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Epigenetic mechanisms affecting chromatin structure contribute to regulate gene expression and assure the inheritance of information, which are essential for the proper expression of key regulatory genes in healthy cells, tissues and organs. In the medical field, an increasing body of evidence indicates that altered gene expression or de-regulated gene function lead to disease. Cancer cells also suffer a profound change in the genomic methylation patterns and chromatin status. Aberrant DNA methylation patterns, changes in chromatin structure and in gene expression are common in all kind of tumor types. However, studies on leukemias have provided paradigmatic examples for the functional implications of the epigenetic alterations in cancer development and progression as well as their relevance for therapeutical targeting.

Alteration of lineage-specific transcriptional programs for hematopoiesis causes differentiation block and promotes leukemia development. Here, we show that AML1/ETO, the most common translocation fusion product in acute myeloid leukemia (AML), counteracts the activity of retinoic acid (RA), a transcriptional regulator of myelopoiesis. AML1/ETO participates in a protein complex with the RA receptor alpha (RARα) at RA regulatory regions on RARβ2, which is a key RA target gene mediating RA activity/resistance in cells. At these sites, AML1/ETO recruits histone deacetylase, DNA methyltransferase, and DNA-methyl-CpG binding activities that promote a repressed chromatin conformation. The link among AML1/ETO, heterochromatic RARβ2 repression, RA resistance, and myeloid differentiation block is indicated by the ability of either siRNA-AML1/ETO or the DNA methylation inhibitor 5-azacytidine to revert these epigenetic alterations and to restore RA differentiation response in AML1/ETO blasts. Finally, RARβ2 is commonly silenced by hypermethylation in primary AML blasts but not in normal hematopoietic precursors, thus suggesting a role for the epigenetic repression of the RA signaling pathway in myeloid leukemogenesis.

ABSTRACTIn eukaryotic organisms cellular fate and tissue specific gene expression are regulated by the activity of proteins known as transcription factors that by interacting with specific DNA sequences direct the activation or repression of target genes. The post genomic era has shown that transcription factors are not the unique key regulators of gene expression. Epigenetic mechanisms such as DNA methylation, post-translational modifications of histone proteins, remodeling of nucleosomes and expression of small regulatory RNAs also contribute to regulation of gene expression, determination of cell and tissue specificity and assurance of inheritance of gene expression levels. The relevant contribution of epigenetic mechanisms to a proper cellular function is highlighted by the effects of their deregulation that cooperate with genetic alterations to the development of various diseases and to the establishment and progression of tumors.

The fucose-mannose ligand (FML) complex of Leishmania donovani is a promising vaccine candidate against murine and canine visceral leishmaniasis, and its main component is a 36-kDa nucleoside hydrolase (NH36). In this study, we tested the immune response and protection induced by the purified FML, the recombinant NH36 (rNH36), and NH36 DNA vaccines against the agents of visceral (L. chagasi) and cutaneous (L. mexicana) leishmaniasis in BALB/c mice. Mice developed weak humoral response to the vaccines alone, except for those immunized with FML. However, all three vaccine groups presented elevated immunoglobulin G (IgG), IgG1, and IgG2a levels after infection with L. chagasi, whereas no differences were observed between vaccine and control groups after infection with L. mexicana. A strong intradermal reaction to L. donovani and L. mexicana antigens was observed in mice immunized with rNH36 or FML, whereas mice immunized with NH36 DNA only reacted against L. donovani antigens. Experimental infection of immunized mice demonstrated that FML and rNH36 induced significant protection against L. chagasi infection with reductions in parasite loads of 79%. FML also conferred partial protection against L. mexicana infection. The best protection was observed in mice immunized with the VR1012-NH36 DNA vaccine, which induced an 88% reduction in L. chagasi parasite load and a 65% reduction in L. mexicana lesion size. Fluorescence-activated cell sorting analysis indicated the DNA vaccine induced a two- to fivefold increase in gamma interferon-producing CD4+ T cells, indicating a Th1-type immune response. Our results showed that the NH36 DNA vaccine induced a strong immunoprotection against visceral and cutaneous leishmaniasis, suggesting that this DNA vaccine represents a very good candidate for use against several Leishmania species.

ABSTRACTThe fucose-mannose ligand (FML) complex of Leishmania donovaniis a promising vaccine candidate against murine and canine visceral leishmaniasis, and its main component is a 36-kDa nucleoside hydrolase (NH36). In this study, we tested the immune response and protection induced by the purified FML, the recombinant NH36 (rNH36), and NH36 DNA vaccines against the agents of visceral (L. chagasi) and cutaneous (L. mexicana) leishmaniasis in BALB/c mice. Mice developed weak humoral response to the vaccines alone, except for those immunized with FML. However, all three vaccine groups presented elevated immunoglobulin G (IgG), IgG1, and IgG2a levels after infection with L. chagasi, whereas no differences were observed between vaccine and control groups after infection with L. mexicana. A strong intradermal reaction to L. donovaniand L. mexicanaantigens was observed in mice immunized with rNH36 or FML, whereas mice immunized with NH36 DNA only reacted against L. donovaniantigens. Experimental infection of immunized mice demonstrated that FML and rNH36 induced significant protection against L. chagasiinfection with reductions in parasite loads of 79%. FML also conferred partial protection against L. mexicanainfection. The best protection was observed in mice immunized with the VR1012-NH36 DNA vaccine, which induced an 88% reduction in L. chagasiparasite load and a 65% reduction in L. mexicanalesion size. Fluorescence-activated cell sorting analysis indicated the DNA vaccine induced a two- to fivefold increase in gamma interferon-producing CD4+T cells, indicating a Th1-type immune response. Our results showed that the NH36 DNA vaccine induced a strong immunoprotection against visceral and cutaneous leishmaniasis, suggesting that this DNA vaccine represents a very good candidate for use against several Leishmaniaspecies.

Objectives: A study was conducted to determine the prevalence of HPV infection in cervical lesions of Brazilian women and to search for specific risk factors associated to progression to malignancy.

Background: The physiology of sheep as small ruminants is remarkably different from monogastric animals especially regarding the forestomach system. Using sheep for surgical procedures during scientific research thereby presents an exceptional setting for the anaesthetist. Long-term anaesthesia generally demands deprivation of food to reduce the risk of bloat in sheep. This might influence the energy and electrolyte balance. In horses and companion animals, close monitoring of mean arterial blood pressure, capnography and blood gas analysis are common procedures during long-term surgery. However, few data are available on reference ranges for blood gas in sheep and these cover only short periods of anaesthesia. To the authors’ knowledge, there is no study available that includes the monitoring of electrolytes and pH in ruminal fluid and kidney function tests in sheep undergoing long term anaesthesia. Thereby, the aim of the present study was to gather data on blood parameters, and data on ruminal fluid and kidney function during long-term anaesthesia in sheep. Data were obtained from eight sheep undergoing the invasive surgical procedure of left pneumonectomy and auto-transplantation or isolated left lung perfusion. After a 19-h fasting period, the animals were administered xylazine and ketamine and then intubated and maintained in general anaesthesia under artificial ventilation using isoflurane in oxygen. Blood samples were evaluated during 9 h of anaesthesia; ruminal fluid and kidney function tests were evaluated during 7 h of anaesthesia. Results: Blood parameters such as electrolytes and partial pressure of carbon dioxide revealed few changes, yet blood glucose decreased and beta-hydroxybutyric acid increased significantly. All animals showed an elevated arterial pH and bicarbonate concentration despite artificial ventilation. In ruminal fluid, the pH significantly decreased and no significant changes in electrolytes occurred. Kidney function tests revealed no significant changes in any of the animals. However, fractional excretion of water and phosphate was slightly increased. One animal showed severe complications due to hypokalaemia. Conclusion: Invasive surgery under long-term anaesthesia in sheep is possible without great imbalances of arterial pH and electrolytes. Nevertheless, potassium concentrations should be monitored carefully, as a deficiency can lead to life-threatening complications. The operated sheep tended not to develop metabolic acidosis and the mean kidney function could be maintained within the physiological range throughout anaesthesia. However, slight elevations in renal fractional water and phosphate excretion could suggest an early tubular reabsorption dysfunction. In ruminal fluid, acidification occurred, though no significant changes were observed in l- and d-lactate levels or in electrolyte concentrations. To our knowledge, the role of the rumen in storing fluids and balancing electrolytes in the blood has not yet been documented during anaesthesia. However, the importance of the rumen for fluid equilibrium in sheep indicates the necessity for routine monitoring and further research.

Several barriers to access hepatocellular carcinoma (HCC) screening in Brazil and the large number of patients diagnosed at advanced disease stages, without prospect of curative treatment, are indicatives that access to early HCC diagnosis and treatment is not equitably guaranteed for entire population.

In this paper, we present a modification in the standard k-ϵturbulence model, together with a new equation to express μtas a function of kand ϵ. In this simplified model, new values of the constants cϵ1, cϵ2and σϵare provided. Using this model, we find an algebraic equation to express the viscosity in the wall (μw) so that the flow remains unchanged throughout the domain. Fitting the numerical solution, we find algebraic equations to approximate the kand ϵprofiles. Using periodic boundary conditions to solve kand ϵequations, this model was applied to simulate the pollutant dispersion for the unstable Praire Grass experiments, obtaining a good agreement with the experimental data.

The defense organization of Crohn's disease and Panic Disorder was studied with a well-validated tachistoscopic paradigm, the Defense Mechanism Test. Three sex- and age-matched groups of 34 subjects (Crohn, Panic, and Nonclinical) were compared on the main codings of defense. Crohn patients were characterized by stereotypy and lack of recognition of the threat. The Panic group presented clearly higher frequencies of protocols with repression, isolation, and disappearance of the threat. Especially was the strongest variant of isolation, barrier-isolation, typical of the defensive substructure of panic patients.

The aim of this paper is to verify whether the control played by poly(ADP‐ribosyl)‐ation on genomic DNA methylation, and in particular on CpG islands, can also be seen on foreign DNA transfected in cells where inhibition of the poly‐(ADP‐ribosyl)ation process was obtained by treating them with 2 mM 3‐aminobenzamide for 24 h. The CpG island‐like pVHCk plasmid containing the bacterial chloramphenicol acyltransferase (CAT) gene under the control of SV40 early promoter was transfected in L929 mouse fibroblast cells. The bisulfite reaction, which is capable of immortalizing the methylation state of cytosine on DNA, was performed before amplification of the plasmid DNA fragment, then used for sequence analysis. Our results have shown that 1)when transfected in control cells, the plasmid maintains its characteristic unmethylated pattern, whereas this pattern is lost when the plasmid is transfected in cells treated with 3‐aminobenzamide; and 2)the presence of new methyl groups on plasmid DNA is paralleled by a decrease of CAT reporter gene expression. These data confirm that poly(ADP‐ribosyl)ation is a process tightly involved in protecting genomic DNA from full methylation and suggest the use of 3‐aminobenzamide as a possible experimental strategy to mime other conditions of DNA hypermethylation in cells.—Zardo, G., Marenzi, S., Perilli, M., Caiafa, P. Inhibition of poly(ADP‐ribosyl)ation introduces an anomalous methylation pattern in transfected foreign DNA. FASEB J. 13, 1518–1522 (1999)

Oligonucleosomal DNA preparations from condensed-inactive chromatin were examined, before and after artificial methylation by bacterial SssI methylase, for their ability to allow cooperative H1–H1 interactions under conditions of different ionic strength. Our results support the conclusion that, within the highly methylated genomic DNA, there are some CpG's whose unmethylated state is critical for chromatin folding. Circular dichroism spectra indicate that artificial overmethylation of native oligonucleosomal DNA reduces its efficiency in inducing an ordered conformation of H1 histone. Temperature melting profiles confirm on the other hand that the native and the artificially overmethylated forms of oligonucleosomal DNA are both able to bind H1 histone.

The unmethylated status of the CpG islands is important for gene expression of correlated housekeeping genes since it is well known that their methylation inhibits transcription process. An interesting question that has been discussed but not solved is how the CpG islands maintain their characteristic unmethylated status even though they are rich in CpG dinucleotides. Our previous in vitro and in vivoresearch has shown that poly(ADP‐ribosyl)ation is involved in protecting CpG dinucleotides from full methylation in genomic DNA and that a block of poly(ADP‐ribosyl)ation is also involved in modifying the methylation pattern in the promoter region of Htf9housekeeping gene. In this study we locked for cytological evidence that in the absence of an active poly(ADP‐ribosyl)ation the DNA methylation pattern in L929 and NIH/3T3 mouse fibroblast cell lines is altered. For this purpose, differences in the methylation levels of interphase nuclei from control and treated cultures of two murine cell lines preincubated with 2 mM 3‐aminobenzamide, an inhibitor of poly(ADP‐ribosyl)ation, were measured in individual cells after indirect immunolabeling with anti‐5MeC antibodies. The quantitative analysis allowed us to demonstrate that blocking of the poly(ADP‐ribosyl)ation results in a higher number, size, and density of antibody binding regions in treated cells when compared to the controls. Analogously, sequential Giemsa staining and indirect immunolabeling of the same slides showed the hetero‐chromatic regions colocalized with the extended methyl‐rich domains.—de Capoa, A., Febbo, F. R., Giovannelli, F., Niveleau, A., Zardo, G., Marenzi, S., Caiafa, P. Reduced levels of poly(ADP‐ribosyl)ation result in chromatin compaction and hypermethylation as shown by cell‐by‐cell computer‐assisted quantitative analysis. FASEB J.13, 89–93 (1999)

In vivo and in vitro experiments carried out on L929 mouse fibroblasts suggested that the poly(ADP-ribosyl) ation process acts somehow as a protecting agent against full methylation of CpG dinucleotides in genomic DNA. Since CpG islands, which are found almost exclusively at the 5'-end of housekeeping genes, are rich in CpG dinucleotides, which are the target of mammalian DNA methyltransferase, we examined the possibility that the poly(ADP-ribosyl)ation reaction is involved in maintaining the unmethylated state of these DNA sequences. Experiments were conducted by two different strategies, using either methylation-dependent restriction enzymes on purified genomic DNA or a sequence-dependent restriction enzyme on an aliquot of the same DNA, previously modified by a bisulfite reaction. With the methylation-dependent restriction enzymes, it was observed that the "HpaII tiny fragments" greatly decreased when the cells were preincubated with 3-aminobenzamide, a well known inhibitor of poly(ADP-ribose) polymerase. The other experimental approach allowed us to prove that, as a consequence of the inhibition of the poly(ADP-ribosyl)ation process, an anomalous methylation pattern could be evidenced in the CpG island of the promoter fragment of the Htf9 gene, amplified from DNA obtained from fibroblasts preincubated with 3-aminobenzamide. These data confirm the hypothesis that, at least for the Htf9 promoter region, an active poly(ADP-ribosyl)ation protects the unmethylated state of the CpG island.

H1e and H1c histone variants were purified from mouse L929 fibroblasts using a reverse phase HPLC, and their effect onin vitroDNA methylation was investigated, together with their ability to bind unmethylated or methylated CpG-rich 44bp oligonucleotides. In a “physiological” range of H1:DNA ratios, only H1e, at variance from H1c, was found to cause a marked inhibition ofin vitroenzymic DNA methylation. It was also shown that both variants have a similar affinity in binding a methylated CpG-rich oligonucleotide, but that the binding to the same oligonucleotide in the unmethylated form occurs preferentially with H1e rather than with H1c. H1e is therefore likely to be directly involved in maintaining CpG-rich sequences in the unmethylated state.

H1 histone somatic variants from L929 mouse fibroblasts were purified by reverse-phase HPLC. We analysed the ability of each H1 histone variant to allow the H1–H1 interactions that are essential for the formation of the higher levels of chromatin structure, and we investigated the role played by the poly(ADP-ribosyl)ation process. Cross-linking analysis showed that H1e is the only somatic variant which, when bound to DNA, is able to produce H1–H1 polymers; the size of polymers was decreased when H1e was enriched in its poly(ADP-ribosyl)ated isoform. Measurement of the methyl-accepting ability in native nuclei compared with nuclei in which poly(ADP-ribosyl)ation was induced showed that the poly(ADP-ribosyl)ated H1 histone had not been removed from linker regions, in spite of its different interaction with DNA.

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Background Though laser guided extractions of cardiac implantable electronic devices leads have become a routine procedure, the severe complications are associated with a high mortality. Here, we report our single center experience using uniportal video-assisted thoracoscopy for laser lead extraction and compare it to stand-alone laser lead extraction.Methods The intraoperative data and postoperative clinical outcomes of patients undergoing laser lead extraction with concomitant thoracoscopy (N= 28) or without (N= 43) in our institution were analyzed retrospectively.Results Neither the median x-ray time (612.0 s for the thoracoscopy group vs. 495.5 s for the non-thoracoscopy group, p= 0.962), length of the operation (112.5 vs. 100.0 min, p= 0.676) or the median length of hospital stay (9.0 vs. 10.0 days, p= 0.990) differed significantly. The mean intensive care unit stay was longer for patients in the non-thoracoscopy group (0.8 vs. 2.5 days, p= 0.005). The 30-day-mortality in the thoracoscopy group was zero, whereas five patients died in the non-thoracoscopy group. Furthermore, four patients in the non-thoracoscopy group had encountered haemothorax, while none were observed in the thoracoscopy group (p= 0.148).Conclusions The adoption of uniportal video-assisted thoracoscopy during laser-guided lead extraction of cardiac implantable electronic devices can be considered safe and does not lengthen the operating time or hospital stay. It might be useful in the detection of severe complications and, in experienced hands, possibly allow direct bleeding control.

The pattern of DNA methylation established during embryonic development is necessary for the control of gene expression and is preserved during the replicative process. DNA regions of about 1–2 kb in size, termed CpG islands and located mostly in the promoter regions of housekeeping genes, are protected from methylation, despite being about 6–10 times richer in the dinucleotide CpG than the rest of DNA. Their unmethylated state guarantees the expression of the corresponding housekeeping genes. At present, the mechanism by which CpG islands remain protected from methylation is not clear. However, some results suggest that poly(ADPribosyl)ation, an enzymatic process that introduces a postsynthetic modification onto chromatin proteins, might be involved. Here we show in L929 mouse fibroblast cells that inhibition of poly(ADP‐ribose) polymerase(s) at different cell‐cycle phases increases the mRNA and protein levels of the major maintenance DNA methyltransferase (DNMT1) in G1/S border. Increase of DNMT1 results in a premature PCNA‐DNMT1 complex formation, which facilitates robust maintenance, as well as de novoDNA methylation processes during the G1/S border, which leads to abnormal hypermethylation.

The clinical, radiological and operative findings in 10 consecutive patients with cervical spine trauma presenting with locked facets (bilaterally in nine patients) are reported. The treatment was cervical traction until reduction of the locked facets was achieved, followed by anterior surgical decompression and ostheosynthesis. Intervertebral disc herniation was present in nine of the patients, and ruptured calcified posterior longitudinal ligament was present in the remaining patient. Four patients improved after treatment (none of these had clinically complete tetraplegia preoperatively), and three patients died. Associated soft tissue injuries producing spinal cord compression after reduction of cervical locked facets were verified in all of the patients of this series, suggesting that systematic surgical exploration should be done in this category of patients.