RESUMEN
We recently reported N4-substituted 3-methylcytidine-5'-α,ß-methylenediphosphates as CD73 inhibitors, potentially useful in cancer immunotherapy. We now expand the structure-activity relationship of pyrimidine nucleotides as human CD73 inhibitors. 4-Chloro (MRS4598 16; Ki = 0.673 nM) and 4-iodo (MRS4620 18; Ki = 0.436 nM) substitution of the N4-benzyloxy group decreased Ki by â¼20-fold. Primary alkylamine derivatives coupled through a p-amido group with a varying methylene chain length (24 and 25) were functionalized congeners, for subsequent conjugation to carrier or reporter moieties. X-ray structures of hCD73 with two inhibitors indicated a ribose ring conformational adaptation, and the benzyloxyimino group (E configuration) binds to the same region (between the C-terminal and N-terminal domains) as N4-benzyl groups in adenine inhibitors. Molecular dynamics identified stabilizing interactions and predicted conformational diversity. Thus, by N4-benzyloxy substitution, we have greatly enhanced the inhibitory potency and added functionality enabling molecular probes. Their potential as anticancer drugs was confirmed by blocking CD73 activity in tumor tissues in situ.
Asunto(s)
5'-Nucleotidasa/antagonistas & inhibidores , Nucleótidos de Citosina/farmacología , Difosfonatos/farmacología , Inhibidores Enzimáticos/farmacología , 5'-Nucleotidasa/metabolismo , Adulto , Nucleótidos de Citosina/síntesis química , Nucleótidos de Citosina/metabolismo , Difosfonatos/síntesis química , Difosfonatos/metabolismo , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/metabolismo , Proteínas Ligadas a GPI/antagonistas & inhibidores , Proteínas Ligadas a GPI/metabolismo , Humanos , Masculino , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Estructura Molecular , Neoplasias/enzimología , Tonsila Palatina/enzimología , Unión Proteica , Relación Estructura-ActividadRESUMEN
Studies on pathophysiology and the therapeutic potential of extracellular ATP and other purines represent an important and rapidly evolving field. The integral response of the cell is determined by multiple factors, including the release of endogenous ATP, co-expression of different types of nucleotide- and adenosine-selective receptors, as well as the specific makeup of ectoenzymes governing the duration and magnitude of purinergic signaling. Current findings support the presence of an extensive network of purine-converting ectoenzymes that are co-expressed to a variable extent among the mammalian tissues and share similarities in substrate specificity. Here, we describe a histochemical approach for simultaneous detection of ecto-nucleotidase and tissue-nonspecific alkaline phosphatase (TNAP) activities in the same tissue slice. Further employment of this technique for staining human palatine tonsil cryosections revealed selective distribution of the key ectoenzymes within certain tonsillar structures, including germinal centers and connective tissues (ecto-5'-nucleotidase/CD73), as well as interfollicular area (TNAP and NTPDase1/CD39).
Asunto(s)
5'-Nucleotidasa/metabolismo , Adenosina Trifosfato/metabolismo , Fosfatasa Alcalina/metabolismo , Antígenos CD/metabolismo , Apirasa/metabolismo , Inmunohistoquímica/métodos , Tonsila Palatina/enzimología , HumanosRESUMEN
BACKGROUND/AIMS: Far-infrared (FIR) irradiation has been reported to exhibit various biological effects including improvement of cardiovascular function. However, its effect on the differentiation of stem cells has not been studied. Using tonsil-derived mesenchymal stem cells (TMSC), we examined whether and how FIR irradiation affects adipogenic or osteogenic differentiation. METHODS: TMSC were exposed to FIR irradiation (3-25 µm wavelength) for various times (0, 30, or 60 min), and then adipogenic or osteogenic differentiation was induced for 14 days with its respective commercially available differentiation medium. At the end of the differentiation, the cells were stained using Oil red O or Alizarin red S solution, and the expression of differentiation-specific proteins was analyzed by western blotting. RESULTS: FIR irradiation did not alter cell viability or the expression of MSC-specific surface antigens (CD14, CD34, CD45, CD73, CD90, and CD105) in TMSC. However, FIR irradiation significantly inhibited adipogenic differentiation of TMSC, as evidenced by decreased Oil red O staining as well as protein expression of peroxisome proliferator-activated receptor γ and fatty acid binding protein 4. In contrast, FIR irradiation induced osteogenic differentiation, as evidenced by increased Alizarin red S staining as well as protein expression of osteocalcin and alkaline phosphatase. Treatment with heat alone did not inhibit the adipogenic differentiation of TMSC, suggesting that the inhibitory effect on adipogenic differentiation was not due to heat induced by FIR irradiation. However, heat alone did stimulate osteogenic differentiation, but to a lesser extent than FIR irradiation. Furthermore, FIR irradiation increased intracellular Ca²âº levels and the activity of protein phosphatase 2B (PP2B) in TMSC. Treatment with cyclosporin A, a specific PP2B inhibitor, reversed the inhibitory effect of FIR irradiation on adipogenic differentiation of TMSC, but had no effect on osteogenic differentiation. CONCLUSION: Our data demonstrate that FIR irradiation inhibits adipogenic differentiation but enhances osteogenic differentiation of TMSC; the inhibitory effect on adipogenic differentiation is non-thermal and mediated at least in part by activation of Ca²âº-dependent PP2B.
Asunto(s)
Adipogénesis , Calcineurina/metabolismo , Diferenciación Celular , Rayos Infrarrojos , Células Madre Mesenquimatosas/enzimología , Osteogénesis , Tonsila Palatina/enzimología , Niño , Preescolar , Femenino , Humanos , Lactante , Masculino , Células Madre Mesenquimatosas/citología , Tonsila Palatina/citologíaRESUMEN
OBJECTIVES: The palatine tonsil is a significant part of the secondary immune system. Tonsillitis and idiopathic tonsillar hypertrophy (ITH) are the most common pathologies of this component. Although there are studies on their pathogenesis, there is insufficient study of the role of antioxidant agents. Glutathione S-transferase (GST) isozymes contribute to the antioxidation reactions in the tissue via the glutathione pathway. The purpose in this study was to reveal the levels of the GST enzyme activity and protein expression of GSTP1 and GSTA1 isozymes in patients with tonsillitis and tonsil hypertrophy, and to investigate their role in the pathogenesis of these diseases. MATERIALS AND METHODS: Sixteen patients with recurrent tonsillitis and 5 patients with ITH and were included in the study. Cytosolic extracts were prepared from post-tonsillectomy tissues of both patient groups and GST enzyme activities were measured. RESULTS: The expression of GSTP1 was found to be significantly higher than GSTA1 in tissue samples of patients with ITH and recurrent tonsillitis (P<0.001). Increased GST activity and GSTP1 isozyme expression were shown in patients with recurrent tonsillitis compared to the idiopathic tonsillar hypertrophy study group. There was a positive correlation between the expressions of GSTP1 (P=0.040; r=0.47). CONCLUSION: Increased GST activity and GSTP1 isozymes were demonstrated histologically in the pathogenesis of ITH and recurrent tonsillitis. We believe that the data of changes in antioxidant capacity, obtained from studies with more extensive and larger samples, would support our findings.
Asunto(s)
Glutatión Transferasa/metabolismo , Hipertrofia/enzimología , Hipertrofia/fisiopatología , Tonsila Palatina/enzimología , Proteínas/metabolismo , Tonsilitis/enzimología , Tonsilitis/fisiopatología , Adolescente , Niño , Preescolar , Femenino , Humanos , Masculino , Tonsila Palatina/patologíaRESUMEN
Primary infection of the immunocompromised host with the oncovirus Epstein-Barr virus (EBV) that targets mainly B-cells is associated with an increased risk for EBV-associated tumors. The early events subsequent to primary infection with potential for B-cell transformation are poorly studied. Here, we modeled in vitro the primary infection by using B-cells isolated from tonsils, the portal of entry of EBV, since species specificity of EBV hampers modeling in experimental animals. Increasing evidence indicates that the host DNA damage response (DDR) can influence and be influenced by EBV infection. Thus, we inoculated tonsillar B-cells (TBCs) with EBV-B95.8 and investigated cell proliferation and the DDR during the first 96 hours thereafter. We identified for the first time that EBV infection of TBCs induces a period of hyperproliferation 48-96 hours post infection characterized by the activation of ataxia telangiectasia and Rad3-releated (ATR) and checkpoint kinase-1 (Chk1). Whereas inhibition of Chk1 did not affect B-cell transformation, the specific inhibition of ATR robustly decreased the transformation efficiency of EBV. Our results suggest that activation of ATR is key for EBV-induced B-cell transformation. Thus, targeting the interaction between ATR/Chk1 and EBV could offer new options for the treatment of EBV-associated malignancies.
Asunto(s)
Linfocitos B/virología , Transformación Celular Viral , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/metabolismo , Infecciones por Virus de Epstein-Barr/virología , Herpesvirus Humano 4/patogenicidad , Tonsila Palatina/enzimología , Tonsila Palatina/virología , Antígenos CD19/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada/análisis , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Linfocitos B/efectos de los fármacos , Linfocitos B/enzimología , Linfocitos B/inmunología , Ligando de CD40/metabolismo , Proliferación Celular , Células Cultivadas , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/análisis , Daño del ADN , Reparación del ADN , Activación Enzimática , Infecciones por Virus de Epstein-Barr/enzimología , Infecciones por Virus de Epstein-Barr/inmunología , Infecciones por Virus de Epstein-Barr/patología , Herpesvirus Humano 4/inmunología , Interacciones Huésped-Patógeno , Humanos , Tonsila Palatina/efectos de los fármacos , Tonsila Palatina/inmunología , Inhibidores de Proteínas Quinasas/farmacología , Transducción de Señal , Factores de TiempoRESUMEN
IMPORTANCE: Cysteinyl leukotrienes (CysLTs) potentially promote adenotonsillar hypertrophy in children with obstructive sleep apnea (OSA). Previous studies have identified CysLTs and their receptors in tonsillar tissue from children with OSA. OBJECTIVE: To demonstrate expression of the leukotriene biosynthetic enzymes 5-lipoxygenase (5-LO), 5-lipoxygenase activating protein (FLAP), leukotriene A(4) hydrolase (LTA(4)H), and leukotriene C(4) synthase (LTC(4)S) in T and B tonsillar lymphocytes from pediatric patients with OSA. It was hypothesized that children with OSA have greater expression of biosynthetic enzymes for CysLTs (5-LO, FLAP, and LTC(4)S) in their tonsillar tissue than do children with recurrent tonsillitis (RT), who were enrolled as controls. DESIGN, SETTING, AND PARTICIPANTS: This prospective, nonrandomized study was performed at a tertiary care university hospital on 13 children with OSA and adenotonsillar hypertrophy undergoing adenotonsillectomy and 12 children without OSA also undergoing tonsillectomy for RT. Tonsillar tissue from children with OSA or RT was examined for 5-LO, FLAP, LTA(4)H, and LTC(4)S expression under real time-quantitative polymerase chain reaction (RT-qPCR), flow cytometry (FC), and confocal laser scanning microscopy (CM). MAIN OUTCOMES AND MEASURES: Expression of biosynthetic enzymes for CysLTs (5-LO, FLAP, and LTC(4)S) was the main outcome measure. Patients with OSA and control patients with RT were compared for numbers of copies of 5-LO, FLAP, and LTC(4)S messenger RNA (by RT-qPCR) in T or B tonsillar lymphocytes and proportions of CD3(+) or CD19(+) tonsillar lymphocytes that expressed 5-LO, FLAP, and LTC(4)S (by FC). RESULTS: Messenger RNA for all 4 enzymes was detected in T and B lymphocytes from both study groups, and expression of all biosynthetic enzymes was demonstrated in participants with OSA and RT by FC. Patients with OSA differed from controls in the proportions (median [10th-90th percentile]) of LTC(4)S(+) CD3(+) T lymphocytes (23.31% [8.64%-50.07%] vs 10.81% [3.48%-23.32%], respectively) (P = .01) and LTC(4)S(+) CD19(+) B lymphocytes (20.66% [14.62%-65.77%] vs 12.53% [2.87%-36.64%], respectively) (P = .01) detected by FC. Immunoreactivity for the 4 enzymes was detected by CM in B lymphocytes of mantle zones and T lymphocytes of extrafollicular areas. CONCLUSIONS AND RELEVANCE: Leukotriene biosynthetic enzymes are expressed in tonsillar lymphocytes, and the previously reported detection of CysLTs in tonsillar tissue from children with OSA may be attributed to endogenous synthesis. Enhanced expression of LTC(4)S is a potential target for pharmacologic interventions in OSA.
Asunto(s)
Cisteína/metabolismo , Leucotrienos/metabolismo , Tonsila Palatina/enzimología , Apnea Obstructiva del Sueño/complicaciones , Apnea Obstructiva del Sueño/enzimología , Proteínas Activadoras de la 5-Lipooxigenasa/metabolismo , Adenoidectomía , Araquidonato 5-Lipooxigenasa/metabolismo , Linfocitos B/enzimología , Niño , Epóxido Hidrolasas/metabolismo , Femenino , Citometría de Flujo , Glutatión Transferasa/metabolismo , Humanos , Hipertrofia , Masculino , Microscopía Confocal , Tonsila Palatina/patología , Tonsila Palatina/cirugía , Polisomnografía , Estudios Prospectivos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Encuestas y Cuestionarios , Linfocitos T/enzimología , TonsilectomíaRESUMEN
The purpose of this study was to investigate the effects of dietary NiCl2 on antioxidant function, apoptosis, and the protein expression, mRNA expression and contents of the bcl-2, bax and caspase-3 in the cecal tonsil of broilers. 280 one-day-old avian broilers were divided into four groups and fed on a corn-soybean basal diet as control diet or the same basal diet supplemented with 300, 600 and 900 mg/kg of NiCl2 for 42 days. The activities of SOD, CAT and GSH-Px, and the ability to inhibit hydroxy radical, and GSH content were significantly decreased in all experimental groups. MDA content was significantly increased. The protein expression, mRNA expression and contents of bcl-2 were decreased, and bax and caspase-3 were increased in all experimental groups. The percentages of apoptotic lymphocytes were significantly increased. In conclusion, dietary NiCl2 in excess of 300 mg/kg caused oxidative stress, and then induced decreased the protein expression, mRNA expression and the contents of bcl-2, and increased protein expression, mRNA expression and the contents of bax and caspase-3 proteins in the cecal tonsil. The local intestinal mucosal immunity could finally be impaired due to the oxidative stress and apoptosis in the cecal tonsil caused by NiCl2.
Asunto(s)
Apoptosis/efectos de los fármacos , Caspasa 3/genética , Níquel/toxicidad , Estrés Oxidativo/efectos de los fármacos , Tonsila Palatina/efectos de los fármacos , Proteína X Asociada a bcl-2/genética , Animales , Catalasa/metabolismo , Pollos , Glutatión Peroxidasa/metabolismo , Malondialdehído/metabolismo , Tonsila Palatina/enzimología , Tonsila Palatina/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Superóxido Dismutasa/metabolismoRESUMEN
BACKGROUND: The concentration and specific activity of N-acetyl-ß-hexosaminidase (HEX) in palatine tonsils with chronic tonsillitis and tonsillar hypertrophy give insight in tonsillar tissue remodeling and constitute a potential marker for diagnosis and treatment of chronic tonsillitis and tonsillar hypertrophy. AIM: Determining the concentration and specific activity of N-acetyl-ß-hexosaminidase in palatine tonsils with hypertrophy and chronic tonsillitis. METHODS: HEX activity was analyzed by the method of Marciniak et al. with p-nitrophenyl N-acetyl-ß-glucosaminepyranoside as a substrate. RESULTS: The concentration and specific activity of HEX in palatine tonsils in patients with tonsillar hypertrophy and chronic tonsillitis both in childhood and adulthood significantly increase in comparison to healthy individuals. CONCLUSIONS: Our data demonstrate the presence of HEX in palatine tonsils and indicate on significant increase of its concentration and specific activity. Based on content and specific HEX activity we suggest that tonsils with hypertrophy and chronic tonsillitis should be treated as identical unit irrespectively of age.
Asunto(s)
Hipertrofia/enzimología , Tonsila Palatina/enzimología , Tonsilitis/enzimología , beta-N-Acetilhexosaminidasas/metabolismo , Adolescente , Adulto , Factores de Edad , Anciano , Biomarcadores/metabolismo , Niño , Preescolar , Enfermedad Crónica , Femenino , Humanos , Masculino , Persona de Mediana Edad , Adulto JovenRESUMEN
After undergoing Ig somatic hypermutation and Ag selection, germinal center (GC) B cells terminally differentiate into either memory or plasma cells (PCs). It is known that the CD40L and IL-21/STAT3 signaling pathways play critical roles in this process, yet it is unclear how the B cell transcription program interprets and integrates these two types of T cell-derived signals. In this study, we characterized the role of STAT3 in the GC-associated PC differentiation using purified human tonsillar GC B cells and a GC B cell-like cell line. When primary GC B cells were cultured under PC differentiation condition, STAT3 inhibition by AG490 prevented the transition from GC centrocytes to preplasmablast, suggesting that STAT3 is required for the initiation of PC development. In a GC B cell-like human B cell line, although IL-21 alone can induce low-level Blimp-1 expression, maximum Blimp-1 upregulation and optimal PC differentiation required both IL-21 and CD40L. CD40L, although having no effect on Blimp-1 as a single agent, greatly augmented the amplitude and duration of IL-21-triggered Jak-STAT3 signaling. In the human PRDM1 locus, CD40L treatment enhanced the ability of STAT3 to upregulate Blimp-1 by removing BCL6, a potent inhibitor of Blimp-1 expression, from a shared BCL6/STAT3 site in intron 3. Thus, IL-21 and CD40L collaborate through at least two distinct mechanisms to synergistically promote Blimp-1 activation and PC differentiation.
Asunto(s)
Adyuvantes Inmunológicos/fisiología , Subgrupos de Linfocitos B/inmunología , Ligando de CD40/fisiología , Diferenciación Celular/inmunología , Interleucinas/fisiología , Células Plasmáticas/inmunología , Proteínas Represoras/biosíntesis , Regulación hacia Arriba/inmunología , Subgrupos de Linfocitos B/enzimología , Subgrupos de Linfocitos B/metabolismo , Línea Celular Tumoral , Humanos , Quinasas Janus/fisiología , Datos de Secuencia Molecular , Técnicas de Cultivo de Órganos , Tonsila Palatina/enzimología , Tonsila Palatina/inmunología , Tonsila Palatina/metabolismo , Células Plasmáticas/enzimología , Células Plasmáticas/metabolismo , Factor 1 de Unión al Dominio 1 de Regulación Positiva , Proteínas Represoras/fisiología , Factor de Transcripción STAT3/fisiologíaRESUMEN
CONCLUSION: The lingual branches of the glossopharyngeal nerve were most likely to bring not only gustatory nerves to the postsulcal part of the tongue but also autonomic nerves to the small glands and vessels. Tonsillectomy may injure the ganglion or reduce its function due to scar formation after surgery. OBJECTIVES: To determine the topographical anatomy of a suggested ganglion cluster along the lingual branches of the glossopharyngeal nerve and to identify the incidence. METHODS: In the human pharynges of 12 donated cadavers, we studied the ganglia using routine procedures for paraffin-embedded histology and immunohistochemistry. RESULTS: Near the palatine tonsil, the lingual branches of the glossopharyngeal nerve often contained ganglion cells (in 9 of 12 specimens). The ganglion cells, 20-40 µ in diameter, were sparsely distributed along a 0.5-3.0 mm length of the nerve course attached to the posterolateral aspect of the superior pharyngeal constrictor. Most of these cells were positive for neuronal nitric oxide synthase, while some were positive for tyrosine hydroxylase. Thus, the ganglion was composed of a mixed population of sympathetic and parasympathetic neurons.
Asunto(s)
Ganglios Autónomos/patología , Nervio Glosofaríngeo/patología , Tonsila Palatina/patología , Anciano , Anciano de 80 o más Años , Cadáver , Femenino , Ganglios Autónomos/enzimología , Nervio Glosofaríngeo/enzimología , Humanos , Masculino , Óxido Nítrico Sintasa de Tipo I/metabolismo , Tonsila Palatina/enzimología , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
OBJECTIVE: The aim of this study was to investigate the status of matrix metalloproteinases (MMP-2, MMP-7, MMP-9) and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) due to dysregulated turnover of connective tissue matrices in children with recurrent tonsillitis (RT). METHODS: Forty-four patients with RT were enrolled in the study. All patients with RT were graded according to the hypertrophy degree of the tonsillar tissue from grade I to grade IV. Patients with grade I tonsillar hypertrophy and grade II tonsillar hypertrophy were accepted as group A, patients with grade III tonsillar hypertrophy and grade IV tonsillar hypertrophy were accepted as group B Tonsillectomy was performed via the usual dissection-snare method. Tonsillar specimens of superficial and core region were evaluated for MMP-2, MMP-7, MMP-9 and TIMP-1 analysis. RESULTS: There was no statistical significance according to the MMP-2, MMP-7, MMP-9 and TIMP-1 activity of superficial part and core regions in both groups individually, MMP-9 level of both the superficial and core regions in group B had statistical significant higher results than group A (p=0.026, p=0.06 respectively). MMP-7 level of the superficial part in group B patients also had statistical significant higher results than group A (p=0.025). However, there was no statistical difference found between superficial and core region MMP-2 and TIMP-1 levels of group A and group B. Related to this, balance between MMP-7-9 and TIMP-1 activities tended to slip MMP-7 and MMP-9 sides with increased tonsillar grade. CONCLUSIONS: Results from this study suggest that the presence of MMPs in tonsil tissue consolidates the involvement of degraded extracellular matrix proteins in the pathophysiology of chronic tonsillitis. MMPs activity showed diffuse dissemination in the tonsillar tissue and especially MMP-9 and MMP-7 are the main promoters of the extracellular matrix that responded to inflammatory changes in the tonsillar tissue. Further studies are needed concerning the possible efficiency of selective MMP inhibitors on tonsillar tissue.
Asunto(s)
Metaloproteinasas de la Matriz/metabolismo , Tonsila Palatina/enzimología , Tonsila Palatina/patología , Tonsilitis/enzimología , Adolescente , Niño , Preescolar , Femenino , Humanos , Hipertrofia , Masculino , Recurrencia , Índice de Severidad de la Enfermedad , TonsilectomíaRESUMEN
RATIONALE: Obstructive sleep apnea (OSA) is a highly prevalent disorder in children, in which enlarged adenotonsillar tissues (AT) play a major pathophysiologic role. Mechanisms leading to the proliferation and hypertrophy of AT in children who subsequently develop OSA remain unknown, and surgical extirpation of AT is associated with potential morbidity and mortality. OBJECTIVES: We hypothesized that a computationally based analysis of gene expression in tonsils from children with OSA and children with recurrent tonsillitis without OSA can identify putative mechanistic pathways associated with tonsillar proliferation and hypertrophy in OSA. METHODS: Palatine tonsils from children with either polysomnographically documented OSA or recurrent infectious tonsillitis were subjected to whole-genome microarray and functional enrichment analyses followed by significance score ranking based on gene interaction networks. The latter enabled identification and confirmation of a candidate list of tonsil-proliferative genes in OSA. MEASUREMENTS AND MAIN RESULTS: In vitro studies using a mixed tonsil cell culture system targeting one of these candidates, phosphoserine phosphatase, revealed that it was more abundantly expressed in tonsils of children with OSA, and that pharmacological inhibition of phosphoserine phosphatase led to marked reductions in T- and B-lymphocyte cell proliferation and increased apoptosis. CONCLUSIONS: A systems biology approach revealed a restricted set of candidate genes potentially underlying the heightened proliferative properties of AT in children with OSA. Furthermore, functional studies confirm a novel role for protein phosphatases in AT hypertrophy, and may provide a promising strategy for discovery of novel, nonsurgical therapeutic targets in pediatric OSA.
Asunto(s)
Tonsila Faríngea/patología , Inhibidores Enzimáticos/farmacología , Tonsila Palatina/patología , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Fosfoproteínas Fosfatasas/genética , Apnea Obstructiva del Sueño/genética , Tonsilitis/genética , Tonsila Faríngea/efectos de los fármacos , Tonsila Faríngea/enzimología , Apoptosis , Estudios de Casos y Controles , Procesos de Crecimiento Celular/efectos de los fármacos , Niño , Preescolar , Sistemas de Liberación de Medicamentos/métodos , Femenino , Perfilación de la Expresión Génica/métodos , Humanos , Hipertrofia/genética , Hipertrofia/patología , Masculino , Tonsila Palatina/efectos de los fármacos , Tonsila Palatina/enzimología , Fosfoproteínas Fosfatasas/biosíntesis , ARN/análisis , Apnea Obstructiva del Sueño/tratamiento farmacológico , Apnea Obstructiva del Sueño/enzimología , Apnea Obstructiva del Sueño/patología , Análisis de Matrices Tisulares , Tonsilitis/tratamiento farmacológico , Tonsilitis/enzimología , Tonsilitis/patologíaRESUMEN
OBJECTIVE: Indications for tonsillectomy in recurrent tonsillitis are defined according to the number of episodes of acute bacterial infections in a year. However, little is known about the tonsil immune competence status in patients presenting with recurrent tonsillitis with either hypertrophied or atrophied tonsils, or in patients presenting with obstructive sleep apnoea. In this study we examined the tonsil immune status in children with 3-5 acute recurrent infections a year and in children with obstructive sleep apnoea by comparing the activity of tonsil and adenoid tissue nonspecific alkaline and acid phosphatase. METHODS: Specific activity of tonsil and adenoid tissue nonspecific alkaline and acid phosphatase was investigated in children who underwent tonsillectomy and adenoidectomy for recurrent infection (72 children) and for obstructive sleep apnoea (10 children). Tissue enzyme activities were measured using p-nitrophenylphosphate as a substrate. Tissue samples were examined by the haematoxylin-eosin histological technique. Statistical analyses were performed using SPSS v. 16 software. RESULTS: The tissue nonspecific alkaline phosphatase activity was similar in hypertrophied tonsils in the recurrent infection group and in the obstructive sleep apnoea group (3.437+/-1.226 and 3.978+/-0.762 U/mg of protein, respectively). The enzyme activity in both hypertrophied tonsil groups was significantly higher as compared to atrophied tonsils in the recurrent tonsillitis group, p=0.021 and p=0.006, respectively. The enzyme activity was significantly higher in the adenoids compared to the tonsils from all three groups. Contrary to this, no significant differences were noticed for tonsil and adenoid acid phosphatase activities among the groups. CONCLUSION: Similar acid phosphatase activity in all three groups implies that all three groups have preserved antigen presenting cell activity. In patients with hypertrophied tonsils similar tissue nonspecific alkaline phosphatase activity suggests preserved B cell tonsil immune activity, regardless of the pathology. Patients with atrophied tonsils had significantly lower alkaline phosphatase activity, indicating relative tonsil B cell immune deficiency. Thus, different immunological status in patients presenting with hypertrophied vs. atrophied tonsils could point to a different underlying pathophysiologic mechanism of the disease.
Asunto(s)
Tonsila Faríngea/enzimología , Tonsila Faríngea/microbiología , Fosfatasa Alcalina/análisis , Fosfatasa Alcalina/metabolismo , Tonsila Palatina/enzimología , Adenoidectomía , Atrofia/enzimología , Atrofia/inmunología , Atrofia/patología , Linfocitos B/enzimología , Linfocitos B/inmunología , Niño , Preescolar , Femenino , Humanos , Concentración de Iones de Hidrógeno , Hipertrofia/enzimología , Hipertrofia/inmunología , Hipertrofia/patología , Macrófagos , Masculino , Obstrucción Nasal/diagnóstico , Obstrucción Nasal/enzimología , Obstrucción Nasal/cirugía , Nitrofenoles , Tonsila Palatina/inmunología , Tonsila Palatina/patología , Recurrencia , Apnea Obstructiva del Sueño/diagnóstico , Apnea Obstructiva del Sueño/enzimología , Tonsilectomía , TonsilitisRESUMEN
Mantle cell lymphoma (MCL) is characterized by genetic instability and a poor prognosis. Many blastoid variants are (hypo)tetraploid and have an even worse prognosis. We investigated the role of signalling by mitogen-activated protein kinases (MAPKs) in MCL. As compared to normal tonsil B cells, MCL cells showed higher activation of the JNK MAPK in both an MAPK array and a sandwich ELISA assay. Immunohistochemistry showed overexpression of phospho (p)-JNK (Thr183/Tyr185) in 30 of 37 MCL cases. Inhibition of p-JNK with SP600125 resulted in growth arrest in all four MCL cell lines (Jeko-1, HBL-2, UPN-1, Granta-519), which could be partly reversed by the addition of CD40L and IL-4. Furthermore, SP600125 led to G2/M phase arrest on day 1 and a striking increase in endoreduplication on day 2 and day 3, which was confirmed by karyotype analysis. G2/M arrest was associated with down-regulation of EGR1 and p21 protein expression. SP600125-induced polyploidy could be blocked by the BCL-2 inhibitor YC137. These data suggest that constitutive JNK activity is necessary to promote proliferation and maintain diploidy in MCL. JNK inhibition leads to cell cycle deregulation and endoreduplication, mimicking the tetraploid state seen in a subset of MCL cases. Thus, our data also provide an experimental model to study polyploid MCL cells.
Asunto(s)
Antracenos/farmacología , Proteínas Quinasas JNK Activadas por Mitógenos/antagonistas & inhibidores , Linfoma de Células del Manto/enzimología , Apoptosis/efectos de los fármacos , Estudios de Casos y Controles , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Ensayo de Inmunoadsorción Enzimática/métodos , Expresión Génica , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/análisis , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Linfoma de Células del Manto/genética , Tonsila Palatina/enzimología , Poliploidía , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Tiazoles/farmacologíaRESUMEN
Sphingolipids serve an important role as effector molecules in signaling pathways bearing on apoptosis and cell survival. The balance between proapoptotic ceramide and prosurvival sphingosine-1-phosphate, sometimes termed the "sphingolipid rheostat," has received particular attention. Less well studied is the role of the follicular lymphoma variant translocation 1 (FVT1) gene, which was identified through its involvement in an atypical follicular lymphoma translocation and which encodes an enzyme in the synthetic pathway of ceramide. We investigated the expression of FVT1 in a variety of B-cell non-Hodgkin lymphomas and found that FVT1 is significantly underexpressed by germinal center-type diffuse large B-cell lymphoma (DLBCL) when compared with non-germinal center-type DLBCL, follicular lymphoma, and normal tonsil control samples. Increased expression of FVT1 correlated with decreased survival, suggesting that changes in the expression of FVT1 and in the concentrations of bioactive sphingolipids may be important in the pathogenesis and treatment of some types of DLBCL.
Asunto(s)
Oxidorreductasas de Alcohol/genética , Linfoma Folicular/genética , Linfoma de Células B Grandes Difuso/genética , Oxidorreductasas de Alcohol/biosíntesis , Ceramidas/biosíntesis , Regulación Neoplásica de la Expresión Génica , Centro Germinal/enzimología , Humanos , Estimación de Kaplan-Meier , Linfoma Folicular/mortalidad , Linfoma Folicular/patología , Linfoma de Células B Grandes Difuso/mortalidad , Linfoma de Células B Grandes Difuso/patología , Tonsila Palatina/enzimología , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Neoangiogenesis and inhibition of apoptosis are two factors considered as major leading causes of tumorigenesis. NO, synthesized by NOS, plays an important role in tumour growth, dissemination and vascularization. Caspase-3 is an executive enzyme of apoptosis. The presented research work has been focused on the comparative evaluation of localization of the angiogenic and proapoptotic cytokines expressed in tonsillar diseases. The immunohistochemical reaction of eNOS, iNOS and caspase-3 in tonsillar cancer (N = 17), chronic tonsillitis (N = 11) and clinically healthy tonsils (N = 8) was detected. High eNOS occurrence in endothelial cells of highly vascularized regions in tonsillar cancer, variable eNOS expression in the vessels of lamina propria in chronic tonsillitis and high expression in the cytoplasm of endothelial cells of small veins in healthy tonsillar tissue was ascertained. Increased iNOS expression was found in cancer tissue in comparison with the healthy tonsils. Nevertheless, the highest expression of iNOS was found in chronic tonsillitis. Higher expression of caspase-3 was discovered in germinal centres of lymphoid follicles of the chronic tonsillitis tissue. However, the positivity in the interfollicular zone and surface squamous epithelium was weak only. Merely isolated caspase-3-positive cells were found in tonsillar cancer. Very low expression of caspase-3 was detected in the lymphatic follicles of the healthy tonsils. Research results showed high expression of eNOS in the carcinomatous tissue. The eNOS expression in chronic tonsillitis confirms its role in regulating the lymphocyte circulation. Low expression of caspase-3 in malignant epithelial cells of tonsillar cancer shows decreased capability of apoptosis compared to chronic tonsillitis tissue, where apoptosis seems to be rather frequent and concentrated in the germinal centres of lymphatic follicles. The differences in localization of eNOS and caspase-3 expression between benign and malignant processes may be a promising tool for precise morphological distinction of chronic inflammation and tumours.
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Caspasa 3/metabolismo , Salud , Óxido Nítrico Sintasa de Tipo III/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , Tonsila Palatina/enzimología , Neoplasias Tonsilares/enzimología , Tonsilitis/enzimología , Enfermedad Crónica , Citocinas/metabolismo , Humanos , Tonsila Palatina/patología , Neoplasias Tonsilares/patología , Tonsilitis/patologíaRESUMEN
Somatic hypermutation (SHM) is a fundamental process in immunoglobulin gene maturation that results in increased affinity of antibodies toward antigens. In one hypothesis explaining SHM in human B cells, the process is initiated by enzymatic deamination of cytosine to uracil in the immunoglobulin gene V-region and this in turn triggers mutation-prone forms of uracil-DNA base excision repair (BER). Yet, an uncertainty with this model is that BER of uracil-DNA in mammalian cells is generally error-free, wherein DNA polymerase beta (pol beta) conducts gap-filling synthesis by insertion of bases according to Watson-Crick rules. To evaluate this inconsistency, we examined pol beta expression in various SHM proficient human BL2 cell line subclones. We report that expression of pol beta in SHM proficient cell lines was strongly down-regulated. In contrast, in other BL2 subclones, we found that SHM was deficient and that pol beta expression was much higher than in the SHM proficient subclones. We also found that overexpression of recombinant human pol beta in a SHM proficient subclone abrogated its capacity for SHM. These results suggest that down-regulation of the normal BER gap-filling DNA polymerase, pol beta, accompanies induced SHM in BL2 cells. This is consistent with the hypothesis that normal error-free BER must be silenced to make way for an error-prone BER process that may be required during somatic hypermutation.
Asunto(s)
ADN Polimerasa beta/metabolismo , Hipermutación Somática de Inmunoglobulina , Adulto , Secuencia de Bases , Línea Celular , ADN Polimerasa beta/genética , Reparación del ADN , Regulación hacia Abajo , Humanos , Región Variable de Inmunoglobulina/genética , Inmunohistoquímica , Técnicas In Vitro , Masculino , Mutagénesis , Oligodesoxirribonucleótidos/genética , Oligodesoxirribonucleótidos/metabolismo , Tonsila Palatina/enzimología , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidad por SustratoRESUMEN
AIM: To study the PKD2 expression, autophosphorylation and localization in reactive lymph nodes and tumors of lymphoid tissues. MATERIALS AND METHODS: Specific antibodies, which recognize PKD1/2 or PKD2 and autophosphorylated PKD1/2, were used for immunohistochemical and biochemical studies of tonsils, reactive lymph nodes, tumor samples of non-Hodgkin's lymphoma (NHL) and Hodgkin's lymphoma (HL). RESULTS: Immunohistochemical and biochemical analysis of PKD1 and PKD2 expression showed PKD2 expression in tonsils, reactive lymph nodes and tumor tissues from patients with NHL and HL. Furthermore, we were not able to reveal PKD1 expression in studied lymphoid tissues. In tonsils and reactive lymph nodes the PKD2 expression was detected in T and B cell zones with highest level in germinal centers of lymphoid follicles and the maximum level of autophosphorylation in the light zones of the germinal centers. We found that low level of PKD2 expression and autophosphorylation was characteristic feature for mantle cell lymphomas, Burkitt's lymphomas, and in 50% of CLL/small lymphocytic lymphomas. Lymphoma cells of germinal center origin and with activated B cell phenotype (diffuse large B cell lymphomas, HL) and anaplastic large cells lymphoma demonstrated the high level of PKD2 expression and autophosphorylation. CONCLUSIONS: The level of PKD2 expression and autophosphorylation in neoplastic cells corresponds to the expression pattern of this kinase in their normal analogs, and to the level of cell differentiation and activation.
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Enfermedad de Hodgkin/enzimología , Linfoma no Hodgkin/enzimología , Proteínas Quinasas/análisis , Enfermedad de Hodgkin/patología , Humanos , Inmunohistoquímica , Ganglios Linfáticos/enzimología , Linfoma no Hodgkin/patología , Tonsila Palatina/enzimología , Fosforilación , Proteína Quinasa C/análisis , Proteína Quinasa D2RESUMEN
The mammalian target of rapamycin (mTOR) is emerging as a promising target for antitumor therapy. However, the mechanism that contributes to its regulation in B lymphomas remains unknown. This study shows that in follicular lymphoma (FL) cells, mTOR is active because the cells displayed rapamycin-sensitive phosphorylation of p70S6 kinase and 4E-BP1. Moreover, immunohistochemistry applied on lymph node tissue sections obtained from patients with FL revealed that, in most cases, p70S6 kinase was highly phosphorylated compared to normal tonsillar tissue. In FL cells, mTOR was under control of both phospholipase D (PLD) and phosphatidylinositol 3-kinase (PI3K). Moreover, we demonstrated that Syk plays a central role in mTOR activation because we found that both expression and activity are elevated compared to normal or chronic lymphocytic leukemia B cells. We also provide evidence that Syk operates through PLD- and PI3K-independent pathways. Finally, Syk inhibition by piceatannol or by siRNA plasmids resulted in a potent inhibition of mTOR activity in FL cells, as well as in mantle cell lymphoma, Burkitt lymphoma, and diffuse large B-cell lymphoma. These findings suggest that the Syk-mTOR pathway has a critical function in FL survival, and therefore, that Syk could be a promising new target for B-lymphoma therapy.
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Péptidos y Proteínas de Señalización Intracelular/metabolismo , Linfoma Folicular/enzimología , Proteínas de Neoplasias/metabolismo , Proteínas Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Transducción de Señal , Linfoma de Burkitt/tratamiento farmacológico , Linfoma de Burkitt/enzimología , Linfoma de Burkitt/patología , Línea Celular Tumoral , Activación Enzimática/efectos de los fármacos , Humanos , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Leucemia Linfocítica Crónica de Células B/enzimología , Leucemia Linfocítica Crónica de Células B/patología , Ganglios Linfáticos/metabolismo , Ganglios Linfáticos/patología , Linfoma de Células B/tratamiento farmacológico , Linfoma de Células B/enzimología , Linfoma de Células B/patología , Linfoma Folicular/tratamiento farmacológico , Linfoma Folicular/patología , Linfoma de Células del Manto/tratamiento farmacológico , Linfoma de Células del Manto/enzimología , Linfoma de Células del Manto/patología , Tonsila Palatina/enzimología , Tonsila Palatina/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfolipasa D/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/farmacología , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Transducción de Señal/efectos de los fármacos , Estilbenos/farmacología , Quinasa Syk , Serina-Treonina Quinasas TORRESUMEN
Activation-induced cytidine deaminase (AID) is necessary for immunoglobulin somatic hypermutation (SHM) and class switch recombination (CSR) in T-dependent immune response in germinal centers (GCs). The structural similarity of AID with RNA-editing enzymes and its largely cytoplasmic location have fueled controversial views of its mode of interaction with DNA. We show that AID, a mature B-cell-restricted cytoplasmic antigen, is relocated into the nucleus in 2.5% of CDKN1B(-), CCNB1(-) GC cells. The GC dark zone and the outer zone (OZ), but not the light zone, contain nuclear and cytoplasmic AID(+) blasts. AID(+) cells in the OZ are in contact with T cells and CD23(-) follicular dendritic cells. In addition, AID is expressed in extrafollicular large proliferating B cells, 14% of which have nuclear AID. GC and extrafollicular AID(+) cells express E47 but not the inhibiting BHLH protein Id2. Outside the GC, AID(+) B cells are in contact with T cells and show partial evidence of CD40 plus bcr stimulation-dependent signature (CCL22, JunB, cMYC, CD30) but lack early and late plasma cell markers. The distribution of nuclear AID is consistent with the topography of SHM and CSR inside the GC and in extrafollicular activated B cells.