RESUMEN
The increasing prevalence of diabetes has resulted in a global epidemic1. Diabetes is a major cause of blindness, kidney failure, heart attacks, stroke and amputation of lower limbs. These are often caused by changes in blood vessels, such as the expansion of the basement membrane and a loss of vascular cells2-4. Diabetes also impairs the functions of endothelial cells5 and disturbs the communication between endothelial cells and pericytes6. How dysfunction of endothelial cells and/or pericytes leads to diabetic vasculopathy remains largely unknown. Here we report the development of self-organizing three-dimensional human blood vessel organoids from pluripotent stem cells. These human blood vessel organoids contain endothelial cells and pericytes that self-assemble into capillary networks that are enveloped by a basement membrane. Human blood vessel organoids transplanted into mice form a stable, perfused vascular tree, including arteries, arterioles and venules. Exposure of blood vessel organoids to hyperglycaemia and inflammatory cytokines in vitro induces thickening of the vascular basement membrane. Human blood vessels, exposed in vivo to a diabetic milieu in mice, also mimic the microvascular changes found in patients with diabetes. DLL4 and NOTCH3 were identified as key drivers of diabetic vasculopathy in human blood vessels. Therefore, organoids derived from human stem cells faithfully recapitulate the structure and function of human blood vessels and are amenable systems for modelling and identifying the regulators of diabetic vasculopathy, a disease that affects hundreds of millions of patients worldwide.
Asunto(s)
Membrana Basal/patología , Vasos Sanguíneos/patología , Angiopatías Diabéticas/patología , Modelos Biológicos , Organoides/patología , Organoides/trasplante , Proteínas Adaptadoras Transductoras de Señales , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Animales , Arterias/citología , Arterias/efectos de los fármacos , Arteriolas/citología , Arteriolas/efectos de los fármacos , Membrana Basal/citología , Membrana Basal/efectos de los fármacos , Vasos Sanguíneos/citología , Vasos Sanguíneos/efectos de los fármacos , Vasos Sanguíneos/crecimiento & desarrollo , Proteínas de Unión al Calcio , Angiopatías Diabéticas/enzimología , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Humanos , Hiperglucemia/complicaciones , Técnicas In Vitro , Mediadores de Inflamación/farmacología , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Ratones , Organoides/citología , Organoides/efectos de los fármacos , Pericitos/citología , Pericitos/efectos de los fármacos , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/efectos de los fármacos , Receptor Notch3/metabolismo , Transducción de Señal , Vénulas/citología , Vénulas/efectos de los fármacosRESUMEN
Prostate cancer (PCa) is a common and fatal type of cancer in men. Metastatic PCa (mPCa) is a major factor contributing to its lethality, although the mechanisms remain poorly understood. PTEN is one of the most frequently deleted genes in mPCa. Here we show a frequent genomic co-deletion of PTEN and STAT3 in liquid biopsies of patients with mPCa. Loss of Stat3 in a Pten-null mouse prostate model leads to a reduction of LKB1/pAMPK with simultaneous activation of mTOR/CREB, resulting in metastatic disease. However, constitutive activation of Stat3 led to high LKB1/pAMPK levels and suppressed mTORC1/CREB pathway, preventing mPCa development. Metformin, one of the most widely prescribed therapeutics against type 2 diabetes, inhibits mTORC1 in liver and requires LKB1 to mediate glucose homeostasis. We find that metformin treatment of STAT3/AR-expressing PCa xenografts resulted in significantly reduced tumor growth accompanied by diminished mTORC1/CREB, AR and PSA levels. PCa xenografts with deletion of STAT3/AR nearly completely abrogated mTORC1/CREB inhibition mediated by metformin. Moreover, metformin treatment of PCa patients with high Gleason grade and type 2 diabetes resulted in undetectable mTORC1 levels and upregulated STAT3 expression. Furthermore, PCa patients with high CREB expression have worse clinical outcomes and a significantly increased risk of PCa relapse and metastatic recurrence. In summary, we have shown that STAT3 controls mPCa via LKB1/pAMPK/mTORC1/CREB signaling, which we have identified as a promising novel downstream target for the treatment of lethal mPCa.
Asunto(s)
Diabetes Mellitus Tipo 2 , Metformina , Neoplasias de la Próstata , Animales , Humanos , Masculino , Ratones , Proteínas Quinasas Activadas por AMP/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Metformina/farmacología , Recurrencia Local de Neoplasia , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismoRESUMEN
Prostate cancer (PCa) has a broad spectrum of clinical behavior; hence, biomarkers are urgently needed for risk stratification. Here, we aim to find potential biomarkers for risk stratification, by utilizing a gene co-expression network of transcriptomics data in addition to laser-microdissected proteomics from human and murine prostate FFPE samples. We show up-regulation of oxidative phosphorylation (OXPHOS) in PCa on the transcriptomic level and up-regulation of the TCA cycle/OXPHOS on the proteomic level, which is inversely correlated to STAT3 expression. We hereby identify gene expression of pyruvate dehydrogenase kinase 4 (PDK4), a key regulator of the TCA cycle, as a promising independent prognostic marker in PCa. PDK4 predicts disease recurrence independent of diagnostic risk factors such as grading, staging, and PSA level. Therefore, low PDK4 is a promising marker for PCa with dismal prognosis.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Recurrencia Local de Neoplasia/genética , Neoplasias Experimentales/patología , Neoplasias de la Próstata/genética , Proteómica/métodos , Piruvato Deshidrogenasa Quinasa Acetil-Transferidora/genética , Factor de Transcripción STAT3/genética , Animales , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Captura por Microdisección con Láser , Masculino , Ratones , Clasificación del Tumor , Recurrencia Local de Neoplasia/metabolismo , Recurrencia Local de Neoplasia/patología , Neoplasias Experimentales/genética , Neoplasias Experimentales/metabolismo , Fosforilación Oxidativa , Pronóstico , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Piruvato Deshidrogenasa Quinasa Acetil-Transferidora/metabolismo , Factor de Transcripción STAT3/metabolismo , Biología de Sistemas , Adulto JovenRESUMEN
The expression of podoplanin, a small mucin-like protein, is upregulated in the invasive front of a number of human carcinomas. We have investigated podoplanin function in cultured human breast cancer cells, in a mouse model of pancreatic beta cell carcinogenesis, and in human cancer biopsies. Our results indicate that podoplanin promotes tumor cell invasion in vitro and in vivo. Notably, the expression and subcellular localization of epithelial markers are unaltered, and mesenchymal markers are not induced in invasive podoplanin-expressing tumor cells. Rather, podoplanin induces collective cell migration by filopodia formation via the downregulation of the activities of small Rho family GTPases. In conclusion, podoplanin induces an alternative pathway of tumor cell invasion in the absence of epithelial-mesenchymal transition (EMT).
Asunto(s)
Actinas/metabolismo , Citoesqueleto/metabolismo , Células Epiteliales/patología , Glicoproteínas de Membrana/metabolismo , Mesodermo/patología , Animales , Cadherinas/genética , Cadherinas/metabolismo , Línea Celular Tumoral , Movimiento Celular , Proteínas de Unión al ADN/metabolismo , Progresión de la Enfermedad , Regulación hacia Abajo , Células Epiteliales/metabolismo , Proteínas Activadoras de GTPasa/genética , Proteínas Activadoras de GTPasa/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Leucemia de Células B/metabolismo , Leucemia de Células B/patología , Mesodermo/metabolismo , Ratones , Ratones Transgénicos , Invasividad Neoplásica/genética , Invasividad Neoplásica/patología , Proteínas de Transporte de Catión Orgánico/metabolismo , Seudópodos/metabolismo , Factores de Transcripción/metabolismo , Proteína de Unión al GTP cdc42/metabolismo , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
Breast cancer (BC) is a major global health issue, affecting a significant proportion of the female population and contributing to high rates of mortality. One of the primary challenges in the treatment of BC is the disease's heterogeneity, which can lead to ineffective therapies and poor patient outcomes. Spatial proteomics, which involves the study of protein localization within cells, offers a promising approach for understanding the biological processes that contribute to cellular heterogeneity within BC tissue. To fully leverage the potential of spatial proteomics, it is critical to identify early diagnostic biomarkers and therapeutic targets, and to understand protein expression levels and modifications. The subcellular localization of proteins is a key factor in their physiological function, making the study of subcellular localization a major challenge in cell biology. Achieving high resolution at the cellular and subcellular level is essential for obtaining an accurate spatial distribution of proteins, which in turn can enable the application of proteomics in clinical research. In this review, we present a comparison of current methods of spatial proteomics in BC, including untargeted and targeted strategies. Untargeted strategies enable the detection and analysis of proteins and peptides without a predetermined molecular focus, whereas targeted strategies allow the investigation of a predefined set of proteins or peptides of interest, overcoming the limitations associated with the stochastic nature of untargeted proteomics. By directly comparing these methods, we aim to provide insights into their strengths and limitations and their potential applications in BC research.
RESUMEN
Systemic lupus erythematosus (SLE) is a complex autoimmune disease affecting various tissues. Involvement of B and T cells as well as increased cytokine levels have been associated with disease manifestation. Recently, we demonstrated that mice with epidermal loss of JunB (JunB(Deltaep)) develop a myeloproliferative syndrome (MPS) due to high levels of G-CSF which are secreted by JunB-deficient keratinocytes. In addition, we show that JunB(Deltaep) mice develop a SLE phenotype linked to increased epidermal interleukin 6 (IL-6) secretion. Intercrosses with IL-6-deficient mice could rescue the SLE phenotype. Furthermore, we show that JunB binds to the IL-6 promoter and transcriptionally suppresses IL-6. Facial skin biopsies of human SLE patients similarly revealed low JunB protein expression and high IL-6, activated Stat3, Socs-1, and Socs-3 levels within lupus lesions. Thus, keratinocyte-induced IL-6 secretion can cause SLE and systemic autoimmunity. Our results support trials to use alpha-IL-6 receptor antibody therapy for treatment of SLE.
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Epidermis/metabolismo , Regulación de la Expresión Génica/fisiología , Queratinocitos/metabolismo , Lupus Eritematoso Sistémico/patología , Proteínas Proto-Oncogénicas c-jun/deficiencia , Animales , Inmunoprecipitación de Cromatina , Cruzamientos Genéticos , Ensayo de Inmunoadsorción Enzimática , Técnica del Anticuerpo Fluorescente Directa , Factor Estimulante de Colonias de Granulocitos/metabolismo , Humanos , Interleucina-6/metabolismo , Luciferasas , Lupus Eritematoso Sistémico/metabolismo , Ratones , Ratones Transgénicos , Microscopía Electrónica , Proteínas Proto-Oncogénicas c-jun/metabolismoRESUMEN
Thyroid hormones are essential metabolic and developmental regulators that exert a huge variety of effects in different organs. Triiodothyronine (T3) and thyroxine (T4) are synthesized in the thyroid gland and constitute unique iodine-containing hormones that are constantly regulated by a homeostatic feedback mechanism. T3/T4 activity in cells is mainly determined by specific transporters, cytosolic binding proteins, deiodinases (DIOs), and nuclear receptors. Modulation of intracellular T3/T4 level contributes to the maintenance of this regulatory feedback. µ-Crystallin (CRYM) is an important intracellular high-affinity T3-binding protein that buffers the amount of T3 freely available in the cytosol, thereby controlling its action. In this review, we focus on the molecular and pathological properties of CRYM in thyroid hormone signaling, with emphasis on its critical role in malignancies.
Asunto(s)
Proteínas Portadoras , Cristalinas , Humanos , Proteínas Portadoras/metabolismo , Cristalinas mu , Triyodotironina/metabolismo , Tiroxina/metabolismo , Hormonas Tiroideas , Cristalinas/metabolismoRESUMEN
We aimed to identify novel markers for aggressive prostate cancer in a STAT3-low proteomics-derived dataset of mitochondrial proteins by immunohistochemical analysis and correlation with transcriptomic data and biochemical recurrence in a STAT3 independent PCa cohort. Formalin-fixed paraffin-embedded tissue (FFPE) sample selection for proteomic analysis and tissue-microarray (TMA) generation was conducted from a cohort of PCa patients. Retrospective data analysis was performed with the same cohort. 153 proteins differentially expressed between STAT3-low and STAT3-high samples were identified. Out of these, 46 proteins were associated with mitochondrial processes including oxidative phosphorylation (OXPHOS), and 45 proteins were upregulated, including NDUFS1/ATP5O. In a STAT3 independent PCa cohort, high expression of NDUFS1/ATP5O was confirmed by immunocytochemistry (IHC) and was significantly associated with earlier biochemical recurrence (BCR). mRNA expression levels for these two genes were significantly higher in intra-epithelial neoplasia and in PCa compared to benign prostate glands. NDUFS1/ATP5O levels are increased both at the mRNA and protein level in aggressive PCa. Our results provide evidence that NDUFS1/ATP5O could be used to identify high-risk PCa patients.
RESUMEN
BACKGROUND: Cross-reactive carbohydrate determinants (CCDs) are probably the most widely occurring IgE epitopes. Approximately one fifth of patients with allergy develop IgE antibodies against such glycans. However, they appear to be of low clinical significance. OBJECTIVE: We wanted to elucidate the reasons for this lack of clinical symptoms on contact with CCD allergens by determination of the binding affinities of patients' IgE and IgG antibodies. METHODS: IgE and IgG against CCDs were affinity-purified from sera of selected patients. The binding affinity to defined glyco-epitopes was measured by surface plasmon resonance. RESULTS: From a pool of CCD-positive sera, we isolated 0.1 and 25 microg CCD-specific IgE and IgG, respectively. The binding affinity of purified IgE antibodies to core alpha1,3-fucosylated glycans was in the 10(-10) mol/L range. The affinity was highest when both fucose and xylose were present, whereas xylosylation alone did not cause IgE binding. CCD-specific IgG exhibited a dissociation constant of approximately 10(-8) mol/L. IgG(4) amounted to only 20% of the CCD-specific IgG (as well as total IgG). CONCLUSION: Low binding affinity of anti-CCD IgE cannot be the reason for the observed clinical insignificance of IgE against plant/insect glycan epitopes. Notably, the affinity of IgG to CCDs is higher than that to protein allergens, and it may therefore function as blocking antibody.
Asunto(s)
Afinidad de Anticuerpos , Venenos de Abeja/química , Carbohidratos/inmunología , Glicoproteínas/inmunología , Inmunoglobulina E/inmunología , Inmunoglobulina G/inmunología , Plantas/química , Animales , Venenos de Abeja/inmunología , Reacciones Cruzadas , Glicoproteínas/metabolismo , Humanos , Inmunoglobulina E/metabolismo , Inmunoglobulina G/metabolismo , Plantas/inmunología , Polen/química , Polen/inmunología , Resonancia por Plasmón de SuperficieRESUMEN
BACKGROUND: Podoplanin is a membrane mucin that, among a series of tissues, is expressed on late osteoblasts and osteocytes. Since recent findings have focussed on podoplanin's potential role as a tumour progression factor, we aimed at identifying regulatory elements conferring PDPN promoter activity. Here, we characterized the molecular mechanism controlling basal PDPN transcription in human osteoblast-like MG63 versus Saos-2 cells. RESULTS: We cloned and sequenced 2056 nucleotides from the 5'-flanking region of the PDPN gene and a computational search revealed that the TATA and CAAT box-lacking promoter possesses features of a growth-related gene, such as a GC-rich 5' region and the presence of multiple putative Sp1, AP-4 and NF-1 sites. Reporter gene assays demonstrated a functional promoter in MG63 cells exhibiting 30-fold more activity than in Saos-2 cells. In vitro DNase I footprinting revealed eight protected regions flanked by DNaseI hypersensitive sites within the region bp -728 to -39 present in MG63, but not in Saos-2 cells. Among these regions, mutation and supershift electrophoretic mobility shift assays (EMSA) identified four Sp1/Sp3 binding sites and two binding sites for yet unknown transcription factors. Deletion studies demonstrated the functional importance of two Sp1/Sp3 sites for PDPN promoter activity. Overexpression of Sp1 and Sp3 independently increased the stimulatory effect of the promoter and podoplanin mRNA levels in MG63 and Saos-2 cells. In SL2 cells, Sp3 functioned as a repressor, while Sp1 and Sp3 acted positively synergistic. Weak PDPN promoter activity of Saos-2 cells correlated with low Sp1/Sp3 nuclear levels, which was confirmed by Sp1/Sp3 chromatin immunoprecipitations in vivo. Moreover, methylation-sensitive Southern blot analyses and bisulfite sequencing detected strong methylation of CpG sites upstream of bp -464 in MG63 cells, but hypomethylation of these sites in Saos-2 cells. Concomitantly, treatment with the DNA methyltransferase inhibitor 5-azaCdR in combination with trichostatin A (TSA) downregulated podoplanin mRNA levels in MG63 cells, and region-specific in vitro methylation of the distal promoter suggested that DNA methylation rather enhanced than hindered PDPN transcription in both cell types. CONCLUSION: These data establish that in human osteoblast-like MG63 cells, Sp1 and Sp3 stimulate basal PDPN transcription in a concerted, yet independent manner, whereas Saos-2 cells lack sufficient nuclear Sp protein amounts for transcriptional activation. Moreover, a highly methylated chromatin conformation of the distal promoter region confers cell-type specific podoplanin upregulation versus Saos-2 cells.
Asunto(s)
Metilación de ADN , Glicoproteínas de Membrana/fisiología , Osteoblastos/metabolismo , Factor de Transcripción Sp1/fisiología , Factor de Transcripción Sp3/fisiología , Activación Transcripcional , Línea Celular Tumoral , Ensayo de Cambio de Movilidad Electroforética , Humanos , Glicoproteínas de Membrana/biosíntesis , Osteosarcoma , Regiones Promotoras Genéticas/fisiología , ARN Mensajero/metabolismoRESUMEN
BACKGROUND: The role of anti-idiotypic antibodies in allergic disease is still poorly understood. According to Jerne, anti-idiotypic antibodies to IgE should represent internal images of an allergen. Our aim was to ultimately prove whether this hypothesis holds true in allergy. Here, we describe the selection of anti-idiotypic antibodies against Phl p 5a-specific IgE directly from the B-cell repertoire of a grass pollen allergic individual. METHODS: Taking Phleum pratense grass pollen allergen Phl p 5 as a model, we selected anti-idiotypic antibodies against allergen-specific IgE directly from the B-cell repertoire of an allergic individual. We screened a combinatorial phage display library of human monovalent antibody heavy and light chain fragments (Fabs) with anti-Phl p 5a-IgE to identify and characterize Fabs with anti-idiotypic specificity. RESULTS: Five different Fab clones with anti-idiotypic specificity for anti-Phl p 5a-IgE were identified. Their hypervariable regions revealed partial sequence homology with solvent accessible antigenic sites of Phl p 5a, which have been identified by our previous mimotope approach. Phagemid DNA derived from the phage clones was used to produce two soluble recombinant anti-idiotypic Fab clones in E. coli. As a proof of molecular mimicry, both Fabs induced anti-Phl p 5a-specific antibodies in immunized BALB/c mice. Molecular modeling of the heavy and light chain hypervariable loops of the anti-idiotypic Fabs illustrated structural similarity with dominant IgE epitopes of Phl p 5a. CONCLUSION: In this straightforward phage technology approach, antibodies with anti-idiotypic specificities could be isolated from a human allergic's repertoire. As predicted by the immune network hypothesis, their hypervariable domains mimic IgE epitopes like internal images and, more importantly, induce allergen-specific immune responses in the absence of the allergen.
Asunto(s)
Anticuerpos Antiidiotipos/inmunología , Epítopos/inmunología , Hipersensibilidad/inmunología , Fragmentos Fab de Inmunoglobulinas/inmunología , Imitación Molecular/inmunología , Proteínas de Plantas/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Antiidiotipos/química , Antígenos de Plantas/inmunología , Regiones Determinantes de Complementariedad/química , Regiones Determinantes de Complementariedad/inmunología , Humanos , Inmunoglobulina E/inmunología , Fragmentos Fab de Inmunoglobulinas/química , Ratones , Ratones Endogámicos BALB C , Modelos Moleculares , Datos de Secuencia Molecular , Biblioteca de Péptidos , Proteínas Recombinantes , Alineación de Secuencia , Análisis de Secuencia de ProteínaRESUMEN
Parvalbumin, the major fish allergen, is recognized by allergen-specific IgE of more than 90% of all fish-allergic patients. A detailed knowledge of allergenic structures is crucial for developing a vaccine inducing blocking antibodies specifically directed towards the IgE binding epitopes. In the present study we aimed to use the phage display technique to generate mimotopes, which mimic epitopes on parvalbumin. Parvalbumin-specific IgE was purified from sera of fish-allergic patients and used for screening of a constrained decamer phage library. After four rounds of biopanning using parvalbumin-specific IgE, five phage clones were selected which were specifically recognized by parvalbumin-specific IgE as well as IgG. DNA sequencing and peptide alignment revealed a high degree of sequence similarities between the mimotopes. Interestingly, on the surface of natural parvalbumin three regions could be defined by computational mimotope matching. In accordance, previously defined allergenic peptides of cod parvalbumin highlighted areas in close proximity or overlapping with the mimotope matching sites. From the presented data we conclude that our approach identified conformational epitopes of parvalbumin relevant for IgE and IgG binding. We suggest that these mimotopes are suitable candidates for an epitope-specific immunotherapy of fish-allergic patients.
Asunto(s)
Proteínas de Peces/química , Proteínas de Peces/inmunología , Peces/inmunología , Parvalbúminas/química , Parvalbúminas/inmunología , Alérgenos/química , Alérgenos/genética , Secuencia de Aminoácidos , Animales , Carpas/genética , Carpas/inmunología , Epítopos/química , Epítopos/genética , Proteínas de Peces/genética , Peces/genética , Hipersensibilidad a los Alimentos/inmunología , Humanos , Inmunoglobulina E/sangre , Inmunoglobulina G/sangre , Modelos Moleculares , Imitación Molecular , Datos de Secuencia Molecular , Parvalbúminas/genética , Biblioteca de Péptidos , Filogenia , Conformación Proteica , Homología de Secuencia de AminoácidoRESUMEN
Allergen-specific IgE and IgG antibodies coexist in allergic individuals, but only IgE has anaphylactogenic capacity. This study aimed to determine the association, dissociation and equilibrium constants for the interaction of allergen-specific IgE and IgG with the major grass and birch pollen allergens Phl p 5a and Bet v 1a. We isolated specific IgE and IgG antibodies from pollen allergic patients' sera by a two-step affinity chromatography protocol and controlled the high purity in a recombinant allergen chip microarray. Surface plasmon resonance measurements of polyclonal IgE and IgG species revealed that their affinities diverge widely, being in the range of 10(-10) and 10(-11) M for IgE, but only 10(-6)-10(-7) M for IgG. Moreover, murine monoclonal IgG1 antibodies against the allergens showed affinities of 10(-7)-10(-8) M. Thus, we conclude from our data that even stringently affinity matured IgG cannot score the superior affinity of IgE antibodies to allergens.
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Afinidad de Anticuerpos/inmunología , Inmunoglobulina E/inmunología , Inmunoglobulina G/inmunología , Proteínas de Plantas/inmunología , Animales , Inmunoglobulina G/aislamiento & purificación , Cinética , Ratones , Análisis de Secuencia por Matrices de OligonucleótidosRESUMEN
The identification of marker molecules specific for blood and lymphatic endothelium may provide new diagnostic tools and identify new targets for therapy of immune, microvascular and cancerous diseases. Here, we used a phage display library expressing human randomized single-chain Fv (scFv) antibodies for direct panning against live cultures of blood (BECs) and lymphatic (LECs) endothelial cells in solution. After six panning rounds, out of 944 sequenced antibody clones, we retrieved 166 unique/diverse scFv fragments, as indicated by the V-region sequences. Specificities of these phage clone antibodies for respective compartments were individually tested by direct cell ELISA, indicating that mainly pan-endothelial cell (EC) binders had been selected, but also revealing a subset of BEC-specific scFv antibodies. The specific staining pattern was recapitulated by twelve phage-independently expressed scFv antibodies. Binding capacity to BECs and LECs and differential staining of BEC versus LEC by a subset of eight scFv antibodies was confirmed by immunofluorescence staining. As one antigen, CD146 was identified by immunoprecipitation with phage-independent scFv fragment. This antibody, B6-11, specifically bound to recombinant CD146, and to native CD146 expressed by BECs, melanoma cells and blood vessels. Further, binding capacity of B6-11 to CD146 was fully retained after fusion to a mouse Fc portion, which enabled eukaryotic cell expression. Beyond visualization and diagnosis, this antibody might be used as a functional tool. Overall, our approach provided a method to select antibodies specific for endothelial surface determinants in their native configuration. We successfully selected antibodies that bind to antigens expressed on the human endothelial cell surfaces in situ, showing that BECs and LECs share a majority of surface antigens, which is complemented by cell-type specific, unique markers.
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Antígenos de Superficie/metabolismo , Células Sanguíneas/metabolismo , Células Endoteliales/metabolismo , Endotelio Linfático/citología , Biblioteca de Péptidos , Anticuerpos de Cadena Única/metabolismo , Animales , Especificidad de Anticuerpos/inmunología , Antígenos/metabolismo , Antígeno CD146/metabolismo , Línea Celular Transformada , Membrana Celular/metabolismo , Células Cultivadas , Cromatografía Liquida , Células Clonales , Biología Computacional , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Humanos , Masculino , Espectrometría de Masas , Ratones , Especificidad de Órganos , Unión Proteica , Proteínas Recombinantes de Fusión/metabolismo , Análisis de Secuencia de Proteína , Anticuerpos de Cadena Única/química , SolubilidadRESUMEN
Type 2 diabetes is associated with microvascular damage that causes frequent infections in the skin and chronic ulcers as a result of impaired wound healing. To trace the pathological changes, we performed a comprehensive analysis of lymphatic vessels in the skin of type 2 diabetic versus nondiabetic patients. The dermis revealed enhanced lymphatic vessel density, and transcriptional profiling of ex vivo isolated lymphatic endothelial cells (LECs) identified 160 genes differentially expressed between type 2 diabetic and nondiabetic LECs. Bioinformatic analysis of deregulated genes uncovered sets functionally related to inflammation, lymphatic vessel remodeling, lymphangiogenesis, and lipid and small molecule transport. Furthermore, we traced CD68(+) macrophage accumulation and concomitant upregulation of tumor necrosis factor-α (TNF-α) levels in type 2 diabetic skin. TNF-α treatment of LECs and its specific blockade in vitro reproduced differential regulation of a gene set that led to enhanced LEC mobility and macrophage attachment, which was mediated by the LEC-derived chemokine CXCL10. This study identifies lymph vessel gene signatures directly correlated with type 2 diabetes skin manifestations. In addition, we provide evidence for paracrine cross-talk fostering macrophage recruitment to LECs as one pathophysiological process that might contribute to aberrant lymphangiogenesis and persistent inflammation in the skin.
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Dermis/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Células Endoteliales/metabolismo , Expresión Génica , Inflamación/metabolismo , Vasos Linfáticos/metabolismo , Adulto , Quimiocina CXCL10/genética , Quimiocina CXCL10/metabolismo , Dermis/patología , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/patología , Células Endoteliales/patología , Humanos , Inflamación/genética , Inflamación/patología , Vasos Linfáticos/patología , Macrófagos/metabolismo , Macrófagos/patología , Transcriptoma , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
In individuals with mammary carcinoma, the most relevant prognostic predictor of distant organ metastasis and clinical outcome is the status of axillary lymph node metastasis. Metastases form initially in axillary sentinel lymph nodes and progress via connecting lymphatic vessels into postsentinel lymph nodes. However, the mechanisms of consecutive lymph node colonization are unknown. Through the analysis of human mammary carcinomas and their matching axillary lymph nodes, we show here that intrametastatic lymphatic vessels and bulk tumor cell invasion into these vessels highly correlate with formation of postsentinel metastasis. In an in vitro model of tumor bulk invasion, human mammary carcinoma cells caused circular defects in lymphatic endothelial monolayers. These circular defects were highly reminiscent of defects of the lymphovascular walls at sites of tumor invasion in vivo and were primarily generated by the tumor-derived arachidonic acid metabolite 12S-HETE following 15-lipoxygenase-1 (ALOX15) catalysis. Accordingly, pharmacological inhibition and shRNA knockdown of ALOX15 each repressed formation of circular defects in vitro. Importantly, ALOX15 knockdown antagonized formation of lymph node metastasis in xenografted tumors. Furthermore, expression of lipoxygenase in human sentinel lymph node metastases correlated inversely with metastasis-free survival. These results provide evidence that lipoxygenase serves as a mediator of tumor cell invasion into lymphatic vessels and formation of lymph node metastasis in ductal mammary carcinomas.
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Lipooxigenasa/metabolismo , Neoplasias Mamarias Animales/metabolismo , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/química , Animales , Araquidonato 12-Lipooxigenasa/metabolismo , Araquidonato 15-Lipooxigenasa/metabolismo , Carcinoma/metabolismo , Carcinoma Ductal de Mama/metabolismo , Línea Celular Tumoral , Técnicas de Cocultivo , Femenino , Humanos , Metástasis Linfática , Ratones , Complejos Multienzimáticos/metabolismo , Metástasis de la Neoplasia , Trasplante de Neoplasias , Recurrencia , Resultado del TratamientoRESUMEN
Cross-linking of IgE antibodies through allergens is a basic event in type I allergy and leads to the immediate release of mediators like histamine, responsible for allergic symptoms like rhino-conjunctivitis or asthma. Critical for the binding of allergens to IgE are the IgE-epitopes, which represent a congregation of several amino acid residues often derived from different regions of the allergen. By means of the mimotope-technology, we isolated peptides from phage libraries, which were able to structurally mimic IgE-epitopes of the plant allergens Bet v 1 (birch) and Phl p 5a (timothy grass). Hence, these are candidates for an epitope-specific immunotherapy. In this mode of immunotherapy, it is the aim to induce IgG antibodies directed exclusively against the IgE-epitopes of allergens without induction of anaphylactogenic IgG species, and without the risk of anaphylaxis through cross-linking of IgE. Immunizing mice, we applied the mimotopes displayed on bacteriophages as well as on alternative carrier systems to enhance their antigenicity. With these systems it was possible to elicit an allergen-specific immune response, which was also accompanied by the appropriate T-cell help. Mimotopes resemble a promising concept for an epitope-tailored immunotherapy of allergic patients.
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Alérgenos , Anticuerpos Bloqueadores/inmunología , Epítopos/inmunología , Hipersensibilidad/inmunología , Hipersensibilidad/terapia , Inmunoglobulina E/inmunología , Inmunoterapia/métodos , Polen/inmunología , Alérgenos/inmunología , Animales , Linfocitos B/inmunología , Bacteriófagos , Betula/inmunología , Sitios de Unión de Anticuerpos , Modelos Animales de Enfermedad , Humanos , Inmunización , Idiotipos de Inmunoglobulinas , Ratones , Ratones Endogámicos BALB C , Imitación Molecular/inmunología , Phleum/inmunología , Proteínas de Plantas/inmunología , Linfocitos T/inmunologíaRESUMEN
Stem cells with certain characteristics have become promising tools for molecular medicine. They have the potential to self-regenerate and to differentiate into specific tissues. Besides their great potential, embryonic stem cells (ESC) run the risk of enhanced tumorigenesis. The use of human embryonic stem cells (hESC) is ethically problematic because their isolation involves the destruction of human embryos. Recently developed methods generate are able to pluripotent stem cells from fibroblasts. Alternatives for ESC are adult stem cells (ASC) derived from bone marrow, cord blood, amniotic fluid and other tissues. The following article is on the basis of testimony of Lukas Kenner for the German Bundestag about the use of ESC for research, therapy and drug development. Ethical aspects are taken into consideration.
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Bioética , Clonación de Organismos , Investigaciones con Embriones/ética , Células Madre Embrionarias , Biología Molecular , Células Madre , Adulto , Células Madre Adultas , Animales , Ensayos Clínicos como Asunto , Modelos Animales de Enfermedad , Células Madre Embrionarias/citología , Células Madre Embrionarias/inmunología , Células Madre Embrionarias/trasplante , Histocompatibilidad , Humanos , Técnicas In Vitro , Ratones , Ratones Transgénicos , Mutagénesis , Neoplasias/etiología , Proyectos Piloto , Células Madre Pluripotentes , Pronóstico , Trasplante de Células Madre , Células Madre/fisiologíaRESUMEN
Xylosylated and core alpha1,3-fucosylated N-glycans from plants are immunogenic, and they play a still obscure role in allergy and in the field of plant-made protein pharmaceuticals. We immunized mice to generate monoclonal antibodies (mAbs) binding plant N-glycans specifically via the epitope containing either the xylose or the core alpha1,3-fucose residue. Splenocytes expressing N-glycan-specific antibodies derived from C57BL/6 mice previously immunized with plant glycoproteins were preselected by cell sorting to generate hybridoma lines producing specific antibodies. However, we obtained only mAbs unable to distinguish fucosylated from xylosylated N-glycans and reactive even with the pentasaccharide core Man3GlcNAc2. In contrast, immunization of rabbits yielded polyclonal sera selectively reactive with either fucosylated or xylosylated N-glycans. Purification of these sera using glyco-modified neoglycoproteins coupled to a chromatography matrix provided polyclonal sera suitable for affinity determination. Surface plasmon resonance measurements using sensor chips with immobilized glyco-modified transferrins revealed dissociation constants of around 10(-9) M. This unexpectedly high affinity of IgG antibodies toward carbohydrate epitopes has repercussions on our conception of the binding strength and significance of antiglycan IgE antibodies in allergy.
Asunto(s)
Anticuerpos Monoclonales/química , Afinidad de Anticuerpos , Inmunoglobulina G/química , Oligosacáridos de Cadena Ramificada/química , Plantas/química , Animales , Anticuerpos Monoclonales/inmunología , Afinidad de Anticuerpos/inmunología , Especificidad de Anticuerpos/inmunología , Glicoproteínas/química , Glicoproteínas/inmunología , Inmunoglobulina G/inmunología , Ratones , Oligosacáridos de Cadena Ramificada/inmunología , Proteínas de Plantas/química , Proteínas de Plantas/inmunología , Plantas/inmunología , Conejos , Especificidad de la Especie , Resonancia por Plasmón de SuperficieRESUMEN
Size and posttranslational modifications are obstacles in the recombinant expression of high-molecular-weight melanoma-associated antigen (HMW-MAA). Creating a tumor antigen mimic via the phage display technology may be a means to overcome this problem for vaccine design. In this study, we aimed to generate an immunogenic epitope mimic of HMW-MAA. Therefore we screened a linear 9mer phage display peptide library, using the anti-HMW-MAA monoclonal antibody (mAb) 225.28S. This antibody mediates antibody-dependent cellular cytotoxicity (ADCC) and has already been used for anti-idiotype therapy trials. Fifteen peptides were selected by mAb 225.28S in the biopanning procedure. They share a consensus sequence, but show only partial homology to the amino acid sequence of the HMW-MAA core protein, indicating mimicry with a conformational epitope. One mimotope was chosen to be fused to albumin binding protein (ABP) as an immunogenic carrier. Immunoassays with 225.28S indicated that the mimotope fusion protein was folded correctly. Subsequently, the fusion protein was tested for immunogenicity in BALB/c mice. The induced anti-mimotope antibodies recognized HMW-MAA of 518A2 human melanoma cells, whereas sera of mice immunized with the carrier ABP alone showed no reactivity. These anti-mimotope antibodies were capable of inducing specific lysis of 518A2 melanoma cells in ADCC assays with murine effector cells. In conclusion, the presented data indicate that mimotopes fused to an immunogenic carrier are suitable tools to elicit epitope-specific anti-melanoma immune responses.