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We explore the physical origin and the general validity of a propensity rule for the conservation of the hyperfine spin state in three-body recombination. This rule was recently discovered for the special case of ^{87}Rb with its nearly equal singlet and triplet scattering lengths. Here, we test the propensity rule for ^{85}Rb for which the scattering properties are very different from ^{87}Rb. The Rb_{2} molecular product distribution is mapped out in a state-to-state fashion using resonance-enhanced multiphoton ionization detection schemes which fully cover all possible molecular spin states. Interestingly, for the experimentally investigated range of binding energies from zero to â¼13 GHz×h we observe that the spin-conservation propensity rule also holds for ^{85}Rb. From these observations and a theoretical analysis we derive an understanding for the conservation of the hyperfine spin state. We identify several criteria to judge whether the propensity rule will also hold for other elements and collision channels.
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In systems with reduced dimensions, quantum fluctuations have a strong influence on the electronic conduction, even at very low temperatures. In superconductors, this is especially interesting, since the coherent state of the superconducting electrons strongly interacts with these fluctuations and therefore is a sensitive tool to study them. In this paper, we report on comprehensive measurements of superconducting nanowires in the quantum phase slip regime. Using an intrinsic electromigration process, we have developed a method to lower the nanowire's resistance in situ and therefore eliminate quantum phase slips in small consecutive steps. We observe critical (Coulomb) blockade voltages and superconducting critical currents, in good agreement with theoretical models. Between these two regimes, we find a continuous transition displaying a nonlinear metallic-like behavior. The reported intrinsic electromigration technique is not limited to low temperatures, as we find a similar change in resistance that spans over 3 orders of magnitude also at room temperature. Aside from superconducting quantum circuits, such a technique to reduce the resistance may also have applications in modern electronic circuits.
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Antibody therapy of solid cancers is well established, but suffers from unsatisfactory tumor penetration of large immunoglobulins or from low serum retention of antibody fragments. Oncolytic viruses are in advanced clinical development showing excellent safety, but suboptimal potency due to limited virus spread within tumors. Here, by developing an immunoRNase-encoding oncolytic adenovirus, we combine viral oncolysis with intratumoral genetic delivery of a small antibody-fusion protein for targeted bystander killing of tumor cells (viro-antibody therapy). Specifically, we explore genetic delivery of a small immunoRNase consisting of an EGFR-binding scFv antibody fragment fused to the RNase Onconase (ONC(EGFR)) that induces tumor cell death by RNA degradation after cellular internalization. Onconase is a frog RNase that combines lack of immunogenicity and excellent safety in patients with high tumor killing potency due to its resistance to the human cytosolic RNase inhibitor. We show that ONC(EGFR) expression by oncolytic adenoviruses is feasible with an optimized, replication-dependent gene expression strategy. Virus-encoded ONC(EGFR) induces potent and EGFR-dependent bystander killing of tumor cells. Importantly, the ONC(EGFR)-encoding oncolytic adenovirus showed dramatically increased cytotoxicity specifically to EGFR-positive tumor cells in vitro and significantly enhanced therapeutic activity in a mouse xenograft tumor model. The latter demonstrates that ONC(EGFR) is expressed at levels sufficient to trigger tumor cell killing in vivo. The established ONC(EGFR)-encoding oncolytic adenovirus represents a novel agent for treatment of EGFR-positive tumors. This viro-antibody therapy platform can be further developed for targeted/personalized cancer therapy by exploiting antibody diversity to target further established or emerging tumor markers or combinations thereof.
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Adenoviridae/genética , Antineoplásicos/administración & dosificación , Antineoplásicos/metabolismo , Vectores Genéticos/genética , Virus Oncolíticos/genética , Ribonucleasas/administración & dosificación , Ribonucleasas/metabolismo , Animales , Anticuerpos Antivirales , Línea Celular Tumoral , Receptores ErbB/metabolismo , Femenino , Terapia Genética/métodos , Humanos , Ratones , Ratones Endogámicos BALB C , Viroterapia Oncolítica/métodos , ARN/metabolismo , Replicación Viral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Oral vaccination with BCG provides protective systemic immunity against pathogenic mycobacterial challenge. In this study, the anatomical distribution of Mycobacterium bovis BCG following oral vaccination was investigated. Replicating bacteria in the Peyer's patches and mesenteric lymph nodes were present as solitary rods or clusters of two to three bacteria, the majority of which were isolated ex vivo as extracellular forms. Only a minority were shown to be associated with typical antigen-presenting cells. Acid-fast staining of mast cell granules in lymphoid tissues revealed a potential pitfall for these analyses and may explain previous reports of acid-fast 'coccoid' forms of mycobacteria in tissues.
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Adenoviruses (Ads), especially HAdV-5, have been genetically equipped with tumor-restricted replication potential to enable applications in oncolytic cancer therapy. Such oncolytic adenoviruses have been well tolerated in cancer patients, but their anti-tumor efficacy needs to be enhanced. In this regard, it should be considered that cancer cells, dependent on their tissue of origin, can differ substantially from the normal host cells to which Ads are adapted by complex virus-host interactions. Consequently, viral replication efficiency, a key determinant of oncolytic activity, might be suboptimal in cancer cells. Therefore, we have analyzed both the replication kinetics of HAdV-5 and the virus-induced transcriptome in human bronchial epithelial cells (HBEC) in comparison to cancer cells. This is the first report on genome-wide expression profiling of Ads in their native host cells. We found that E1A expression and onset of viral genome replication are most rapid in HBEC and considerably delayed in melanoma cells. In squamous cell lung carcinoma cells, we observed intermediate HAdV-5 replication kinetics. Infectious particle production, viral spread and lytic activity of HAdV-5 were attenuated in melanoma cells versus HBEC. Expression profiling at the onset of viral genome replication revealed that HAdV-5 induced the strongest changes in the cellular transcriptome in HBEC, followed by lung cancer and melanoma cells. We identified prominent regulation of genes involved in cell cycle and DNA metabolism, replication and packaging in HBEC, which is in accord with the necessity to induce S phase for viral replication. Strikingly, in melanoma cells HAdV-5 triggered opposing regulation of said genes and, in contrast to lung cancer cells, no weak S phase induction was detected when using the E2F promoter as reporter. Our results provide a rationale for improving oncolytic adenoviruses either by adaptation of viral infection to target tumor cells or by modulating tumor cell functions to better support viral replication.
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Adenovirus Humanos/genética , Adenovirus Humanos/fisiología , Neoplasias/virología , Virus Oncolíticos/genética , Virus Oncolíticos/fisiología , Transcriptoma/genética , Replicación Viral/genética , Infecciones por Adenovirus Humanos/virología , Bronquios/patología , Línea Celular Tumoral , Análisis por Conglomerados , Efecto Citopatogénico Viral , Regulación hacia Abajo/genética , Factor de Transcripción E2F1/genética , Células Epiteliales/metabolismo , Células Epiteliales/virología , Regulación Viral de la Expresión Génica , Genes Reporteros/genética , Genoma Viral/genética , Humanos , Anotación de Secuencia Molecular , Regiones Promotoras Genéticas/genética , Fase S , Regulación hacia Arriba/genéticaRESUMEN
Cancer-specificity is the key requirement for a drug or treatment regimen to be effective against malignant disease--and has rarely been achieved adequately to date. Therefore, targeting strategies need to be implemented for future therapies to ensure efficient activity at the site of patients' tumors or metastases without causing intolerable side-effects. Gene therapy and viral oncolysis represent treatment modalities that offer unique opportunities for tumor targeting. This is because both the transfer of genes with anti-cancer activity and viral replication-induced cell killing, respectively, facilitate the incorporation of multiple mechanisms restricting their activity to cancer. To this end, cellular mechanisms of gene regulation have been successfully exploited to direct therapeutic gene expression and viral cell lysis to cancer cells. Here, transcriptional targeting has been the role model and most widely investigated. This approach exploits cellular gene regulatory elements that mediate cell type-specific transcription to restrict the expression of therapeutic genes or essential viral genes, ideally to cancer cells. In this review, we first discuss the rationale for such promoter targeting and its limitations. We then give an overview how tissue-/tumor-specific promoters are being identified and characterized. Strategies to apply and optimize such promoters for the engineering of targeted viral gene transfer vectors and oncolytic viruses-with respect to promoter size, selectivity and activity in the context of viral genomes-are described. Finally, we discuss in more detail individual examples for transcriptionally targeted virus drugs. First highlighting oncolytic viruses targeted by prostate-specific promoters and by the telomerase promoter as representatives of tissue-targeted and pan-cancer-specific virus drugs respectively, and secondly recent developments of the last two years.
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Marcación de Gen , Terapia Genética/métodos , Neoplasias/genética , Neoplasias/terapia , Viroterapia Oncolítica/métodos , Animales , Humanos , Especificidad de Órganos , Regiones Promotoras Genéticas/genética , Virus/genéticaRESUMEN
The attachment, entry, and fusion of Kaposi's sarcoma-associated herpesvirus (KSHV) with target cells are mediated by complex machinery containing, among others, viral glycoprotein H (gH) and its alleged chaperone, gL. We observed that KSHV gH, in contrast to its homologues in several other herpesviruses, is transported to the cytoplasm membrane independently from gL, but not vice versa. Mutational analysis revealed that the N terminus of gH is sufficient for gL interaction. However, the entire extracellular part of gH is required for efficient gL secretion. The soluble ectodomain of gH was sufficient to interact with the surfaces of potential target cells in a heparin-dependent manner, and binding was further enhanced by coexpression of gL. Surface plasmon resonance revealed a remarkably high affinity of gH for glycosaminoglycans. Heparan sulfate (HS) proteoglycans of the syndecan family act as cellular receptors for the gH/gL complex. They promoted KSHV infection, and expression of gH/gL on target cells inhibited subsequent KSHV infection. Whereas gH alone was able to bind to HS, we observed that only the gH/gL complex adhered to heparan sulfate-negative cells at lamellipodium-like structures.
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Herpesvirus Humano 8/fisiología , Receptores Virales/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Proteínas Virales/metabolismo , Internalización del Virus , Línea Celular , Membrana Celular/química , Análisis Mutacional de ADN , Glicosaminoglicanos/metabolismo , Proteoglicanos de Heparán Sulfato/metabolismo , Humanos , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Mapeo de Interacción de Proteínas , Transporte de Proteínas , Resonancia por Plasmón de Superficie , Proteínas del Envoltorio Viral/genéticaRESUMEN
Oral vaccination of mice with lipid-encapsulated Mycobacterium bovis bacille Calmette-Guérin (BCG) expands a subset of interferon-gamma (IFN-gamma)-secreting T cells and mediates protection against aerosol mycobacterial challenge. We have traced the movement of the live vaccine through the regional lymphatics of mice and monitored the resultant immune response. Six hours after oral vaccination BCG was detected in low numbers systemically and in draining lymphatic tissue. However, after 48 h, BCG was predominantly associated with alimentary tract lymphatic tissues, such as the cervical and mesenteric lymph nodes and Peyer's patches. Lymphocytes that produced IFN-gamma in response to PPD-B or BCG-pulsed dendritic cells predominated in the spleen and were almost exclusively CD4(+), CD44(+) and CD62L(-), thus resembling an effector memory T cell population. Despite the fact that an oral route was used for immunization, splenic IFN-gamma-secreting T cells in vaccinated mice did not express the mucosal homing antigens alpha(4)beta(7) integrin or alphaIEL (CD103). However, a proportion of BCG-specific CD4(+) T cells expressed the CD29 integrin (beta(1)) chain, potentially involved in lung homing function. Thus, oral priming with M. bovis BCG appears to induce a subset of spleen-resident CD4(+) T cells with the potential to provide protective immunity in the lung.