CLPFFD-PEG functionalized NIR-absorbing hollow gold nanospheres and gold nanorods inhibit ß-amyloid aggregation.

Gold nanoparticles with specific optical properties in combination with the CLPFFD peptide that exhibits selectivity for ß-amyloid (Aß) aggregates are promising photothermal absorbers for application in Alzheimer's disease therapy. We report on hollow gold nanospheres (HAuNS) and gold nanorods (AuNR), which exhibit strong plasmonic near infrared (NIR) absorbance in the optical window of biological tissue and which are functionalized with CLPFFD in two different ways. Therefore the peptide was either directly bound to the particle surface or indirectly to a particle-protecting polyethylene glycol (PEG) ligand shell, thereby reducing the CLPFFD density on the surfaces of both types of particles. Fully PEGylated particles were used for comparison. The effects on cell viability and the fundamental suitability of the HAuNS and AuNR conjugates as photothermal absorbers to inhibit Aß-fibrillation are analysed in vitro. The positive influence of the use of PEG ligands on the reduced cytotoxicity of the conjugates and on the Aß-disaggregation is discussed.

Hydrophobic superparamagnetic FePt nanoparticles in hydrophilic poly(N-vinylcaprolactam) microgels: a new multifunctional hybrid system.

We report the synthesis of a new multifunctional colloidal hybrid system consisting of thermoresponsive amphiphilic biocompatible poly(N-vinylcaprolactam) microgels loaded with hydrophobic superparamagnetic FePt nanoparticles (NPs). In the first step, water swellable poly(N-vinylcaprolactam) microgels were mixed with hydrophobically coated sub-10 nm superparamagnetic FePt NPs in a tetrahydrofuran (THF) solution. In the second step, changing the surrounding solvent from THF to water forces the FePt NPs to migrate into the amphiphilic microgels. These new hybrid microgels (i) are colloidally stable in water and their thermo-responsive properties in terms of volume phase transition are retained, (ii) exhibit superparamagnetic characteristics introduced by FePt NPs, (iii) show a drastically reduced cytotoxicity compared to water-soluble FePt NPs of similar size, as known from the literature. This makes the new hybrid microgels suitable e.g. as biocompatible containers for drug delivery or for imaging.

Multivalency of PEG-thiol ligands affects the stability of NIR-absorbing hollow gold nanospheres and gold nanorods.

In this work the effect of multivalency on the stability of NIR-absorbing HAuNSs and AuNRs functionalized by mono-, bi- and tridentate polyethyleneglycol (PEG) thiol ligands is reported. Comparison of commercially-available monodentate and self-synthesized bi- and tridentate methoxy terminated thiol-polyethyleneglycol ligands having molecular weights of around 5000 Da shows the stability increase of HAuNSs and AuNRs for bi- and tridentate ligands, attributed to the multivalency of the ligands. The stability was explored according to three different aspects: (1) stability towards competition reactions with the strong binding ligand dithiothreitol, (2) resistance towards oxidative Au dissolution with potassium cyanide, and (3) colloidal stability, tested by the addition of NaCl. Our PEGylation approach leads to AuNRs where the CTAB concentration is below the detection limit of the performed analytical methods, which is vital for any clinical applications. Furthermore, we found strikingly high biocompatibility after PEGylation for both particle types whereby we observed no significant difference in cytotoxicity comparing the mono-, bi- and tridentate PEGylated species.

Down-regulation of a host microRNA by a viral noncoding RNA.

Primate herpesviruses express more noncoding RNAs (ncRNAs) than any other class of mammalian viruses during either latency or the lytic phase of the viral life cycle. T cells transformed by the monkey virus Herpesvirus saimiri (HVS) express seven viral U-rich ncRNAs called HSURs. Conserved sequences in HSURs1 and 2 exhibit complementarity to three host-cell microRNAs (miRNAs). The predicted interactions of HSURs1 and 2 with these miRNAs were confirmed by coimmuno-precipitation experiments performed on extracts of marmoset T cells transformed by a wild-type or a mutant HVS lacking these two HSURs. Mutational analyses demonstrated that the binding of miR-27 to HSUR1 and that of miR-16 to HSUR2 involves base pairing. One of these miRNAs, miR-27, is dramatically lowered in abundance in HVS-transformed cells, with consequent effects on the expression of miR-27 target genes. Transient knockdown and ectopic expression of HSUR1 demonstrated that degradation of mature miR-27 occurs in a sequence-specific and binding-dependent manner but does not occur by AU-rich element (ARE)-mediated decay, which controls the intracellular level of HSUR1 itself. This viral strategy exemplifies the use of an ncRNA to control host-cell gene expression via the miRNA pathway and has potential applications both experimentally and therapeutically.

Balb/c EGFP mice are tolerant against immunization utilizing recombinant adenoviral-based vectors encoding EGFP: a novel model for the study of tolerance mechanisms and vaccine efficacy.

Enhanced green fluorescent protein (EGFP) is a marker gene product which is readily detectable using the techniques of fluorescence microscopy, flow cytometry, or macroscopic imaging. Previous studies have demonstrated the immunogenicity of EGFP in Balb/c mice, identifying an immunodominant H2-K(d) restricted CTL epitope. To model immunological tolerance and vaccine efficiency against self-antigens, we generated a stable transgenic BALB/c mouse expressing EGFP (Balb/c EGFP) through back-crossing C57Bl/6-TG(ACTbEGFP)10sb more than ten times with Balb/c wildtype (wt) mice. High level EGFP expression was detected in the skin and heart, whereas low level expression was observed in the kidney, liver, gut, lung, and spleen. To characterize the immune reactivity to self-antigen, we immunized Balb/c EGFP and Balb/c wt mice with recombinant adenoviral-based vectors encoding EGFP (Ad-EGFP) or beta-galactosidase (Ad-betagal) as a control. Immunization utilizing the Ad-betagal vector expressing 'foreign' antigen induced robust humoral and cellular transgene-specific immunity, whereas Balb/c EGFP mice presented no reactivity following Ad-EGFP immunization against the 'self-antigen' EGFP. These findings describe the creation of a transgenic mouse line tolerant against the common protein marker EGFP, providing a novel system for the evaluation of methods of tolerance disruption and vaccine efficacy.

Comparison of recombinant adenovirus and synthetic peptide for DC-based melanoma vaccination.

Optimal strategies for antigen-specific melanoma vaccination are currently being defined in experimental mouse models. Using a single H2-K(b)-binding peptide epitope derived from the melanosomal enzyme tyrosinase-related protein 2 (TRP2) in C57BL/6 mice, we show that adenovirus-transduced dendritic cells (DC) are clearly superior to peptide-pulsed DC for the induction of CD8+ T cells and antimelanoma immunity. Vaccine efficacy strictly depended on the presence of linked CD4+ T-cell help during the priming but not the effector phase of the immune response. These results provide important information for the translation of melanoma vaccine strategy in future clinical applications.

The challenge of viral snRNPs.

Some gammaherpesviruses encode nuclear noncoding RNAs (ncRNAs) that assemble with host proteins. Their conservation and abundance implies that they serve important functions for the virus. This paper focuses on our studies of three classes of nuclear noncoding herpesvirus RNAs. (1) EBERs 1 and 2 are expressed by Epstein-Barr virus in latent infection of human B lymphocytes. Recent studies revealed three sites on EBER1 that associate with ribosomal protein L22. In addition, heterokaryon assays have definitively shown that both EBERs are confined to the nucleus, arguing that their contribution to viral latency is purely nuclear. (2) HSURs 1-7 are U RNAs encoded by Herpesvirus saimiri, which causes aggressive T-cell leukemias and lymphomas. Comparison of monkey T cells transformed with wild-type or mutant virus lacking HSURs 1 and 2 revealed significant changes in host mRNAs implicated in T-cell signaling. (3) PAN is a 1-kb polyadenylated RNA that accumulates in the nucleus of Kaposi's sarcoma-associated herpesvirus lytically infected cells. A novel element, the ENE, is essential for its high accumulation. Recent results indicate that the ENE functions to counteract poly(A)-dependent RNA degradation, which we propose contributes to nuclear surveillance of mRNA transcripts in mammalian cells. Continuing studies of these viral RNAs will provide insights into both cellular and viral gene expression.

The expanding universe of noncoding RNAs.

The 71st Cold Spring Harbor Symposium on Quantitative Biology celebrated the numerous and expanding roles of regulatory RNAs in systems ranging from bacteria to mammals. It was clearly evident that noncoding RNAs are undergoing a renaissance, with reports of their involvement in nearly every cellular process. Previously known classes of longer noncoding RNAs were shown to function by every possible means-acting catalytically, sensing physiological states through adoption of complex secondary and tertiary structures, or using their primary sequences for recognition of target sites. The many recently discovered classes of small noncoding RNAs, generally less than 35 nucleotides in length, most often exert their effects by guiding regulatory complexes to targets via base-pairing. With the ability to analyze the RNA products of the genome in ever greater depth, it has become clear that the universe of noncoding RNAs may extend far beyond the boundaries we had previously imagined. Thus, as much as the Symposium highlighted exciting progress in the field, it also revealed how much farther we must go to understand fully the biological impact of noncoding RNAs.

Absence of CD26 expression on skin-homing CLA+ CD4+ T lymphocytes in peripheral blood is a highly sensitive marker for early diagnosis and therapeutic monitoring of patients with Sézary syndrome.

Patients with Sézary syndrome (SS) show clonal expansion in the peripheral blood of skin-homing CD4+ T-helper cells expressing cutaneous lymphocyte antigen (CLA). However, an increase of CLA+ CD4+ T cells can also be observed in various inflammatory dermatoses. To facilitate early diagnosis and therapeutic monitoring of SS using flow cytometry, we evaluated the expression of CD7 and CD26 on the CLA+ CD4+ lymphocyte subset. Peripheral lymphocytes from 7 patients with SS, 16 patients with mycosis fungoides (MF) and 11 healthy controls were analysed by flow cytometry for the expression of CD4, CD7, CD26, CLA and CCR4. In addition, a longitudinal study was performed over 16 months in two patients with SS. Absence of CD7 and CD26 on CLA+ CD4+ T cells was highly specific for SS. Importantly, the absence of CD26 on CLA+ CD4+ T cells was very sensitive for SS, at 100% in our patient cohort. The number of CD26- CLA+ CD4+ T cells closely correlated with therapeutic interventions in the longitudinal analysis of two patients over more than 1 year. We conclude that the absence of CD26 expression on skin-homing CLA+ CD4+ T-helper cells is a very sensitive and highly specific parameter for early diagnosis and therapeutic monitoring of patients with SS.

Transcriptional targeting of dendritic cells for gene therapy using the promoter of the cytoskeletal protein fascin.

Strong cell-type-specific promoters are basic tools in gene therapy allowing for novel applications and focused strategies by transcriptionally targeting gene expression to selected cells. In immunotherapy, dendritic cells (DC) are of central importance, since they represent the principal inducers of immune responses. Here we describe isolation and use of the promoter of the murine actin-bundling protein fascin to target transcriptionally gene expression to cutaneous DC. Using the reporter gene enhanced green fluorescent protein (EGFP), we demonstrate that the fascin promoter mediates a strong antigen expression that is restricted to mature DC. DNA vaccination with antigen-encoding expression vectors under control of the fascin promoter using a gene gun resulted, consistently, in limited antigen expression by few directly transfected DC. Nevertheless, nearly as many antigen-specific CD8+ T cells directed against the encoded antigens EGFP and beta-galactosidase, respectively, were induced as with expression constructs under control of the ubiquitously expressed CMV promoter. This result impressively underlines the pivotal role of directly transfected DC in DNA vaccination. Immunization using the fascin promoter induced markedly lower levels of antigen-specific antibodies following single or repeated immunization. Thus, our DC-targeted DNA vaccination approach induces qualitatively distinct, predominantly cellular immune responses and provides new opportunities for immunotherapy.

Efficacy of recombinant adenovirus as vector for allergen gene therapy in a mouse model of type I allergy.

DNA-based immunization represents an attractive alternative approach to the current treatment of allergic diseases by specific immunotherapy with allergen extracts. In this study, we used a replication-deficient adenovirus vector (AdCMV), to examine the in vivo efficacy of preventive and therapeutic genetic immunization in a mouse model of type I allergy. Primary immunization with a recombinant adenovirus expressing the model antigen beta-galactosidase (AdCMV-(beta)gal) induced a Th1 immune response (predominance of IgG2a antibodies, high frequency of IFN-gamma producing T cells) and large numbers of cytotoxic T lymphocytes. Prophylactic vaccination with AdCMV-(beta)gal abolished the production of specific IgE following subsequent immunization with (beta)gal-protein, and skewed the Th2-biased immune response to a Th1-orientated response. In contrast, therapeutic administration of AdCMV-(beta)gal after priming with (beta)gal-protein neither significantly inhibited ongoing IgE production nor modulated a manifest Th2 immune response. Thus, allergen gene transfer via recombinant adenovirus represents an effective method to establish protection against the development of allergic disorders, but does not qualify as a therapeutic tool to interfere with ongoing high IgE production.

Genetic immunization with a melanocytic self-antigen linked to foreign helper sequences breaks tolerance and induces autoimmunity and tumor immunity.

Mechanisms maintaining peripheral tolerance to self-antigens present a major obstacle for the development of antigen-specific melanoma vaccines, presumably because self-antigens are not able to stimulate a CD4 T-helper response. Using the melanosomal enzyme tyrosinase-related protein 2 (TRP2) expressed by melanocytes and most melanoma cells as a model self-antigen, we investigated whether linkage with a foreign immunogenic protein providing strong CD4 helper sequences would be able to circumvent tolerance and enhance the induction of antigen-specific tumor immunity. We found that genetic immunization of mice with cDNA encoding a fusion protein between enhanced green fluorescent protein (EGFP) from jellyfish and autologous murine TRP2 (EGFP.mTRP2) resulted in the stimulation of TRP2-reactive T cells in vivo. Importantly, immunization with EGFP.mTRP2 effectively protected mice against metastatic growth of B16 melanoma in the lungs and was associated with fur depigmentation as a sign of autoimmune-mediated destruction of melanocytes. Our results show that tumor vaccines consisting of self-antigens linked to immunogenic helper sequences can be successfully applied to the immunotherapy of melanoma and provide a scientific basis for the translation of this strategy in future clinical investigations.

Delineation of mRNA export pathways by the use of cell-permeable peptides.

The transport of messenger RNAs (mRNAs) from the nucleus to the cytoplasm involves adapter proteins that bind the mRNA as well as receptor proteins that interact with the nuclear pore complex. We demonstrate the utility of cell-permeable peptides designed to interfere with interactions between potential adapter and receptor proteins to define the pathways accessed by particular mRNAs. We show that HuR, a protein implicated in the stabilization of short-lived mRNAs containing AU-rich elements (AREs), serves as an adapter for c-fos mRNA export through two pathways. One involves the HuR shuttling domain, HNS, which exhibits a heat shock-sensitive interaction with transportin 2 (Trn2); the other involves two protein ligands of HuR-pp32 and APRIL-which contain leucine-rich nuclear export signals (NES) recognized by the export receptor CRM1. Heterokaryon and in situ hybridization experiments reveal that the peptides selectively block the nucleocytoplasmic shuttling of their respective adapter proteins without perturbing the overall cellular distribution of polyadenylated mRNAs.

Depletion of CD25(+) CD4(+) T cells and treatment with tyrosinase-related protein 2-transduced dendritic cells enhance the interferon alpha-induced, CD8(+) T-cell-dependent immune defense of B16 melanoma.

Transduction of B16 melanoma cells with IFN alpha (B16-IFN alpha) enhances CD8(+) T-cell-dependent tumor immunity in mice, resulting in delayed outgrowth in vivo. Here we provide evidence that CD4(+) T cells down-regulate the IFN alpha-induced tumor immune defense. Importantly, depletion of regulatory CD25(+) CD4(+) T cells prevented growth of B16-IFN alpha in most mice and promoted long-lasting protective tumor immunity. Rejection of B16-IFN alpha could also be achieved with therapeutic injections of dendritic cells genetically engineered to express the melanoma antigen tyrosinase-related protein 2. These results support the development of novel strategies for the immunotherapy of melanoma using IFN alpha in combination with elimination of regulatory T cells or antigen-specific immunization.

Position within the host intron is critical for efficient processing of box C/D snoRNAs in mammalian cells.

In mammalian cells, all small nucleolar RNAs (snoRNAs) that guide rRNA modification are encoded within the introns of host genes. A database analysis of human box C/D snoRNAs revealed conservation of their intronic location, with a preference for 70-80 nt upstream of the 3' splice site. Transfection experiments showed that synthesis of gas5-encoded U75 and U76 snoRNAs dropped significantly for mutant constructs possessing longer or shorter spacers between the snoRNA and the 3' splice site. However, the position of the snoRNA did not affect splicing of the host intron. Substitution mutations within the spacer indicated that the length, but not the specific sequence, is important. A in vitro system that couples pre-mRNA splicing and processing of U75 has been developed. U75 synthesis in vitro depends on its box C and D sequences and requires an appropriate spacer length. Further mutational analyses both in vivo and in vitro, with subsequent mapping of the branch points, revealed that the critical distance is from the snoRNA coding region to the branch point, suggesting synergy between splicing and snoRNA release.

Communication of the position of exon-exon junctions to the mRNA surveillance machinery by the protein RNPS1.

In mammalian cells, splice junctions play a dual role in mRNA quality control: They mediate selective nuclear export of mature mRNA and they serve as a mark for mRNA surveillance, which subjects aberrant mRNAs with premature termination codons to nonsense-mediated decay (NMD). Here, we demonstrate that the protein RNPS1, a component of the postsplicing complex that is deposited 5' to exon-exon junctions, interacts with the evolutionarily conserved human Upf complex, a central component of NMD. Significantly, RNPS1 triggers NMD when tethered to the 3' untranslated region of beta-globin mRNA, demonstrating its role as a subunit of the postsplicing complex directly involved in mRNA surveillance.

Protein ligands mediate the CRM1-dependent export of HuR in response to heat shock.

AU-rich elements (AREs) located in the 3' UTRs of the messenger RNAs (mRNAs) of many mammalian early response genes promote rapid mRNA turnover. HuR, an RRM-containing RNA-binding protein, specifically interacts with AREs, stabilizing these mRNAs. HuR is primarily nucleoplasmic, but shuttles between the nucleus and the cytoplasm via a domain called HNS located between RRM2 and RRM3. We recently showed that HuR interacts with two protein ligands, pp32 and APRIL, which are also shuttling proteins, but rely on NES domains recognized by CRM1 for export. Here we show that heat shock induces increased association of HuR with pp32 and APRIL through protein-protein interactions and that these ligands partially colocalize with HuR in cytoplasmic foci. HuR associations with the hnRNP complex also increase, but through RNA links. CRM1 coimmunoprecipitates with HuR only after heat shock, and nuclear export of HuR becomes sensitive to leptomycin B, an inhibitor of CRM1. Export after heat shock requires the same domains of HuR (HNS and RRM3) that are essential for binding pp32 and APRIL. In situ hybridization and coimmunoprecipitation experiments show that LMB treatment blocks both hsp70 mRNA nuclear export and its cytoplasmic interaction with HuR after heat shock. Together, our results argue that upon heat shock, HuR switches its export pathway to that of its ligands pp32 and APRIL, which involves the nuclear export factor CRM1. HuR and its ligands may be instrumental in the nuclear export of heat-shock mRNAs.

Adenovirus-transduced dendritic cells stimulate cellular immunity to melanoma via a CD4(+) T cell-dependent mechanism.

We previously showed that genetic immunization of C57BL/6 mice with recombinant adenovirus encoding human TRP2 (Ad-hTRP2) was able to circumvent tolerance and induce cellular and humoral immune responses to murine TRP2 associated with protection against metastatic growth of B16 melanoma. In the present study we compared delivery of Ad-hTRP2 with cultured dendritic cells (DC) and direct injections of Ad-hTRP2. We show that application of Ad-hTRP2 with cultured DC enhanced protective immunity to B16 melanoma cells. Most importantly, delivery of recombinant adenovirus with DC alters the character of the immune response resulting in preferential stimulation of strong cellular immunity in the absence of significant humoral immunity to the encoded antigen. Adoptive transfer of lymphocytes from mice immunized with Ad-hTRP2-transduced DC confirmed that cellular components of the immune response were responsible for rejection of B16 melanoma. The protective efficacy of Ad-hTRP2-transduced DC clearly depended on the presence of CD4(+) T helper cells. Furthermore, AD-hTRP2-transduced DC, but not direct injection of Ad-hTRP2, were effective in the presence of neutralizing anti-adenoviral antibodies. These preclinical studies demonstrate the superiority of melanoma vaccines consisting of cultured DC transduced with recombinant adenoviruses encoding melanoma antigens.

Proximity of the invariant loop of U5 snRNA to the second intron residue during pre-mRNA splicing.

A photoactivatable azidophenacyl group has been introduced into seven positions in the backbone of the 11 nucleotide invariant loop of U5 snRNA. By reconstituting depleted splicing extracts with reassembled U5 snRNP particles, molecular neighbors were assessed as a function of splicing. All cross-links to the pre-mRNA mapped to the second nucleotide downstream of the 5' splice site, and formed most readily when the reactive group was at the phosphate between U5 positions 42 and 43 or 43 and 44. Both their kinetics of appearance and sensitivity to oligonucleotide inhibition suggest that these cross-links capture a late state in spliceosome assembly occurring immediately prior to the first step. A later forming, second cross-linked species is a splicing product of the first cross-link, suggesting that the U5 loop backbone maintains this position through the first step. The proximity of the U5 loop backbone to the intron's 5' end provides sufficient restrictions to develop a three-dimensional model for the arrangement of RNA components in the spliceosome during the first step of pre-mRNA splicing.