Search

How SARS-CoV-2 penetrates the airway barrier of mucus and periciliary mucins to infect nasal epithelium remains unclear. Using primary nasal epithelial organoid cultures, we found that the virus attaches to motile cilia via the ACE2 receptor. SARS-CoV-2 traverses the mucus layer, using motile cilia as tracks to access the cell body. Depleting cilia blocks infection for SARS-CoV-2 and other respiratory viruses. SARS-CoV-2 progeny attach to airway microvilli 24 h post-infection and trigger formation of apically extended and highly branched microvilli that organize viral egress from the microvilli back into the mucus layer, supporting a model of virus dispersion throughout airway tissue via mucociliary transport. Phosphoproteomics and kinase inhibition reveal that microvillar remodeling is regulated by p21-activated kinases (PAK). Importantly, Omicron variants bind with higher affinity to motile cilia and show accelerated viral entry. Our work suggests that motile cilia, microvilli, and mucociliary-dependent mucus flow are critical for efficient virus replication in nasal epithelia.

Understanding the mechanisms of HIV tissue persistence necessitates the ability to visualize tissue microenvironments where infected cells reside; however, technological barriers limit our ability to dissect the cellular components of these HIV reservoirs. Here, we developed protein and nucleic acid in situ imaging (PANINI) to simultaneously quantify DNA, RNA, and protein levels within these tissue compartments. By coupling PANINI with multiplexed ion beam imaging (MIBI), we measured over 30 parameters simultaneously across archival lymphoid tissues from healthy or simian immunodeficiency virus (SIV)-infected nonhuman primates. PANINI enabled the spatial dissection of cellular phenotypes, functional markers, and viral events resulting from infection. SIV infection induced IL-10 expression in lymphoid B cells, which correlated with local macrophage M2 polarization. This highlights a potential viral mechanism for conditioning an immunosuppressive tissue environment for virion production. The spatial multimodal framework here can be extended to decipher tissue responses in other infectious diseases and tumor biology.

A thorough understanding of complex spatial host-disease interactions in situ is necessary in order to develop effective preventative measures and therapeutic strategies. Here, we developed Protein And Nucleic acid IN situ Imaging (PANINI) and coupled it with Multiplexed Ion Beam Imaging (MIBI) to sensitively and simultaneously quantify DNA, RNA, and protein levels within the microenvironments of tissue compartments. The PANINI-MIBI approach was used to measure over 30 parameters simultaneously across large sections of archival lymphoid tissues from non-human primates that were healthy or infected with simian immunodeficiency virus (SIV), a model that accurately recapitulates human immunodeficiency virus infection (HIV). This enabled multiplexed dissection of cellular phenotypes, functional markers, viral DNA integration events, and viral RNA transcripts as resulting from viral infection. The results demonstrated immune coordination from an unexpected upregulation of IL10 in B cells in response to SIV infection that correlated with macrophage M2 polarization, thus conditioning a potential immunosuppressive environment that allows for viral production. This multiplexed imaging strategy also allowed characterization of the coordinated microenvironment around latently or actively infected cells to provide mechanistic insights into the process of viral latency. The spatial multi-modal framework presented here is applicable to deciphering tissue responses in other infectious diseases and tumor biology.

A schematic of a biological system, i.e., a representation of its pieces, how they are combined, and what they do, would facilitate understanding its essential organization and alteration in pathogenesis or evolution. We present a computational approach for constructing tissue schematics (TSs) from high-parameter imaging data and a biological model for interpreting them. TSs map the spatial assembly of cellular neighborhoods into tissue motifs, whose modular composition, we propose, enables the generation of complex outputs. We developed our approach in human lymphoid tissue (HLT), identifying the follicular outer zone as a potential relay between neighboring zones and a core lymphoid assembly with modifications characteristic of each HLT type. Applying the TS approach to the tumor microenvironment in human colorectal cancer identified a higher-order motif, whose mutated assembly was negatively associated with patient survival. TSs may therefore elucidate how immune architectures can be specialized and become vulnerable to reprogramming by tumors.

Interaction between the activating NKG2D receptor on lymphocytes and its ligands MICA, MICB, and ULBP1–6 modulate T and NK cell activity and may contribute to the pathogenesis of Crohn’s disease (CD). NKG2D ligands are generally not expressed on the cell surface of normal, non-stressed cells, but expression of MICA and MICB in CD intestine has been reported. In this exploratory study, we further characterize the expression of NKG2D and its ligands, including the less well-described ULBP4–6, in CD, and test if NKG2D ligand interactions are involved in the migration of activated T cells into the affected mucosal compartments. Intestinal tissue from CD patients and healthy controls were analyzed by flow cytometry, mass cytometry, and immunohistochemistry for expression of NKG2D and ligands, and for cytokine release. Furthermore, NKG2D-dependent chemotaxis of activated CD8+ T cells across a monolayer of ligand-expressing human intestinal endothelial cells was examined. Activated lymphocytes down-regulated NKG2D expression upon accumulation in inflamed CD intestine. NKG2D expression on CD56+ T and γδ T cells from inflamed tissue seemed inversely correlated with CRP levels and cytokine release. B cells, monocytes, mucosal epithelium, and vascular endothelium expressed NKG2D ligands in inflamed CD intestine. The expression of NKG2D ligands was correlated with cytokine release, but was highly variable between patients. Stimulation of vascular intestinal endothelial cells in vitro induced expression of NKG2D ligands, including MICA/B and ULBP2/6. Blockade of NKG2D on CD8+ T cells inhibited the migration over ligand-expressing endothelial cells. Intestinal induction of NKG2D ligands and ligand-induced down-regulation of NKG2D in CD suggest that the NKG2D-ligand interaction may be involved in both the activation and recruitment of NKG2D+ lymphocytes into the inflamed CD intestine.

Systemic Lupus Erythematosus (SLE) is a heterogeneous autoimmune disease with heightened disease severity in children. The incomplete understanding of the precise cellular and molecular events that drive disease activity pose a significant hurdle to the development of targeted therapeutic agents. Here, we performed single-cell phenotypic and functional characterization of pediatric SLE patients and healthy controls blood via mass cytometry. We identified a distinct CD14hi monocyte cytokine signature, with increased levels of monocyte chemoattractant protein-1 (MCP1), macrophage inflammatory protein-1β (Mip1β), and interleukin-1 receptor antagonist (IL-1RA). This signature was shared by every clinically heterogeneous patient, and reproduced in healthy donors' blood upon ex-vivo exposure to plasma from clinically active patients only. This SLE-plasma induced signature was abrogated by JAK1/JAK2 selective inhibition. This study demonstrates the utility of mass cytometry to evaluate immune dysregulation in pediatric autoimmunity, by identification of a multi-parametric immune signature that can be further dissected to delineate the events that drive disease pathogenesis.

Summary Antitumoral immunity requires organized, spatially nuanced interactions between components of the immune tumor microenvironment (iTME). Understanding this coordinated behavior in effective versus ineffective tumor control will advance immunotherapies. We re-engineered co-detection by indexing (CODEX) for paraffin-embedded tissue microarrays, enabling simultaneous profiling of 140 tissue regions from 35 advanced-stage colorectal cancer (CRC) patients with 56 protein markers. We identified nine conserved, distinct cellular neighborhoods (CNs)—a collection of components characteristic of the CRC iTME. Enrichment of PD-1+CD4+ T cells only within a granulocyte CN positively correlated with survival in a high-risk patient subset. Coupling of tumor and immune CNs, fragmentation of T cell and macrophage CNs, and disruption of inter-CN communication was associated with inferior outcomes. This study provides a framework for interrogating how complex biological processes, such as antitumoral immunity, occur through concerted actions of cells and spatial domains., Graphical Abstract, Highlights • FFPE-CODEX multiplexed tissue imaging of 56 markers in 140 tissues of 35 CRC patients • Cellular neighborhoods reveal spatial organization of the tumor microenvironment • Altered organization of tumor and immune components in low- versus high-risk patients • Local enrichment of PD-1+CD4+ T cells correlates with survival in high-risk patients, A multiplexed tissue imaging and computational analysis framework applied to colorectal cancer allows interrogation of how spatial organization of the immune tumor microenvironment is linked to clinical outcomes.

A cytometric imaging approach, called CO-Detection by indEXing (CODEX), that enables high parameter multiplexing of antibody-tagged target epitopes is used here to create high parameter imaging datasets of normal mouse and lupus (MRL/lpr) spleens. In this procedure, antibody binding events are rendered iteratively using DNA barcodes, fluorescent dNTP analogs, and an in-situ polymerization-based indexing procedure. Fluorescent signals from multiple rounds of indexing are computationally combined into a multi-channel image stack and subjected to image segmentation and quantification. A segmentation and linear model algorithm was developed to accurately quantify membrane antigen levels on dissociated cells as well as tissue sections. Leveraging the spatially resolved nature of CODEX multiplexed single-cell imaging data, quantitative de novo characterization of lymphoid tissue architecture was enabled and overlaid onto previously described morphological features. We observed an unexpected, profound impact of the cellular neighborhood on the expression of protein receptors on immune cells. By comparing normal murine spleen to spleens from animals with systemic autoimmune disease (MRL/lpr), extensive and previously uncharacterized splenic cell interaction dynamics in the healthy versus diseased state was observed. The fidelity of multiplexed imaging data analysis demonstrated here will allow deep proteomic analysis and systematic characterization of complex tissue architecture in normal and clinically aberrant samples.

Summary Although pathogens must infect differentiated host cells that exhibit substantial diversity, documenting the consequences of infection against this heterogeneity is challenging. Single-cell mass cytometry permits deep profiling based on combinatorial expression of surface and intracellular proteins. We used this method to investigate varicella-zoster virus (VZV) infection of tonsil T cells, which mediate viral transport to skin. Our results indicate that VZV induces a continuum of changes regardless of basal phenotypic and functional T cell characteristics. Contrary to the premise that VZV selectively infects T cells with skin trafficking profiles, VZV infection altered T cell surface proteins to enhance or induce these properties. Zap70 and Akt signaling pathways that trigger such surface changes were activated in VZV-infected naive and memory cells by a T cell receptor (TCR)-independent process. Single-cell mass cytometry is likely to be broadly relevant for demonstrating how intracellular pathogens modulate differentiated cells to support pathogenesis in the natural host.

Single cell mass cytometry facilitates high-dimensional, quantitative analysis of the effects of bioactive molecules on cell populations at single-cell resolution. Datasets are generated with antibody panels (upwards of 40) in which each antibody is conjugated to a polymer chelated with a stable metal isotope, usually in the Lanthanide series of the periodic table. Isotope labelled antibodies recognize surface markers to delineate cell types and intracellular signaling molecules to provide a measure of the network state—and thereby demarcating multiple cell state functions such as apoptosis, DNA damage and cell cycle. By measuring all these parameters simultaneously, the signaling state of an individual cell can be measured at its network state. This review will cover the basics of mass cytometry as well as outline steps already taken to allow it to stand aside traditional fluorescence based cytometry in the immunologist’s analytical arsenal in their study of immune states during infection.

In recent years, advances in technology have provided us with tools to quantify the expression of multiple genes in individual cells. The ability to simultaneously measure multiple genes on the same cell is necessary to resolve the incredible diversity of cell subsets, as well as to define their function in the host. Fluorescence-based flow cytometry is the benchmark for this; with it, we can quantify 18 proteins per cell, at >10,000 cells per second. “Mass cytometry” is a new technology that promises to significantly extend these capabilities. Immunophenotyping by mass spectrometry provides the ability to measure more than three dozen proteins at a rate of 1,000 cells per second. We review these cytometric technologies, capable of high-content, high-throughput single-cell assays.

Cytotoxic CD8(+) T lymphocytes directly kill infected or aberrant cells and secrete proinflammatory cytokines. By using metal-labeled probes and mass spectrometric analysis (cytometry by time-of-flight, or CyTOF) of human CD8(+) T cells, we analyzed the expression of many more proteins than previously possible with fluorescent labels, including surface markers, cytokines, and antigen specificity with modified peptide-MHC tetramers. With 3-dimensional principal component analysis (3D-PCA) to display phenotypic diversity, we observed a relatively uniform pattern of variation in all subjects tested, highlighting the interrelatedness of previously described subsets and the continuous nature of CD8(+) T cell differentiation. These data also showed much greater complexity in the CD8(+) T cell compartment than previously appreciated, including a nearly combinatorial pattern of cytokine expression, with distinct niches occupied by virus-specific cells. This large degree of functional diversity even between cells with the same specificity gives CD8(+) T cells a remarkable degree of flexibility in responding to pathogens.

While flow cytometry once enabled researchers to examine 10--15 cell surface parameters, new mass flow cytometry technology enables interrogation of up to 45 parameters on a single cell. This new technology has increased understanding of cell expression and how cells differentiate during hematopoiesis. Using this information, knowledge of leukemia cell biology has also increased. Other new technologies, such as SPADE analysis and single cell network profiling (SCNP), are enabling researchers to put different cancers into more biologically similar categories and have the potential to enable more personalized medicine.

We demonstrate the use of electron microscopy as a powerful characterization tool to identify and locate antibody-conjugated composite organic-inorganic nanoparticle (COINs) surface enhanced Raman scattering (SERS) nanoparticles on cells. U937 leukemia cells labeled with antibody CD54-conjugated COINs were characterized in their native, hydrated state using wet scanning electron microscopy (SEM) and in their dehydrated state using high-resolution SEM. In both cases, the backscattered electron (BSE) detector was used to detect and identify the silver constituents in COINs due to its high sensitivity to atomic number variations within a specimen. The imaging and analytical capabilities in the SEM were further complemented by higher resolution transmission electron microscopy (TEM) images and scanning Auger electron spectroscopy (AES) data to give reliable and high-resolution information about nanoparticles and their binding to cell surface antigens.

Liver-directed gene therapy has the potential for treatment of numerous inherited diseases affecting metabolic functions. The aim of this study was to evaluate gene expression in hepatocytes using feline immunodeficiency virus-based lentiviral vectors, which may be potentially safer than those based on human immunodeficiency virus. In vitro studies revealed that gene expression was stable for up to 24 days post-transduction and integration into the host cell genome was suggested by Alu PCR and Southern blot analyses. Systemic in vivo administration of viral particles by the hydrodynamics method resulted in high levels of gene expression exclusively in the liver for over 7 months whereas injection of plasmid DNA by the same method led to transient expression levels. Our studies suggest that feline immunodeficiency-based lentiviral vectors specifically transduce liver cells and may be used as a novel vehicle of gene delivery for treatment of metabolic disease.

A library of constrained peptides that form stable folded structures was screened for aptamers that bind with high affinity to the fluorescent dye Texas red. Two selected clones had binding constants to Texas red of 25 and 80 pM as phage and binding had minimal effects on the fluorescence of Texas red. The peptides interact with distinct but overlapping regions of Texas red. One peptide bound to X-rhod calcium sensors, which share the same core fluorophore as Texas red. These dyes retained calcium sensitivity when bound to the peptide. This peptide was used to label a fusion protein with X-rhod-5F in vivo, and X-rhod sensed changes in calcium locally. Thus, minimal, constrained peptides can functionally bind to environmentally sensitive dyes or other organic agents in biological contexts, suggesting tools for in vivo imaging and analysis.

In this study, we demonstrate that vaccination of rabbits with murine endothelial cells yields polyclonal immunoglobulin (IgG) with potent antiangiogenic activity. The mechanism of this response appears to be through apoptosis of endothelial cells in vitro. Induction of polyclonal IgG in a xenogeneic host may be useful in passive immunotherapy of a variety of cancers. In fact, the antibody showed antitumor activity in three mouse tumor models (murine B16F10 melanoma, murine SVR angiosarcoma, and human DLD-1 colorectal adenocarcinoma). The polyclonal antibody generated here demonstrated utility in radioimaging of tumors in vivo, using positron emission tomography (PET) imaging, and suggested an antitumor effect in vivo. The results suggest that the antitumor effect in vivo may be related to antiangiogenic effects. Furthermore, anti-endothelial cell antibodies such as these could be useful reagents in isolating specific targets that comprise and induce the antiangiogenic effect.

We identified intracellular adhesion molecule-2 (ICAM-2) in a genetic screen as an activator of the PI3K/AKT pathway leading to inhibition of apoptosis. ICAM-2 induced tyrosine phosphorylation of ezrin and PI3K kinase membrane translocation, resulting in phosphatidylinositol 3,4,5 production, PDK-1 and AKT activation, and subsequent phosphorylation of AKT targets BAD, GSK3, and FKHR. ICAM-2 clustering protected primary human CD19+ cells from TNFα- and Fas-mediated apoptosis as determined by single-cell analysis. ICAM-2 engagement by CD19+ cells of its natural receptor, LFA-1, on CD4+ naive cells specifically induced AKT activity in the absence of an MHC-peptide interaction. These results attribute a novel signaling function to ICAM-2 that might suggest mechanisms by which ICAM-2 signals intracellular communication at various immunological synapses.

HIV-1 budding appears to require Vps4 and Tsg101-two proteins that have links to endosomal sorting machinery. A picture emerges wherein divergent viruses recruit endosomal proteins like Tsg101 to gain access to ubiquitin processes that play a crucial role during viral budding.

The Type II EBV malignancies nasopharyngeal carcinoma and EBV(+) Hodgkin's disease express three subdominant antigens, latency membrane protein (LMP) 1, LMP2, and EBNA-1. While adoptive immunotherapy with T cell lines for Type III EBV malignancy (such as posttransplant lymphoma, PTLD, which expresses the immunodominant EBNA-3 antigens) has been used to prevent and treat PTLD, the generation of class I MHC-restricted CTL suitable for the immunotherapy of Type II EBV malignancy is difficult. This is primarily due to the lack of anti-LMP or EBNA-1 CTL activity in many healthy volunteers. We have engineered, by retroviral transduction of the TCR, CTL that have the potential to recognize subdominant EBV latency antigens. Using the SAMEN retroviral vector we demonstrate the ability to transfer CTL activity from a LMP2 peptide-specific CTL clone to a stimulated PBMC population. TCR-transduced PBMC also secrete IFN-gamma upon coculture with LMP2 targets and maintain expression of the transduced TCR during subsequent mitogenic expansion.

Angiostatin and endostatin are potent endothelial cell growth inhibitors that have been shown to inhibit angiogenesis in vivo and tumor growth in mice. However, tumor shrinkage requires chronic delivery of large doses of these proteins. Here we report synergistic antitumor activity and survival of animals when these factors are delivered in combination to tumors by retroviral gene transfer. We have demonstrated this efficacy in both murine leukemia and melanoma models. Complete loss of tumorigenicity was seen in 40% of the animals receiving tumors transduced by the combination of angiostatin and endostatin in the leukemia model. The synergy was also demonstrated in vitro on human umbilical vein endothelial cell differentiation and this antiangiogenic activity may suggest a mechanism for the antitumor activity in vivo. These findings imply separate pathways by which angiostatin and endostatin mediate their antiangiogenic effects. Together, these data suggest that a combination of antiangiogenic factors delivered by retroviral gene transfer may produce synergistic antitumor effects in both leukemia and solid tumors, thus avoiding long-term administration of recombinant proteins. The data also suggest that novel combinations of antiangiogenic factors delivered into tumors require further investigation as therapeutic modalities.

Background: Fluorophore dyes are used extensively in biomedical research to sensitively assay cellular constituents and physiology. We have created, as proof of principle, fluorophore dye binding peptides that could have applications in fluorescent dye-based approaches in vitro and in vivo . Results: A panel of Texas red, Rhodamine red, Oregon green 514 and fluorescein binding peptides, termed here ‘fluorettes', was selected via biopanning of a combinatorial library of 12-mer peptides fused to a minor coat plll protein of the filamentous bacteriophage M 13, The ‘best' fluorette sequences from each of the groups were subjected to further mutagenesis, followed by a second biopanning to select a new generation of improved fluorettes. Phage were selected that had higher avidity for each fluorophore except Rhodamine red, Of these, peptides were characterized that could specifically and with high affinity bind at least one dye, Texas red, in solution, In addition, the binding of certain peptides to Texas red shifted the peak excitation and/or the emission spectra of the bound dye. Conclusions: Peptides in the context of phage display could readily be selected that could bind to small-molecule fluorophores. The affinities of selected mutant fluorettes could be increased by mutation and further selection, Only a subset of the free peptides could bind free dyes in solution, suggesting that phage context contributed to the selection and ability of certain peptidic regions to independently bind the dyes, Future screens might lead to the creation of other dye-binding peptides with novel characteristics or Texas red derivatives with cross-linking substituents might be designed to increase the utility of the system.

Fas is a surface receptor that can transmit signals for apoptosis. Using retroviral cDNA library–based functional cloning we identified a gene toso, that blocks Fas-mediated apoptosis. Toso expression was confined to lymphoid cells and was enhanced after cell-specific activation processes in T cells. Toso appeared limited to inhibition of apoptosis mediated by members of the TNF receptor family and was capable of inhibiting T cell self-killing induced by TCR activation processes that up-regulate Fas ligand. We mapped the effect of Toso to inhibition of caspase-8 processing, the most upstream caspase activity in Fas-mediated signaling, potentially through activation of cFLIP. Toso therefore serves as a novel regulator of Fas-mediated apoptosis and may act as a regulator of cell fate in T cells and other hematopoietic lineages.

Since several genes expressed in the pituitary can bind the transcription factor NF-KB, its presence and regulation was examined in the GH3 pituitary cell line. An electrophoretic mobility shift assay using nuclear extracts and an oligonucleotide probe corresponding to the Ig KB binding site was employed to identify activated NF-KB. One complex possessed properties characteristic of NF-KB: co-migration with an NF-KB complex and binding specificity restricted to NF-KB binding DNA sequences. Antibodies to the NF-KB subunits NFKB1p50 (p50) and RelA (p65) interacted with the extract-DNA complex. Activation of NF-KB in GH3 cells was increased by PMA or the cytokine tumor necrosis factor alpha. A synergy between PMA and TNF or a calcium mobilizing agent was seen in NF-KB activation. Further TNF activation was enhanced by TRH. These observations indicate the presence of NF-KB in GH3 cells and demonstrate its activation by hormones/second messengers that act on pituitary cells.

Advances are needed in random-display technologies to more tightly link drug actions and functions to the genes that control physiological processes. The reports discussed here explore two sides of these issues—generating new library formats and identifying the targets of drug ligands.

The sequence and biochemical properties of the product of the cloned cDNA for the p65 subunit of nuclear factor κB (NF-κB) have been determined. The cDNA has an open reading frame of 549 amino acids capable of encoding a 60 kd protein. NF-κB p65 contains an amino-terminal region of 320 amino acids with extensive similarity to the oncogene c- rel and lesser similarity to NF-κB p50. In vitro translated p65 forms a DNA-binding complex with NF-κB p50, and the binding of this complex can be specifically inhibited by purified IκB. Progressive carboxy-terminal deletions of p65 show that, contrary to previous assumptions, p65 does include a DNA-binding domain that in vivo might become activated only through hetero-oligomerization with p50. DNA binding by truncated p65 is inhibited by IκB, thus mapping the IκB interaction domain to the rel -homologous region and suggesting that IκB exerts its inhibitory effect upon NF-κB primarily through interaction with p65.

The DNA binding subunit of the transcription factor NF-kappa B, p50, has been cloned. p50 appears to be synthesized as a larger protein that is then processed to its functional size. Sequence analysis reveals remarkable homology for over 300 amino acids at the amino-terminal end to the oncogene v-rel, its cellular homolog c-rel, and the Drosophila maternal effect gene dorsal. This establishes NF-kappa B as a member of the rel family of proteins, all of which display nuclear-cytosolic translocation. Protein sequence from the p65 polypeptide has established that it is not encoded in the same mRNA as p50. However, p65 appears homologous to c-rel, suggesting that c-rel may form heterodimers with p50 and rel may include a homodimerization motif.

Background/Aims: Salivary glands have been suggested as an accessible organ for introduction of transgenes enabling expression of recombinant proteins in the saliva, as well as their secretion to the blood circulation. Introduction of viral vectors to the salivary glands is facilitated by direct cannulation via their ducts, which open into the oral cavity. FIV-based lentiviral vectors have the ability to infect non-dividing cells, and are therefore well-suited for transduction of the characteristically slow-replicating salivary gland cells. Our goal was to evaluate the efficacy of FIV vector-based gene transfer to murine salivary glands.

Spliceosome Mediated RNA Trans-splicing (SMaRT™) is a platform technology to reprogram genes at the pre-mRNA level. Applications include RNA therapy and real-time molecular imaging, with each application dependent upon the nature of the sequences encoded by the pre-trans-splicing molecule (PTM). SMaRT utilizes native spliceosomes to carry out trans-splicing reactions between two RNA molecules: a pre-mRNA target present in the cell and a delivered PTM.

The gene families encoding the proteins NF-kappa B, c-Rel and Dorsal, in conjunction with their respective inhibitors l kappa B, pp40, and Cactus, achieve specificity in gene regulation by means of common principles. The related activities of NF-kappa B and Dorsal are mediated by heterodimeric or homodimeric complexes of proteins containing the conserved dimerization and DNA-binding domain termed Rel. The l kappa Bs and Cactus, which share a core series of structural repeats termed ankyrin, inhibit cognate activators through differential interactions with the Rel-homology domain. Together, the inhibitory ankyrin proteins and their cognate Rel dimers probably define specific signalling pathways able to activate specific gene expression. Both gene families include proto-oncogenes, thus broadly implicating Rel/l kappa B in the control of both normal gene expression and the aberrant gene expression that makes cells cancerous.