our data also indicate that the observed effects of PMA on RANTES secretion are probably due to activation of protein kinase C (PKC) isoenzymes, since RANTES secretion was unaffected by the non-PKC activating 4alpha-phorbol ester, whilst the general PKC inhibitor Ro-32-0432 inhibits PMA-stimulated RANTES secretion. Moreover, the effect of PMA appears to be chemokine-specific because PMA was unable to increase secretion of the related CC chemokine MIP-1alpha. Under stimulation conditions where increases in [Ca2+]i occur (e.g. PMA plus ionomycin or CD3 plus CD28 ligation) RANTES secretion can be severely reduced compared with the levels observed in response to the phorbol ester PMA. Hence, whilst PKC-dependent pathways are sufficient for strong RANTES secretion, a calcium-dependent factor is activated which negatively regulates RANTES secretion. This correlates well with the observation that ligation of cytolytic T lymphocyte-associated antigen-4 (CTLA-4) (expression of which has been reported to be dependent on a sustained calcium signal), inhibits RANTES secretion induced by CD3/CD28, but has no effect on PMA-stimulated RANTES secretion.

Interactions between phosphatidylinositol 3-kinase and nitric oxide: explaining the paradox.

Nitric oxide (NO) and the many derivatives and reactive oxygen intermediates thereof are all molecules that are utilised by mammalian cells in the war against microbial pathogens and tumours. They are potentially toxic molecules and, with damage control being crucial, the production and metabolism of nitric oxide is a tightly regulated process. The duality of NO is well documented. On the one hand, beneficial effects include normal healing in the skin and intestinal mucosa, killing of certain bacteria, regulating T cell proliferation and differentiation (Th1 vs Th2), and regulating leukocyte recruitment, by affecting adhesion molecule expression. On the other hand, persistent high levels of NO can lead to the production of toxic metabolites (peroxynitrite and hydroxyls), which can have detrimental effects, such as increased microvascular and epithelial permeability, increased oxidative stress (which can damage DNA), and damage to iron-sulphur proteins in mitochondria. NO has been reported to modulate its own production and the mechanisms involved in this self-regulation are being hotly pursued. The purpose of this review is to update recent intriguing advances in our understanding of the interaction of the phosphatidylinositol (PI) 3-kinase-dependent signal transduction pathway in regulating the activity of the enzymes that generate NO, namely, the nitric oxide synthases.

Phosphoinositide 3-kinase: a key biochemical signal for cell migration in response to chemokines.

Chemokines can couple to distinct signalling pathways that have been demonstrated to mediate not only migration, but also cell growth and transcriptional activation. One particular signalling pathway, namely that controlled by the lipid kinase phosphoinositide 3-kinase (PI3K), has been the focus of much attention with respect to its activation by chemokine receptors and the role it plays in regulating cell migration. Identification of PI3K is arguably one of the most exciting recent developments in biochemical signalling. Pharmacological and genetic studies have now convincingly shown that both CC and CXC chemokines stimulate PI3K-dependent chemotaxis of inflammatory cells such as eosinophils, macrophages, neutrophils and T lymphocytes. This review considers the role of specific sub-classes of PI3Ks (e.g. the p85/p110 heterodimer, PI3Kgamma and PI3K-C2alpha) as well as their downstream effector targets in mediating chemokine-stimulated cell migration.

CD28 stimulates tyrosine phosphorylation, cellular redistribution and catalytic activity of the inositol lipid 5-phosphatase SHIP.

The D-3 phosphoinositide lipids phosphatidylinositol 3,4, 5-trisphophate [PtdIns(3,4,5)P(3)] and phosphatidylinositol 3, 4-bisphosphate [PtdIns(3,4)P(2)] represent upstream components of a major signaling pathway that is strongly activated by the T cell costimulatory molecule CD28. A major route for degradation of PtdIns(3,4,5)P(3) (and hence, regulation of PtdIns(3,4,5)P(3)-driven effector pathways), involves its conversion to PtdIns(3,4)P(2) by the 145-kDa SH2-containing inositol (poly)phosphate 5-phosphatase (SHIP). In this study, we demonstrate using the murine T cell hybridoma DC27.1, that SHIP is strongly tyrosine phosphorylated after ligation of CD28 by either mAb or the natural ligand B7.1. Ligation of CD3 also stimulates SHIP tyrosine phosphorylation and an additive effect on tyrosine phosphorylation of SHIP is observed when both CD3 and CD28 are ligated. The tyrosine phosphorylation of SHIP in response to CD28 ligation correlates with a marked redistribution of SHIP from the cytosol to the plasma membrane, as well as an increase in the in vitro 5-phosphatase activity associated with SHIP immunoprecipitates derived from CD28-stimulated cells. However, we have been unable to detect a direct association between CD28 and SHIP, so the mechanisms by which CD28 exerts the observed effects on SHIP remain unclear. This is the first demonstration that SHIP is a biochemical target for CD28 and suggests that SHIP may be involved in the regulation of T cell activation.

The CXC chemokine stromal cell-derived factor activates a Gi-coupled phosphoinositide 3-kinase in T lymphocytes.

The cellular effects of stromal cell-derived factor-1 (SDF-1) are mediated primarily by binding to the CXC chemokine receptor-4. We report in this study that SDF-1 and its peptide analogues induce a concentration- and time-dependent accumulation of phosphatidylinositol-(3,4,5)-trisphosphate (PtdIns(3,4,5)P3) in Jurkat cells. This SDF-1-stimulated generation of D-3 phosphoinositide lipids was inhibited by pretreatment of the cells with an SDF-1 peptide antagonist or an anti-CXCR4 Ab. In addition, the phosphoinositide 3 (PI 3)-kinase inhibitors wortmannin and LY294002, as well as the Gi protein inhibitor pertussis toxin, also inhibited the SDF-1-stimulated accumulation of PtdIns(3,4,5)P3. The effects of SDF-1 on D-3 phosphoinositide lipid accumulation correlated well with activation of the known PI 3-kinase effector protein kinase B, which was also inhibited by wortmannin and pertussis toxin. Concentrations of PI 3-kinase inhibitors, sufficient to inhibit PtdIns(3,4,5)P3 accumulation, also inhibited chemotaxis of Jurkat and peripheral blood-derived T lymphocytes in response to SDF-1. In contrast, SDF-1-stimulated actin polymerization was only partially inhibited by PI 3-kinase inhibitors, suggesting that while chemotaxis is fully dependent on PI 3-kinase activation, actin polymerization requires additional biochemical inputs. Finally, SDF-1-stimulated extracellular signal-related kinase (ERK)-1/2 mitogen-activated protein kinase activation was inhibited by PI 3-kinase inhibitors. In addition, the mitogen-activated protein/ERK kinase inhibitor PD098059 partially attenuated chemotaxis in response to SDF-1. Hence, it appears that ERK1/2 activation is dependent on PI 3-kinase activation, and both biochemical events are involved in the regulation of SDF-1-stimulated chemotaxis.

Cytokine-induced apoptosis in epithelial HT-29 cells is independent of nitric oxide formation. Evidence for an interleukin-13-driven phosphatidylinositol 3-kinase-dependent survival mechanism.

A combination of the pro-inflammatory cytokines interleukin (IL)-1alpha, interferon (IFN)-gamma, and tumor necrosis factor (TNF)-alpha induces nitric oxide synthase mRNA expression and nitric oxide (NO) generation in the human colon carcinoma cell line HT-29. This can be inhibited by pretreatment with IL-13 via a phosphatidylinositol (PI) 3-kinase-dependent mechanism (Wright, K., Ward, S. G., Kolios, G., and Westwick, J. (1997) J. Biol. Chem. 272, 12626-12633). Since NO has been implicated in regulating mechanisms leading to cell death, while activation of PI 3-kinase-dependent signaling cascades are thought to be involved with promoting cell survival events, we have investigated the outcome of these cytokine treatments on apoptosis and cell survival of HT-29 cells. Initiation of apoptosis can be achieved by the combinations of IFN-gamma/TNF-alpha, IFN-gamma/CD95, IL-1alpha/IFN-gamma, and IL-1alpha/IFN-gamma/TNF-alpha to varying extents. Induction of apoptotic markers by HT-29 cells in response to cytokine treatment is not dependent on NO production. Pretreatment with IL-13 protects against IL-1alpha/IFN-gamma/TNF-alpha- and IFN-gamma/TNF-alpha- as well as IFN-gamma/CD95-induced (but not IL-1alpha/IFN-gamma-induced) cell death. In addition, IFN-gamma/TNF-alpha and IL-1alpha/IFN-gamma/TNF-alpha stimulate activation of caspase-8 and caspase-3, which IL-13 pretreatment was able to partially inhibit and delay. IL-13 also stimulates activation of the major PI 3-kinase effector, protein kinase B. The PI 3-kinase inhibitors wortmannin and LY294002 inhibit IL-13 stimulation of protein kinase B as well as the cell survival effects of IL-13. These data demonstrate that cytokine-induced apoptosis of HT-29 cells is NO-independent and that the activation of a PI 3-kinase-dependent signaling cascade by IL-13 is a key signal responsible for the inhibition of apoptosis.

The complexities of CD28 and CTLA-4 signalling: PI3K and beyond.

A successful immune response requires a set of non-cognate cell-cell interactions which provide the second "costimulatory" signal to the T cells. The best characterized costimulatory receptor expressed on resting T cells is CD28 which provides poorly-defined cyclosporin-resistant biochemical signal(s) that promote expression of several cytokines/chemokines. Another major effect of CD28 ligation is the promotion of cell survival which is thought to occur via the up-regulation of Bcl-xL expression, CD28 shares its ligands B7.1 and B7.2 with the related CTLA-4, which plays an inhibitory role in T cell activation. Manipulation of CD28/CTLA-4 interactions with their natural ligands has provided exciting results in transplantation and tumor therapy settings and also has potential in the treatment of several diseases such as arthritis and multiple sclerosis, asthma and protection against HIV infection. The biochemical basis for the different functional outcomes of CD28 and CTLA-4 ligation has been the subject of intense investigation over the past few years. This review will focus on our current understanding of the biochemical signals that may be involved in regulating the different functional outcomes of CD28 and CTLA-4, with particular emphasis on the role played by the PI3K-dependent signalling cascade.