RESUMEN
Upregulation of the proto-oncogene T-cell leukemia/lymphoma 1A (TCL1A) is causally implicated in various B-cell and T-cell malignancies. High-level TCL1A correlates with aggressive disease features and inferior clinical outcomes. However, the molecular and cell biological consequences of, particularly nuclear, TCL1A are not fully elucidated. We observed here in mouse models of subcellular site-specific TCL1A-induced lymphomagenesis that TCL1A exerts a strong transforming impact via nuclear topography. In proteomic screens of TCL1A-bound molecules in chronic lymphocytic leukemia (CLL) cells and B-cell lymphoma lines, we identified regulators of cell cycle and DNA repair pathways as novel TCL1A interactors, particularly enriched under induced DNA damage and mitosis. By functional mapping and in silico modeling, we specifically identified the mitotic checkpoint protein, cell division cycle 20 (CDC20), as a direct TCL1A interactor. According to the regulatory impact of TCL1A on the activity of the CDC20-containing mitotic checkpoint and anaphase-promoting complexes during mitotic progression, TCL1A overexpression accelerated cell cycle transition in B-cell lymphoma lines, impaired apoptotic damage responses in association with pronounced chromosome missegregation, and caused cellular aneuploidy in Eµ-TCL1A mice. Among hematopoietic cancers, CDC20 levels seem particularly low in CLL. CDC20 expression negatively correlated with TCL1A and lower expression marked more aggressive and genomically instable disease and cellular phenotypes. Knockdown of Cdc20 in TCL1A-initiated murine CLL promoted aneuploidy and leukemic acceleration. Taken together, we discovered a novel cell cycle-associated effect of TCL1A abrogating controlled cell cycle transition. This adds to our concept of oncogenic TCL1A by targeting genome stability. Overall, we propose that TCL1A acts as a pleiotropic adapter molecule with a synergistic net effect of multiple hijacked pathways.
Asunto(s)
Leucemia Linfocítica Crónica de Células B , Linfoma de Células B , Ratones , Animales , Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfocítica Crónica de Células B/patología , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteómica , Linfoma de Células B/genética , Ciclo Celular/genética , Proto-Oncogenes , Proteínas de Ciclo Celular/genética , MitosisRESUMEN
In chronic lymphocytic leukemia (CLL), epigenetic alterations are considered to centrally shape the transcriptional signatures that drive disease evolution and underlie its biological and clinical subsets. Characterizations of epigenetic regulators, particularly histone-modifying enzymes, are very rudimentary in CLL. In efforts to establish effectors of the CLL-associated oncogene T-cell leukemia 1A (TCL1A), we identified here the lysine-specific histone demethylase KDM1A to interact with the TCL1A protein in B cells in conjunction with an increased catalytic activity of KDM1A. We demonstrate that KDM1A is upregulated in malignant B cells. Elevated KDM1A and associated gene expression signatures correlated with aggressive disease features and adverse clinical outcomes in a large prospective CLL trial cohort. Genetic Kdm1a knockdown in Eµ-TCL1A mice reduced leukemic burden and prolonged animal survival, accompanied by upregulated p53 and proapoptotic pathways. Genetic KDM1A depletion also affected milieu components (T, stromal, and monocytic cells), resulting in significant reductions in their capacity to support CLL-cell survival and proliferation. Integrated analyses of differential global transcriptomes (RNA sequencing) and H3K4me3 marks (chromatin immunoprecipitation sequencing) in Eµ-TCL1A vs iKdm1aKD;Eµ-TCL1A mice (confirmed in human CLL) implicate KDM1A as an oncogenic transcriptional repressor in CLL which alters histone methylation patterns with pronounced effects on defined cell death and motility pathways. Finally, pharmacologic KDM1A inhibition altered H3K4/9 target methylation and revealed marked anti-B-cell leukemic synergisms. Overall, we established the pathogenic role and effector networks of KDM1A in CLL via tumor-cell intrinsic mechanisms and its impacts in cells of the microenvironment. Our data also provide rationales to further investigate therapeutic KDM1A targeting in CLL.
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Leucemia Linfocítica Crónica de Células B , Humanos , Ratones , Animales , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Histonas/metabolismo , Lisina , Estudios Prospectivos , Histona Demetilasas/genética , Histona Demetilasas/metabolismo , Microambiente TumoralRESUMEN
T-cell prolymphocytic leukemia (T-PLL) is a poor-prognostic mature T-cell malignancy. It typically presents with exponentially rising lymphocyte counts, splenomegaly, and bone marrow infiltration. Effective treatment options are scarce and a better understanding of TPLL's pathogenesis is desirable. Activation of the TCL1 proto-oncogene and loss-of-function perturbations of the tumor suppressor ATM are TPLL's genomic hallmarks. The leukemic cell reveals a phenotype of active T-cell receptor (TCR) signaling and aberrant DNA damage responses. Regulatory networks based on the profile of microRNA (miR) have not been described for T-PLL. In a combined approach of small-RNA and transcriptome sequencing in 46 clinically and moleculary well-characterized T-PLL, we identified a global T-PLL-specific miR expression profile that involves 34 significantly deregulated miR species. This pattern strikingly resembled miR-ome signatures of TCR-activated T cells. By integrating these T-PLL miR profiles with transcriptome data, we uncovered regulatory networks associated with cell survival signaling and DNA damage response pathways. Despite a miR-ome that discerned leukemic from normal T cells, there were also robust subsets of T-PLL defined by a small set of specific miR. Most prominently, miR-141 and the miR- 200c-cluster separated cases into two major subgroups. Furthermore, increased expression of miR-223-3p as well as reduced expression of miR-21 and the miR-29 cluster were associated with more activated Tcell phenotypes and more aggressive disease presentations. Based on the implicated pathobiological role of these miR deregulations, targeting strategies around their effectors appear worth pursuing. We also established a combinatorial miR-based overall survival score for T-PLL (miROS-T-PLL), that might improve current clinical stratifications.
Asunto(s)
Leucemia Prolinfocítica de Células T , MicroARNs , Daño del ADN , Humanos , Leucemia Prolinfocítica de Células T/tratamiento farmacológico , Leucemia Prolinfocítica de Células T/genética , Leucemia Prolinfocítica de Células T/patología , Activación de Linfocitos , MicroARNs/genética , Linfocitos TRESUMEN
T-cell receptor (TCR) ß repertoire analysis can distinguish monoclonal from polyclonal T-cell proliferations and crucially aid in the diagnosis of T-cell malignancies. TCR repertoire can be assessed either by flow cytometry (FCM), or by molecular genetic techniques. We compared the results of parallel analyses of Vß expression by FCM and TRB rearrangements by DNA-based next-generation sequencing (NGS) in 80 diagnostic peripheral blood samples of patients with T-cell prolymphocytic leukemia (T-PLL) for (1) the diagnosis of clonality and (2) the assessment of dominant Vß usage. FCM-based analysis of the surface expression was performed using the IOTest Beta Mark kit. The NGS-based analysis employed the multiplex Biomed-2 VB-JB primers. In all the samples, one or two clonal TRB rearrangements were detected by NGS. Although a dominant Vß domain usage was detected by FCM in only 41/80 (51%) samples, clonality was suspected in all of them. In a total of 12 cases, the FCM missed the clone detected by NGS, despite theoretical coverage by the antibodies, the functionality of the rearrangement, and the expression of TCRαß on the cell surface. Partly overlapping with those cases, FCM discovered predominant Vß usage in the T-PLL population that differed from the one detected by NGS in 10 cases. Overall, the concordant NGS and FCM results were obtained on 61/80 (76%) of samples. We conclude that NGS-based TRB analysis can overcome certain limitations of FCM-based analysis by the identification of both productive and nonproductive rearrangements and by covering the whole Vß spectrum. Currently available FCM analysis of Vß expression lacks this breadth but has advantages, such as parallel immunophenotyping and a more accurate quantification of the Vß usage. © 2018 International Society for Advancement of Cytometry.
Asunto(s)
Leucemia Prolinfocítica de Células T/genética , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Linfocitos T/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Citometría de Flujo/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Inmunofenotipificación/métodos , Activación de Linfocitos/genética , Masculino , Persona de Mediana EdadRESUMEN
BACKGROUND: Redox stress is a hallmark of the rewired metabolic phenotype of cancer. The underlying dysregulation of reactive oxygen species (ROS) is interconnected with abnormal mitochondrial biogenesis and function. In chronic lymphocytic leukemia (CLL), elevated ROS are implicated in clonal outgrowth and drug resistance. The pro-survival oncogene T-cell leukemia 1 (TCL1) is causally linked to the high threshold towards classical apoptosis in CLL. We investigated how aberrant redox characteristics and bioenergetics of CLL are impacted by TCL1 and if this is therapeutically exploitable. METHODS: Bio-organometallic chemistry provided compounds containing a cytosine nucleobase, a metal core (ferrocene, ruthenocene, Fe(CO)3), and a 5'-CH2O-TDS substituent. Four of these metal-containing nucleoside analogues (MCNA) were tested for their efficacy and mode of action in CLL patient samples, gene-targeted cell lines, and murine TCL1-transgenic splenocytes. RESULTS: The MCNA showed a marked and selective cytotoxicity towards CLL cells. MCNA activity was equally observed in high-risk disease groups, including those of del11q/del17p cytogenetics and of clinical fludarabine resistance. They overcame protective stromal cell interactions. MCNA-evoked PARP-mediated cell death was non-autophagic and non-necrotic as well as caspase- and P53-independent. This unconventional apoptosis involved early increases of ROS, which proved indispensible based on mitigation of MCNA-triggered death by various scavengers. MCNA exposure reduced mitochondrial respiration (oxygen consumption rate; OCR) and induced a rapid membrane depolarization (∆ΨM). These characteristics distinguished the MCNA from the alkylator bendamustine and from fludarabine. Higher cellular ROS and increased MCNA sensitivity were linked to TCL1 expression. The presence of TCL1 promoted a mitochondrial release of in part caspase-independent apoptotic factors (AIF, Smac, Cytochrome-c) in response to MCNA. Although basal mitochondrial respiration (OCR) and maximal respiratory capacity were not affected by TCL1 overexpression, it mediated a reduced aerobic glycolysis (lactate production) and a higher fraction of oxygen consumption coupled to ATP-synthesis. CONCLUSIONS: Redox-active substances such as organometallic nucleosides can confer specific cytotoxicity to ROS-stressed cancer cells. Their P53- and caspase-independent induction of non-classical apoptosis implicates that redox-based strategies can overcome resistance to conventional apoptotic triggers. The high TCL1-oncogenic burden of aggressive CLL cells instructs their particular dependence on mitochondrial energetic flux and renders them more susceptible towards agents interfering in mitochondrial homeostasis.
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Leucemia Linfocítica Crónica de Células B/patología , Mitocondrias/metabolismo , Nucleósidos/farmacología , Oncogenes , Compuestos Organometálicos/farmacología , Proteínas Proto-Oncogénicas/genética , Especies Reactivas de Oxígeno/metabolismo , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Caspasa 3/metabolismo , Línea Celular Tumoral , Metabolismo Energético/efectos de los fármacos , Homeostasis/efectos de los fármacos , Humanos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Necrosis , Nucleósidos/química , Compuestos Organometálicos/química , Factores de Riesgo , Células del Estroma/efectos de los fármacos , Células del Estroma/patología , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
The cell-surface glycoprotein CD44 is expressed in chronic lymphocytic leukemia (CLL), but its functional role in this disease is poorly characterized. We therefore investigated the contribution of CD44 to CLL in a murine disease model, the Eµ-TCL1 transgenic mouse, and in CLL patients. Surface CD44 increased during murine CLL development. CD44 expression in human CLL was induced by stimulation with interleukin 4/soluble CD40 ligand and by stroma cell contact. Engagement of CD44 by its natural ligands, hyaluronic acid or chondroitin sulfate, protected CLL cells from apoptosis, while anti-CD44 small interfering RNAs impaired tumor cell viability. Deletion of CD44 during TCL1-driven murine leukemogenesis reduced the tumor burden in peripheral blood and spleen and led to a prolonged overall survival. The leukemic cells from these CD44 knockout animals revealed lower levels of antiapoptotic MCL1, a higher propensity to apoptosis, and a diminished B-cell receptor kinase response. The inhibitory anti-CD44 antibodies IM7 and A3D8 impaired the viability of CLL cells in suspension cultures, in stroma contact models, and in vivo via MCL1 reduction and by effector caspase activation. Taken together, CD44 expression in CLL is mediated by the tumor microenvironment. As a coreceptor, CD44 promotes leukemogenesis by regulating stimuli of MCL1 expression. Moreover, CD44 can be addressed therapeutically in CLL by specific antibodies.
Asunto(s)
Apoptosis/genética , Transformación Celular Neoplásica/genética , Receptores de Hialuranos/fisiología , Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfocítica Crónica de Células B/patología , Proteínas Proto-Oncogénicas c-bcl-2/genética , Animales , Células Cultivadas , Progresión de la Enfermedad , Femenino , Regulación Leucémica de la Expresión Génica , Humanos , Receptores de Hialuranos/genética , Receptores de Hialuranos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Microambiente Tumoral/genética , Microambiente Tumoral/fisiologíaRESUMEN
Purpose: Togu Jumper is a both sides utilized balance training device, which consists of an inflated rubber hemisphere attached to a rigid platform. It has been shown to be effective in improving postural control but there are no recommendations for the usage of the sides. Our aim was to examine leg muscle activity and kinematics in response to a single-leg stance on the two sides of the Togu Jumper and the floor. Methods: In 14 female subjects, linear acceleration of leg segments, segmental angular sway, and myoelectric activity of 8 leg muscles were recorded in the three stance conditions. Results: Except gluteus medius and gastrocnemius medialis, all muscles were more active when balancing on either Togu Jumper side compared to the floor (p < 0.001), but there was no difference between the two sides in any muscles. Linear acceleration was the greatest in the frontal plane on the flat Togu side in the case of the foot (p < 0.001). Pelvis acceleration was unaffected by the balance conditions. Segmental angular sway was the greatest in the frontal plane, on the bladder side in the foot segment (p < 0.001). No difference was found among the three conditions (all p > 0.05) in the case of the shank, thigh, and pelvis. Conclusion: The use of the two Togu Jumper sides produced different balance strategies in the foot segment and induced no difference in equilibrium procedures at the level of the pelvis.
RESUMEN
BACKGROUND: Delayed wound healing is a serious side effect of mTOR inhibitor-based immunosuppression after solid organ transplantation. The aim of this study was to test the hypothesis that the mTOR inhibitor everolimus interferes with the inflammatory phase of healing in experimental colonic anastomoses. MATERIALS AND METHODS: Thirty male Sprague-Dawley rats received a colonic anastomosis. Then, animals were randomized to three groups of daily treatment with either vehicle or everolimus in two different dosages (1.0mg/kg or 3.0mg/kg). After 7 d, rats were sacrificed, and mechanical, histologic, and biochemical parameters of intestinal healing were assessed. RESULTS: Anastomotic bursting pressure was significantly decreased by everolimus in both dosages, whereas hydroxyproline content was reduced only by the high everolimus dosage. Everolimus diminished cellular proliferation and new vessel growth. Furthermore, both quantity as well as quality of newly synthesized collagen fibers in the anastomotic granulation tissue was reduced. On the other hand, myeloperoxidase-positive (MPO) cells and interleukin-6 (IL-6) concentrations were increased, as was the activity of matrix-metalloproteinases MMP-2 and MMP-9. CONCLUSION: Everolimus interferes with the inflammatory phase of healing. However, it remains unclear whether this phenomenon is involved in everolimus impairment of experimental anastomotic repair.
Asunto(s)
Colon/cirugía , Inmunosupresores/farmacología , Inflamación/prevención & control , Sirolimus/análogos & derivados , Cicatrización de Heridas/efectos de los fármacos , Anastomosis Quirúrgica , Animales , Colon/metabolismo , Colon/patología , Everolimus , Hidroxiprolina/metabolismo , Inflamación/fisiopatología , Interleucina-6/metabolismo , Masculino , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Modelos Animales , Antígeno Nuclear de Célula en Proliferación/metabolismo , Ratas , Ratas Sprague-Dawley , Sirolimus/farmacología , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Cicatrización de Heridas/fisiologíaRESUMEN
Understanding antibody-based SARS-CoV-2 immunity is critical for overcoming the COVID-19 pandemic and informing vaccination strategies. We evaluated SARS-CoV-2 antibody dynamics over 10 months in 963 individuals who predominantly experienced mild COVID-19. Investigating 2,146 samples, we initially detected SARS-CoV-2 antibodies in 94.4% of individuals, with 82% and 79% exhibiting serum and IgG neutralization, respectively. Approximately 3% of individuals demonstrated exceptional SARS-CoV-2 neutralization, with these "elite neutralizers" also possessing SARS-CoV-1 cross-neutralizing IgG. Multivariate statistical modeling revealed age, symptomatic infection, disease severity, and gender as key factors predicting SARS-CoV-2-neutralizing activity. A loss of reactivity to the virus spike protein was observed in 13% of individuals 10 months after infection. Neutralizing activity had half-lives of 14.7 weeks in serum versus 31.4 weeks in purified IgG, indicating a rather long-term IgG antibody response. Our results demonstrate a broad spectrum in the initial SARS-CoV-2-neutralizing antibody response, with sustained antibodies in most individuals for 10 months after mild COVID-19.
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Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , COVID-19/inmunología , Adolescente , Adulto , Anciano , Estudios de Cohortes , Femenino , Humanos , Inmunoglobulina G/inmunología , Masculino , Persona de Mediana Edad , SARS-CoV-2 , Factores de Tiempo , Adulto JovenRESUMEN
Clinical trials in T-cell prolymphocytic leukemia (T-PLL) are scarce. Based on a precursor study testing fludarabine, mitoxantrone, and cyclophosphamide followed by alemtuzumab (FMC-A), we aimed to improve this regimen by upfront combining subcutaneous (s.c.) alemtuzumab with FMC for four cycles followed by an alemtuzumab-maintenance (FMCA + A). This prospective multicenter phase-II trial assessed response, survival, and toxicity of that regimen administered to pretreated (n = 4) and treatment-naïve (n = 12) T-PLL patients. The best overall response rate after FMCA was 68.8% (n = 11) including five CRs (31.3%) and six PRs (37.5%). Six patients entered the alemtuzumab-maintenance. Median overall and progression-free survival was 16.7 and 11.2 months, respectively. Hematologic toxicities were the most frequent grade 3/4 side effects. A reduced incidence of CMV-reactivations was attributed to the prophylactic administration of valganciclovir. Overall, FMCA + A did not improve the efficacy of the FMC-A-regimen or of single i.v. alemtuzumab. It suggests that a chemotherapy backbone prevents efficient alemtuzumab dosing and confirms that intravenous alemtuzumab is to be preferred over its s.c. route in T-PLL. ClinicalTrials.gov identifier: NCT01186640.
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Alemtuzumab/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Leucemia Prolinfocítica de Células T/tratamiento farmacológico , Adulto , Anciano , Alemtuzumab/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Ciclofosfamida/administración & dosificación , Femenino , Humanos , Incidencia , Quimioterapia de Inducción , Estimación de Kaplan-Meier , Leucemia Prolinfocítica de Células T/diagnóstico , Leucemia Prolinfocítica de Células T/mortalidad , Quimioterapia de Mantención , Masculino , Persona de Mediana Edad , Mitoxantrona/administración & dosificación , Resultado del Tratamiento , Vidarabina/administración & dosificación , Vidarabina/análogos & derivadosRESUMEN
Deciphering the evolution of cancer cells under therapeutic pressure is a crucial step to understand the mechanisms that lead to treatment resistance. To this end, we analyzed whole-exome sequencing data of eight chronic lymphocytic leukemia (CLL) patients that developed resistance upon BCL2-inhibition by venetoclax. Here, we report recurrent mutations in BTG1 (2 patients) and homozygous deletions affecting CDKN2A/B (3 patients) that developed during treatment, as well as a mutation in BRAF and a high-level focal amplification of CD274 (PD-L1) that might pinpoint molecular aberrations offering structures for further therapeutic interventions.
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Compuestos Bicíclicos Heterocíclicos con Puentes/uso terapéutico , Resistencia a Antineoplásicos , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Sulfonamidas/uso terapéutico , Inhibidor p15 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p15 de las Quinasas Dependientes de la Ciclina/metabolismo , Inhibidor p16 de la Quinasa Dependiente de Ciclina , Inhibidor p18 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p18 de las Quinasas Dependientes de la Ciclina/metabolismo , Femenino , Humanos , Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfocítica Crónica de Células B/metabolismo , Masculino , Mutación , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismoRESUMEN
The serine/threonine death-associated protein kinases (DAPK) provide pro-death signals in response to (oncogenic) cellular stresses. Lost DAPK expression due to (epi)genetic silencing is found in a broad spectrum of cancers. Within B-cell lymphomas, deficiency of the prototypic family member DAPK1 represents a predisposing or early tumorigenic lesion and high-frequency promoter methylation marks more aggressive diseases. On the basis of protein studies and meta-analyzed gene expression profiling data, we show here that within the low-level context of B-lymphocytic DAPK, particularly CLL cells have lost DAPK1 expression. To target this potential vulnerability, we conceptualized B-cell-specific cytotoxic reconstitution of the DAPK1 tumor suppressor in the format of an immunokinase. After rounds of selections for its most potent cytolytic moiety and optimal ligand part, a DK1KD-SGIII fusion protein containing a constitutive DAPK1 mutant, DK1KD, linked to the scFv SGIII against the B-cell-exclusive endocytic glyco-receptor CD22 was created. Its high purity and large-scale recombinant production provided a stable, selectively binding, and efficiently internalizing construct with preserved robust catalytic activity. DK1KD-SGIII specifically and efficiently killed CD22-positive cells of lymphoma lines and primary CLL samples, sparing healthy donor- or CLL patient-derived non-B cells. The mode of cell death was predominantly PARP-mediated and caspase-dependent conventional apoptosis as well as triggering of an autophagic program. The notoriously high apoptotic threshold of CLL could be overcome by DK1KD-SGIII in vitro also in cases with poor prognostic features, such as therapy resistance. The manufacturing feasibility of the novel CD22-targeting DAPK immunokinase and its selective antileukemic efficiency encourage intensified studies towards specific clinical application. Mol Cancer Ther; 15(5); 971-84. ©2016 AACR.
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Antineoplásicos/administración & dosificación , Apoptosis/efectos de los fármacos , Proteínas Quinasas Asociadas a Muerte Celular/metabolismo , Leucemia Linfocítica Crónica de Células B/metabolismo , Linfoma de Células B/metabolismo , Proteínas Recombinantes de Fusión/administración & dosificación , Lectina 2 Similar a Ig de Unión al Ácido Siálico/antagonistas & inhibidores , Línea Celular Tumoral , Proteínas Quinasas Asociadas a Muerte Celular/antagonistas & inhibidores , Proteínas Quinasas Asociadas a Muerte Celular/química , Proteínas Quinasas Asociadas a Muerte Celular/genética , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfocítica Crónica de Células B/patología , Linfoma de Células B/genética , Linfoma de Células B/patología , Familia de Multigenes , Mutación , Fosforilación , Dominios y Motivos de Interacción de Proteínas/genética , Anticuerpos de Cadena Única/administración & dosificaciónRESUMEN
Cholesterol oxidation products (oxysterols) are markers for in vitro LDL oxidation. They are potent inducers of programmed cell death and are also found in high concentrations inside atherosclerotic lesions. Among physiologically occurring oxysterols, 7beta-OH-cholesterol suggests an increase of lipid peroxidation in vivo. In the underlying study, we quantified free plasma oxysterols by means of gas chromatography in patients with stable coronary artery disease (CAD). Total free plasma oxysterols were elevated more than 2-fold in patients with stable CAD (233 +/- 49 vs 108 +/- 19 ng/ml, n = 22, P < 0.05) compared to a control group (n = 20) with similar atherogenic risk profile and angiographically normal coronary arteries. We found that 7-ketocholesterol, as well as the beta-isomers of epoxide (25.7 +/- 10.0 vs 7.3 +/- 1.4 ng/ml, P = 0.07) and 7beta-OH-cholesterol (65.1 +/- 15.7 vs 19.4 +/- 8.9 ng/ml, P < 0.01), was mainly responsible for this increase. To elucidate a potential relevance of oxysterol stereospecificity in regard to endothelial damage, we further conducted in vitro experiments using human arterial endothelial cells (HAECs). Surprisingly, beta-isomers exerted an up to 10-fold higher amount of cell death in equivalent doses when compared to alpha-isomers. The greater cytotoxic potential of beta-isomers was due to increased apoptosis, preceded by mitochondrial release of cytochrome c with subsequent caspase-3 activation. Stereospecific release of cytochrome c depended on the presence of an intact cytoplasmic membrane, hinting at the existence of a putative oxysterol receptor or a direct stereospecific effect on membrane biology. Finally, both isoforms of oxysterols directly released cytochrome c only in conjunction with protein containing cytosol and endoplasmatic reticulum. Free plasma oxysterol levels, particularly 7-ketocholesterol, beta-epoxide and 7beta-OH-cholesterol, are elevated in patients with stable CAD, independent of their LDL cholesterol levels. Due to the highly increased cytotoxicity of oxysterol beta-isomers in vitro, they may represent important atherogenic risk factors.
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Colesterol/química , Colesterol/metabolismo , Enfermedad de la Arteria Coronaria/metabolismo , Caspasa 3 , Caspasas/metabolismo , Muerte Celular/efectos de los fármacos , Extractos Celulares , Células Cultivadas , Colesterol/farmacología , Enfermedad de la Arteria Coronaria/patología , Citocromos c/metabolismo , Activación Enzimática/efectos de los fármacos , Femenino , Humanos , Masculino , Persona de Mediana Edad , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Oxidación-Reducción , Electricidad Estática , Estereoisomerismo , Especificidad por SustratoRESUMEN
Deep brain stimulation (DBS) of the subthalamic nucleus (STN) has become a popular treatment option for patients suffering from severe Parkinson's disease (PD). Yet the long-term outcome of subthalamic DBS is unknown. A total of 27 patients suffering from severe PD underwent bilateral stereotactic implantation of high-frequency stimulators in the STN. Before surgery and at least annually after surgery they were examined with the Unified Parkinson's Disease Rating Scale (UPDRS). This study presents the results of a mean 30 months (range 23 to 55) follow-up of these patients. We found stable and significant off medication improvement of motor function by DBS (between 40% and 44% in the UPDRS part III). While on medication there was no significant change in the motor function by DBS. UPDRS part III worsened gradually during the follow-up period, suggesting disease progression. Thirty months postsurgery the UPDRS part II (ADL) was still improved by 17%. There was a lasting decrease in fluctuations by more than 50%, and dyskinesias were reduced by about 70%. Freezing was reduced significantly from 2.2 in the UPDRS part II to 1.2 at the endpoint. The daily levodopa-equivalent dose was reduced by 39% at 12 months and by 30% at 30 months after STN stimulator implantation. Subthalamic DBS improves sustainable motor function in patients with severe Parkinson's disease and leads to a lasting reduction of medication. Limitations of this procedure were found for disturbances of speech and swallowing.
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Terapia por Estimulación Eléctrica/métodos , Enfermedad de Parkinson/fisiopatología , Enfermedad de Parkinson/terapia , Núcleo Subtalámico/fisiopatología , Adulto , Anciano , Terapia por Estimulación Eléctrica/efectos adversos , Estudios de Seguimiento , Humanos , Persona de Mediana Edad , Actividad Motora , Inhibición Neural , Resultado del TratamientoRESUMEN
Cell survival in chronic lymphocytic leukemia (CLL) largely depends on B-cell receptor-induced AKT activation. Gain-of-function genomic lesions of PI3K-AKT-mTOR pathway components are usually absent in CLL. We previously established that a BCR-mediated growth response in CLL is determined by the oncogene T-cell leukemia 1 (TCL1) through a sensitizer effect on AKT phospho-activation. Despite high clinical response rates following AKT-cascade inhibition in CLL, resistances in a substantial proportion of patients call for reliable pre- and post-exposure strata to better predict compound responses. Using a panel of inhibitors with differential vertical affinities in the PI3K-AKT-mTOR axis, we describe distinct patterns and determinants of sensitivities in 75 CLL samples. The compounds specifically impacted the BCR-induced physical TCL1-AKT interaction. In general, there was an efficient and tumor-selective abrogation of cell survival in suspension or protective stromal-cell cultures. However, biochemical and survival responses were heterogeneous across CLL and showed only incomplete overlap across inhibitors. Sensitivity clusters could be defined by differential responses to selective pan-PI3K inhibition vs. compounds acting more down-stream. An elevated PI3K/AKT/mTOR activation state conferred sensitivity or resistance, depending on the applied inhibitor. In fact, down-stream interception by mTOR or dual mTOR/PI3K inhibition appears more efficient in cases expressing the BCR-response and poor-risk determinants of ZAP70 or TCL1. Finally, exploiting the TCL1-AKT interaction, peptide-based TCL1-interphase mimics were potent in steric AKT antagonization and in reducing CLL cell survival. Overall, this study provides informative response relationships in AKT-pathway interception that can help refining predictive models in BCR-pathway inhibition in CLL.
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Leucemia Linfocítica Crónica de Células B/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Linfocitos B/metabolismo , Supervivencia Celular/fisiología , Humanos , Fosfatidilinositol 3-Quinasas/metabolismo , Transducción de Señal/fisiología , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
To date, there are few reports of paroxysmal exercise-induced dystonia associated with familial epilepsy. We describe a family with 4 affected members spanning 3 generations, suggestive of autosomal-dominant inheritance, who exhibited typical exercise-induced dystonia, different types of epilepsy (absence and primary generalized seizures), developmental delay, and migraine in variable combinations. Linkage of the disease to loci on chromosome 2 (paroxysmal nonkinesigenic dyskinesia) and chromosome 16 (paroxysmal kinesigenic choreoathetosis, infantile convulsions with choreoathetosis) was excluded, suggesting an as yet unidentified underlying genetic basis.
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Epilepsia/genética , Tolerancia al Ejercicio , Ejercicio Físico , Trastornos del Movimiento/genética , Adolescente , Adulto , Anciano , Niño , Mapeo Cromosómico , Cromosomas Humanos Par 2 , Epilepsia/etiología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Trastornos del Movimiento/etiología , LinajeRESUMEN
BACKGROUND: Insulin-like growth factor-I (IGF-I) is accepted as a potent stimulus of wound healing when applied in combination with its binding proteins. However, there is only one study published that has investigated the effect of repeated topical application of unbound IGF-I on ischemic wound healing. The aim of this study was to show the effect of daily topical IGF-I therapy on cutaneous ulcer healing in a steroid-suppressed wound model. MATERIALS AND METHODS: Full-thickness wounds were created on the back of 40 male Sprague-Dawley rats. Before surgery, animals received depot-steroids subcutaneously. Wounds were treated daily with either a standard hydrogel dressing (control), topical IGF-I dissolved in 0.2% methylcellulose gel (IGF-I gel), or a hydrogel dressing containing IGF-I (IGF-I dressing). After 7 days of treatment, wounds were excised and measured by photoplanimetry. SMA- and PCNA-expression as well as the formation of granulation tissue were assessed in tissue sections. Results are given as median(min-max). Differences between groups were calculated by the Mann-Whitney U test. RESULTS: Subcutaneous injection of depot-steroids induced a significant delay in healing, as shown by an enlarged wound size [44(33-65) versus 25(20-35)] mm(2); P = 0.001). In steroid-treated rats, both IGF-I gel and IGF-I dressing enhanced excisional healing, as shown by a significant reduction in wound size (P = 0.0001), with IGF-I released from the dressing being even more effective than IGF-I gel (P = 0.03). However, in these animals only IGF-I released from the hydrogel dressing stimulated SMA- (P = 0.03) as well as PCNA-expression (P = 0.001) and increased granulation tissue formation (P = 0.018). CONCLUSIONS: Our data indicate that a repeated application of topical IGF-I enhances cutaneous ulcer healing. In addition, only the controlled release of IGF-I from the hydrogel dressing is capable of reversing the steroid-induced delay of healing, suggesting different mechanisms of action with respect to the mode of IGF-I delivery.
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Factor I del Crecimiento Similar a la Insulina/administración & dosificación , Úlcera Cutánea/fisiopatología , Esteroides/farmacología , Cicatrización de Heridas/efectos de los fármacos , Actinas/metabolismo , Administración Tópica , Animales , Vendajes , Sistemas de Liberación de Medicamentos , Implantes de Medicamentos , Geles , Tejido de Granulación/patología , Hidrogel de Polietilenoglicol-Dimetacrilato , Inmunohistoquímica , Inyecciones Subcutáneas , Factor I del Crecimiento Similar a la Insulina/farmacología , Masculino , Metilcelulosa , Músculo Liso/metabolismo , Antígeno Nuclear de Célula en Proliferación/metabolismo , Ratas , Ratas Sprague-Dawley , Retratamiento , Úlcera Cutánea/metabolismo , Úlcera Cutánea/patología , Esteroides/administración & dosificaciónRESUMEN
BACKGROUND: Abdominal sepsis is frequently the cause of severe pulmonary dysfunction. Via the thoracic duct, the lung is the first organ exposed to gut-derived mediators released into the mesenteric lymph. AIM: The aim of this study is to investigate whether an enteral immunonutrition with long chain triglycerides prevents septic pulmonary dysfunctions. MATERIALS AND METHODS: Mesenteric lymph was obtained from lymph fistula donor rats during sepsis (lipopolysaccharides [LPS], 5 mg/kg i.p.) with or without enteral immunonutrition (1% of olive oil or 1% of fish oil). Sepsis lymph was then reinfused into the jugular vein of separate recipient rats. Thereafter, the lung tissue was analyzed for the distance of oxygen diffusion, inflammatory response, and cell apoptosis. RESULTS: Sepsis significantly increased TNFalpha release into the mesenteric lymph, whereas an enteral immunonutrition with olive oil significantly reduced the TNFalpha release into the mesenteric lymph by more than five-fold. Sepsis lymph induced a significant increase in alveolar wall thickness, inflammatory reaction, and apoptosis; whereas sepsis lymph collected during olive oil resorption prevented the thickening of the alveolar walls and induced only a mild inflammation, being more potent than fish oil to reduce septic pulmonary dysfunction. CONCLUSIONS: Mediators in the sepsis lymph induce pulmonary dysfunction. The lung may be protected by an enteral immunonutrition containing long chain triglycerides such as olive oil.
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Aceites de Pescado/uso terapéutico , Enfermedades Pulmonares/prevención & control , Linfa/fisiología , Aceites de Plantas/uso terapéutico , Sepsis/terapia , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Apoptosis/fisiología , Pulmón/enzimología , Pulmón/patología , Enfermedades Pulmonares/metabolismo , Enfermedades Pulmonares/patología , Linfa/química , Vasos Linfáticos/fisiología , Masculino , Modelos Animales , Aceite de Oliva , Peroxidasa/análisis , Aceites de Plantas/química , Ratas , Ratas Sprague-Dawley , Sepsis/metabolismo , Sepsis/fisiopatologíaRESUMEN
IL-8 is an important mediator of leukocyte trafficking and activation, participating in tumor angiogenesis, inflammatory processes and coronary atherosclerosis. Under flow conditions IL-8, in conjunction with MCP-1, triggers the firm adhesion of monocytes to the vascular endothelium. While previous studies have suggested the requirement of NF-kappaB for IL-8 secretion by endothelial cells, we investigated the possibility of IL-8 transactivation under conditions of NF-kappaB suppression. Inhibition of the proteasome by MG-132 or lactacystin completely blocked TNF-alpha-induced IkappaBalpha degradation as well as NF-kappaB activity in human arterial endothelial cells. Surprisingly, basal secretion of IL-8 protein was eight- to tenfold induced by proteasome inhibitors, while MCP-1 expression was, as expected, completely down-regulated. IL-8 was up-regulated at the transcriptional level, and promoter studies proved a more than ninefold induction of transcription factor AP-1 activity to be the cause of increased IL-8 transcription. Mutation of the AP-1 binding site in an IL-8 promoter construct completely abrogated this effect, while mutation of the NF-kappaB motif did not influence IL-8 transactivation by proteasome inhibitors. With DNA binding assays we found a seven- to eightfold induction of phosphorylated c-Jun and hence JNK kinase activity under MG-132 treatment. Induction of JNK kinase appeared independent of the cell type, even in tumor cell lines not responding to proteasome inhibitors. Since neither inactivation of p53 in wild-type p53 cells nor reintroduction of functional p53 into p53(-/-) cells affected MG-132-inducible IL-8 secretion, a direct influence of p53 on IL-8 regulation could be excluded. These results show that proteasome inhibitors can not only lead to functional AP-1 induction by enhanced c-Jun phosphorylation, but also transactivate the IL-8 gene in human endothelial cells despite complete suppression of NF-kappaB activity.