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Here we present 2shRNA, a shRNA-based nanobinder, which can simultaneously attack two therapeutic targets involved in drug resistance pathways and can additionally bind accessory molecules such as cell targeting peptides or fluorophores. We create 2shRNAs designed to specifically kill HER2+ breast cancer cells in the absence of a transfecting agent.

Pattern recognition receptors (PRRs) are generally recognized as the initiators of all immune responses. PRRs bind molecular patterns associated with microorganisms or endogenous mediators released by stressed tissues. Upon ligand binding, PRRs induce the activation of an inflammatory process that ultimately leads to pathogen clearance or restoration of tissue homeostasis. PRRs govern these processes, regulating the activation of a complex network of transcription factors able to induce the appropriate immune response to a specific ligand. Toll-like-receptors (TLRs) are the first and best characterized PRR family, and for a long period of time they were believed to be autonomous proteins able to recognize and initiate all the immune response to a given stimulus. Recently this view was challenged by the discovery that so-called TLR co-receptors, such as CD14 and CD36, not only favor TLR-dependent signaling but can also transduce their own signal in a TLR-independent manner. Here we will discuss the capacity of TLR co-receptors to bind different microbial and endogenous ligands and to integrate TLR functions inducing specific signaling modules.

Inappropriate activation of Toll-like receptor 4 (TLR4) on resident fibroblasts, through the binding of damage-associated molecular patterns, is a potential driver of fibrosis in systemic sclerosis. New evidence suggests that targeting fibroblast-specific TLR4 or an accessory molecule MD2 could have therapeutic value.

Congenital myasthenic syndromes are genetic disorders of neuromuscular transmission that should be considered in the differential diagnosis of seronegative myasthenia gravis and other neuromuscular disorders. They are present at birth but may not manifest until childhood or adult life. A classification system of congenital myasthenic syndromes based on molecular genetics is under evolution. Clinical and neurophysiological correlations with molecular studies have defined diagnostic criteria that assist the clinician in identifying specific clinical myasthenic syndromes. Some types of congenital myasthenia (e.g., slow-channel and fast-channel syndrome, acetylcholinesterase deficiency, and choline acetyltransferase deficiency) can be identified by clinical features, response to cholinesterase inhibitors, and standard electrodiagnostic studies. The molecular genetics, pathogenesis, clinical features, differential diagnosis, natural history, and treatment of well-characterized congenital myasthenic syndromes are discussed.

Mouse T cells fail to respond to xenogeneic pig and human antigens using the direct antigen-presenting pathway. The poor response by mouse CD8 cells is because of multiple defects in the molecular interactions between mouse CD8 cells and xenogeneic antigen-presenting cells (APCs). Using human CD4/ DR3 + , mouse CD4 - /major histocompatibility complex (MHC) class II - mice, we investigated the defects in molecular interaction responsible for the poor response to xenogeneic antigens by naive mouse CD4 + cells. Mouse CD4 cells failed to respond to human leucocyte antigen (HLA)-DR3 expressed on mouse APCs but developed a strong response to alloantigens, indicating a defect in the interaction between mouse CD4 and HLA-DR3 molecules. Human CD4 + / mouse CD4 - mouse T cells respond poorly to human and pig APCs but not allogeneic APCs, indicating that accessory molecular interactions are deficient across highly separated species. Adding mouse interleukin-2 (IL-2) to the mixed lymphocyte reaction system did not improve the poor response to human or pig antigens by mouse or human CD4 + /mouse CD4 - mouse T cells. Therefore, multiple molecular interactions deficient between mouse CD4 cells and human or pig APCs may lead to the poor response to xenogeneic antigens by naive mouse CD4 cells.

Our understanding of the mechanisms governing of Rab and Arf protein function has exploded in recent years with a convergence of information from model genetic organisms, biochemical studies, cell biological observations and protein structural information. However, the list of known Rab and Arf interacting factors still remains small relative to the number of these small GTPases that have been identified through complete genomic sequencing. It can be anticipated that the factors listed and discussed in this review probably represent a small fraction of the Rab and Arf accessory molecules that remain to be discovered. The identification of regulators and accessory molecules for the Rab and Arf families has allowed investigators to elaborate themes and develop a framework for a mechanistic understanding of these proteins. The themes are highlighted in this review, which aims to concentrate on Rab and Arf function in exocytosis.

IgA, the principal ligand for FcαRI, exists in serum as monomeric IgA and at mucosal sites as secretory IgA (SIgA). SIgA consists of dimeric IgA linked by joining chain and secretory components. Human polymorphonuclear leukocytes (PMN) and mouse PMN transgenic for human FcαRI exhibited spreading and elicited respiratory burst activity upon interaction with either serum or SIgA. However, PMN devoid of the β2 integrin Mac-1 (Mac-1−/−) were unable to bind SIgA, despite expression of FcαRI. Consistent with this, serum IgA stimulated Mac-1−/− PMN oxygen radical production, in contrast to SIgA. Binding studies showed the secretory component, by itself, to interact with Mac-1-expressing PMN, but not with Mac-1−/− PMN. These data demonstrate an essential role for Mac-1 in establishing SIgA-FcαRI interactions.

A rat IgM monoclonal antibody, PA3-795, inhibits the antigen-specific responses of mouse T-cell hybridomas. It recognizes a heavily glycosylated cell-surface protein, designated Lgp55, that is detectable after activation on mature T cells. During fetal life, Lgp55 is found at high levels on newly immigrant thymic T-cell precursors prior to surface expression of other T-lineage molecules. High levels of expression are also found on thymocytes in the outer cortex of adult mice. Thymocytes at later stages of differentiation bear decreasing amounts of surface Lgp55, and none is detectable on “single-positive” thymocytes in the thymic medulla or on resting mature T cells from the periphery. Addition of monoclonal anti-Lgp55 to fetal thymus organ culture decreases the output of “mature” CD4 singlepositive thymocytes when it is begun before fetal day 13.5. These findings suggest that Lgp55 contributes to cell-cell interactions that regulate very early steps in T-cell development in the mouse.

Given a choice, a fruit fly will seek a temperature optimum of 18°C. This behavior requires a biochemical signaling pathway apparently initiated by a heterotrimeric guanine nucleotide–binding protein (G protein)–coupled receptor. Given the similarity of these signaling events to those initiated by rhodopsin, the G protein–coupled receptor that senses light, Shen et al. (see the Perspective by Minke and Peters) tested the effects of rhodopsin mutations on temperature selection in flies and, surprisingly, found that it resulted in discrimination between temperatures within a few degrees of the optimal 18°C, independent of light sensation. One or more accessory molecules may also be required, as the rhodopsin molecule itself does not seem to be the temperature sensor. W. L. Shen, Y. Kwon, A. A. Adegbola, J. Luo, A. Chess, C. Montell, Function of rhodopsin in temperature discrimination in Drosophila . Science 331 , 1333–1336 (2011). [Abstract][Full Text] B. Minke, M. Peters, Rhodopsin as thermosensor? Science 331 , 1272–1273 (2011). [Abstract][Full Text]

Recognition of microbial infection and initiation of immune responses are controlled by multiple mechanisms. Toll-like receptors (TLRs) are key components of the innate immune system that detect microbial infection. TLR activation helps to eliminate the invading pathogens, coordinate systemic defenses, and initiate adaptive immune responses. Despite progress elucidating the TLR signaling aspects and the physiological relevance of TLRs in microbial infections, the molecular basis of microbial recognition by TLRs is still not fully understood. In this article we focus on the availability of microbial ligands to regulate presentation to TLRs and assist in our understanding of TLR- mediated microbial recognition.

Numerous novel therapies are in development for rheumatoid arthritis as a consequence of tremendous progress in understanding pathogenic mechanisms and defining relevant disease cells and molecules. Anti-tumour necrosis factor alpha (TNFα) therapies have been particularly successful and set a new standard for symptom control and attenuation of structural damage to joints. Other potential therapeutic targets across a variety of disease pathways have yet to be fully validated. These include pro-inflammatory cytokines, inflammatory cells and accessory molecules. © 2004 Elsevier Ltd. All rights reserved.

Salmonellalipid A is inactive in human macrophages despite being potently active in murine macrophages. We investigated the molecular basis for this species-specific action ofSalmonellalipid A. When murine CD14 (mCD14), mTLR4, and mMD-2 were all expressed in human monocytic THP-1 cells, these cells were capable of responding toSalmonellalipid A. Expressing each of these proteins separately did not impart such responsiveness. Expression of mTLR4 plus mMD-2, but not mCD14 plus mTLR4 or mCD14 plus mMD-2, conferred this responsiveness. In THP-1 cells expressing mCD14, mTLR4, and mMD-2, replacing mCD14 with human CD14 had no effect on responsiveness toSalmonellalipid A or syntheticSalmonella-type lipid A (compound 516). When mTLR4 was replaced with human TLR4, the responses to these lipid A preparations were decreased to half, and the replacement of mMD-2 decreased responsiveness to one-third, although the responses toEscherichia colilipid A or syntheticE. coli-type lipid A (compound 506) were not affected. These results indicate that both TLR4 and MD-2 participate in the species-specific action ofSalmonellalipid A.

There is now much evidence that tumours can be immunogenic, ie. that they frequently do express antigens in a form recognizable by the host immune system. This has been shown not only in experimental animals but also for spontaneously arising human tumours. Tumour progression may therefore require the evolution of variants which can “escape” the immune response. Mechanisms for the escape of tumours from immune responses (endogenous or therapeutic) include: downregulation of immune responses by the tumour and / or associated T cells and their products altered expression of MHC and/or tumour antigens by tumour cells altered expression of adhesion or accessory molecules by tumour and / or dendritic cells usurpation of the immune response to the advantage of the tumour (“immunostimulation”) The purpose of this chapter is to consider the current status of knowledge concerning these different tumour escape strategies, focussing on clinical studies wherever possible. Animal models will only be considered to illustrate points of potential importance to studies in humans where data in the latter are scarce or absent. This area is very fast-moving and for this reason, predominantly literature from the last 5 years will be reviewed. Some of the overall concepts have been recently discussed in a “Symposiumin-Writing” published in October 1999 (see ref.[1]). Weight will be given to those studies that not only contribute to defining the nature of the event associated with tumour escape, but point to possible ways for preventing this occurence.

Dendritic cells are critical initiators of immune responses mediated by T and B lymphocytes. Dendritic cells process antigens captured in the periphery, then emigrate to lymphoid organs. There they complete their maturation by upregulating important accessory molecules and secreting cytokines, all of which support potent stimulation of antigen-specific lymphocytes. These lymphocytes return to the periphery to complete the immune response. Investigators have discovered culture systems that use exogenous hematopoietic cytokines to support the growth, differentiation, and maturation of dendritic cells in larger numbers and greater purity than was ever before possible. This has rendered dendritic cells accessible to detailed experimental evaluations and clinical applications. Dendritic cells provide a powerful means of controlling both normal and pathologic immunity.

Stephen R. Sprang Guanosine triphosphatases (GTPases) are critical regulatory molecules in cells. In this issue, Scheffzek et al. report the structure of the key GTPase Ras complexed with its GAP, an accessory molecule necessary for efficient catalysis. In his Perspective, Sprang compares this new structure with the structure of Gabound to RGS, another important GTPase regulatory system.

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Publisher Summary There has been remarkable progress in the elucidation of many of the key processes of class I peptide loading. In addition, gp48, the factor missing in the .220 cell line promises to be a critical contributor to class I assembly with β 2 m and loading with peptide. Using both chemical and genetic approaches, many of the details of peptide loading onto class I have been resolved. This chapter discusses the stoichiometric relationship and precise individual contributions of each of these accessory molecules. Some authors share their discovery illustrating that proteins lacking signals that target the secretory compartment can contribute peptide epitopes for presentation to the immune system via class I molecules at the cell surface. The chapter discusses recent evidence elucidating the mechanism of β 2 m assembly and peptide loading and the accessory molecules involved in these processes.

The immunological properties of endothelial cells suggest they perform a pivotal role in acute and chronic rejection following solid organ transplantation. Their constitutive expression of MHC class II molecules (which initiate allograft rejection by activating CD4 T cells) and accessory molecules allows them to present foreign antigen by both the direct and indirect route to the recipient's immune system. The costimulatory molecules used by endothelial cells appear to differ from those used by traditional antigen-presenting cells such as B cells and dendritic cells. Release of non-HLA antigens from damaged endothelial cells results in a chronic antibody response — possibly contributing to graft vasculopathy and chronic rejection. Further understanding of the factors that regulate MHC class II and accessory molecule expression on endothelial cells could lead to novel strategies of therapeutic intervention.

Paraneoplastic MG is probably caused by sensitisation of T-cells to muscle-like epitopes presented on the neoplastic epithelial cells. Accessory molecules on antigen-presenting cells and T-cells are important for the outcome of the immunisation process. In this study, cryostat sections from 13 thymomas (11 with MG, 2 without; 10 non-invasive, 3 invasive; 3 spindle-cell, 10 polygonal-cell thymomas) and 6 normal thymuses from children were examined for the accessory molecules ICAM-1, VCAM-1, LFA-3 and CD28 using commercially available mouse monoclonal antibodies and immunoperoxidase staining.

Summary We report a case of mid-dermal elastolysis in which dermal inflammation was a mild but definite feature. The aetiology of this condition remains unknown, although inflammatory destruction of elastic fibres appears to be the final result. In this study, areas of inflammation exhibited features of a specific immune response, with induction of a number of immune accessory molecules.