RESUMEN
The additional author support information was erroneously omitted from the Supplementary Information. This has been corrected online.
RESUMEN
Patients with glioblastoma currently do not sufficiently benefit from recent breakthroughs in cancer treatment that use checkpoint inhibitors1,2. For treatments using checkpoint inhibitors to be successful, a high mutational load and responses to neoepitopes are thought to be essential3. There is limited intratumoural infiltration of immune cells4 in glioblastoma and these tumours contain only 30-50 non-synonymous mutations5. Exploitation of the full repertoire of tumour antigens-that is, both unmutated antigens and neoepitopes-may offer more effective immunotherapies, especially for tumours with a low mutational load. Here, in the phase I trial GAPVAC-101 of the Glioma Actively Personalized Vaccine Consortium (GAPVAC), we integrated highly individualized vaccinations with both types of tumour antigens into standard care to optimally exploit the limited target space for patients with newly diagnosed glioblastoma. Fifteen patients with glioblastomas positive for human leukocyte antigen (HLA)-A*02:01 or HLA-A*24:02 were treated with a vaccine (APVAC1) derived from a premanufactured library of unmutated antigens followed by treatment with APVAC2, which preferentially targeted neoepitopes. Personalization was based on mutations and analyses of the transcriptomes and immunopeptidomes of the individual tumours. The GAPVAC approach was feasible and vaccines that had poly-ICLC (polyriboinosinic-polyribocytidylic acid-poly-L-lysine carboxymethylcellulose) and granulocyte-macrophage colony-stimulating factor as adjuvants displayed favourable safety and strong immunogenicity. Unmutated APVAC1 antigens elicited sustained responses of central memory CD8+ T cells. APVAC2 induced predominantly CD4+ T cell responses of T helper 1 type against predicted neoepitopes.
Asunto(s)
Vacunas contra el Cáncer/inmunología , Vacunas contra el Cáncer/uso terapéutico , Glioblastoma/diagnóstico , Glioblastoma/terapia , Medicina de Precisión/métodos , Adulto , Anciano , Antígenos de Neoplasias/inmunología , Linfocitos T CD8-positivos/inmunología , Epítopos de Linfocito T/inmunología , Femenino , Glioblastoma/inmunología , Antígenos HLA-A/inmunología , Humanos , Memoria Inmunológica/inmunología , Masculino , Persona de Mediana Edad , Linfocitos T Colaboradores-Inductores/inmunología , Resultado del TratamientoRESUMEN
Knowledge about the peptide repertoire presented by human leukocyte antigens (HLA) holds the key to unlock target-specific cancer immunotherapies such as adoptive cell therapies or bispecific T cell engaging receptors. Therefore, comprehensive and accurate characterization of HLA peptidomes by mass spectrometry (immunopeptidomics) across tissues and disease states is essential. With growing numbers of immunopeptidomics datasets and the scope of peptide identification strategies reaching beyond the canonical proteome, the likelihood for erroneous peptide identification as well as false annotation of HLA-independent peptides as HLA ligands is increasing. Such "fake ligands" can lead to selection of nonexistent targets for immunotherapeutic development and need to be recognized as such as early as possible in the preclinical pipeline. Here we present computational and experimental methods that enable the identification of "fake ligands" that might be introduced at different steps of the immunopeptidomics workflow. The statistics presented herein allow discrimination of true HLA ligands from coisolated HLA-independent proteolytic fragments. In addition, we describe necessary steps to ensure system suitability of the chromatographic system. Furthermore, we illustrate an algorithm for detection of source fragmentation events that are introduced by electrospray ionization during mass spectrometry. For confirmation of peptide sequences, we present an experimental pipeline that enables high-throughput sequence verification through similarity of fragmentation pattern and coelution of synthetic isotope-labeled internal standards. Based on these methods, we show the overall high quality of existing datasets but point out limitations and pitfalls critical for individual peptides and how they can be uncovered in order to identify true ligands.
Asunto(s)
Antígenos HLA , Péptidos , Humanos , Ligandos , Proteolisis , Proteoma , ProteómicaRESUMEN
Cancer cells are subjected to constant selection by the immune system, meaning that tumors that become clinically manifest have managed to subvert or hide from immunosurveillance. Immune control can be facilitated by induction of autophagy, as well as by polyploidization of cancer cells. While autophagy causes the release of ATP, a chemotactic signal for myeloid cells, polyploidization can trigger endoplasmic reticulum stress with consequent exposure of the "eat-me" signal calreticulin on the cell surface, thereby facilitating the transfer of tumor antigens into dendritic cells. Hence, both autophagy and polyploidization cause the emission of adjuvant signals that ultimately elicit immune control by CD8+ T lymphocytes. We investigated the possibility that autophagy and polyploidization might also affect the antigenicity of cancer cells by altering the immunopeptidome. Mass spectrometry led to the identification of peptides that were presented on major histocompatibility complex (MHC) class I molecules in an autophagy-dependent fashion or that were specifically exposed on the surface of polyploid cells, yet lost upon passage of such cells through immunocompetent (but not immunodeficient) mice. However, the preferential recognition of autophagy-competent and polyploid cells by the innate and cellular immune systems did not correlate with the preferential recognition of such peptides in vivo. Moreover, vaccination with such peptides was unable to elicit tumor growth-inhibitory responses in vivo. We conclude that autophagy and polyploidy increase the immunogenicity of cancer cells mostly by affecting their adjuvanticity rather than their antigenicity.
Asunto(s)
Adyuvantes Inmunológicos , Antígenos de Neoplasias/inmunología , Muerte Celular , Vigilancia Inmunológica , Neoplasias/inmunología , Adenosina Trifosfato/metabolismo , Animales , Estrés del Retículo Endoplásmico , Humanos , Ratones , Monitorización Inmunológica , Transducción de SeñalRESUMEN
Human tumours typically harbour a remarkable number of somatic mutations. If presented on major histocompatibility complex class I molecules (MHCI), peptides containing these mutations could potentially be immunogenic as they should be recognized as 'non-self' neo-antigens by the adaptive immune system. Recent work has confirmed that mutant peptides can serve as T-cell epitopes. However, few mutant epitopes have been described because their discovery required the laborious screening of patient tumour-infiltrating lymphocytes for their ability to recognize antigen libraries constructed following tumour exome sequencing. We sought to simplify the discovery of immunogenic mutant peptides by characterizing their general properties. We developed an approach that combines whole-exome and transcriptome sequencing analysis with mass spectrometry to identify neo-epitopes in two widely used murine tumour models. Of the >1,300 amino acid changes identified, â¼13% were predicted to bind MHCI, a small fraction of which were confirmed by mass spectrometry. The peptides were then structurally modelled bound to MHCI. Mutations that were solvent-exposed and therefore accessible to T-cell antigen receptors were predicted to be immunogenic. Vaccination of mice confirmed the approach, with each predicted immunogenic peptide yielding therapeutically active T-cell responses. The predictions also enabled the generation of peptide-MHCI dextramers that could be used to monitor the kinetics and distribution of the anti-tumour T-cell response before and after vaccination. These findings indicate that a suitable prediction algorithm may provide an approach for the pharmacodynamic monitoring of T-cell responses as well as for the development of personalized vaccines in cancer patients.
Asunto(s)
Exoma/genética , Fenómenos Inmunogenéticos/genética , Espectrometría de Masas , Mutación , Neoplasias/genética , Animales , Linfocitos T CD8-positivos/inmunología , Vacunas contra el Cáncer/inmunología , Línea Celular Tumoral , Femenino , Perfilación de la Expresión Génica , Inmunidad Celular/inmunología , Inmunoprecipitación , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , Neoplasias/inmunología , Péptidos/genética , Estructura Terciaria de ProteínaRESUMEN
Immunotherapy is revolutionizing cancer treatment and has shown success in particular for tumors with a high mutational load. These effects have been linked to neoantigens derived from patient-specific mutations. To expand efficacious immunotherapy approaches to the vast majority of tumor types and patient populations carrying only a few mutations and maybe not a single presented neoepitope, it is necessary to expand the target space to non-mutated cancer-associated antigens. Mass spectrometry enables the direct and unbiased discovery and selection of tumor-specific human leukocyte antigen (HLA) peptides that can be used to define targets for immunotherapy. Combining these targets into a warehouse allows for multi-target therapy and accelerated clinical application. For precise personalization aimed at optimally ensuring treatment efficacy and safety, it is necessary to assess the presence of the target on each individual patient's tumor. Here we show how LC-MS paired with gene expression data was used to define mRNA biomarkers currently being used as diagnostic test IMADETECT™ for patient inclusion and personalized target selection within two clinical trials (NCT02876510, NCT03247309). Thus, we present a way how to translate HLA peptide presentation into gene expression thresholds for companion diagnostics in immunotherapy considering the peptide-specific correlation to its encoding mRNA.
Asunto(s)
Antígenos de Neoplasias/metabolismo , Antígenos HLA/metabolismo , Inmunoterapia , Neoplasias/metabolismo , Fragmentos de Péptidos/metabolismo , Medicina de Precisión , Proteogenómica/métodos , Presentación de Antígeno/inmunología , Antígenos de Neoplasias/análisis , Antígenos de Neoplasias/inmunología , Toma de Decisiones , Epítopos/inmunología , Epítopos/metabolismo , Antígenos HLA/análisis , Antígenos HLA/inmunología , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Espectrometría de Masas/métodos , Neoplasias/inmunología , Fragmentos de Péptidos/análisis , Fragmentos de Péptidos/inmunología , ARN Mensajero/análisis , ARN Mensajero/genética , ARN Mensajero/inmunologíaRESUMEN
BACKGROUND: In a phase 2 study in patients with metastatic renal cell carcinoma, overall survival was associated with T-cell responses against IMA901, a vaccine consisting of ten tumour-associated peptides. In this phase 3 trial, we aimed to determine the clinical effect of adding IMA901 to sunitinib, the standard first-line treatment in metastatic renal cell carcinoma with postulated favourable immunomodulatory effects. METHODS: The IMPRINT study is an open-label, randomised, controlled, phase 3 trial done at 124 clinical sites in 11 countries. HLA-A*02-positive patients (aged ≥18 years) with treatment-naive, histologically confirmed metastatic or locally advanced (or both) clear-cell renal cell carcinoma were randomly assigned (3:2) to receive sunitinib plus up to ten intradermal vaccinations of IMA901 (4·13 mg) and granulocyte macrophage colony-stimulating factor (75 µg), with one dose of cyclophosphamide (300 mg/m2) 3 days before the first vaccination, or to receive sunitinib alone. Sunitinib (50 mg) was given orally once daily, with each cycle defined as 4 weeks on treatment followed by 2 weeks off treatment, until progression of disease as determined by the investigator, death, or withdrawal of consent. Block randomisation (block size five) was done centrally using an interactive web response system, stratified by prognostic risk, geographical region, and previous nephrectomy. Patients and investigators were not masked to treatment allocation. The primary endpoint was overall survival from randomisation until death of any cause as determined by the investigator, analysed by intention to treat. This study is registered with ClinicalTrials.gov, number NCT01265901. FINDINGS: Between Dec 22, 2010, and Dec 15, 2012, we screened 1171 patients, of whom 339 were randomly assigned to receive sunitinib plus IMA901 (n=204) or sunitinib monotherapy (n=135). Patients had a median follow-up of 33·27 months (IQR 29·92-35·64). Median overall survival did not differ significantly between the groups (33·17 months [95% CI 27·81-41·36] in the sunitinib plus IMA901 group vs not reached [33·67-not reached] in the sunitinib monotherapy group; hazard ratio 1·34 [0·96-1·86]; p=0·087). 116 (57%) of 202 patients in the sunitinib plus IMA901 group and 62 (47%) of 132 in the sunitinib group had grade 3 or worse adverse events, the most common of which were hypertension, neutropenia, and anaemia in both groups, and mild-to-moderate transient injection-site reactions (eg, erythema, pruritus) were the most frequent IMA901-related side-effect in the sunitinib plus IMA901 group. Serious adverse events leading to death occurred in four (2%) patients (one respiratory failure and circulatory collapse [possibly related to sunitinib], one oesophageal varices haemorrhage [possibly related to sunitinib], one cardiac arrest [possibly related to sunitinib], and one myocardial infarction) and eight (6%) patients in the sunitinib group (one case each of renal failure, oesophageal varices haemorrhage, circulatory collapse, wound infection, ileus, cerebrovascular accident [possibly treatment related], and sepsis). INTERPRETATION: IMA901 did not improve overall survival when added to sunitinib as first-line treatment in patients with metastatic renal cell carcinoma. The magnitude of immune responses needs to be improved before further development of IMA901 in this disease is indicated. FUNDING: Immatics Biotechnologies.
Asunto(s)
Vacunas contra el Cáncer/administración & dosificación , Carcinoma de Células Renales/terapia , Indoles/uso terapéutico , Neoplasias Renales/terapia , Pirroles/uso terapéutico , Anciano , Vacunas contra el Cáncer/efectos adversos , Carcinoma de Células Renales/mortalidad , Carcinoma de Células Renales/patología , Femenino , Humanos , Indoles/administración & dosificación , Indoles/efectos adversos , Neoplasias Renales/mortalidad , Neoplasias Renales/patología , Masculino , Persona de Mediana Edad , Metástasis de la Neoplasia , Pirroles/administración & dosificación , Pirroles/efectos adversos , SunitinibRESUMEN
Peptides presented at the cell surface reflect the protein content of the cell; those on HLA class I molecules comprise the critical peptidome elements interacting with CD8 T lymphocytes. We hypothesize that peptidomes from ex vivo tumour samples encompass immunogenic tumour antigens. Here, we uncover >6000 HLA-bound peptides from HLA-A*02(+) glioblastoma, of which over 3000 were restricted by HLA-A*02. We prioritized in-depth investigation of 10 glioblastoma-associated antigens based on high expression in tumours, very low or absent expression in healthy tissues, implication in gliomagenesis and immunogenicity. Patients with glioblastoma showed no T cell tolerance to these peptides. Moreover, we demonstrated specific lysis of tumour cells by patients' CD8(+) T cells in vitro. In vivo, glioblastoma-specific CD8(+) T cells were present at the tumour site. Overall, our data show the physiological relevance of the peptidome approach and provide a critical advance for designing a rational glioblastoma immunotherapy. The peptides identified in our study are currently being tested as a multipeptide vaccine (IMA950) in patients with glioblastoma.
Asunto(s)
Antígenos de Neoplasias/inmunología , Neoplasias Encefálicas/inmunología , Glioblastoma/inmunología , Péptidos/inmunología , Presentación de Antígeno/fisiología , Antígenos CD/metabolismo , Antígenos de Neoplasias/química , Antígenos de Neoplasias/uso terapéutico , Neoplasias Encefálicas/patología , Neoplasias Encefálicas/terapia , Linfocitos T CD8-positivos/inmunología , Cromatografía Liquida , Citocinas/metabolismo , Citometría de Flujo , Perfilación de la Expresión Génica , Proteína Ácida Fibrilar de la Glía/metabolismo , Glioblastoma/patología , Glioblastoma/terapia , Antígenos HLA-A/análisis , Antígenos HLA-A/química , Antígenos HLA-A/inmunología , Humanos , Espectrometría de Masas , Análisis de Secuencia por Matrices de Oligonucleótidos , Péptidos/análisis , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , ARN Mensajero/metabolismo , Proteínas Tirosina Fosfatasas Clase 5 Similares a Receptores/metabolismo , Análisis de Secuencia de ProteínaRESUMEN
IMA101 is an actively personalized, multi-targeted adoptive cell therapy (ACT), whereby autologous T cells are directed against multiple novel defined peptide-HLA (pHLA) cancer targets. HLA-A*02:01-positive patients with relapsed/refractory solid tumors expressing ≥1 of 8 predefined targets underwent leukapheresis. Endogenous T cells specific for up to 4 targets were primed and expanded in vitro. Patients received lymphodepletion (fludarabine, cyclophosphamide), followed by T-cell infusion and low-dose IL2 (Cohort 1). Patients in Cohort 2 received atezolizumab for up to 1 year (NCT02876510). Overall, 214 patients were screened, 15 received lymphodepletion (13 women, 2 men; median age, 44 years), and 14 were treated with T-cell products. IMA101 treatment was feasible and well tolerated. The most common adverse events were cytokine release syndrome (Grade 1, n = 6; Grade 2, n = 4) and expected cytopenias. No patient died during the first 100 days after T-cell therapy. No neurotoxicity was observed. No objective responses were noted. Prolonged disease stabilization was noted in three patients lasting for 13.7, 12.9, and 7.3 months. High frequencies of target-specific T cells (up to 78.7% of CD8+ cells) were detected in the blood of treated patients, persisted for >1 year, and were detectable in posttreatment tumor tissue. Individual T-cell receptors (TCR) contained in T-cell products exhibited broad variation in TCR avidity, with the majority being low avidity. High-avidity TCRs were identified in some patients' products. This study demonstrates the feasibility and tolerability of an actively personalized ACT directed to multiple defined pHLA cancer targets. Results warrant further evaluation of multi-target ACT approaches using potent high-avidity TCRs. See related Spotlight by Uslu and June, p. 865.
Asunto(s)
Inmunoterapia Adoptiva , Neoplasias , Adulto , Femenino , Humanos , Masculino , Linfocitos T CD8-positivos , Estudios de Factibilidad , Inmunoterapia Adoptiva/efectos adversos , Inmunoterapia Adoptiva/métodos , Neoplasias/terapia , Neoplasias/etiología , Receptores de Antígenos de Linfocitos T/genéticaRESUMEN
T cell receptor (TCR)-based immunotherapy has emerged as a promising therapeutic approach for the treatment of patients with solid cancers. Identifying peptide-human leukocyte antigen (pHLA) complexes highly presented on tumors and rarely expressed on healthy tissue in combination with high-affinity TCRs that when introduced into T cells can redirect T cells to eliminate tumor but not healthy tissue is a key requirement for safe and efficacious TCR-based therapies. To discover promising shared tumor antigens that could be targeted via TCR-based adoptive T cell therapy, we employed population-scale immunopeptidomics using quantitative mass spectrometry across ~1500 tumor and normal tissue samples. We identified an HLA-A*02:01-restricted pan-cancer epitope within the collagen type VI α-3 (COL6A3) gene that is highly presented on tumor stroma across multiple solid cancers due to a tumor-specific alternative splicing event that rarely occurs outside the tumor microenvironment. T cells expressing natural COL6A3-specific TCRs demonstrated only modest activity against cells presenting high copy numbers of COL6A3 pHLAs. One of these TCRs was affinity-enhanced, enabling transduced T cells to specifically eliminate tumors in vivo that expressed similar copy numbers of pHLAs as primary tumor specimens. The enhanced TCR variants exhibited a favorable safety profile with no detectable off-target reactivity, paving the way to initiate clinical trials using COL6A3-specific TCRs to target an array of solid tumors.
Asunto(s)
Inmunoterapia Adoptiva , Receptores de Antígenos de Linfocitos T , Linfocitos T , Antígenos de Neoplasias , Línea Celular Tumoral , Tratamiento Basado en Trasplante de Células y Tejidos , Humanos , Inmunoterapia Adoptiva/métodos , Proteómica , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores de Antígenos de Linfocitos T/uso terapéuticoRESUMEN
OBJECTIVE: To evaluate the effect of UV-blocking soft contact lenses in treatment for chronic superficial keratitus (CSK). METHODS: Twenty six dogs with CSK were treated continuously with UV-blocking contact lenses for 6 months. A contact lens was placed on one eye of each dog; the other eye remained without a lens as a control eye. After this primary study, five of the dogs were further treated and they wore then contact lenses in both eyes. Continuously, all patients were concurrently treated topically with cyclosporine. The contact lenses were changed every 4 weeks and an ophthalmic examination performed. Evaluation criteria included corneal alterations as pigmentation, edema, pannus and vascularization. To determine the transmittance characteristics of the contact lenses before and after use, 32 contact lenses were measured with a UV-vis-NIR spectrophotometer. RESULTS: Pigmentation increased in eyes wearing lenses and in control eyes over the evaluation period of 6 months. Corneal edema increased in the eyes wearing lenses, but remained unaffected in the control eyes. A significant difference in the incidence of pannus and the extent of corneal vascularisation could not be evaluated. Adverse effects were noted in six cases (corneal edema and vascularisation, conjunctivitis, blepharospasm). All new lenses studied reduced UV-radiation to a safe level, whereas used lenses did not maintain their transmittance characteristics. CONCLUSIONS: No positive effect of UV-blocking contact lenses could be proven with the study design used.
Asunto(s)
Lentes de Contacto de Uso Prolongado/veterinaria , Enfermedades de los Perros/terapia , Queratitis/veterinaria , Rayos Ultravioleta , Animales , Enfermedad Crónica , Perros , Femenino , Queratitis/terapia , MasculinoRESUMEN
The goal of this study was the application of a novel, fully automatic column-switching approach in a metabonomics study combining the orthogonal selectivities of hydrophilic interaction chromatography (HILIC) and reversed-phase chromatography. The temporal, pharmacodynamic effects of the ginsenoside Rg3 on the metabonome in urine of healthy and liver-tumor-bearing rats have been investigated. Within a total analysis time of 52 min we detected 5686 polar, and on the second column an additional 1808 apolar, urinary metabolite ions. The administration of a single, high dose of Rg3 in a beta-cyclodextrin-based formulation led to a considerable change of the metabolic pattern in cancer rats during 3 days studied. Seventeen biomarker candidates including three apolar metabolites, which were not retained on the HILIC column, were detected. Overall, the results suggest that the developed liquid chromatography-mass spectrometry strategy is a promising tool in metabonomics studies for global analysis of highly complex biosamples. It may not only increase the number of discovered biomarkers but consequently improve the comprehensive information on metabolic changes in a fully automatic manner.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Ginsenósidos/orina , Glicómica/métodos , Interacciones Hidrofóbicas e Hidrofílicas , Neoplasias/metabolismo , Espectrometría de Masa por Ionización de Electrospray/métodos , beta-Ciclodextrinas/orina , Animales , Biomarcadores , Línea Celular Tumoral , Salud , Masculino , Estructura Molecular , Ratas , Ratas Sprague-DawleyRESUMEN
Sudden acquired retinal degeneration syndrome (SARDS) is a disease characterised by sudden and bilateral vision loss of dogs. Previous studies failed to identify the underlying cause [Mattson, A., Roberts, S.M., Isherwood, J.M.E., 1992. Clinical features suggesting hyperadrenocorticism associated with sudden acquired retinal degeneration syndrome in a dog. J. Am. Anim. Hosp. Assoc. 28, 199-202; Van der Woerdt, A., Nasisse, M.P., Davidson, M.G., 1991. Sudden acquired retinal degeneration in the dog: clinical and laboratory findings in 36 cases. Prog. Vet. Comp. Ophthamol. 1, 11-18] and earlier investigations about the occurrence of anti-retinal antibodies in SARDS patients showed inconsistent results. To provide a novel approach to those findings we designed a more detailed study. Autoantibodies of SARDS patients and normal controls were tested against the purified autoantigens S-antigen and cellular retinaldehyde binding protein (CRALBP) that play a role in human autoimmune uveitis. Next we tested the autoantibody binding pattern to whole retinal lysate. No difference in the incidence of autoantibodies could be found between SARDS patients and healthy controls while testing the well-known autoantigens S-antigen and CRALBP. Potential novel, yet unknown autoantigens were identified by a screening test using the retinal proteome as an autoantigenic source. In SARDS patients and normal controls, several retinal proteins were bound by IgG antibodies, but one band was strongly marked by SARDS patients. That band was excised, subjected to mass spectrometry (matrix-assisted laser desorption/ionisation-time of flight (MALDI-TOF/TOF)) and identified as neuron-specific enolase. Binding of the IgG autoantibodies of SARDS-affected dogs to this protein was verified using purified NSE, revealing 25% of NSE autoantibody-positive SARDS patients and 0% of negative controls. Our findings indicate that at least some dogs with SARDS have autoantibodies against NSE, although it is unclear whether these play a causative role in SARDS or whether they are the result of retinal destruction by another mechanism.
Asunto(s)
Arrestina/inmunología , Proteínas Portadoras/inmunología , Enfermedades de los Perros/inmunología , Fosfopiruvato Hidratasa/inmunología , Degeneración Retiniana/veterinaria , Animales , Arrestina/sangre , Autoantígenos/sangre , Western Blotting/veterinaria , Proteínas Portadoras/sangre , Enfermedades de los Perros/enzimología , Perros , Femenino , Masculino , Fosfopiruvato Hidratasa/sangre , Degeneración Retiniana/enzimología , Degeneración Retiniana/inmunología , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/veterinariaRESUMEN
OBJECTIVE: To determine the density of the canine and feline corneal neural network in healthy dogs and cats using in vivo confocal microscopy (IVCM). ANIMALS EXAMINED: A total of 16 adult dogs (9 Mesocephalic breeds, 7 Brachycephalic breeds) and 15 cats (9 Domestic Short-haired cats (DSH), 6 Persian cats) underwent IVCM. PROCEDURE: Animals were examined with a confocal corneal microscope (HRTII/RCM; Heidelberg Retina Tomograph II/Rostock Cornea Module, Heidelberg Engineering, Dossenheim, Germany). The investigations focused on the distribution of the corneal nerves and quantification of central subepithelial and subbasal nerve plexus. RESULTS: The corneal stromal nerve trunks, subepithelial and subbasal nerve plexus were observed. The nerve fiber density (NFD) quantified in nerve fiber length in mesocephalic dogs were 12.39 +/- 5.25 mm/mm(2) in the subepithelial nerve plexus and 14.87 +/- 3.08 mm/mm(2) in the subbasal nerve plexus. The NFD of the subepithelial nerve plexus in DSH cats was 15.49 +/- 2.7 and 18.4 +/- 3.84 mm/mm(2) in the subbasal nerve plexus. The subbasal NFD of DSH cats was significantly higher than in mesocephalic dogs (P = 0.037). The subepithelial NFD in brachycephalic dogs, and Persian cats were 10.34 +/- 4.71 and 9.50 +/- 2.3 mm/mm(2), respectively. The subbasal NFD measured 11.80 +/- 3.73 mm/mm(2) in brachycephalic dogs, and 12.28 +/- 4.3 mm/mm(2) NFD in Persian cats, respectively. The subepithelial and subbasal NFD in Persian cats were significantly lower than in DSH cats (P = 0.028, respectively, P = 0.031), in contrast to brachycephalic vs. mesocephalic dogs. CONCLUSION: The noninvasive IVCM accurately detects corneal innervation and provides a reliable quantification of central corneal nerves.
Asunto(s)
Gatos/anatomía & histología , Córnea/anatomía & histología , Córnea/inervación , Perros/anatomía & histología , Microscopía Confocal/veterinaria , Animales , Cruzamiento , Femenino , Masculino , Valores de Referencia , Especificidad de la EspecieRESUMEN
In addition to genomic mutations, RNA editing is another major mechanism creating sequence variations in proteins by introducing nucleotide changes in mRNA sequences. Deregulated RNA editing contributes to different types of human diseases, including cancers. Here we report that peptides generated as a consequence of RNA editing are indeed naturally presented by human leukocyte antigen (HLA) molecules. We provide evidence that effector CD8+ T cells specific for edited peptides derived from cyclin I are present in human tumours and attack tumour cells that are presenting these epitopes. We show that subpopulations of cancer patients have increased peptide levels and that levels of edited RNA correlate with peptide copy numbers. These findings demonstrate that RNA editing extends the classes of HLA presented self-antigens and that these antigens can be recognised by the immune system.
Asunto(s)
Antígenos de Neoplasias/inmunología , Epítopos/inmunología , Sistema Inmunológico/inmunología , Neoplasias/inmunología , Edición de ARN/inmunología , Presentación de Antígeno/inmunología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Línea Celular Tumoral , Células Cultivadas , Ciclina I/genética , Ciclina I/inmunología , Ciclina I/metabolismo , Citotoxicidad Inmunológica/inmunología , Antígenos HLA/inmunología , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Péptidos/genética , Péptidos/inmunología , Péptidos/metabolismo , Proteogenómica/métodosRESUMEN
The adult mammalian CNS has a limited capacity for nerve regeneration and structural plasticity. The presence of glia-derived inhibitory factors myelin-associated glycoprotein (MAG) and Nogo-A have been suggested to provide a nonpermissive environment for elongating nerve fibers. In particular, Nogo-A, an integral membrane protein predominantly expressed by oligodendrocytes, has been demonstrated to impair neurite growth in vitro and in vivo. Structure function analysis revealed that Nogo-A protein contains at least two active domains, NiG and Nogo-66, with diverse effects on neurite outgrowth and cell spreading. We now provide evidence that these inhibitory domains mediate their effects via an antagonistic regulation of the small GTPases RhoA and Rac1, resulting in activation of RhoA and suppression of Rac1. By inactivating RhoA with C3 transferase or the downstream effector Rho-kinase ROCK with, the inhibitory effects of both Nogo-A fragments and MAG on neurite outgrowth and oligodendrocyte-mediated growth cone collapse were abolished. Furthermore, we show that the recently cloned receptor for Nogo-66 and MAG, NgR, is not necessary for either NiG- or MAG-induced RhoA activation.
Asunto(s)
Proteínas de la Mielina/metabolismo , Glicoproteína Asociada a Mielina/metabolismo , Neuritas/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Amidas/farmacología , Animales , Células CHO , Comunicación Celular/efectos de los fármacos , Comunicación Celular/fisiología , Diferenciación Celular/fisiología , Células Cultivadas , Técnicas de Cocultivo , Cricetinae , Inhibidores Enzimáticos/farmacología , Proteínas Ligadas a GPI , Regulación de la Expresión Génica/fisiología , Conos de Crecimiento/efectos de los fármacos , Conos de Crecimiento/fisiología , Humanos , Péptidos y Proteínas de Señalización Intracelular , Riñón/citología , Riñón/metabolismo , Microscopía por Video , Proteínas de la Mielina/química , Proteínas de la Mielina/genética , Proteínas de la Mielina/farmacología , Glicoproteína Asociada a Mielina/farmacología , Neuritas/efectos de los fármacos , Neuronas/citología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Proteínas Nogo , Receptor Nogo 1 , Oligodendroglía/citología , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Estructura Terciaria de Proteína/fisiología , Piridinas/farmacología , Ratas , Receptores de Superficie Celular/metabolismo , Quinasas Asociadas a rho , Proteína de Unión al GTP rhoA/antagonistas & inhibidoresRESUMEN
IMA901 is the first therapeutic vaccine for renal cell cancer (RCC) consisting of multiple tumor-associated peptides (TUMAPs) confirmed to be naturally presented in human cancer tissue. We treated a total of 96 human leukocyte antigen A (HLA-A)*02(+) subjects with advanced RCC with IMA901 in two consecutive studies. In the phase 1 study, the T cell responses of the patients to multiple TUMAPs were associated with better disease control and lower numbers of prevaccine forkhead box P3 (FOXP3)(+) regulatory T (T(reg)) cells. The randomized phase 2 trial showed that a single dose of cyclophosphamide reduced the number of T(reg) cells and confirmed that immune responses to multiple TUMAPs were associated with longer overall survival. Furthermore, among six predefined populations of myeloid-derived suppressor cells, two were prognostic for overall survival, and among over 300 serum biomarkers, we identified apolipoprotein A-I (APOA1) and chemokine (C-C motif) ligand 17 (CCL17) as being predictive for both immune response to IMA901 and overall survival. A randomized phase 3 study to determine the clinical benefit of treatment with IMA901 is ongoing.
Asunto(s)
Vacunas contra el Cáncer/uso terapéutico , Carcinoma de Células Renales/terapia , Ciclofosfamida/uso terapéutico , Inmunosupresores/uso terapéutico , Inmunoterapia Activa , Neoplasias Renales/terapia , Linfocitos T Reguladores/inmunología , Vacunas de Subunidad/uso terapéutico , Antígenos de Neoplasias/inmunología , Apolipoproteína A-I/sangre , Biomarcadores , Linfocitos T CD8-positivos/inmunología , Carcinoma de Células Renales/sangre , Carcinoma de Células Renales/tratamiento farmacológico , Carcinoma de Células Renales/inmunología , Quimiocina CCL17/sangre , Terapia Combinada , Ciclofosfamida/administración & dosificación , Ciclofosfamida/farmacología , Femenino , Factor Estimulante de Colonias de Granulocitos y Macrófagos/uso terapéutico , Antígeno HLA-A2/inmunología , Humanos , Inmunosupresores/administración & dosificación , Inmunosupresores/farmacología , Estimación de Kaplan-Meier , Neoplasias Renales/sangre , Neoplasias Renales/tratamiento farmacológico , Neoplasias Renales/inmunología , Depleción Linfocítica , Masculino , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Pronóstico , Linfocitos T Reguladores/efectos de los fármacos , Resultado del TratamientoRESUMEN
Impaired glucose tolerance (IGT) which precedes overt type 2 diabetes (T2DM) for decades is associated with multiple metabolic alterations in insulin sensitive tissues. In an UPLC-qTOF-mass spectrometry-driven non-targeted metabonomics approach we investigated plasma as well as spot urine of 51 non-diabetic, overnight fasted individuals aiming to separate subjects with IGT from controls thereby identify pathways affected by the pre-diabetic metabolic state. We could clearly demonstrate that normal glucose tolerant (NGT) and IGT subjects clustered in two distinct groups independent of the investigated metabonome. These findings reflect considerable differences in individual metabolite fingerprints, both in plasma and urine. Pre-diabetes associated alterations in fatty acid-, tryptophan-, uric acid-, bile acid-, and lysophosphatidylcholine-metabolism, as well as the TCA cycle were identified. Of note, individuals with IGT also showed decreased levels of gut flora-associated metabolites namely hippuric acid, methylxanthine, methyluric acid, and 3-hydroxyhippuric acid. The findings of our non-targeted UPLC-qTOF-MS metabonomics analysis in plasma and spot urine of individuals with IGT vs NGT offers novel insights into the metabolic alterations occurring in the long, asymptomatic period preceding the manifestation of T2DM thereby giving prospects for new intervention targets. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11306-010-0203-1) contains supplementary material, which is available to authorized users.
RESUMEN
BACKGROUND: Exercise is an extreme physiological challenge for skeletal muscle energy metabolism and has notable health benefits. We aimed to identify and characterize metabolites, which are components of the regulatory network mediating the beneficial metabolic adaptation to exercise. METHODOLOGY AND PRINCIPAL FINDINGS: First, we investigated plasma from healthy human subjects who completed two independent running studies under moderate, predominantly aerobic conditions. Samples obtained prior to and immediately after running and then 3 and 24 h into the recovery phase were analyzed by a non-targeted (NT-) metabolomics approach applying liquid chromatography-qTOF-mass spectrometry. Under these conditions medium and long chain acylcarnitines were found to be the most discriminant plasma biomarkers of moderately intense exercise. Immediately after a 60 min (at 93% V(IAT)) or a 120 min run (at 70% V(IAT)) a pronounced, transient increase dominated by octanoyl-, decanoyl-, and dodecanoyl-carnitine was observed. The release of acylcarnitines as intermediates of partial beta-oxidation was verified in skeletal muscle cell culture experiments by probing (13)C-palmitate metabolism. Further investigations in primary human myotubes and mouse muscle tissue revealed that octanoyl-, decanoyl-, and dodecanoyl-carnitine were able to support the oxidation of palmitate, proving more effective than L-carnitine. CONCLUSIONS: Medium chain acylcarnitines were identified and characterized by a functional metabolomics approach as the dominating biomarkers during a moderately intense exercise bout possessing the power to support fat oxidation. This physiological production and efflux of acylcarnitines might exert beneficial biological functions in muscle tissue.
Asunto(s)
Carnitina/análogos & derivados , Ejercicio Físico/fisiología , Metabolismo de los Lípidos/fisiología , Músculo Esquelético/metabolismo , Adulto , Animales , Carnitina/sangre , Carnitina/química , Carnitina/metabolismo , Células Cultivadas , Cromatografía Líquida de Alta Presión , Humanos , Técnicas In Vitro , Espectroscopía de Resonancia Magnética , Masculino , Espectrometría de Masas , Ratones , Fibras Musculares Esqueléticas/metabolismo , Oxidación-Reducción , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The oral glucose tolerance test (oGTT) is a common tool to provoke a metabolic challenge for scientific purposes, as well as for diagnostic reasons, to monitor the kinetics of glucose and insulin. Here, we aimed to follow the variety of physiological changes of the whole metabolic pattern in plasma during an oGTT in healthy subjects in a nontargeted reversed-phase ultra performance liquid chromatography coupled to electrospray ionization quadrupole time of flight mass spectrometric metabolomics approach. We detected 11,500 metabolite ion masses/individual. Applying multivariate data analysis, four major groups of metabolites have been detected as the most discriminating oGTT biomarkers: free fatty acids (FFA), acylcarnitines, bile acids, and lysophosphatidylcholines. We found in detail 1) a strong decrease of all saturated and monounsaturated FFA studied during the oGTT; 2) a significant faster decline of palmitoleate (C16:1) and oleate (C18:1) FFA levels than their saturated counterparts; 3) a strong relative increase of polyunsaturated fatty acids in the fatty acid pattern at 120 min; and 4) a clear decrease in plasma C10:0, C12:0, and C14:1 acylcarnitine levels. These data reflect the switch from beta-oxidation to glycolysis and fat storage during the oGTT. Moreover, the bile acids glycocholic acid, glycochenodeoxycholic acid, and glycodeoxycholic acid were highly discriminative, showing a biphasic kinetic with a maximum of a 4.5- to 6-fold increase at 30 min after glucose ingestion, a significant decrease over the next 60 min followed by an increase until the end of the oGTT. Lysophosphatidylcholines were also increased significantly. The findings of our metabolomics study reveal detailed insights in the complex physiological regulation of the metabolism during an oGTT offering novel perspectives of this widely used procedure.