Optical projection tomography implemented for accessibility and low cost (OPTImAL).

Optical projection tomography (OPT) is a three-dimensional mesoscopic imaging modality that can use absorption or fluorescence contrast, and is widely applied to fixed and live samples in the mm-cm scale. For fluorescence OPT, we present OPT implemented for accessibility and low cost, an open-source research-grade implementation of modular OPT hardware and software that has been designed to be widely accessible by using low-cost components, including light-emitting diode (LED) excitation and cooled complementary metal-oxide-semiconductor (CMOS) cameras. Both the hardware and software are modular and flexible in their implementation, enabling rapid switching between sample size scales and supporting compressive sensing to reconstruct images from undersampled sparse OPT data, e.g. to facilitate rapid imaging with low photobleaching/phototoxicity. We also explore a simple implementation of focal scanning OPT to achieve higher resolution, which entails the use of a fan-beam geometry reconstruction method to account for variation in magnification. This article is part of the Theo Murphy meeting issue 'Open, reproducible hardware for microscopy'.

Notch signalling and voltage-gated Na+ channel activity in human prostate cancer cells: independent modulation of in vitro motility.

This study tested the possible functional relationship of two signalling mechanisms shown previously to be involved in human prostate cancer (PCa), Notch and voltage-gated sodium channel. Notch1 and Notch2 were differentially expressed in PCa cell lines of varying metastatic potential (LNCaP, PC-3, PC-3M) in comparison to a normal prostate cell line (PNT2), whereas Notch3 and Notch4 were not expressed. The Notch ligand Jagged1, but not Jagged2, was increased in all cell lines, whereas the Notch downstream target Deltex was not expressed. In comparison to the LNCaP cell line, Hes1, another downstream target, showed elevated expression in the metastatic PC-3 and PC-3M cells and promoted lateral motility. In contrast, the Notch ligand Delta-like1 (Dll1) levels were higher in LNCaP compared with PC-3 and PC-3M cells. Importantly, decreasing Dll1 expression increased the lateral motility of PC-3 cells, whereas blocking voltage-gated Na(+) channel activity with tetrodotoxin decreased motility. However, the effect of Dll1 was independent of Notch signalling through Hes1 and voltage-gated Na(+) channel expression/activity.

Notch signalling in the regulation of peripheral immunity.

Notch signalling plays a critical role in embryogenesis, influencing the differentiation and growth of a variety of cell types across the species. In the mammalian immune system, Notch signalling operates at various levels; it controls the differentiation of haematopoietic stem cells and directs the early development of the T and B-cell lineages. It is also involved in the maturation of both CD4+ and CD8+ T cells in the thymus. The biological activities of this pathway extend beyond lymphocyte ontogeny; recent evidence has shown that it also contributes to the regulation of the peripheral immune system through its ability to influence cell survival and growth. In fulfilling this function, Notch signalling appears to act in conjunction with defined immunological signals such as cytokines, T-cell antigen receptor and co-stimulatory receptor-mediated signalling. In this review we discuss the potential of the Notch signalling pathway in the maintenance of homeostasis within the immune system affecting both peripheral tolerance and the negative feedback controlling productive immunity. The therapeutic manipulation of this pathway is likely to have broad application in a range of immunologically based diseases.

Murine IL-10 gene transfer inhibits established collagen-induced arthritis and reduces adenovirus-mediated inflammatory responses in mouse liver.

The effects of homologous IL-10 administration during an established autoimmune disease are controversial, given its reported immunostimulatory and immunosuppressive properties. Studies of collagen-induced arthritis have shown efficacy with repeated administrations of IL-10; however, when the EBV IL-10 homologue was administered via adenovirus gene transfer technology the results were equivocal. Therefore, the present study was undertaken to elucidate the effects of prolonged homologous IL-10 administration via adenovirus-mediated gene delivery on the progression of established arthritis. Collagen type II (CII)-immunized mice received i.v. injections of 10(7) or 10(8) PFU of an E1-deleted adenoviral vector containing the murine IL-10 gene (AdIL-10), after arthritis onset. Mice were monitored for 3 wk for disease progression, and gene transduction was assessed by quantification of serum mIL-10. CII-specific cell-mediated and humoral immune responses were analyzed by lymph node cell proliferation, cytokine production, and anti-CII Ab responses. Furthermore, because adenoviral vectors have been reported to induce organ dysfunction due to cell-mediated immune responses to the viral Ags, we have also evaluated delayed-type hypersensitivity responses and reactive hepatitis to the systemically delivered adenovirus and whether the IL-10 produced could influence those responses. Sustained suppression of autoimmune arthritis and elevated serum levels of IL-10 were achieved in our study. AdIL-10 treatment reduced cell-mediated immune reactivity, but did not affect humoral responses. Furthermore, IL-10 was able to reduce, but not totally abrogate, adenovirus-induced hepatic inflammation. These findings provide further insights into the diverse interplay of immune processes involved in autoimmune inflammation and the mechanism of cytokine immunotherapy.

In situ expression of soluble B7-1 in the context of oncolytic herpes simplex virus induces potent antitumor immunity.

In vivo delivery of immunomodulatory genes is a promising strategy for solid tumor vaccination. A drawback is that it necessitates induction of a large effect from transgene expression in a small percentage of tumor cells. Although the B7 family is known to be the most potent of the costimulatory molecules, gene transduction of B7 alone has not been effective in inducing antitumor immunity in nonimmunogenic tumors by ex vivo methods, much less in vivo. We have developed a novel approach where a gene encoding soluble B7-1, a fusion protein of the extracellular domain of murine B7-1 and the Fc portion of human IgG1, is delivered to tumor cells in vivo in the context of an oncolytic replication-competent herpes simplex virus, and the gene product is secreted by tumor cells rather than expressed on the cell surface. Defective herpes simplex virus vectors containing the B7-1-immunoglobulin (B7-1-Ig) fusion transgene (dvB7Ig) were generated using G207 as a helper virus and tested in the poorly immunogenic murine neuroblastoma, Neuro2a, in syngeneic A/J mice. Intraneoplastic inoculation of dvB7Ig/G207 at a low titer successfully inhibited the growth of established s.c. tumors, despite the expression of B7-1-Ig being detected in only 1% or fewer of tumor cells at the inoculation site, and prolonged the survival of mice bearing intracerebral tumors. Immunohistochemistry of dvB7Ig/G207-inoculated tumors revealed a significant increase in CD4+ and CD8+ T-cell infiltration compared with control tumors inoculated with defective vector expressing alkaline phosphatase (dvAP/G207). The antitumor effect of dvB7Ig/G207 was not manifested in athymic mice. In vivo depletion of immune cell subsets in A/J mice further revealed that CD8+ T cells, but not CD4+ T cells, were required. Animals cured of their tumors by dvB7Ig/G207 treatment were protected against rechallenge with a lethal dose of Neuro2a cells but not SaI/N cells. The results demonstrate that the use of soluble B7-1 for immune gene therapy is a potent and clinically applicable means of in situ cancer vaccination.

Selective silencing of full-length CD80 but not IgV-CD80 leads to impaired clonal deletion of self-reactive T cells and altered regulation of immune responses.

Co-stimulation provided by the B7 family of proteins underpins the development of protective immunity. There are three identified members of this family: CD80, its splice variant IgV-CD80 and CD86. It has hitherto been difficult to analyze the expression and function of IgV-CD80 since there are no appropriate reagents capable of distinguishing it from CD80. We have generated mice, by gene targeting, the lack CD80 whilst maintaining expression of IgV-CD80. Mutant animals did not delete T cells bearing mammary tumor virus-reactive TCR as efficiently as wild-type animals. We also demonstrate the importance of IgV-CD80 in the responses of recently activated cells and reveal a role for CD80 in sustaining T cell responses. CD86, whilst critical to primary T cell activation, made only a minor contribution to re-activation of normal cells.

A fast and efficient method for transiently transfecting ES cells: application to the development of systems for conditional gene expression.

Classically, mouse embryonic stem (ES) cells are transfected by electroporation, a method that requires a large number of cells. Here we describe a protocol using a liposome based transfection agent that is a very simple, rapid and cost effective way of transiently transfecting very low numbers of ES cells. We found this method very useful in screening a large number of ES clones when working with inducible expression systems in which at least two elements are required for regulated expression of the gene of interest. After stable transfection of the first component, clones can be easily and rapidly screened for expression of the gene of interest by transiently transfecting the second component of the system using this protocol.

Costimulation of T cells.

Costimulation is critical to T cell activation. On the antigen-presenting cell the key players are found in the extended family of B7 genes comprising cd80, cd86, B7h/B7RP-1 and B7-H1. cd80 and cd86 encode proteins that bind to CD28 and CTLA4 on T cells. Blocking this pathway with the potent CTLA4-Ig fusion protein shows encouraging potential as a therapeutic agent. While cd80 and cd86 pathways act mainly on naive T cells, B7h/B7RP-1 and B7-H1 seem to exert their effects on antigen-experienced lymphocytes.

T-cell regulation of peripheral tolerance and immunity: the potential role for Notch signalling.

Recognition of antigen by T cells in the periphery may lead either to the generation of productive immunity or the induction of tolerance. These two functional outcomes are a consequence of distinct pathways of T-cell differentiation. T cells are selected to become regulatory cells and their function is to maintain homeostasis with the immune system. In this review we discuss the cell-fate decisions that T cells might make allowing them to promote immunity or induce tolerance in the context of the role that Notch signalling may play in this process.

Adenovirus-mediated gene transfer of CTLA-4Ig fusion protein in the suppression of experimental autoimmune arthritis.

OBJECTIVE: Blockade of CD28-B7 interactions with soluble CTLA-4Ig fusion protein (which binds and blocks both B7-1 and B7-2 costimulatory molecules on antigen-presenting cells) has been shown to ameliorate experimental autoimmune diseases such as lupus, experimental autoimmune encephalomyelitis, diabetes, and, in our laboratory, collagen-induced arthritis (CIA). Because prolonged inhibition of this costimulatory pathway may be required, and the adenovirus-mediated gene-transfer technology is very efficient in achieving sustained expression of proteins in vivo, we examined the effects of adenovirally delivered CTLA-4Ig in established murine CIA. METHOD: Replication-deficient recombinant adenoviruses encoding a chimeric CTLA-4Ig fusion protein, or beta-galactosidase as control, were injected intravenously into male DBA/1 mice once at arthritis onset. Disease activity was monitored by the assessment of clinical score, paw thickness, and type II collagen (CII)-specific cellular and humoral responses for 3 weeks. Groups of mice were also serially injected with a CTLA-4Ig fusion protein and an anti-cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) monoclonal antibody (mAb), and disease activity was compared with that in the adenovirally transfused groups. RESULTS: Both the adenovirally delivered and the recombinant CTLA-4Ig fusion protein suppressed established CIA, whereas anti-CTLA-4 mAb and the control beta-galactosidase adenovirus did not significantly affect the disease course. CII-specific lymphocyte proliferation, interferon-gamma production, and anti-CII antibody levels, both IgG1 and IgG2a, were significantly reduced by CTLA-4Ig treatment. CONCLUSION: Blockade of the B7-CD28 costimulatory pathway by adenovirus-mediated CTLA-4Ig gene transfer is as effective as the recombinant fusion protein in treating established CIA, without the need for repeated administrations. Significant reduction in pathogenic cellular and humoral responses is achieved even after the onset of arthritis, thus suggesting the valuable therapeutic potential of this gene-transfer method in human rheumatoid arthritis.

Impaired antiviral response and alpha/beta interferon induction in mice lacking beta interferon.

We have generated mice lacking the gene for beta interferon and report that they are highly susceptible to vaccinia virus infection. Furthermore, in cultured embryo fibroblasts, viral induction of alpha interferon and of 2-5A synthetase genes is impaired. We also show that beta interferon does not prime its own expression.

Serrate1-induced notch signalling regulates the decision between immunity and tolerance made by peripheral CD4(+) T cells.

Signals derived from antigen-presenting cells (APC) influence the functional differentiation of CD4(+) T cells. We report here that Serrate1 (Jagged1), a ligand for the Notch1 receptor, may contribute to the differentiation of peripheral CD4(+) T cells into either helper or regulatory cells. Our findings demonstrate that antigen presented by murine APC overexpressing human Serrate1 induces naive peripheral CD4(+) T cells to become regulatory cells. These cells can inhibit primary and secondary immune responses, and transfer antigen-specific tolerance to recipient mice. Our results show that Notch signalling may help explain 'linked' suppression in peripheral tolerance, whereby tolerance induced to one epitope encompasses all epitopes on that antigen during the course of an immune response.

CD4(+) T cells induced by virus-like particles expressing a major T cell epitope down-regulate IL-5 production in an ongoing immune response to Der p 1 independently of IFN-gamma production.

It has been previously demonstrated that hybrid Ty virus-like particles (VLP) prime effective CD8(+) and CD4(+) T cell responses. In this study, we investigated the effect of treating mice with Ty VLP carrying the immunodominant epitope of Der p 1 after sensitizing them to the group 1 allergen of the house dust mite Dermatophagoides pteronyssinus (Der p 1), under conditions that induce T(h)2 immunity. We show that i.p. treatment with the hybrid VLP abrogated allergen-specific IL-5 production and reduced allergen-specific cell proliferation. This suppression of the response was mediated by CD4(+) T cells and was not accompanied by an increase in IFN-gamma production.

Linked suppression in peripheral T cell tolerance to the house dust mite derived allergen Der p 1.

BACKGROUND: Peripheral tolerance is required to maintain balance within the immune system. A feature of peripheral tolerance is linked suppression, in which tolerance induced to a single T cell epitope inhibits the response to all epitopes in the same protein. It is suggested that this phenomenon is mediated by regulatory T cells through either the activity of immunopressive cytokines or direct cell contact. In previous experiments we failed to detect inhibitory cytokines when T cells from mice rendered tolerant by intranasal delivery of the immunodominant peptide of Der p 1 (p 1, 110-131) were restimulated with peptide in vitro. Therefore, the aim of this study was to determine if cognate interactions between T cells mediated by Notch/Delta signaling induce and maintain peripheral T cell tolerance. METHODS: Using in situ hybridization and viral mediated gene transfer, the expression and function of Delta1 were investigated in a murine model of T cell tolerance to Der p 1 in vivo. RESULTS: Delta1 expression is increased on peripheral T cells during the induction of tolerance with high-dose peptide delivered intranasally and when tolerant animals are rechallenged under immunogenic conditions. Peptide p 1, 110-131-specific CD4+ T cells transfected with Delta1 inhibited the response of antigen-primed T cells and induced linked suppression. CONCLUSIONS: High-dose peptide delivered intranasally induces transient expression of Delta 1 on inhibitory CD4+ T cells. Ligation of the Notch1 receptor on neighbouring T cells by Delta1+ regulatory T cells inhibits clonal expansion of the former and mediates linked suppression.

Local gene therapy with CTLA4-immunoglobulin fusion protein in experimental allergic encephalomyelitis.

It has been reported previously that the induction phase of experimental allergic encephalomyelitis (EAE) is highly sensitive to systemic blockade of stimulation via MHC class II molecules and co-stimulation via the CD28:CD80/CD86 pathways. In contrast, the effector phases of EAE were relatively unaffected by similar treatments using MHC class II antigen (Ag)-specific mAb and cytotoxic T lymphocyte antigen (CTLA)4-Ig fusion proteins in some studies. This has been attributed to different sensitivities of effector cell function or the poor penetrance of inhibitory proteins into the central nervous system (CNS). To examine this question further, MHC class II Ag-specific mAb and CTLA4-Ig were delivered directly into the CNS following EAE induction, and both were found to inhibit disease. While it was found that systemic administration of mouse CTLA4-Ig could also inhibit the progression of effector immune responses when administered shortly before or during clinical disease, these were significantly more active when delivered directly into the CNS, which probably involved an action on both CD28 ligands, CD80 and CD86. Although mouse CTLA4-human Ig was therapeutically less efficient than mouse CTLA4-mouse Ig protein, probably due to the enhanced immunogenicity and lower functional activity, gene delivery of CTLA4-human Ig into the CNS using a non-replicating adenoviral vector was more effective than a single injection of CTLA4-human Ig protein. Gene delivery significantly ameliorated the development of EAE, without necessarily inhibiting unrelated peripheral immune responsiveness. Local gene delivery of CTLA4-Ig may thus be an important target for immunotherapy of human autoimmune conditions such as multiple sclerosis.

Independent contributions of HLA epitopes and killer inhibitory receptor expression to the functional alloreactive specificity of natural killer cells.

Human NK cells express receptors (KIR) which inhibit lysis through binding to HLA class I on target cells. KIR expression in different individuals has not been intensively investigated and it is not known how the KIR repertoire relates to HLA type or influences the overall activity of NK populations. This may be important in the response of NK cells to HLA-mismatched organ transplants since the ligands for KIR are supertypic epitopes shared between certain HLA alleles. We studied the effect of matching for HLA on the cytotoxicity of NK cells from individuals homozygous or heterozygous for relevant HLA class I epitopes and correlated this with KIR expression and genotype. Considerable variation in the KIR repertoire of different donors was evident, including functional KIR expressed in the absence of specific HLA ligands. We confirmed the predominant influence of HLA-C in a hierarchy of inhibitory effects mediated by HLA class I loci. In certain individuals, inhibition patterns are more complicated and may be due to the relative expression of the CD94/NKG2 receptors. Our study reveals the separate contributions of HLA and KIR molecules to NK cell alloreactivity and provides a basis for consideration of the functional diversity of KIR genes in transplantation.

HLA-DRB1 amino acid disparity is the major stimulus of interleukin-2 production by alloreactive helper T-lymphocytes.

The generation of interleukin-2 (IL-2)-mediated helper activity is a central step in the immune response induced by allogeneic histocompatibility antigens, and IL-2-producing helper T-lymphocyte precursor (HTLp) frequencies have been proposed as a measure of alloreactivity in transplant recipients. We analyzed the influence of HLA-matching on the alloresponse of HTLp in limiting dilution assays derived from healthy individuals. Mean HTLp frequencies were significantly higher in HLA-DR antigen-mismatched than HLA-DR-matched combinations. Significant differences in the effect of one or two mismatched HLA-DR antigens on mean HTLp frequencies were also detected. Mean HLA class I (HLA-A, -B, -Cw) mismatches were not significantly different in each group and had no apparent influence on HTLp frequencies. Analysis of HLA protein sequence disparities revealed no significant difference in the number of mismatched amino acid residues at the HLA-DRB1 locus between one and two HLA-DR antigen-mismatched combinations but correlated strongly with HTLp frequency. The positive correlation was evident with mismatched residues in the beta sheet and alpha helical regions of the HLA-DRB1 molecule, suggesting a predominant influence of bound peptides in the stimulation of alloreactive helper cells. This finding was supported by analysis of the location of individual residue mismatches. Evidence of an effect of polymorphism in the CD4-binding region in the beta-2 domain of HLA-DRB1 molecules was also found. Our results demonstrate the major influence of HLA-DR amino acid sequence mismatching on alloreactive HTLp frequencies but also suggest that additional genetic or environmental influences affect the alloreactive helper T-cell repertoire.

Semi-quantitative polymerase chain reaction analysis of cytokine and cytokine receptor gene expression during thymic ontogeny.

Semi-quantitative, polymerase chain reaction (PCR) is used to uncover the patterns of cytokine transcription in the mouse thymus from day 14 to day 20 of gestation, a time period which includes many of the important events in thymic ontogeny. Interleukin 4 (IL-4), IL-7 and interferon gamma (IFN-gamma) mRNA is abundant from fetal day (Fd) 14-16, corresponding with the period of rapid proliferation of immature thymocytes in vivo. As the level of mRNA for these cytokines diminishes, the induction and increased expression of IL-3 and IL-2 occurs. The transcription of these cytokines correlates temporally with the period of proliferation-dependent phenotypic differentiation between Fd 16 and 20. The thymic epithelium (TE)-derived cytokines including IL-1alpha, IL-6 and granulocyte-macrophage colony-stimulating factor (GM-CSF) begin to be transcribed between Fd 14-15 and show peak mRNA abundance from Fd 16-20. IL-5, tumour necrosis factor alpha (TNF-alpha) and LT (lymphotoxin or TNF-beta) constitute a fourth group of cytokines, along with the IL-4 receptor (IL-4R), which are transcribed at an even level throughout the fetal period. The IL-2 receptor beta chain (IL-2Rbeta) and IL-10 show abundant mRNA from Fd 14-20 and have a peak level of mRNA content on Fd 16. Taken together, these studies uncover complex, overlapping patterns of cytokine gene expression. The mRNA abundance and pattern of expression of each cytokine or cytokine receptor may indicate the relative contribution that it makes to different stages of fetal thymic ontogeny.