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Enfermedades de los Bovinos , Lactancia , Femenino , Bovinos , Animales , Granjas , Marcha , Dolor/veterinaria , Cojera Animal , Industria LecheraRESUMEN
BACKGROUND: The extent to which preventive hoof trimming is implemented on Great Britain's (GB) dairy farms is unknown. The aims of this study were to determine common practices and capture producers' input on key areas that require further research. METHODS: An online survey was conducted over a 4-week period from March to April 2017. RESULTS: A total of 338 valid survey responses were received. The majority of farmers undertook preventive hoof trimming (82.4%), and this was significantly positively associated with increased herd yield (p < 0.001). Drying off was the most common time trimming was undertaken, with 72.2% of farmers who implemented preventive trimming doing so at this point in the management cycle. Of those undertaking preventive trimming, 46.4% solely used an external hoof trimmer, 31.7% solely used farm staff and the remainder (21.9%) used a combination of operators. Four over-arching themes were identified within the key research questions; when to trim, why we should trim, how to trim and who to trim. The most frequent questions related to optimal trimming timing, frequency and method. CONCLUSION: This study highlighted that preventive hoof trimming is a widespread practice on GB dairy farms, undertaken by both external hoof trimmers and farm staff. Despite this, farmers still want to know when they should undertake preventive hoof trimming and which technique they should use.
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Enfermedades de los Bovinos , Enfermedades del Pie , Pezuñas y Garras , Animales , Bovinos , Enfermedades de los Bovinos/prevención & control , Industria Lechera/métodos , Agricultores , Enfermedades del Pie/veterinaria , HumanosRESUMEN
Human Vγ9Vδ2 T cells are a unique T-cell type, and data from recent studies of Vγ9Vδ2 T cells emphasize their potential relevance to cancer immunotherapy. Vγ9Vδ2 T cells exhibit dual properties since they are both antigen-presenting cells and cytotoxic toward cancer cells. The majority of Vγ9Vδ2 T cells are double-negative for the co-receptors CD4 and CD8, and only 20-30% express CD8. Even though they are mostly neglected, a small fraction of Vγ9Vδ2 T cells also express the co-receptor CD4. Here the authors show that CD4+ Vγ9Vδ2 T cells comprise 0.1-7% of peripheral blood Vγ9Vδ2 T cells. These cells can be expanded in vitro using zoledronic acid, pamidronic acid or CD3 antibodies combined with IL-2 and feeder cells. Unlike most conventional CD4+ αß T cells, CD4+ Vγ9Vδ2 T cells are potently cytotoxic and can kill cancer cells, which is here shown by the killing of cancer cell lines of different histological origins, including breast cancer, prostate cancer and melanoma cell lines, upon treatment with zoledronic acid. Notably, the killing capacity of CD4+ Vγ9Vδ2 T cells correlates with co-expression of CD56.
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Neoplasias , Linfocitos T , Células Presentadoras de Antígenos , Linfocitos T CD4-Positivos , Humanos , Activación de Linfocitos , Masculino , Neoplasias/terapia , Receptores de Antígenos de Linfocitos T gamma-delta , Ácido Zoledrónico/farmacologíaRESUMEN
Infusion of pituitary adenylate cyclase activating peptide-38 (PACAP-38) provokes migraine attacks in migraineurs and headache in non-migraineurs. Adverse events like long-lasting flushing and heat sensation can be terminated with oral antihistamine treatment, indicating the involvement of mast cell activation after PACAP-infusion. Degranulation of rat peritoneal mast cells was provoked by several isoforms of PACAP via previously unknown receptor pharmacology. The effect might thus be mediated either via specific splice variants of the PAC1-receptor or via an unknown receptor for PACAP-38. In the present study, we characterize degranulation of rat meningeal mast cells in response to PACAP-receptor ligands. Furthermore, we investigate if PACAP-38-induced mast cell degranulation is mediated via PAC1-receptor splice variants and/or via the orphan Mas-related G-protein coupled member B3 (MrgB3)-receptor. To address this, the pharmacological effect of different PACAP isoforms on meningeal mast cell degranulation was investigated in the hemisected skull model after toluidine blue staining followed by microscopic quantification. Presence of mRNA encoding PAC1-receptor splice variants and the MrgB3-receptor in rat mast cells was investigated by Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) analysis. The effect of PACAP isoforms on PAC1- and MrgB3-receptor-expressing Xenopus laevis oocytes were performed by two-electrode voltage-clamp (TEVC) electrophysiology. PACAP-38 is a more potent mast cell degranulating agent than Pituitary Adenylate Cyclase Activating Peptide-27 (PACAP-27) in the meninges. Presence of mRNA encoding the PAC1-receptor and its different splice variants could not be detected in peritoneal mast cells by RT-PCR, whereas the orphan MrgB3-receptor, recently suggested to be a mediator of basic secretagogues-induced mast cell degranulation, was widely present. In PAC1-receptor-expressing Xenopus laevis oocytes both PACAP-38, PACAP-27 and the specific PAC1-receptor agonist maxadilan were equipotent, however, only PACAP-38 showed a significant degranulatory effect on mast cells. We confirmed Pituitary Adenylate Cyclase Activating Peptide(6-38) [PACAP(6-38)] to be a PAC1-receptor antagonist, and we demonstrated that it is a potent mast cell degranulator and have an agonistic effect on MrgB3-receptors expressed in oocytes. The present study provides evidence that PACAP-induced mast cell degranulation in rat is mediated through a putative new PACAP-receptor with the order of potency being: PACAP-38 = PACAP(6-38) > > PACAP-27 = maxadilan. The results suggest that the observed responses are mediated via the orphan MrgB3-receptor.
RESUMEN
BACKGROUND: In migraineurs pituitary adenylate cyclase activating peptide1-38 (PACAP1-38) is a potent migraine provoking substance and the accompanying long lasting flushing suggests degranulation of mast cells. Infusion of the closely related vasoactive intestinal peptide (VIP) either induces headache or flushing. This implicates the pituitary adenylate cyclase activating peptide type I receptor (PAC1) to be involved in the pathophysiology of PACAP1-38 provoked headaches. Here we review studies characterizing the effects of mainly PACAP but also of VIP on cerebral and meningeal arteries and mast cells. DISCUSSION: PACAP1-38, PACAP1-27 and VIP dilate cerebral and meningeal arteries from several species including man. In rat cerebral and meningeal arteries the dilation seems to be mediated preferably via vasoactive intestinal peptide receptor type 1 (VPAC1) receptors while, in human, middle meningeal artery dilation induced via vasoactive intestinal peptide receptor type 2 (VPAC2) receptors cannot be ruled out. PACAP1-38 is a strong degranulator of peritoneal and dural mast cells while PACAP1-27 and VIP only have weak effects. More detailed characterization studies suggest that mast cell degranulation is not mediated via the known receptors for PACAP1-38 but rather via a still unknown receptor coupled to phospholipase C. CONCLUSION: It is suggested that PACAP1-38 might induce migraine via degranulation of dural mast cells via a yet unknown receptor.
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Degranulación de la Célula/fisiología , Mastocitos/metabolismo , Arterias Meníngeas/metabolismo , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/metabolismo , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria/metabolismo , Animales , Degranulación de la Célula/efectos de los fármacos , Humanos , Mastocitos/efectos de los fármacos , Arterias Meníngeas/efectos de los fármacos , Trastornos Migrañosos/metabolismo , Trastornos Migrañosos/fisiopatología , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/farmacología , Hipófisis/efectos de los fármacos , Hipófisis/metabolismo , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria/metabolismo , Receptores de Tipo II del Péptido Intestinal Vasoactivo/metabolismo , Receptores de Tipo I del Polipéptido Intestinal Vasoactivo/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Péptido Intestinal Vasoactivo/metabolismo , Péptido Intestinal Vasoactivo/farmacologíaRESUMEN
Background A common characteristic of migraine-inducing substances is that they cause headache and no pain in other areas of the body. Few studies have compared pain mechanisms in the trigeminal and spinal systems and, so far, no major differences have been noted. We compared signalling molecules in the trigeminal and spinothalamic system after infusion of the migraine-provoking substance glyceryltrinitrate. Method A catheter was placed in the femoral vein of rats and one week later glyceryltrinitrate 4 µg/kg/min was infused for 20 min. Protein expression in the dura mater, trigeminal ganglion, nucleus caudalis, dorsal root ganglion and the dorsal horn of the thoracic spinal cord was analysed at different time points using western blotting and immunohistochemistry. Results Glyceryltrinitrate caused a threefold increase in expression of phosphorylated extracellular signal-regulated kinases at 30 min in the dura mater and nucleus caudalis ( P < 0.05) and at 2 h in the trigeminal ganglion with very few expressions in the dorsal root ganglion. In the nucleus caudalis, expression of phosphorylated extracellular signal-regulated kinases and Cam KII increased 2.6-fold and 3.2-fold, respectively, at 2 h after glycerytrinitrate infusion ( P < 0.01). p-CREB/ATF-1 upregulation was observed only at 30 min ( P < 0.05) in the nucleus caudalis. None of these markers showed increased expression in the regions of thoracic spinal cord dorsal horn. Conclusion The dura, trigeminal ganglion and nucleus caudalis are activated shortly after glycerytrinitrate infusion with long-lasting expression of phosphorylated extracellular signal-regulated kinases observed in the nucleus caudalis. These activations were not observed at the spinal level.
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Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/biosíntesis , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/biosíntesis , Quinasas MAP Reguladas por Señal Extracelular/biosíntesis , Núcleo Caudal del Trigémino/efectos de los fármacos , Ganglio del Trigémino/efectos de los fármacos , Animales , Duramadre/efectos de los fármacos , Masculino , Trastornos Migrañosos/inducido químicamente , Trastornos Migrañosos/metabolismo , Trastornos Migrañosos/fisiopatología , Nitroglicerina/toxicidad , Ratas , Ratas Sprague-Dawley , Médula Espinal/efectos de los fármacos , Médula Espinal/metabolismo , Núcleo Caudal del Trigémino/metabolismo , Ganglio del Trigémino/metabolismo , Regulación hacia Arriba , Vasodilatadores/toxicidadRESUMEN
The trigeminal ganglia (TG) subserving the head and the dorsal root ganglia (DRG) subserving the rest of the body are homologous handling sensory neurons. Differences exist, as a number of signaling substances cause headache but no pain in the rest of the body. To date, very few genes involved in this difference have been identified. We aim to reveal basal gene expression levels in TG and DRG and detect genes that are differentially expressed (DE) between TG and DRG. RNA-Sequencing from six naïve rats describes the whole transcriptome expression profiles of TG and DRG. Differential expression analysis was followed by pathway analysis to identify DE processes between TG and DRG. In total, 64 genes had higher and 55 genes had lower expressed levels in TG than DRG. Higher expressed genes, including S1pr5 and Gjc2, have been related to phospholipase activity. The lower expressed genes, including several Hox genes and Slc5a7, have been related to tyrosine and phenylalanine metabolism. Tissue-specific expression was identified for Gabra6 and Gabrd in TG, and for several Hox genes in DRG. Furthermore, genes that were known to be associated with headache/migraine were mostly moderately to highly expressed in one or both tissues. We present a comprehensive overview of the expression profiles of whole tissue comparison of TG and DRG. Further, we showed DE genes/pathways between TG and DRG, including several known migraine-associated genes. This study provides a basis for further pain-related studies using TG and DRG in rats.
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Ganglios Espinales/metabolismo , Expresión Génica/fisiología , Células Receptoras Sensoriales/metabolismo , Transcriptoma/fisiología , Ganglio del Trigémino/metabolismo , Animales , Masculino , Ratas Wistar , Análisis de Secuencia de ARN/métodosRESUMEN
Recently, we showed that combined intranasal and subcutaneous immunization with a non-replicating adenoviral vector expressing NP of influenza A, strain PR8, induced long-standing protection against a range of influenza A viruses. However, H-2b mice challenged with an influenza A strain mutated in the dominant NP366 epitope were not efficiently protected. To address this problem, we envision the use of a cocktail of adenovectors targeting different internal proteins of influenza A virus. Consequently, we investigated the possibility of using PB1 as a target for an adenovector-based vaccine against influenza A. Our results showed that PB1 is not as immunogenic as the NP protein. However, by tethering PB1 to the murine invariant chain we were able to circumvent this problem and raise quite high numbers of PB1-specific CD8+ T cells in the circulation. Nevertheless, mice immunized against PB1 were not as efficiently protected against influenza A challenge as similarly NP-vaccinated animals. The reason for this is not a difference in the quality of the primed cells, nor in functional avidity. However, under similar conditions of immunization fewer PB1-specific cells were recruited to the airways, and surface expression of the dominant PB1 peptide, PB1703, was less stable than in the case of NP366.
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Antígenos de Diferenciación de Linfocitos B/inmunología , Linfocitos T CD8-positivos/metabolismo , Antígenos de Histocompatibilidad Clase II/inmunología , Virus de la Influenza A/inmunología , Vacunas contra la Influenza/administración & dosificación , Proteínas Virales/metabolismo , Animales , Antígenos de Diferenciación de Linfocitos B/genética , Dependovirus/genética , Dependovirus/inmunología , Femenino , Antígenos de Histocompatibilidad Clase II/genética , Humanos , Virus de la Influenza A/genética , Vacunas contra la Influenza/genética , Vacunas contra la Influenza/inmunología , Ratones , Mutación , Proteínas de la Nucleocápside , Proteínas de Unión al ARN/genética , Proteínas del Núcleo Viral/genéticaRESUMEN
INTRODUCTION: Infusion of glyceryl trinitrate (GTN), a donor of nitric oxide, induces immediate headache in humans that in migraineurs is followed by a delayed migraine attack. In order to achieve increased knowledge of mechanisms activated during GTN-infusion this present study aims to investigate transcriptional responses to GTN-infusion in the rat trigeminal ganglia. METHODS: Rats were infused with GTN or vehicle and trigeminal ganglia were isolated either 30 or 90 minutes post infusion. RNA sequencing was used to investigate transcriptomic changes in response to the treatment. Furthermore, we developed a novel method for Gene Set Analysis Of Variance (GSANOVA) to identify gene sets associated with transcriptional changes across time. RESULTS: 15 genes displayed significant changes in transcription levels in response to GTN-infusion. Ten of these genes showed either sustained up- or down-regulation in the 90-minute period after infusion. The GSANOVA analysis demonstrate enrichment of pathways pointing towards an increase in immune response, signal transduction, and neuroplasticity in response to GTN-infusion. Future functional in-depth studies of these mechanisms are expected to increase our understanding of migraine pathogenesis.
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Trastornos Migrañosos/inducido químicamente , Trastornos Migrañosos/genética , Nitroglicerina/efectos adversos , Ganglio del Trigémino/efectos de los fármacos , Ganglio del Trigémino/metabolismo , Vasodilatadores/efectos adversos , Animales , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Infusiones Intraventriculares , Masculino , Trastornos Migrañosos/metabolismo , Nitroglicerina/administración & dosificación , Ratas , Ratas Sprague-Dawley , Análisis de Secuencia de ARN , Vasodilatadores/administración & dosificaciónRESUMEN
The threat from unpredictable influenza virus pandemics necessitates the development of a new type of influenza vaccine. Since the internal proteins are highly conserved, induction of T cells targeting these antigens may provide the solution. Indeed, adenoviral (Ad) vectors expressing flu nucleoprotein have previously been found to induce short-term protection in mice. In this study we confirm that systemic (subcutaneous (s.c.) immunization rapidly induced heterosubtypic protection predominantly mediated by CD8 T cells, but within three months clinical protection completely disappeared. Local (intranasal (i.n.)) immunization elicited delayed, but more lasting protection despite relatively inefficient immunization. However, by far, the most robust protection was induced by simultaneous, combined (i.n. + s.c.) vaccination, and, notably, in this case clinical protection lasted at least 8 months without showing any evidence of fading. Interestingly, the superior ability of the latter group to resist reinfection correlated with a higher number of antigen-specific CD8 T cells in the spleen. Thus, detailed analysis of the underlying CD8 T cell responses highlights the importance of T cells already positioned in the lungs prior to challenge, but at the same time underscores an important back-up role for circulating antigen-specific cells with the capacity to expand and infiltrate the infected lungs.
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Linfocitos T CD8-positivos/inmunología , Inmunidad , Inmunización , Virus de la Influenza A/inmunología , Infecciones por Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/virología , Adenoviridae/metabolismo , Animales , Antígenos Virales/inmunología , Vías de Administración de Medicamentos , Femenino , Memoria Inmunológica , Vacunas contra la Influenza/inmunología , Ratones Endogámicos C57BL , Infecciones por Orthomyxoviridae/prevención & control , Fenotipo , Especificidad de la Especie , Factores de Tiempo , VacunaciónRESUMEN
The aim of the present study was to identify influenza A-derived peptides which bind to both HLA class I and -II molecules and by immunization lead to both HLA class I and class II restricted immune responses. Eight influenza A-derived 9-11mer peptides with simultaneous binding to both HLA-A*02:01 and HLA-DRB1*01:01 molecules were identified by bioinformatics and biochemical technology. Immunization of transgenic HLA-A*02:01/HLA-DRB1*01:01 mice with four of these double binding peptides gave rise to both HLA class I and class II restricted responses by CD8 and CD4 T cells, respectively, whereas four of the double binding peptides did result in HLA-A*02:01 restricted responses only. According to their cytokine profile, the CD4 T cell responses were of the Th2 type. In influenza infected mice, we were unable to detect natural processing in vivo of the double restricted peptides and in line with this, peptide vaccination did not decrease virus titres in the lungs of intranasally influenza challenged mice. Our data show that HLA class I and class II double binding peptides can be identified by bioinformatics and biochemical technology. By immunization, double binding peptides can give rise to both HLA class I and class I restricted responses, a quality which might be of potential interest for peptide-based vaccine development.
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Antígeno HLA-A2/inmunología , Cadenas HLA-DRB1/inmunología , Subtipo H1N1 del Virus de la Influenza A/inmunología , Oligopéptidos/inmunología , Secuencia de Aminoácidos , Animales , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Citocinas/inmunología , Citocinas/metabolismo , Ensayo de Immunospot Ligado a Enzimas , Epítopos de Linfocito T/inmunología , Antígeno HLA-A2/genética , Cadenas HLA-DRB1/genética , Humanos , Inmunización , Subtipo H1N1 del Virus de la Influenza A/fisiología , Vacunas contra la Influenza/inmunología , Ratones Noqueados , Ratones Transgénicos , Infecciones por Orthomyxoviridae/genética , Infecciones por Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/virología , Vacunas de Subunidad/inmunologíaRESUMEN
BACKGROUND: Migraine patients develop attacks several hours after intravenous infusion of glyceryl trinitrate. Due to the short half-life of nitric oxide, this delayed migraine cannot be caused by a direct action of nitric oxide derived from glyceryl trinitrate. The involvement of meningeal inflammation and dural mast cell degranulation is supported by the effectiveness of prednisolone on glyceryl trinitrate-induced delayed headache. METHODS: Using a newly developed rat model mimicking the human glyceryl trinitrate headache model, we have investigated the occurrence of dural mast cell degranulation after a clinically relevant dose of glyceryl trinitrate. RESULTS: A 6-fold increase in degranulation was observed starting at 2 hours after glyceryl trinitrate infusion. Interestingly, pre-treatment with the effective anti-migraine substances L-nitro-arginine methyl ester and sumatriptan prevented glyceryl trinitrate-induced mast cell degranulation whereas the calcitonin gene-related peptide-receptor antagonist olcegepant and the substance P receptor antagonist L-733,060 did not affect mast cell degranulation. However, topical application of two different nitric oxide donors did not cause mast cell degranulation ex vivo. CONCLUSIONS: Direct application of an exogenous nitric oxide donor on dural mast cells does not cause mast cell degranulation ex vivo. In vivo application of the nitric oxide donor glyceryl trinitrate leads to a prominent level of degranulation via a yet unknown mechanism. This effect can be completely blocked by inhibition of the endogenous nitric oxide production and by 5-HT1B/1D receptor agonists but is unaffected by calcitonin gene-related peptide and substance P receptor antagonists.
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Degranulación de la Célula/efectos de los fármacos , Mastocitos/efectos de los fármacos , Mastocitos/metabolismo , Donantes de Óxido Nítrico/toxicidad , Nitroglicerina/toxicidad , Animales , Degranulación de la Célula/fisiología , Cefalea/inducido químicamente , Cefalea/metabolismo , Masculino , Mastocitos/fisiología , Ratas , Ratas Sprague-DawleyRESUMEN
PROBLEM: The chemokine IL-8 recruits neutrophils to sites of infection, including the endometrium of the bovine uterus. However, quantification of bovine IL-8 often yields lower concentrations than for other species, which may reflect impaired innate immune responses by bovine cells or inaccurate measurement of IL-8 using the current human IL-8 ELISA method. METHOD OF STUDY: An ELISA was developed and validated for detection of bovine IL-8. Utility of the assay was tested by measuring the response of bovine endometrium and cells to bacteria and pathogen-associated molecular patterns. RESULTS: The developed ELISA detected 62.5-2000 pg/mL IL-8, with minimal cross-reactivity to other inflammatory mediators. Concentrations of bovine IL-8 were measured more accurately by the bovine than human IL-8 ELISA. Bovine endometrial IL-8 responses to pathogen-associated molecules were quantitatively similar to other species. CONCLUSION: A bovine-specific IL-8 ELISA was developed, which accurately measured IL-8 secretion from endometrial cells.
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Genitales Femeninos/inmunología , Genitales Femeninos/microbiología , Interleucina-8/inmunología , Infecciones del Sistema Genital/inmunología , Animales , Bovinos , Endometrio/inmunología , Endometrio/microbiología , Ensayo de Inmunoadsorción Enzimática/métodos , FemeninoRESUMEN
The strong UV chromophores thymine (Thy) and uracil (Ura) have identical heteroaromatic rings that only differ by one methyl substituent. While their photophysics has been elucidated in detail, the effect on the excited states of base protonation and single water molecules is less explored. Here we report gas-phase absorption spectra of ThyH(+) and UraH(+) and monohydrated ions and demonstrate that the substituent is not only responsible for spectral shifts but also influences the tautomer distribution, being different for bare and monohydrated ions. Spectra interpretation is aided by calculations of geometrical structures and transition energies. The lowest free-energy tautomer (denoted 178, enol-enol form) accounts for 230-280 nm (ThyH(+)) and 225-270 nm (UraH(+)) bands. ThyH(+) hardly absorbs above 300 nm, whereas a discernible band is measured for UraH(+) (275-320 nm), ascribed to the second lowest free-energy tautomer (138, enol-keto form) comprising a few percent of the UraH(+) population at room temperature. Band widths are similar to those measured of cold ions in support of very short excited-state lifetimes. Attachment of a single water increases the abundance of 138 relative to 178, 138 now clearly present for ThyH(+). 138 resembles more the tautomer present in aqueous solution than 178 does, and 138 may indeed be a relevant transition structure. The band of ThyH(+)(178) is unchanged, that of UraH(+)(178) is nearly unchanged, and that of UraH(+)(138) blue-shifts by about 10 nm. In stark contrast to protonated adenine, more than one solvating water molecule is required to re-establish the absorption of ThyH(+) and UraH(+) in aqueous solution.
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Gases/química , Protones , Timina/química , Uracilo/química , Agua/química , Adenina/química , Iones , Modelos Químicos , Transición de Fase , Teoría Cuántica , Espectrofotometría Ultravioleta , Estereoisomerismo , Temperatura , TermodinámicaRESUMEN
Skin cancer is the most common type of all cancers. However, it comprises several different types of cancers, one of which is malignant melanoma. Even though melanomas only make up about 5% of skin cancers, they are responsible for the majority of skin cancer deaths due to the poor chance of survival once the tumor has metastasized. In the present study, we have developed a new assay for quantitative analysis of B16 melanoma metastasis in the lungs. We have used a triplex Q-PCR to determine the expression of the melanoma genes GP100/Pmel and tyrosinase-related protein 2 (TRP-2), and found that B16.F10gp cells were detectable in the lungs as early as 2 hours after intravenous challenge with ≥ 10(4) tumor cells. When investigating the gene expression as a function of time, we observed a gradual decrease from 2-24 hours post tumor challenge followed by an increase of approximately 2 log10 on day 11. The early decrease was accelerated in the presence of activated NK cells. To further evaluate our assay, we also investigated the level of metastasis in the context of vaccination with replication defective adenoviral vectors, Ad-Ii-GP and Ad-GP, previously found to significantly delay the outgrowth of subcutaneous melanomas. Results obtained using Q-PCR were compared to conventional counting of metastatic foci under a dissection microscope. A marked reduction in gene expression was observed in the lungs after vaccination with both vectors; however, Ad-Ii-GP showed the highest protection, and matching results were obtained by enumeration of visible tumor nodules on the lung surfaces. Finally, we could show that inhibition of tumor metastasis required antigen-specific CD8 T cells and IFNγ, but not perforin. In conclusion, the presented results validate triplex Q-PCR as a fast, objective, and quantitative method for analysis of melanoma metastasis in the lungs.
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Regulación Neoplásica de la Expresión Génica , Oxidorreductasas Intramoleculares/biosíntesis , Neoplasias Pulmonares/metabolismo , Reacción en Cadena de la Polimerasa Multiplex/métodos , Antígeno gp100 del Melanoma/biosíntesis , Animales , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/patología , Femenino , Interferón gamma/inmunología , Interferón gamma/metabolismo , Oxidorreductasas Intramoleculares/inmunología , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/secundario , Melanoma , Ratones , Metástasis de la Neoplasia , Antígeno gp100 del Melanoma/inmunologíaRESUMEN
Microsolvation of chromophore ions commonly has large effects on their electronic structure and as a result on their optical absorption spectra. Here spectroscopy of protonated adenine (AdeH(+)) and its complex with one water molecule isolated in vacuo was done using a home-built mass spectrometer in combination with a tuneable pulsed laser system. Experiments also included the protonated adenosine 5'-monophosphate nucleotide (AMPH(+)). In the case of bare AdeH(+) ions, one-photon absorption leads to four dominant fragment ions corresponding to ammonium and ions formed after loss of either NH3, HCN, or NH2CN. The yields of these were measured as a function of the wavelength of the light from 210 nm to 300 nm, and they were combined to obtain the total photoinduced dissociation at each wavelength (i.e., action spectrum). A broad band between 230 nm and 290 nm and the tail of a band with maximum below 210 nm (high-energy band) are seen. In the case of AdeH(+)(H2O), the dominant dissociation channel after photoexcitation in the low-energy band was simply loss of H2O while photodissociation of protonated AMP revealed two dominant dissociation channels associated with the formation of either AdeH(+) or loss of H3PO4. The action spectra of AdeH(+), AdeH(+)(H2O), and AMPH(+) are almost identical in the 230-290 nm region, and they resemble the absorption spectrum of protonated adenine in aqueous solution recorded at low pH. Hence from our work it is firmly established that the lowest-energy transitions are independent of the surroundings.
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Adenina/química , Adenosina Monofosfato/química , Protones , Análisis Espectral , Agua/químicaRESUMEN
It is generally accepted that CD8 T cells play a major role in tumor control, yet vaccination aimed at eliciting potent CD8 T cell responses are rarely efficient in clinical trials. To try and understand why this is so, we have generated potent adenoviral vectors encoding the endogenous tumor Ags (TA) tyrosinase-related protein-2 (TRP-2) and glycoprotein 100 (GP100) tethered to the invariant chain (Ii). Using these vectors, we sought to characterize the self-TA-specific CD8 T cell response and compare it to that induced against non-self-Ags expressed from a similar vector platform. Prophylactic vaccination with adenoviral vectors expressing either TRP-2 (Ad-Ii-TRP-2) or GP100 (Ad-Ii-GP100) had little or no effect on the growth of s.c. B16 melanomas, and only Ad-Ii-TRP-2 was able to induce a marginal reduction of B16 lung metastasis. In contrast, vaccination with a similar vector construct expressing a foreign (viral) TA induced efficient tumor control. Analyzing the self-TA-specific CD8 T cells, we observed that these could be activated to produce IFN-γ and TNF-α. In addition, surface expression of phenotypic markers and inhibitory receptors, as well as in vivo cytotoxicity and degranulation capacity matched that of non-self-Ag-specific CD8 T cells. However, the CD8 T cells specific for self-TAs had a lower functional avidity, and this impacted on their in vivo performance. On the basis of these results and a low expression of the targeted TA epitopes on the tumor cells, we suggest that low avidity of the self-TA-specific CD8 T cells may represent a major obstacle for efficient immunotherapy of cancer.
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Antígenos de Neoplasias/inmunología , Linfocitos T CD8-positivos/inmunología , Vacunas contra el Cáncer/inmunología , Neoplasias/inmunología , Adenoviridae/genética , Animales , Autoantígenos/inmunología , Degranulación de la Célula/inmunología , Línea Celular Tumoral , Citotoxicidad Inmunológica , Epítopos de Linfocito T/inmunología , Epítopos de Linfocito T/metabolismo , Femenino , Vectores Genéticos/genética , Inmunoterapia , Interferón gamma/biosíntesis , Oxidorreductasas Intramoleculares/inmunología , Oxidorreductasas Intramoleculares/metabolismo , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/secundario , Neoplasias Pulmonares/terapia , Melanocitos/inmunología , Ratones , Neoplasias/terapia , Fenotipo , Unión Proteica/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T Reguladores/inmunología , Transducción Genética , Antígeno gp100 del Melanoma/inmunología , Antígeno gp100 del Melanoma/metabolismoRESUMEN
It has previously been found that combination therapy with anti-CTLA-4 and anti-4-1BB antibodies may enhance tumor immunity. However, this treatment is not efficient against all tumors, and it has been suggested that variations in tumor control may reflect differences in the immunogenicity of different tumors. In the present report, we have formally tested this hypothesis. Comparing the efficiency of combination antibody therapy against two antigenically distinct variants of the B16.F10 melanoma cell line, we observed that antibody therapy delayed the growth of a variant expressing an exogenous antigen (P<0.0001), while this treatment failed to protect against the non-transfected parental line (Pâ=â0.1850) consistent with published observations. As both cell lines are poorly immunogenic in wild type mice, these observations suggested that the magnitude of the tumor targeting T-cell repertoire plays a major role in deciding the efficiency of this antibody treatment. To directly test this assumption, we made use of mice expressing the exogenous antigen as a self-antigen and therefore carrying a severely purged T-cell repertoire directed against the major tumor antigen. Notably, combination therapy completely failed to inhibit tumor growth in the latter mice (Pâ=â0.8584). These results underscore the importance of a functionally intact T-cell population as a precondition for the efficiency of treatment with immunomodulatory antibodies. Clinically, the implication is that this type of antibody therapy should be attempted as an early form of tumor-specific immunotherapy before extensive exhaustion of the tumor-specific T-cell repertoire has occurred.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Antígeno CTLA-4/inmunología , Neoplasias/inmunología , Subgrupos de Linfocitos T/inmunología , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/inmunología , Animales , Anticuerpos Monoclonales/administración & dosificación , Antineoplásicos/administración & dosificación , Antígeno CTLA-4/genética , Terapia Combinada , Modelos Animales de Enfermedad , Inmunoterapia , Melanoma Experimental , Ratones , Neoplasias/mortalidad , Neoplasias/terapia , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/genéticaRESUMEN
The degree of electronic coupling between DNA bases is a topic being up for much debate. Here we report on the intrinsic electronic properties of isolated DNA strands in vacuo free of solvent, which is a good starting point for high-level excited states calculations. Action spectra of DNA single strands of adenine reveal sign of exciton coupling between stacked bases from blueshifted absorption bands (~3 nm) relative to that of the dAMP mononucleotide (one adenine base). The bands are blueshifted by about 10 nm compared to those of solvated strands, which is a shift similar to that for the adenine molecule and the dAMP mononucleotide. Desolvation has little effect on the bandwidth, which implies that inhomogenous broadening of the absorption bands in aqueous solution is of minor importance compared to, e.g., conformational disorder. Finally, at high photon energies, internal conversion competes with electron detachment since dissociation of the bare photoexcited ions on the microsecond time scale is measured.