RESUMEN
The comet assay is a useful technique for the study of genetic damage in humans exposed to environmental mutagens and carcinogens. In this study the effects of hydrogen peroxide (H(2)O(2)) and ultraviolet (UV) irradiation on 80 healthy individuals living in urban and rural areas with different smoking habits were investigated. Endonuclease III (endo III) treatment was also used to reveal the level of oxidized pyrimidine formation in these groups. The extent of damage and subsequent repair appear to be influenced by the living conditions (urban or rural areas). Smoking, however, was shown to have the most significant effect on DNA damage on all groups studied.
Asunto(s)
Contaminación del Aire , Daño del ADN , Reparación del ADN , Peróxido de Hidrógeno/toxicidad , Linfocitos/citología , Fumar , Rayos Ultravioleta , Adulto , Células Cultivadas , Ensayo Cometa , Criopreservación , Grecia , Humanos , Linfocitos/efectos de los fármacos , Linfocitos/efectos de la radiación , Masculino , Persona de Mediana Edad , Población Rural , Población UrbanaRESUMEN
The pesticides in use in Greek greenhouses include a number of agents known to be mutagens and carcinogens. In the present study, we evaluated whether occupational exposure of agricultural workers to a complex mixture of pesticides resulted in a significant increase in DNA damage in human peripheral blood lymphocytes (PBLs). A total of 116 healthy individuals were divided into groups based on exposure to pesticides, smoking status, and gender. Alkaline comet assays performed on PBLs from these individuals indicated no statistically significant differences in basal DNA damage between the study groups. In addition, exposure of PBLs to a dose of hydrogen peroxide led to a similar degree of DNA damage and subsequent repair for all the study populations. The results of the study indicate that the agricultural workers who participated in this study had no detectable increase in DNA damage or alteration in the cellular response to DNA damage.
Asunto(s)
Daño del ADN , Monitoreo del Ambiente , Linfocitos/efectos de los fármacos , Exposición Profesional/efectos adversos , Plaguicidas/efectos adversos , Adulto , Ensayo Cometa , Reparación del ADN , Femenino , Grecia , Humanos , Peróxido de Hidrógeno/farmacología , Masculino , Persona de Mediana Edad , Exposición Profesional/análisis , Oxidantes/farmacologíaRESUMEN
This report is an analysis of data relating to the epitopes of 28 murine monoclonal antibodies reactive with the protein core of human carcinoma-associated MUC1 mucins. All anti-MUC1 antibodies define epitopes of linear sequences of 3, 4 or 5 amino acids within the hydrophilic domain, APDTRPAP, which is expressed multiple times in a highly conserved 20 amino acid repeat sequence of the MUC1 core. The R residue is present in the epitopes defined by all of the 28 anti-MUC1 monoclonal antibodies. Epitopes of antibodies originally prepared against immunogens containing human milk fat globule membranes include the motif DTR in over 90% of the examples studied.
RESUMEN
Many murine monoclonal antibodies against MUC1 mucin have been analysed for reactivity against short overlapping peptides with sequences based upon that of the 20 amino acid tandem repeat of the MUC1 protein core, in order to identify the minimum binding units or epitopes. This report summarises the findings using a set of overlapping heptamers to identify the epitopes of 5 established anti-MUC1 antibodies. The results are evaluated and reviewed in comparison to those obtained using other length peptides and other assay formats for epitope mapping using the Pepscan technique.
Asunto(s)
Anticuerpos Monoclonales , Epítopos/análisis , Mucina-1/inmunología , Fragmentos de Péptidos/química , Secuencia de Aminoácidos , Animales , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/patología , Membrana Celular/inmunología , Epítopos/química , Femenino , Glucolípidos/química , Glucolípidos/inmunología , Glicoproteínas/química , Glicoproteínas/inmunología , Humanos , Inmunoglobulina G , Gotas Lipídicas , Ratones , Leche Humana/inmunología , Datos de Secuencia Molecular , Mucina-1/química , Mucina-1/orina , Fragmentos de Péptidos/síntesis químicaRESUMEN
BU combined with CY, the preferred preparatory regimen for thalassemic patients, is associated with a substantial incidence of graft rejection especially in patients with advanced disease stage. This study retrospectively analyzes the outcome of 75 consecutive pediatric patients with ß-thalassemia who underwent HLA-matched sibling transplantation after anti-thymocyte globulin (ATG)-containing myeloablative conditioning regimens. With a median follow-up of 9 years (range 1-15 years), the overall survival (OS) and thalassemia free survival (TFS) rates were 96% and 92%, respectively. Both the estimated TRM and the cumulative incidence of rejection/failure were 4%. The cumulative incidences of acute GVHD grade II-III and grade III were 20% and 5.3%, respectively. No patient developed acute GVHD grade IV. Only two patients developed extensive chronic GVHD. The estimated OS and TFS for patients with Class 1 and 2 disease according to Pesaro criteria were 96.3% and 94.4%, whereas for patients with Class 3 disease they were 94.1% and 88.2%, respectively. In our series, the use of myeloablative conditioning regimens, which include ATG for the transplantation of thalassemic children from matched sibling donors, resulted in excellent outcomes with very low incidences of TRM and rejection.
Asunto(s)
Suero Antilinfocítico/administración & dosificación , Inmunosupresores/administración & dosificación , Donadores Vivos , Hermanos , Trasplante de Células Madre , Acondicionamiento Pretrasplante/métodos , Talasemia beta/mortalidad , Talasemia beta/terapia , Adolescente , Niño , Preescolar , Supervivencia sin Enfermedad , Femenino , Estudios de Seguimiento , Rechazo de Injerto/etiología , Rechazo de Injerto/mortalidad , Rechazo de Injerto/terapia , Enfermedad Injerto contra Huésped/etiología , Enfermedad Injerto contra Huésped/mortalidad , Enfermedad Injerto contra Huésped/terapia , Grecia/epidemiología , Prueba de Histocompatibilidad , Humanos , Lactante , Masculino , Estudios Retrospectivos , Tasa de Supervivencia , Trasplante HomólogoAsunto(s)
Células de la Médula Ósea/citología , Células del Estroma/citología , Quimera por Trasplante , Molécula 1 de Adhesión Celular Vascular , Antígenos CD , Trasplante de Médula Ósea/métodos , Técnicas de Cultivo de Célula , Endoglina , Humanos , Separación Inmunomagnética , Receptores de Superficie Celular , Trasplante HomólogoRESUMEN
BACKGROUND: Inflammation due to perinatal infection (PI) and perinatal asphyxia (PA) may cause damage to various tissues and very often to the immature brain of the fetus and the newborn. Previously, we have shown that the neonatal immune system has the ability to produce increased chemokine protein levels in the serum during the inflammatory response caused by PI and PA. AIM: The aim of our present study was to investigate mRNA levels of the proinflammatory chemokines interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1) in peripheral blood leukocytes from infected and asphyxiated neonates. METHODS: Forty-two premature neonates were studied; 11 with PI, 16 with PA and 15 without PA and PI, were used as controls. IL-8 and MCP-1 mRNA levels were investigated in whole blood and in phytohemagglutinin-activated lymphocytes using semi-quantitative polymerase chain reaction and real-time polymerase chain reaction, respectively. RESULTS: IL-8 mRNA levels were significantly increased in whole blood both during PA and PI, while MCP-1 mRNA levels were not. In vitro activated lymphocytes expressed significantly increased IL-8 mRNA levels during PI, whereas no increase was observed during PA. MCP-1 mRNA levels were significantly increased in activated lymphocytes during PA, while no increase was observed during PI. CONCLUSIONS: Our data show that chemokine mRNA levels expressed by activated lymphocytes during inflammation caused by PIs are different to those expressed during PAs. These findings might have important implications during the administration of specific chemokine antagonists in order to prevent or reduce tissue damage caused by inflammation.
Asunto(s)
Asfixia Neonatal/sangre , Quimiocina CCL2/biosíntesis , Infección Hospitalaria/sangre , Interleucina-8/biosíntesis , Leucocitos Mononucleares/metabolismo , ARN Mensajero/metabolismo , Quimiocina CCL2/sangre , Quimiocina CCL2/genética , Expresión Génica , Humanos , Recién Nacido , Interleucina-8/sangre , Interleucina-8/genética , Leucocitos Mononucleares/efectos de los fármacos , Activación de Linfocitos , Mitógenos/farmacología , Fitohemaglutininas/farmacologíaRESUMEN
BACKGROUND: Sunlight precipitates a series of genetic events that lead to the development of skin cancers such as basal cell carcinoma (BCC) and squamous cell carcinoma (SCC). The p53 tumour suppressor gene, which plays a pivotal role in cell division and apoptosis, is frequently found mutated in sunlight-induced skin tumours. OBJECTIVE: To investigate the immunoreactivity of the p53 gene in non-melanoma skin cancers and to correlate its expression with apoptotic and cell proliferation markers. METHODS: We analysed 35 non-melanoma tumours including 19 BCCs and 16 SCCs from sun-exposed skin areas. p53 protein expression was studied immunohistochemically using the DO7 monoclonal antibody against wild-type and mutant p53 forms. The percentage of p53-immunopositive nuclei was measured by image analysis. Cell proliferation and apoptosis were also assessed by image analysis following Ki-67 immunostaining and application of the TUNEL method on paraffin sections, respectively. RESULTS: The percentage of p53-expressing cells varied from 3.5 to 90 in BCCs (median value 54.4%) and from 3.7 to 94 in SCCs (median value 40.3%). The mean value of Ki-67-positive cells was comparable in both groups of tumours with a mean value of 40.6% in BCCs and 34.6% in SCCs. Conversely, the TUNEL assay showed sporadic staining of apoptotic cells within the tumours with a mean value of 1.12% in BCCs and 1.8% in SCCs. p53 protein expression was correlated positively with cell proliferation (r = 0.75, P = 0.000001) and negatively with apoptosis (r = -0.23, P = 0.05). CONCLUSION: p53 immunoreactivity was high in the majority of the skin carcinomas examined and correlated positively with cell proliferation and negatively with apoptosis. The p53 protein overexpression appears to be related to an inactivated protein resulting from mutations of the p53 gene or other unclear molecular mechanisms.
Asunto(s)
Carcinoma Basocelular/genética , Carcinoma de Células Escamosas/genética , Genes p53/genética , Neoplasias Cutáneas/genética , Proteína p53 Supresora de Tumor/genética , Anciano , Apoptosis , Carcinoma Basocelular/patología , Carcinoma de Células Escamosas/patología , División Celular , Femenino , Humanos , Inmunohistoquímica , Inmunofenotipificación , Masculino , Mutación , Neoplasias Cutáneas/patología , Luz Solar , Proteína p53 Supresora de Tumor/biosíntesisRESUMEN
A panel of 56 murine monoclonal antibodies against the MUC1 mucin was analysed for reactivity against purified MUC1 mucin and synthetic MUC1 mucin protein core-related peptides. In an ELISA assay, with purified normal urinary mucin as the target antigen, 48/56 (86%) of antibodies showed positive reactivity. A smaller proportion of antibodies, 31/56 (55%), reacted with a bovine serum albumin conjugate containing the synthetic peptide, APDTRPAPG. This peptide represents the hydrophilic region of the tandem repeat sequence of the MUC1 mucin core. These 31 anti-mucin core antibodies were then evaluated for reactivity with a set of overlapping heptamers with sequences based upon that of the 20-amino acid tandem repeat of the MUC1 protein core. For this purpose, the 'Pepscan' procedure was employed to detect antibody binding to peptides tethered at their carboxy termini to the polypropylene pins. Synthetic peptides binding to these antibodies were identified for 29 of the samples provided and epitopes or 'minimum binding units' were deduced for these. Most antibodies bound to epitopes of 4 amino acid residues with the sequence RPAP occurring most frequently. The results confirm that the short hydrophilic region PDTRPAP in the MUC1 mucin core is immunodominant in the induction of antibodies to the MUC1 mucin since half of the antibodies submitted reacted with this peptide.
Asunto(s)
Anticuerpos Monoclonales/análisis , Mapeo Epitopo , Epítopos Inmunodominantes/inmunología , Mucina-1/inmunología , Fragmentos de Péptidos/inmunología , Secuencia de Aminoácidos , Animales , Especificidad de Anticuerpos/inmunología , Bovinos , Ensayo de Inmunoadsorción Enzimática , Humanos , Ratones , Datos de Secuencia MolecularRESUMEN
The aim of this work was to investigate the relationship between mechanisms of DNA repair and apoptosis induced by oxidative stress (H2O2) in human lymphocytes. Using the comet assay, fluorescent microscopy, and DNA electrophoresis, we studied the DNA damage induced by hydrogen peroxide (H2O2) treatment, the time and the amount of repair of strand breaks, the type of cell death, and the influence of inhibitors of repair (nicotinamide). When lymphocytes were treated with H2O2, we observed an increased in necrosis compared to apoptosis. However, when nicotinamide (which inhibits DNA repair) was added, the mode of death reversed to increased apoptosis. These results indicate that nicotinamide "protects" resting lymphocytes exposed to H2O2 from necrosis but not from apoptosis.
Asunto(s)
Apoptosis/efectos de los fármacos , Peróxido de Hidrógeno/toxicidad , Linfocitos/efectos de los fármacos , Niacinamida/farmacología , Supervivencia Celular/efectos de los fármacos , Ensayo Cometa , Daño del ADN/efectos de los fármacos , Fragmentación del ADN/efectos de los fármacos , Reparación del ADN/efectos de los fármacos , Electroforesis en Gel de Agar , Humanos , Linfocitos/patología , NecrosisRESUMEN
Antibodies (Abs) to MUC1 occur naturally in both healthy subjects and cancer patients and can be induced by MUC1 peptide vaccination. We compared the specificity of natural and induced MUC1 Abs with the objective of defining an effective MUC1 vaccine for active immunotherapy of adenocarcinoma patients. Serum samples, selected out of a screened population of 492 subjects for their high levels of IgG and/or IgM MUC1 Abs, were obtained from 55 control subjects and from 26 breast cancer patients before primary treatment, as well as from 19 breast cancer patients immunized with MUC1 peptides coupled to keyhole limpet hemocyanin (KLH) and mixed with QS-21. The samples were tested with enzyme-linked immunoassays for reactivity with (1) overlapping hepta- and 20-mer peptides spanning the MUC1 tandem repeat sequence; (2) two modified 60-mer peptides with substitutions in the PDTR (PDTA) or in the STAPPA (STAAAA) sequence of each tandem repeat; and (3) four 60-mer glycopeptides with each 1, 2, 3 and 5 mol N-acetylgalactosamine (GalNAc) per repeat. More than one minimal epitopic sequence could be defined, indicating that Abs directed to more than one region of the MUC1 peptide core can coexist in one and the same subject. The most frequent minimal epitopic sequence of natural MUC1 IgG and IgM Abs was RPAPGS, followed by PPAHGVT and PDTRP. MUC1 peptide vaccination induced high titers of IgM and IgG Abs predominantly directed, respectively, to the PDTRPAP and the STAPPAHGV sequences of the tandem repeat. Natural MUC1 Abs from breast cancer patients reacted more strongly with the N-acetylgalactosamine (GalNAc) peptides than with the naked 60-mer peptide, while reactivity with the GalNAc-peptides was significantly reduced (2-tailed p < 0.0001) in the MUC1 IgG and IgM Abs induced by MUC1 peptide vaccination. Whereas in cancer patients glycans appear to participate in epitope conformation, the epitope(s) recognized by MUC1 Abs induced by peptide vaccination are already masked by minimal glycosylation. Therefore, our results indicate that a MUC1 glycopeptide would be a better vaccine than a naked peptide.
Asunto(s)
Acetilgalactosamina/inmunología , Mucina-1/inmunología , Fragmentos de Péptidos/inmunología , Acetilgalactosamina/química , Formación de Anticuerpos , Mapeo Epitopo , Femenino , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Inmunoglobulina M/sangre , Masculino , Mucina-1/química , Fragmentos de Péptidos/química , Péptidos/inmunologíaRESUMEN
Sixteen research groups participated in the ISOBM TD-4 Workshop in which the reactivity and specificity of 56 monoclonal antibodies against the MUC1 mucin was investigated using a diverse panel of target antigens and MUC1 mucin-related synthetic peptides and glycopeptides. The majority of antibodies (34/56) defined epitopes located within the 20-amino acid tandem repeat sequence of the MUC1 mucin protein core. Of the remaining 22 antibodies, there was evidence for the involvement of carbohydrate residues in the epitopes for 16 antibodies. There was no obvious relationship between the type of immunogen and the specificity of each antibody. Synthetic peptides and glycopeptides were analyzed for their reactivity with each antibody either by assay of direct binding (e.g. by ELISA or BiaCore) or by determining the capacity of synthetic ligands to inhibit antibody binding interactions. There was good concordance between the research groups in identifying antibodies reactive with peptide epitopes within the MUC1 protein core. Epitope mapping tests were performed using the Pepscan analysis for antibody reactivity against overlapping synthetic peptides, and results were largely consistent between research groups. The dominant feature of epitopes within the MUC1 protein core was the presence, in full or part, of the hydrophilic sequence of PDTRAPAP. Carbohydrate epitopes were less easily characterized and the most useful reagents in this respect were defined oligosaccharides, rather than purified mucin preparations enriched in particular carbohydrate moieties. It was evident that carbohydrate residues were involved in many epitopes, by regulating epitope accessibility or masking determinants, or by stabilizing preferred conformations of peptide epitopes within the MUC1 protein core. Overall, the studies, highlight concordance between groups rather than exposing inconsistencies which gives added confidence to the results of analyses of the specificity of antimucin monoclonal antibodies.