Cyclic dinucleotides modulate induced type I IFN responses in innate immune cells by degradation of STING.

The cyclic dinucleotides, GMP-AMP (cGAMP) and c-di-AMP [bis-(3',5')-cyclic dimeric AMP], are potent type I IFN inducers via STING-TBK1-IRF3 cascade. They are promising adjuvants that promote antigen-specific humoral and cellular immune responses in different preclinical models; however, an optimal outcome of vaccination depends on a balanced immune activation. Here, we characterize the process of IFN-ß induction by c-di-AMP and cGAMP in an in vitro model on the basis of primary mouse dendritic cells. Results obtained show decreased IFN-ß production upon prolonged cell stimulation. We demonstrate that this effect depends on c-di-AMP/cGAMP-mediated down-regulation of stimulator of IFN gene (STING) protein levels. These results were confirmed by using human peripheral blood mononuclear cell-derived dendritic cells. Studies performed to explore the potential mechanism of STING modulation suggested proteolytic degradation to be a contributing factor to the observed decrease in cellular STING levels. Our work contributes to the elucidation of the molecular mode of action of vaccine constituents, which, in turn, is a prerequisite for the rational design of vaccines with predictable efficacy and safety profiles-Rueckert, C., Rand, U., Roy, U., Kasmapour, B., Strowig, T., Guzmán, C. A. Cyclic dinucleotides modulate induced type I IFN responses in innate immune cells by degradation of STING.

HIV-1 matrix protein p17 promotes lymphangiogenesis and activates the endothelin-1/endothelin B receptor axis.

OBJECTIVE: AIDS-related lymphomas are high grade and aggressively metastatic with poor prognosis. Lymphangiogenesis is essential in supporting proliferation and survival of lymphoma, as well as tumor dissemination. Data suggest that aberrant lymphangiogenesis relies on action of HIV-1 proteins rather than on a direct effect of the virus itself. HIV-1 matrix protein p17 was found to accumulate and persist in lymph nodes of patients even under highly active antiretroviral therapy. Because p17 was recently found to exert a potent proangiogenic activity by interacting with chemokine (C-X-C motif) receptors 1 and 2, we tested the prolymphangiogenic activity of the viral protein. APPROACH AND RESULTS: Human primary lymph node-derived lymphatic endothelial cells were used to perform capillary-like structure formation, wound healing, spheroids, and Western blot assays after stimulation with or without p17. Here, we show that p17 promotes lymphangiogenesis by binding to chemokine (C-X-C motif) receptor-1 and chemokine (C-X-C motif) receptor-2 expressed on lymph node-derived lymphatic endothelial cells and activating the Akt/extracellular signal-regulated kinase signaling pathway. In particular, it was found to induce capillary-like structure formation, sprout formation from spheroids, and increase lymph node-derived lymphatic endothelial cells motility. The p17 lymphangiogenic activity was, in part, sustained by activation of the endothelin-1/endothelin receptor B axis. A Matrigel plug assay showed that p17 was able to promote the outgrowth of lymphatic vessels in vivo, demonstrating that p17 directly regulates lymphatic vessel formation. CONCLUSIONS: Our results suggest that p17 may generate a prolymphangiogenic microenvironment and plays a role in predisposing the lymph node to lymphoma growth and metastasis. This finding offers new opportunities to identify treatment strategies in combating AIDS-related lymphomas.

Vaccines: from empirical development to rational design.

Infectious diseases are responsible for an overwhelming number of deaths worldwide and their clinical management is often hampered by the emergence of multi-drug-resistant strains. Therefore, prevention through vaccination currently represents the best course of action to combat them. However, immune escape and evasion by pathogens often render vaccine development difficult. Furthermore, most currently available vaccines were empirically designed. In this review, we discuss why rational design of vaccines is not only desirable but also necessary. We introduce recent developments towards specifically tailored antigens, adjuvants, and delivery systems, and discuss the methodological gaps and lack of knowledge still hampering true rational vaccine design. Finally, we address the potential and limitations of different strategies and technologies for advancing vaccine development.

The oncogenic TBC domain protein USP6/TRE17 regulates cell migration and cytokinesis.

BACKGROUND INFORMATION: Cancer cells are characterized by their intrinsic ability to rapidly divide and migrate and to invade other tissues. How these processes are regulated at a molecular level is largely unknown. RESULTS: Here, we identify the oncogenic TBC (Tre-2/Bub2/Cdc16) domain protein USP6 (also termed TRE17) as a regulator of both cell migration and division. We show that manipulating USP6 expression levels alters the ability of cells to migrate and to divide. Furthermore, we observe that cell proliferation and progression through cytokinesis depend on USP6 expression via a pathway that involves the small GTPase Arf6 and its GTPase-activating protein ACAP1. CONCLUSIONS: Our data suggest a model whereby the oncogenic potential of USP6 is linked to its ability to integrate cell migration and cytokinesis by regulating Arf6/ACAP1.

The hinge region of the scaffolding protein of cell contacts, zonula occludens protein 1, regulates interacting with various signaling proteins.

Zonula occludens protein 1 (ZO-1) is a ubiquitous scaffolding protein, but it is unknown why it functions in very different cellular contacts. We hypothesized that a specific segment, the unique hinge region, can be bound by very different regulatory proteins. Using surface plasmon resonance spectroscopy and binding assays to peptide libraries, we show, for the first time, that the hinge region directly interacts with disparate signal elements such as G-proteins alpha 12 and alpha i2, the regulator of G-protein signaling 5, multifunctional signaling protein ahnak1, and L-type Ca2+-channel beta-2-subunit. The novel binding proteins specifically bound to a coiled coil-helix predicted in the hinge region of ZO-. The interactions were modulated by phosphorylation in the hinge helix. Activation of the G-proteins influenced their association to ZO-1. In colon cells, G alpha i2 and ZO-1 were associated, as shown by coimmunoprecipitation. After cotransfection in kidney cells, G alpha i2 barely colocalized with ZO-1; the colocalization coefficient was significantly increased when epinephrine activated G-protein signaling. In conclusion, proteins with different regulatory potential adhere to and influence cellular functions of ZO-proteins, and the interactions can be modulated via its hinge region and/or the binding proteins.

A novel sandwich ELISA for alpha1 domain based detection of soluble HLA-G heavy chains.

The detection of soluble human leukocyte antigen G (HLA-G) has been a technically demanding task for several years now and various enzyme linked immunosorbent assay (ELISA) formats have been designed. However, no ELISA test has been described so far which is able to detect all possible kinds of soluble HLA-G (sHLA-G) molecules that might occur in bio fluids. Here we describe a new ELISA approach able to recognize soluble alpha1 domain containing heavy chains of all HLA-G isoforms. The detection limit is shown to be at about 150 pg soluble recombinant HLA-G1 heavy chain per milliliters. Detectable HLA-G fragments are shown to occur in the supernatants of different HLA-G transfected cell lines and appear to be particularly abundant in supernatant of trophoblast derived choriocarcinoma cell lines. The novel ELISA employs the well characterized HLA-G mAbs 4H84 and MEM-G1 which ensure high HLA-G specificity. A negative control ELISA format, designed against non-existing analytes, has been established to reveal non-specific signal interference.

Cyclic GMP-AMP displays mucosal adjuvant activity in mice.

The recently discovered mammalian enzyme cyclic GMP-AMP synthase produces cyclic GMP-AMP (cGAMP) after being activated by pathogen-derived cytosolic double stranded DNA. The product can stimulate STING-dependent interferon type I signaling. Here, we explore the efficacy of cGAMP as a mucosal adjuvant in mice. We show that cGAMP can enhance the adaptive immune response to the model antigen ovalbumin. It promotes antigen specific IgG and a balanced Th1/Th2 lymphocyte response in immunized mice. A characteristic of the cGAMP-induced immune response is the slightly reduced induction of interleukin-17 as a hallmark of Th17 activity--a distinct feature that is not observed with other cyclic di-nucleotide adjuvants. We further characterize the innate immune stimulation activity in vitro on murine bone marrow-derived dendritic cells and human dendritic cells. The observed results suggest the consideration of cGAMP as a candidate mucosal adjuvant for human vaccines.

The mucosal adjuvant cyclic di-AMP exerts immune stimulatory effects on dendritic cells and macrophages.

The cyclic di-nucleotide bis-(3',5')-cyclic dimeric adenosine monophosphate (c-di-AMP) is a candidate mucosal adjuvant with proven efficacy in preclinical models. It was shown to promote specific humoral and cellular immune responses following mucosal administration. To date, there is only fragmentary knowledge on the cellular and molecular mode of action of c-di-AMP. Here, we report on the identification of dendritic cells and macrophages as target cells of c-di-AMP. We show that c-di-AMP induces the cell surface up-regulation of T cell co-stimulatory molecules as well as the production of interferon-ß. Those responses were characterized by in vitro experiments with murine and human immune cells and in vivo studies in mice. Analyses of dendritic cell subsets revealed conventional dendritic cells as principal responders to stimulation by c-di-AMP. We discuss the impact of the reported antigen presenting cell activation on the previously observed adjuvant effects of c-di-AMP in mouse immunization studies.

Association between segments of zonula occludens proteins: live-cell FRET and mass spectrometric analysis.

The tight junction protein ZO-1 (zonula occludens protein 1) has recruiting/scaffolding functions in the junctional complex of epithelial and endothelial cells. Homodimerization was proposed to be crucial for ZO-1 function. Here, we investigated the ability of ZO-1 domains to mediate self-interaction in living cells. We expressed ZO-1 truncation mutants as fusions with derivatives of green fluorescent protein in tight junction-free HEK-293 cells and determined self-association by means of fluorescence resonance energy transfer measurements using live-cell imaging. We show that both an SH3-hinge-GuK fusion protein and the PDZ2 domain self-associate in our test system. The recombinant PDZ2 domain also binds to ZO-1 and ZO-2 in tight junction-forming HT29/B6 cell lysates, as demonstrated by coprecipitation. Both interaction types are of relevance for the function of ZO-1 in the regulation of the junctional complex in polar cells.

The oligomerization of the coiled coil-domain of occludin is redox sensitive.

The transmembrane tight junction protein occludin is sensitive to oxidative stress. Occludin oligomerizes; however, its function in the tight junction is unknown. The cytosolic C-terminal tail contains a coiled coil-domain and forms dimers contributing to the oligomerization. The regulation of the oligomerization remains unclear. As the domain area contains sulfhydryl residues, we tested the hypothesis that the dimerization of the coiled coil-domain depends on these residues. We showed that the dimerization is modulated by the thiol concentration in the low-millimolar range, which is relevant both for physiological and pathophysiological conditions. Masking the sulfhydryl residues in the fragment by covalent binding of 4-vinyl pyridine prevented the dimerization but did not affect its helical structure and cylindric shape. The data demonstrate, for the first time, that disulfide bridge formation of murine cystein 408 is involved in the dimerization. This process is redox-sensitive but the secondary structure of the domain is not. It is concluded that the dimerization of occludin may play a regulatory role in the tight junction assembly under physiological and pathological conditions.

Individual return to Leydig cell function after GnRH-immunization of boars.

Immunization of boars against GnRH inhibits synthesis of testicular anabolic steroids and the sex odour androstenone. Time between second vaccination and return of Leydig cell function is unknown. Six catheterized boars received the second dose (Improvac) at 26 weeks of age (day 0). Titre, LH, testicular steroids, and IGF-I were determined in blood until testosterone exceeded 0.5 ng/mL again in all boars. At week 10 the titre was low again. Return of testicular function was preceded by increased pulsatile secretion of LH but onset of steroid synthesis was delayed and highly variable (10-24 weeks) and was not related to the initial antibody titre. A major reason for delayed onset of Leydig cell function apparently is a variable refractoriness against LH.

Natural MHC class I polymorphism controls the pathway of peptide dissociation from HLA-B27 complexes.

Analysis of antigen dissociation provides insight into peptide presentation modes of folded human leukocyte antigen (HLA) molecules, which consist of a heavy chain, beta2-microglobulin (beta2m), and an antigenic peptide. Here we have monitored peptide-HLA interactions and peptide dissociation kinetics of two HLA-B27 subtypes by fluorescence depolarization techniques. A single natural amino-acid substitution distinguishes the HLA-B*2705 subtype that is associated with the autoimmune disease ankylosing spondylitis from the non-disease-associated HLA-B*2709 subtype. Peptides with C-terminal Arg or Lys represent 27% of the natural B*2705 ligands. Our results show that dissociation of a model peptide with a C-terminal Lys (GRFAAAIAK) follows a two-step mechanism. Final peptide release occurs in the second step for both HLA-B27 subtypes. However, thermodynamics and kinetics of peptide-HLA interactions reveal different molecular mechanisms underlying the first step, as indicated by different activation energies of 95+/-8 kJ/mol (HLA-B*2705) and 150+/-10 kJ/mol (HLA-B*2709). In HLA-B*2709, partial peptide dissociation probably precedes fast final peptide release, while in HLA-B*2705 an allosteric mechanism based on long-range interactions between beta2m and the peptide binding groove controls the first step. The resulting peptide presentation mode lasts for days at physiological temperature, and determines the peptide-HLA-B*2705 conformation, which is recognized by cellular ligands such as T-cell receptors.