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INTRODUCTION: Serum calcification propensity is emerging as an independent predictor for cardiovascular outcomes in high-risk populations. Calcification propensity can be monitored by the maturation time of calciprotein particles in serum (T50 test). A low T50 value is an independent determinant of cardiovascular morbidity and mortality in various populations. Aim was to investigate the T50 and its relationship to type 2 diabetes mellitus. RESEARCH DESIGN AND METHODS: Using nephelometry, serum T50 was cross-sectionally measured in 932 stable patients with type 2 diabetes mellitus (55% male) with a median age of 66 (62-75) years, diabetes duration of 6.5 (3.0-10.2) years and hemoglobin A1c (HbA1c) of 49 (44-54) mmol/mol. RESULTS: Serum T50 was normally distributed with a mean value of 261±66 min. In linear regression, serum T50 was lower in women and current smokers. A lower T50 value was found in patients with a higher HbA1c or higher systolic blood pressure, insulin users and patients with a longer history of diabetes. The association with HbA1c was independent of other determinants in multivariable analysis. There was no association between T50 and previous macrovascular events or the presence of microvascular disease. CONCLUSIONS: Serum calcification propensity is independently associated with glycemic control, suggesting that a lower HbA1c may be associated with better cardiovascular outcomes. Retrospective analysis could not establish an association between a history of macrovascular events and T50, and prospective studies will have to be performed to address this hypothesis. TRIAL REGISTRATION NUMBER: NCT01570140.
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Calcinosis , Diabetes Mellitus Tipo 2 , Anciano , Diabetes Mellitus Tipo 2/epidemiología , Femenino , Hemoglobina Glucada , Humanos , Masculino , Estudios Prospectivos , Estudios RetrospectivosRESUMEN
Hyalinosis is a vascular lesion affecting the renal vasculature and contributing to aging-related renal function decline. We assessed whether arteriolar hyalinosis is caused by Klotho deficiency, a state known to induce both renal and vascular phenotypes associated with aging. Histochemistry was used to assess hyalinosis in Klotho-/- kidneys, compared with Klotho+/- and wild-type littermates. Immunohistochemistry was used to investigate vascular lesion composition and the different layers of the vascular wall. Finally, spironolactone was used to inhibit calcification in kl/kl mice, and vascular lesions were characterized in the kidney. Arteriolar hyalinosis was detected in Klotho-/- mice, which was present up to the afferent arterioles. Hyalinosis was accompanied by loss of α-smooth muscle actin expression, whereas the endothelial lining was mostly intact. Hyalinous lesions were positive for IgM and iC3b/c/d, indicating subendothelial leakage of plasma proteins. The presence of extracellular matrix proteins suggested increased production by smooth muscle cells (SMCs). Finally, in Klotho-/- mice with marked vascular calcification, treatment with spironolactone allowed for replacement of calcification by hyalinosis. Klotho deficiency potentiates both endothelial hyperpermeability and SMC dedifferentiation. In the absence of a calcification-inducing stimulus, SMCs assume a synthetic phenotype in response to subendothelial leakage of plasma proteins. In the kidney, this results in arteriolar hyalinosis, which contributes to the decline in renal function. Klotho may play a role in preventing aging-related arteriolar hyalinosis.
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Arterioloesclerosis/patología , Glucuronidasa/fisiología , Riñón/patología , Músculo Liso Vascular/patología , Calcificación Vascular/patología , Animales , Arterioloesclerosis/metabolismo , Células Cultivadas , Riñón/metabolismo , Proteínas Klotho , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Liso Vascular/metabolismo , Calcificación Vascular/metabolismoRESUMEN
AIMS: Oxidative stress is a driver in the development of type 2 diabetes (T2DM) complications. As thiols (R-SH) are oxidized by reactive oxygen and sulfur species, circulating concentrations may directly reflect systemic redox status. We hypothesized that high serum R-SH concentrations are a reflection of a favourable redox status and may therefore positively associate with disease status. METHODS: R-SH were measured in serum of 943 T2DM outpatients (55% males, 65â¯years and HbA1c of 6.7% (50â¯mmol/mol)) with a follow-up period of 1.2â¯years. RESULTS: In the highest R-SH tertile patients were younger, more often men, had less microvascular complications, lower HbA1c and were more often treated nutritionally or with oral glucose-lowering drugs. Age- and sex adjusted hazard ratios for developing micro-, macro- or any complication plus death were 0.994, 0.992 and 0.993: even after adjustment for potential confounders. The Harrell's C statistic to predict microvascular complications or any complication plus death was higher in the models with R-SH than in those without R-SH. CONCLUSIONS: Although R-SH concentrations were associated with a favourable disease status, it did not add to the predictive capacity for long-term complications. Based on the current data R-SH seems unsuitable as a prognostic marker in T2DM.
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Klotho is a renal protein involved in phosphate homeostasis, which is downregulated in renal disease. It has long been considered an antiaging factor. Two Klotho gene transcripts are thought to encode membrane-bound and secreted Klotho. Indeed, soluble Klotho is detectable in bodily fluids, but the relative contributions of Klotho secretion and of membrane-bound Klotho shedding are unknown. Recent advances in RNA surveillance reveal that premature termination codons, as present in alternative Klotho mRNA (for secreted Klotho), prime mRNAs for degradation by nonsense-mediated mRNA decay (NMD). Disruption of NMD led to accumulation of alternative Klotho mRNA, indicative of normally continuous degradation. RNA IP for NMD core factor UPF1 resulted in enrichment for alternative Klotho mRNA, which was also not associated with polysomes, indicating no active protein translation. Alternative Klotho mRNA transcripts colocalized with some P bodies, where NMD transcripts are degraded. Moreover, we could not detect secreted Klotho in vitro. These results suggest that soluble Klotho is likely cleaved membrane-bound Klotho only. Furthermore, we found that, especially in acute kidney injury, splicing of the 2 mRNA transcripts is dysregulated, which was recapitulated by various noxious stimuli in vitro. This likely constitutes a novel mechanism resulting in the downregulation of membrane-bound Klotho.
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Glucuronidasa/metabolismo , Degradación de ARNm Mediada por Codón sin Sentido/fisiología , ARN Mensajero/metabolismo , Insuficiencia Renal Crónica/metabolismo , Línea Celular , Codón sin Sentido , Cicloheximida/antagonistas & inhibidores , Silenciador del Gen , Glucuronidasa/genética , Humanos , Hibridación in Situ , Proteínas Klotho , Degradación de ARNm Mediada por Codón sin Sentido/efectos de los fármacos , ARN Helicasas/genética , ARN Helicasas/metabolismo , ARN Mensajero/genética , Transactivadores/genética , Transactivadores/metabolismoRESUMEN
AIMS: Cardiovascular disease (CVD) is the leading cause of death in patients with chronic kidney disease (CKD), a disease state that is strongly associated with loss of renal and systemic (alpha-)Klotho. Reversely, murine Klotho deficiency causes marked medial calcification. It is therefore thought that Klotho conveys a vasculoprotective effect. Klotho expression in the vessel wall, however, is disputed. METHODS AND RESULTS: We assessed Klotho expression in healthy human renal donor arteries (n = 9), CKD (renal graft recipient) arteries (n = 10), carotid endarterectomy specimens (n = 8), other elastic arteries (three groups of n = 3), and cultured human aortic smooth muscle cells (HASMCs) (three primary cell lines), using immunohistochemistry (IHC), immunofluorescence, quantitative reverse transcriptase-polymerase chain reaction, and western blotting (WB). We have extensively validated anti-Klotho antibody KM2076 by comparing staining patterns with other anti-Klotho antibodies (SC-22220, SC-22218, and AF1819), competition assays with recombinant Klotho, IHC on Klotho-deficient kl/kl mouse kidney, and WB with recombinant Klotho. Using KM2076, we could not detect full-length Klotho in vascular tissues or HASMCs. On the mRNA level, using primers against all four exon junctions, klotho expression could not be detected either. Fibroblast growth factor 23 (FGF23) injections in mice induced FGF23 signalling in kidneys but not in the aorta, indicating the absence of Klotho-dependent FGF23 signalling in the aorta. CONCLUSION: Using several independent and validated methods, we conclude that full-length, membrane-bound Klotho is not expressed in healthy or uraemic human vascular tissue.
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Glucuronidasa/metabolismo , Riñón/metabolismo , Arteria Renal/metabolismo , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Animales , Aorta/citología , Aorta/metabolismo , Western Blotting , Células Cultivadas , Factor-23 de Crecimiento de Fibroblastos , Factores de Crecimiento de Fibroblastos/metabolismo , Células HEK293 , Humanos , Arteria Ilíaca/metabolismo , Inmunohistoquímica , Fallo Renal Crónico/metabolismo , Trasplante de Riñón , Proteínas Klotho , Ratones , Persona de Mediana Edad , Miocitos del Músculo Liso/metabolismo , Uremia/metabolismo , Adulto JovenRESUMEN
BACKGROUND: Flat epithelial atypia (FEA) of the breast is characterised by a few layers of mildly atypical luminal epithelial cells. Genetic changes found in ductal carcinoma in situ (DCIS) and invasive ductal breast cancer (IDC) are also found in FEA, albeit at a lower concentration. So far, miRNA expression changes associated with invasive breast cancer, like miR-21, have not been studied in FEA. METHODS: We performed miRNA in-situ hybridization (ISH) on 15 cases with simultaneous presence of normal breast tissue, FEA and/or DCIS and 17 additional cases with IDC. Expression of the miR-21 targets PDCD4, TM1 and PTEN was investigated by immunohistochemistry. RESULTS: Two out of fifteen cases showed positive staining for miR-21 in normal breast ductal epithelium, seven out of fifteen cases were positive in the FEA component and nine out of twelve cases were positive in the DCIS component. A positive staining of miR-21 was observed in 15 of 17 IDC cases. In 12 cases all three components were present in one tissue block and an increase of miR-21 from normal breast to FEA and to DCIS was observed in five cases. In three cases the FEA component was negative, whereas the DCIS component was positive for miR-21. In three other cases, normal, FEA and DCIS components were negative for miR-21 and in the last case all three components were positive. Overall we observed a gradual increase in percentage of miR-21 positive cases from normal, to FEA, DCIS and IDC. Immunohistochemical staining for PTEN revealed no obvious changes in staining intensities in normal, FEA, DCIS and IDC. Cytoplasmic staining of PDCD4 increased from normal to IDC, whereas, the nuclear staining decreased. TM1 staining decreased from positive in normal breast to negative in most DCIS and IDC cases. In FEA, the staining pattern for TM1 was similar to normal breast tissue. CONCLUSION: Upregulation of miR-21 from normal ductal epithelial cells of the breast to FEA, DCIS and IDC parallels morphologically defined carcinogenesis. No clear relation was observed between the staining pattern of miR-21 and its previously reported target genes.
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Proteínas Reguladoras de la Apoptosis/genética , Neoplasias de la Mama/genética , Carcinoma Ductal de Mama/genética , Epitelio/metabolismo , Expresión Génica , MicroARNs/genética , Fosfohidrolasa PTEN/genética , Proteínas de Unión al ARN/genética , Tropomiosina/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Carcinoma Ductal de Mama/metabolismo , Carcinoma Ductal de Mama/patología , Femenino , Humanos , MicroARNs/metabolismo , Invasividad Neoplásica , Fosfohidrolasa PTEN/metabolismo , Proteínas de Unión al ARN/metabolismo , Tropomiosina/metabolismoRESUMEN
MicroRNAs (miRNAs) are an important class of small RNAs that regulate gene expression at the post-transcriptional level. It has become evident that miRNAs are involved in hematopoiesis, and that deregulation of miRNAs may give rise to hematopoietic malignancies. The aim of our study was to establish miRNA profiles of naïve, germinal center (GC) and memory B cells, and validate their expression patterns in normal lymphoid tissues. Quantitative (q) RT-PCR profiling revealed that several miRNAs were elevated in GC B cells, including miR-17-5p, miR-106a and miR-181b. One of the most abundant miRNAs in all three B-cell subsets analyzed was miR-150, with a more than 10-fold lower level in GC B cell as compared with the other two subsets. miRNA in situ hybridization (ISH) in tonsil tissue sections confirmed the findings from the profiling work. Interestingly, gradual decrease of miR-17-5p, miR-106a and miR-181b staining intensity from the dark to the light zone was observed in GC. A strong cytoplasmic staining of miR-150 was observed in a minority of the centroblasts in the dark zone of the GC. Inverse staining pattern of miR-150 against c-Myb and Survivin was observed in tonsil tissue sections, suggesting possible targeting of these genes by miR-150. In line with this, the experimental induction of miR-150 lead to reduced c-Myb, Survivin and Foxp1 expression levels in the Burkitt's lymphoma cell line, DG75. In conclusion, miRNA profiles of naïve, GC and memory B cells were established and validated by miRNA ISH. Within the GC cells, a marked difference was observed between the light and the dark zone.
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Subgrupos de Linfocitos B/metabolismo , Centro Germinal/metabolismo , MicroARNs/metabolismo , Tonsila Palatina/metabolismo , Adolescente , Subgrupos de Linfocitos B/inmunología , Línea Celular Tumoral , Niño , Preescolar , Perfilación de la Expresión Génica , Centro Germinal/inmunología , Humanos , Hibridación in Situ , Tonsila Palatina/inmunología , Tonsila Palatina/patología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tonsilitis/inmunología , Tonsilitis/metabolismo , Tonsilitis/patologíaRESUMEN
Recent studies have shown that certain non-coding short RNAs, called miRNAs, play an important role in diffuse large B-cell lymphomas. Patients with diffuse large B-cell lymphoma have great diversity in both clinical characteristics, site of presentation and outcome. The aim of our study is to validate the differential expression in germinal center and non-germinal center diffuse large B-cell lymphoma,s and to study to the extent to which the primary site of differentiation is associated with the miRNA expression profile. We studied 50 cases of de novo diffuse large B-cell lymphoma for the expression of 15 miRNAs (miR-15a, miR-15b, miR-16, miR-17-3p, miR-17-5p, miR-18a, miR-19a, miR-19b, miR-20a, miR-21, miR-92, miR-127, miR-155, miR-181a and miR-221). Apart from 19 nodal cases without extranodal dissemination (stages I and II), we selected two groups with unambiguous stages I and II extranodal presentation; 9 cases of primary central nervous system, 11 cases of primary testicular and 11 cases of other primary extranodal diffuse large B-cell lymphomas. All cases were analyzed with qRT-PCR. In situ hybridization for the most differentially expressed miRNAs was performed to show miRNA expression in tumor cells, but not in background cells. MiR-21 and miR-19b showed the highest expression levels. No significant differences were seen between germinal center and non-germinal center diffuse large B-cell lymphomas in either the total or the nodal group for any of the 15 miRNAs. Two miRNAs showed significant differences in expression levels for diffuse large B-cell lymphoma subgroups according to the site of presentation. MiR-17-5p showed a significant higher expression level in the central nervous system compared with testicular and nodal diffuse large B-cell lymphomas (P<0.05). MiR-127 levels were significantly higher in testicular than in central nervous system and in nodal diffuse large B-cell lymphomas (P<0.05). We conclude that the location of diffuse large B-cell lymphoma is an important factor in determining the differential expression of miRNAs.
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Neoplasias del Sistema Nervioso Central/genética , Linfoma de Células B Grandes Difuso/genética , MicroARNs/biosíntesis , Neoplasias Testiculares/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Neoplasias del Sistema Nervioso Central/patología , Femenino , Centro Germinal/patología , Humanos , Hibridación in Situ , Linfoma de Células B Grandes Difuso/patología , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Neoplasias Testiculares/patología , Análisis de Matrices TisularesRESUMEN
Hodgkin lymphoma (HL) is derived from preapoptotic germinal center B cells, although a general loss of B cell phenotype is noted. Using quantitative reverse transcription-polymerase chain reaction and miRNA microarray, we determined the microRNA (miRNA) profile of HL and compared this with the profile of a panel of B-cell non-Hodgkin lymphomas. The two methods showed a strong correlation for the detection of miRNA expression levels. The HL-specific miRNA included miR-17-92 cluster members, miR-16, miR-21, miR-24, and miR-155. Using a large panel of cell lines, we found differential expression between HL and other B-cell lymphoma-derived cell lines for 27 miRNA. A significant down-regulation in HL compared to non-Hodgkin lymphoma was observed only for miR-150. Next, we performed target gene validation of predicted target genes for miR-155, which is highly expressed in HL and is differentially expressed between HL and Burkitt lymphoma. Using luciferase reporter assays, we validated 11 predicted miR-155 target genes in three different HL cell lines. We demonstrated that AGTR1, FGF7, ZNF537, ZIC3, and IKBKE are true miR-155 target genes in HL.
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Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Enfermedad de Hodgkin/genética , Enfermedad de Hodgkin/patología , MicroARNs/análisis , MicroARNs/genética , Línea Celular Tumoral , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Little is known about the gene expression profile and significance of the rosetting CD4+CD26- T cells in classical Hodgkin's lymphoma (cHL). To characterize these T cells, CD4+CD26- and CD4+CD26+ T-cell populations were sorted from lymph node (LN) cell suspensions from nodular sclerosis HL (NSHL) and reactive LNs. mRNA profiles of stimulated and resting cell subsets were evaluated with quantitative RT-PCR for 46 genes. We observed a higher percentage of CD4+CD26- T cells in NSHL than in reactive LNs. The resting CD4+CD26- T cells in NSHL showed higher mRNA levels of CD25, CTLA4, OX40 and CCR4 compared with in LNs, supporting a regulatory T-cell (Treg) type, and this was validated by immunohistochemistry. Moreover, these cells showed low or no expression of the Th1- or Th2-related cytokines IL-2, IFN-gamma, IL-13, IL-12B, IL-4, and IL-5, and the chemoattractant receptor CRTH2. Besides Tregs, Th17 cells may exist in NSHL based on the significantly higher IL-17 mRNA level for both T-cell populations in NSHL. Upon stimulation in vitro, lack of upregulation of mRNA levels of most cytokine genes indicated an anergic character for the CD4+CD26- T-cell subset. Anergy fits with the Treg profile of these cells, probably explaining the immunosuppressive mechanism involved in NSHL.
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Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/patología , Dipeptidil Peptidasa 4/metabolismo , Enfermedad de Hodgkin/patología , Linfocitos T Reguladores/patología , Adolescente , Adulto , Linfocitos T CD4-Positivos/efectos de los fármacos , Niño , Femenino , Citometría de Flujo , Expresión Génica , Enfermedad de Hodgkin/genética , Enfermedad de Hodgkin/metabolismo , Humanos , Inmunohistoquímica/métodos , Ionomicina/farmacología , Ganglios Linfáticos/patología , Masculino , ARN Mensajero/metabolismo , Reproducibilidad de los Resultados , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Esclerosis , Coloración y Etiquetado , Subgrupos de Linfocitos T/metabolismo , Subgrupos de Linfocitos T/patología , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Galectin-1 is the homodimeric form of a protein, which is present in a dynamic equilibrium with the beta-galactoside monomeric form and has potent anti-inflammatory and immunomodulating effects. These favorable effects are probably related to the induction of apoptosis in activated T cells and the induction of IL-10, which have been demonstrated to be characteristic for the dimeric form of the protein. Based on these findings it can be speculated that the in vivo effects of galectin-1 can be improved by the generation of stable galectin-1 homodimers (dGal). To test this hypothesis we produced leucine-zipper based stable galectin-1 homodimers and tested its apoptosis inducing effects on MOLT-4 cells and its immunomodulatory effects in vitro on PBMC of five independent donors. Phosphatidylserine exposure and a drop in mitochondrial membrane potential was strongly enhanced on MOLT-4 cells upon treatment with dGal as compared to wtGal. The minimal effective concentration was 20-fold reduced as compared to the minimal effective wtGal concentration. dGal showed enhanced induction of IL-10 on total PBMC as compared to treatment with wild-type protein (wtGal). The minimal effective dGal concentration was 100-fold lower than that of wtGal. Of the purified cell populations monocytes are the strongest IL-10 producers, whereas T cells induce IL-10 at a lower level and no induction is observed in B cells. Besides induction of IL-10, dGal caused an increase in IL-1beta production in all donors and a reduction of IL-2 production in 3 out of 5 donors, whereas no consistent changes were observed for other inflammatory cytokines. In summary, we demonstrated that dGal shows enhanced effects at strongly reduced concentrations. Application of dGal may therefore serve as an improved treatment of chronic inflammatory diseases.
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Apoptosis/efectos de los fármacos , Galectina 1/farmacología , Interleucina-10/biosíntesis , Leucocitos Mononucleares/metabolismo , Proteínas Recombinantes/farmacología , Secuencia de Aminoácidos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Galectina 1/genética , Humanos , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/patología , Datos de Secuencia Molecular , Proteínas Recombinantes/genética , Regulación hacia Arriba/efectos de los fármacosRESUMEN
To identify genes involved in the pathogenesis of classical Hodgkin lymphoma (cHL), we performed serial analysis of gene expression (SAGE) and array-based comparative genomic hybridization (aCGH). Comparison of SAGE libraries of cHL cell lines L428 and L1236 with that of germinal centre B cells revealed consistent overexpression of only 14 genes. In contrast, 141 genes were downregulated in both cHL cell lines, including many B cell and HLA genes. aCGH revealed gain of 2p, 7p, 9p, 11q and Xq and loss of 4q and 11q. Eighteen percent of the differentially expressed genes mapped to regions with loss or gain and a good correlation was observed between underexpression and loss or overexpression and gain of DNA. Remarkably, gain of 2p and 9p did not correlate with increased expression of the proposed target genes c-REL and JAK2. Downregulation of many genes within the HLA region also did not correlate with loss of DNA. FSCN1 and IRAK1 mapping at genomic loci (7p and Xq) that frequently showed gain were overexpressed in cHL cell lines and might be involved in the pathogenesis of cHL.
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Perfilación de la Expresión Génica/métodos , Genes Relacionados con las Neoplasias/genética , Enfermedad de Hodgkin/genética , Proteínas Portadoras/genética , Línea Celular Tumoral , Regulación hacia Abajo , Antígenos HLA/genética , Enfermedad de Hodgkin/etiología , Humanos , Quinasas Asociadas a Receptores de Interleucina-1/genética , Janus Quinasa 2/genética , Proteínas de Microfilamentos/genética , Hibridación de Ácido Nucleico , Proteínas Proto-Oncogénicas c-rel/genética , ARN Neoplásico/análisis , Regulación hacia ArribaRESUMEN
DEV is the only cell line derived from nodular lymphocyte predominance type of Hodgkin's lymphoma (NLPHL); however, a comprehensive report about the genetic and immunophenotypic profile of this unique cell line is lacking. We analyzed DEV with respect to immunophenotype and genetic aberrations. The immunostaining revealed positivity for CD45, CD20, CD22, CD79a, IgA2, CD80, CD86, CD74, and BCL6. Cytogenetically, DEV has complex chromosome 3 translocations involving chromosomes 7, 14, and 22. A detailed analysis of the 3q27 breakpoint of the der(3)t(3;14)(p14;q32)t(3;22)(q27;q11.2) revealed a break in the BCL6 alternative breakpoint region. Using array comparative genomic hybridization, a 3-megabase homozygous deletion at 17q24.1-24.2 was identified. Fluorescence in situ hybridization indicated the presence of 2 chromosome 17 homologues, each of which carried a small interstitial deletion. Eight microsatellite markers flanking the homozygously deleted region all showed a homozygous pattern suggesting loss of one of the parental alleles. D17S1809 and D17S1816 could not be amplified using DEV DNA, in keeping with a location within the homozygously deleted segment. In conclusion, DEV has an immunophenotype that is consistent with the neoplastic cells of NLPHL cases, the lymphocytic and histiocytic cells. We demonstrated involvement of the BCL6 gene based on the presence of a breakpoint in the alternative breakpoint region and nuclear staining for BCL6 protein and identified a homozygously deleted region at 17q24.
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Deleción Cromosómica , Cromosomas Humanos Par 17 , Proteínas de Unión al ADN/genética , Enfermedad de Hodgkin/genética , Linfocitos/fisiología , Línea Celular Tumoral , Rotura Cromosómica , Mapeo Cromosómico , Análisis Citogenético , Marcadores Genéticos , Enfermedad de Hodgkin/patología , Homocigoto , Humanos , Inmunohistoquímica , Inmunofenotipificación , Hibridación Fluorescente in Situ , Masculino , Repeticiones de Microsatélite , Persona de Mediana Edad , Proteínas Proto-Oncogénicas c-bcl-6RESUMEN
Anaplastic large cell lymphomas (ALCL) are characterised by the presence of CD30-positive large cells, which usually are of T-cell type. Based on the presence or absence of translocations involving the anaplastic lymphoma kinase (ALK) locus, ALCL cases can be divided into two groups. To gain more insight in the biology of ALCL, we applied serial analysis of gene expression (SAGE) on the Karpas299 cell line and identified 25 up- and 19 downregulated genes. Comparison of the differentially expressed genes with DNA copy number changes in Karpas299 revealed that two overexpressed genes, S100A10 and S100A11, were located in an amplicon suggesting that the increased mRNA levels were caused by DNA amplification. Quantitative reverse transcription polymerase chain reaction on 5 ALCL cell lines and 12 ALCL tissues confirmed the SAGE data for 13 out of 14 up- and one out of four downregulated genes. Immunohistochemical staining confirmed the presence of S100A10, a calcium-binding protein, in three out of five ALK+ and all 7 ALK- ALCL cases. S100A11 staining was confirmed in all ALK+ and six of seven ALK- ALCL cases. Three of the upregulated genes represented calcium-binding proteins, which suggest that altered intracellular signaling might be associated with the oncogenesis of ALCL.
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Anexina A2/genética , Señalización del Calcio/genética , Calmodulina/genética , Regulación Neoplásica de la Expresión Génica , Linfoma de Células B Grandes Difuso/genética , Proteínas S100/genética , Anexina A2/análisis , Linfocitos T CD4-Positivos/metabolismo , Línea Celular Tumoral , Células Cultivadas , Regulación hacia Abajo , Galectina 1/análisis , Galectina 1/genética , Perfilación de la Expresión Génica , Humanos , Inmunohistoquímica/métodos , Linfoma de Células B Grandes Difuso/metabolismo , Proteínas de la Membrana/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas S100/análisisRESUMEN
In a previous study we demonstrated high expression of the non-coding BIC gene in the vast majority of Hodgkin's lymphomas (HLs). Evidence suggesting that BIC is a primary microRNA transcript containing the mature microRNA-155 (miR-155) as part of a RNA hairpin is now accumulating. We therefore analysed HL cell lines and tissue samples to determine whether miR-155 is also expressed in HL. High levels of miR-155 could be demonstrated, indicating that BIC is processed into a microRNA in HL. Most non-HL subtypes were negative for BIC as determined by RNA-ISH. However, in diffuse large B cell lymphoma (DLBCL) and primary mediastinal B cell lymphoma (PMBL), significant percentages of positive tumour cells were observed in 12/18 and 8/8 cases. A higher proportion of tumour cells were positive for BIC in DLBCL with activated B cell-like phenotype than in DLBCL with germinal centre B cell-like phenotype. Differential BIC expression was confirmed by qRT-PCR analysis. Northern blot analysis showed expression of miR-155 in all DLBCL and PMBL derived cell lines and tissue samples analysed. In summary, we demonstrate expression of primary microRNA BIC and its derivative miR-155 in HL, PMBL and DLBCL.
Asunto(s)
Linfoma/genética , Neoplasias del Mediastino/genética , MicroARNs/genética , Receptores de Antígenos de Linfocitos B/genética , Línea Celular Tumoral , Enfermedad de Hodgkin/genética , Humanos , Inmunohistoquímica/métodos , Hibridación in Situ/métodos , Linfoma de Células B/genética , Linfoma de Células B Grandes Difuso/genética , ARN Neoplásico/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodosRESUMEN
Anaplastic large cell lymphomas (ALCLs) can be subdivided into two subgroups on the basis of their expression of the ALK protein. ALK protein expression leads to activation of signal transducer and activator of transcription (STAT) 3, which is more commonly expressed in ALK-positive than in ALK-negative tumours. Activated STAT3 leads to the induction of several genes such as Mcl-1, Bcl-2 and Bcl-X(L), and tissue inhibitor of metalloproteinase (TIMP)-1. In this study, we analysed TIMP-1 expression in five ALCL cell lines and 11 tumours by quantitative RT-PCR and immunohistochemistry. We identified high-level TIMP-1 expression by RT-PCR in three ALK-positive ALCL-derived cell lines and in all ALK-positive ALCLs, whereas ALK-negative ALCLs generally demonstrated a lower level of TIMP-1 expression. Concordant with these results, we observed TIMP-1 immunostaining in all ALK-positive ALCLs and in only two of six ALK-negative ALCLs. No relationship was observed between the levels of ALK and TIMP-1 expression in the ALK-positive tumours. STAT3 expression levels were similar in all ALCL samples. Double staining with either CD30 or CD68 demonstrated that TIMP-1 expression was restricted to macrophages in the majority of TIMP-1-positive tumours. Expression of the TIMP-1 substrate MMP-2 was more prominent in ALK-negative tumours, while MMP-9 levels were low in all cases. Expression levels of IL-6 and TGF-beta1, which are cytokines known to induce TIMP-1, were higher in ALK-negative ALCLs and moderate in ALK-positive tumours. No clear relationship was observed between IL-10 expression and ALK positivity. Overall, no correlation was seen in ALCLs between the expression of TIMP-1 and that of cytokines that induce TIMP-1. Lack of TIMP-1 expression in the tumour cells of ALK-positive ALCLs argues against a direct role for ALK-induced activation of STAT3 in the regulation of TIMP-1 expression in ALCL.
Asunto(s)
Linfoma de Células B Grandes Difuso/química , Macrófagos/química , Inhibidor Tisular de Metaloproteinasa-1/análisis , Quinasa de Linfoma Anaplásico , Antígenos CD/análisis , Línea Celular Tumoral , Citocinas/análisis , Proteínas de Unión al ADN/análisis , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Inmunohistoquímica/métodos , Linfoma de Células B Grandes Difuso/genética , Linfoma de Células B Grandes Difuso/patología , Metaloproteinasas de la Matriz/análisis , Proteínas de Neoplasias/análisis , Proteínas Tirosina Quinasas/análisis , Proteínas Tirosina Quinasas Receptoras , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Factor de Transcripción STAT3 , Transactivadores/análisisRESUMEN
Since Hodgkin and Reed-Sternberg (HRS) cells of Hodgkin lymphoma (HL) generally have immunoglobulin gene rearrangements, they are considered to be of B-cell origin. One of the characteristics of HRS cells is a prominent production of various cytokines and chemokines. Cytokine production is generally driven by expression of T-cell transcription factors (TFs). Only limited information is available on the expression of T-cell TFs in HL. Expression of four T-cell TFs and the target cytokine spectrum of these TFs were analyzed in six HL and three large B-cell lymphoma (LBCL) cell lines using quantitative PCR. ERM expression was observed in all HL and LBCL cell lines. Out of HL cell lines, T-bet was expressed in five, GATA-3 in four, and c-Maf in two cell lines. Immunohistochemistry in HL tissues revealed that in 11 of 12 (92%) of the classical HL cases HRS cells were GATA-3 and/or T-bet positive. In three of six cases of nodular lymphocyte predominance type of HL, the neoplastic cells were T-bet positive. Overall, the T-cell TF and cytokine profiles of the HL cell lines showed a considerable degree of consistency. The expression of T-cell TFs may explain the production of various cytokines by HL cell lines and HRS cells.
Asunto(s)
Proteínas de Unión al ADN/genética , Enfermedad de Hodgkin/genética , Transactivadores/genética , Factores de Transcripción/genética , Linfocitos B/patología , Secuencia de Bases , Línea Celular Tumoral , Cartilla de ADN , Factor de Transcripción GATA3 , Reordenamiento Génico de Linfocito B/genética , Enfermedad de Hodgkin/patología , Humanos , Inmunohistoquímica , Interleucinas/genética , Reacción en Cadena de la Polimerasa , Células de Reed-Sternberg/patología , Proteínas de Dominio T Box , Linfocitos T/patologíaRESUMEN
Galectin-1, a beta-galactoside binding protein that can occur as both a monomer and a homodimer, binds to leucocyte membrane antigens such as CD7, CD43, and CD45, and has immune-regulatory functions in several animal models of autoimmune disease. However, its mechanism of action is only partially understood. In this study, a marked increase in IL-10 mRNA and protein levels was demonstrated in non-activated and activated CD4(+) and CD8(+) T-cells, following treatment with a high concentration (dimeric form), but not a low concentration (monomeric form), of recombinant galectin-1 protein. IL-10 is known to suppress TH1 type immune responses and upregulation of IL-10 may thus contribute to the immune-regulatory function of galectin-1. Galectin-1 was strongly expressed on the endothelial cells of human kidney allografts, suggesting a role in the regulation of immune responses in transplantation. Administration of high concentrations of galectin-1 may be a useful tool in the treatment of T-cell-mediated diseases.