In Vitro Apoptotic Effects of Farnesyltransferase blockade in Acute Myeloid Leukemia Cells.

Farnesyltransferase inhibitors (FTIs) are a class of oral anti-cancer drugs currently tested in phase I-II clinical trials for treatment of hematological malignancies. The in vitro effects of various FTIs (alpha-hydroxyfarnesylphosphonic acid, manumycin-A and SCH66336) were tested on CD34+ KG1a cell line and in primary acute myeloid leukemia (AML) cells from 64 patients. By cell viability and clonogeneic methylcellulose assays, FTIs showed a significant inhibitory activity in CD34+ KG1a and primary bone marrow (BM) leukemic cells from 56% of AML patients. FTIs also induced activation of caspase-3 and Fas-independent apoptosis, confirmed by the finding that inhibition of caspase-8 was not associated with the rescue of FTI-treated cells. We concluded that other cellular events induced by FTIs may trigger activation of caspase-3 and subsequent apoptosis, but the expression of proapoptotic molecules, as Bcl-2 and Bcl-XL, and antiapoptotic, as Bcl-X(s), were not modified by FTIs. By contrast, expression of inducible nitric oxide synthase (iNOS) was increased in FTI-treated AML cells. Our results suggest a very complex mechanism of action of FTIs that require more studies for a better clinical use of the drugs alone or in combination in the treatment of hematological malignancies.

Prolonged complete hematologic response in relapsed/refractory T-large granular lymphocyte leukemia after bendamustine treatment.

T-large granular lymphocyte leukemia (T-LGLL) is a chronic clonal proliferation of effector memory cytotoxic CD3+CD57+CD56- T cells and the current guidelines suggest immunosuppressive therapy as first-line therapy, but the treatment of refractory/relapsed patients is still challenging due to the lack of prospective studies. We describe a series of two refractory/relapsed T-LGLL patients successfully treated with bendamustine, a chemotherapeutic agent largely used for B-cell neoplasms, but poorly investigated for the treatment of T-cell diseases. Complete remission (CR) was achieved in 3 and 6 months, respectively, and maintained for at least 20 months. One patient relapsed after a 20-month CR, but she was responsive to bendamustine therapy again, obtaining a further prolonged CR. Bendamustine as single agent or in combination could be a feasible therapeutic option in refractory/relapsed T-LGLL, especially for elderly patients because of its safety profile.

Risk of having BRCA1 mutation in high-risk women with triple-negative breast cancer: a meta-analysis.

Testing for BRCA1 mutation has important clinical implications such as identifying risk of second primary cancers and risk of cancer in the family. This study seeks to quantify the risk of having BRCA1 mutation in female breast cancer patients with triple-negative phenotype compared with those with other phenotypes. We undertook a search of MEDLINE and EMBASE databases for relevant studies through 10 May 2013. Outcomes were calculated and reported as risk ratio and risk difference. 12 studies comprising 2533 breast cancer patients were included in the analysis. It was found that almost all eligible studies were performed on high-risk population with breast cancer. By analyzing the incidence rates of BRCA1 mutation in patients with triple-negative breast cancer (TNBC) and non-TNBC, our meta-analysis provides a relative risk of 5.65 [95% confidence interval (CI), 4.15-7.69] and risk difference of 0.22 (95% CI, 0.15-0.29). This implies that, in selected population with high-risk features, women with TNBC are approximately five and a half times more likely to have BRCA1 mutation compared with non-TNBC phenotype, and approximately two in nine women with TNBC harbor BRCA1 mutation. Triple-negative phenotype significantly increases the risk of having BRCA1 mutation in high-risk breast cancer patients compared with non-TNBC.

A new xenicane norditerpene from the Indian marine gorgonian Acanthogorgia turgida.

Chemical investigation of the liposoluble extract of the gorgonian Acanthogorgia turgida, from Indian coasts, led us to isolate a new xenicane-based norditerpene, isoacalycixeniolide-A (1), along with the known structurally related compounds 2-6. The structure of the norditerpene (1) was elucidated by spectral methods (mainly by NMR techniques), whereas the absolute stereochemistry was suggested by the application of circular dicroism methodology.

Sponsorship and conflicts of interests in CME: the Italian experience.

Pursuant to the Italian healthcare framework, sponsorship of Continuing Medical Education (CME) for healthcare professionals governs the relationship between the medical industry and the healthcare sector, as public institutions are directly involved in it. Sponsorship is based on a voluntarily sharing of mutual benefits between two contracting parties, namely the sponsor and the sponsorship beneficiary, whose interests are relevant to the same degree. To avoid conflicts of interests from occurring, sponsorship shall comply with two ethical standards: 1) the contracting parties shall verify if their interests about CME activities converge or conflict; 2) the sponsorship contract shall be published and advertised to disclose what kind of commitment the contracting parties undertook. When entering a CME sponsorship contract as sponsorship beneficiary, Italian local health authorities may rely on a code of conduct which lays down all principles, criteria and proceedings that shall apply.

Tomografía de 64 cortes: una nueva aproximación al diagnóstico de enfermedad coronaria / Sixty four slice computed tomography: a new approach to the diagnosis of coronary artery disease

Originalmente las arterias coronarias podían ser visualizadas directamente al abrir el tórax, luego mediante la cinecoronariografía e indirectamente con las pruebas no invasivas, que se desarrollaron para inferir el grado de obstrucción coronaria de acuerdo a la isquemia miocárdica inducida por el aumento de los requerimientos miocárdicos de oxígeno. La tomografía de las arterias coronarias permite su observación directa de una manera no invasiva, con las limitaciones propias de la irradiación y del contraste iodado. Sus indicaciones se están estableciendo en forma acelerada y su valor predictivo negativo es cercano al 100 por ciento, lo cual hace a esta nueva técnica una excelente herramienta para el diagnóstico de la enfermedad coronaria, a la vez de poder observar la función ventricular izquierda y órganos adyacentes al corazón.

Originally the coronary arteries could only be visualized when a thoracotomy was performed and thereafter with coronary arteriography. With the purpose of inferring the degree of coronary artery narrowing the so called non invasive test were developed to induce myocardial ischemia by an increase of myocardial oxygen demands. Multislice compared tomography offers the possibility of "seeing" the coronary arteries for the first time non-invasively, with the proper limitations of radiation and iodine contrast administration. Its indications are rapidly changing with a negative predictive value close to 100%, which makes this diagnostic tool unique for a better assessment not only of the coronary arteries but also of the left ventricular function and anatomy of its surrounding structures.

Multidisciplinary and multisetting team management programme in heart failure patients affects hospitalisation and costing.

BACKGROUND: We evaluated whether multidisciplinary disease management programme developed with collaboration of physicians and nurses inside and outside general district hospital settings can affect clinical outcomes in heart failure population over a 12-month period. METHODS: 571 patients hospitalised with CHF were referred to our unit and 509 patients agreed to participation. The intervention team included physicians and nurses from Internal Medicine and Cardiac Dept., and the patient's general practitioners. Contacts were on a pre-specified schedule, included a computerised programme of hospital visits and phone calls; in case of NYHA functional class III and IV patients, home visits were also planned. RESULTS: The median age of patients was 77.7+/-9 years (43.3% women). At baseline the percentage of patients with NYHA class III and IV was 56.0% vs. 26.0% after 12 months (P<0.05). Programme enrolment reduced total hospital admissions (82 vs. 190, -56%, P<0.05), number of patients hospitalised (62 vs. 146, 57%, P<0.05). All NYHA functional class benefited (class I=75%, class IV=67%), with reduction in the costing (-48%, P<0.05). Improvement in symptoms (-9.0+/-3.2) and signs (-5.2+/-3.1) scores was measured (P<0.01). Therapy optimisation was obtained by 20.5% increase in patients taking betablockade and 21.0% increase in those on anti-aldosterone drugs. CONCLUSIONS: Multidisciplinary approach to CHF management can improve clinical management, reducing hospitalisation rate and costing.

Escherichia coli RecA promotes strand invasion with cisplatin-damaged DNA.

The antitumor drug cisplatin causes intrastrand cross-linking of adjacent guanine residues that severely distorts the DNA backbone. These DNA adducts impede the progress of the replisome and may result in replication fork arrest. In Escherichia coli, the response to cisplatin involves the action of the prototypic recombinase RecA. Here we show that RecA can utilize, albeit at reduced levels, oligonucleotides that bear site-specific cisplatin-induced 1,2 d(GpG) intrastrand cross-links in strand invasion reactions. Binding of RecA to cisplatin-damaged oligonucleotides was not affected, indicating that the impediment was in the pairing step. The cognate E. coli single-strand DNA-binding protein specifically stimulated strand invasion particularly with cisplatin-damaged DNA. These results indicate that RecA is capable of processing the major cisplatin-induced lesion via a recombination mechanism.

Analysis of Sanfilippo A gene mutations in a large pedigree.

Mucopolysaccharidosis type IIIA, also known as Sanfilippo A disease, results from mutations in the sulfamidase gene. To date, a total of 62 mutations have been described underlying this lysosomal disorder. Expression studies on missense mutations have shown that each alteration was disease-causing and helped to clarify the genotype-phenotype correlation. In this report we describe a large pedigree where the mutations have been identified in two second cousins: one with severe disease (E369K/R433Q) and the other with a mild form of the illness (E369K/P128L). This study places R433Q as a severe mutation underlying Sanfilippo A disease.

Uptake of recombinant iduronate-2-sulfatase into neuronal and glial cells in vitro.

Mucopolysaccharidosis type II (MPS II, Hunter syndrome) is a congenital storage disorder resulting from mutations on the iduronate-2-sulfatase (IDS) gene. The disease shows variable clinical phenotypes from severe to mild with progressive neurological dysfunction. The therapeutic options for treatment of MPS II are limited and currently no specific therapies are available; the problem is further compounded by difficulties in delivering therapeutic agents to the central nervous system (CNS). In this work, as a potential treatment for this disease, the transfer of the recombinant IDS enzyme into brain cells has been studied in vitro. Two different approaches to obtain recombinant IDS have been utilized: production of the recombinant enzyme by a transfected human clone (Bosc 23 cells); production of the recombinant enzyme by adenoviral transduction of neuronal (SK-N-BE) or glial (C6) cells. Our data indicate that the transfected as well as the infected cells produce a large amount of the IDS enzyme, which is efficiently endocytosed into neuronal and glial cells through the mannose 6-phosphate (M6P) receptor system. Somatic gene therapy appears therefore to be suitable to correct IDS deficiency in brain cells.

Reconstitution of the base excision repair pathway for 7,8-dihydro-8-oxoguanine with purified human proteins.

In mammalian cells, repair of the most abundant endogenous premutagenic lesion in DNA, 7,8-dihydro-8-oxoguanine (8-oxoG), is initiated by the bifunctional DNA glycosylase OGG1. By using purified human proteins, we have reconstituted repair of 8-oxoG lesions in DNA in vitro on a plasmid DNA substrate containing a single 8-oxoG residue. It is shown that efficient and complete repair requires only hOGG1, the AP endonuclease HAP1, DNA polymerase (Pol) beta and DNA ligase I. After glycosylase base removal, repair occurred through the AP lyase step of hOGG1 followed by removal of the 3'-terminal sugar phosphate by the 3'-diesterase activity of HAP1. Addition of PCNA had a slight stimulatory effect on repair. Fen1 or high concentrations of Pol beta were required to induce strand displacement DNA synthesis at incised 8-oxoG in the absence of DNA ligase. Fen1 induced Pol beta strand displacement DNA synthesis at HAP1-cleaved AP sites differently from that at gaps introduced by hOGG1/HAP1 at 8-oxoG sites. In the presence of DNA ligase I, the repair reaction at 8-oxoG was confined to 1 nt replacement, even in the presence of high levels of Pol beta and Fen1. Thus, the assembly of all the core proteins for 8-oxoG repair catalyses one major pathway that involves single nucleotide repair patches.

The effect of four mutations on the expression of iduronate-2-sulfatase in mucopolysaccharidosis type II.

Mucopolysaccharidosis type II (Hunter syndrome; OMIM 309900) is a rare X-linked recessive lysosomal storage disorder caused by the deficiency of the enzyme iduronate-2-sulfatase (IDS; EC 3.1.6.13). Different alterations at the IDS locus, mostly missense mutations, have been demonstrated, by expression study, as deleterious, causing significant consequences on the enzyme function or stability. In the present study we report on the results of the transient expression of the novel K347T, 533delTT, N265I and the already described 473delTCC (previously named DeltaS117) mutations in the COS 7 cells proving their functional consequence on IDS activity. This type of information is potentially useful for genotype-phenotype correlation, prognosis and possible therapeutic intervention.

Okazaki fragment processing: modulation of the strand displacement activity of DNA polymerase delta by the concerted action of replication protein A, proliferating cell nuclear antigen, and flap endonuclease-1.

DNA polymerase (pol) delta is essential for both leading and lagging strand DNA synthesis during chromosomal replication in eukaryotes. Pol delta has been implicated in the Okazaki fragment maturation process for the extension of the newly synthesized fragment and for the displacement of the RNA/DNA segment of the preexisting downstream fragment generating an intermediate flap structure that is the target for the Dna2 and flap endonuclease-1 (Fen 1) endonucleases. Using a single-stranded minicircular template with an annealed RNA/DNA primer, we could measure strand displacement by pol delta coupled to DNA synthesis. Our results suggested that pol delta alone can displace up to 72 nucleotides while synthesizing through a double-stranded DNA region in a distributive manner. Proliferating cell nuclear antigen (PCNA) reduced the template dissociation rate of pol delta, thus increasing the processivity of both synthesis and strand displacement, whereas replication protein A (RP-A) limited the size of the displaced fragment down to 20-30 nucleotides, by generating a "locked" flap DNA structure, which was a substrate for processing of the displaced fragment by Fen 1 into a ligatable product. Our data support a model for Okazaki fragment processing where the strand displacement activity of DNA polymerase delta is modulated by the concerted action of PCNA, RP-A and Fen 1.

Extraneurologic symptoms as presenting signs of Sanfilippo disease.

Sanfilippo disease, or mucopolysaccharidosis type III, results from the deficiency of lysosomal hydrolases, which impairs heparan sulfate metabolism. Clinically, the disease is characterized by a mild somatic phenotype combined with early severe neurodegenerative illness with prominent behavioral disturbance. We report clinical and molecular findings of a child with Sanfilippo disease type B (alpha-N>-acetylglucosaminidase deficiency) who presented at age 18 months with marked systemic involvement and normal initial psychomotor development. These findings suggest that atypical mucopolysaccharidosis type III patients may present with early somatic changes preceding the onset of overt neurologic symptoms and ensuring an early diagnosis and possible therapeutic intervention.

Ultrasound biomicroscopy of the peripheral retina and the ciliary body in degenerative retinoschisis associated with pars plana cysts.

AIM: To evaluate the ciliary body and peripheral retina in degenerative retinoschisis associated with pars plana cysts using ultrasound biomicroscopy (UBM). METHODS: 18 eyes of 12 patients with degenerative retinoschisis associated with pars plana cysts were selected through binocular indirect ophthalmoscopy and Goldmann three mirror lens examination, both with scleral depression. These patients were studied in detail with UBM. RESULTS: Study of the ciliary body with UBM showed pars plana cysts of different size and uneven shape. In cross sections the morphology of pars plana cysts in detail and the close relation of the cysts with the oral region and the peripheral retina, where areas of cystoid degeneration and retinoschisis were present, were observed. In transverse sections three main morphological aspects of pars plana cysts could be differentiated ("isolated," "confluent," and "clustered" cysts). Furthermore, ultrabiomicroscopy allowed differential diagnosis between retinoschisis and associated retinal detachment in six eyes. CONCLUSIONS: The study of peripheral degenerative retinoschisis and pars plana cysts is possible in vivo by means of UBM, showing the detailed morphology of the lesions (not otherwise evident through ophthalmoscopic examination) and the close relation between pars plana cysts, cystoid degeneration, and peripheral retinoschisis.

Biventricular pacing in patients with heart failure and intraventricular conduction delay.

In patients with advanced chronic heart failure, characterized by prolonged QRS duration and by decreased cardiac contractility, decreasing dysynchrony by biventricular pacing seems to improve exercise tolerance (6-min walk distance), symptoms (New York Health Association class), and quality of-life scores. Although the results of several reports were consistent, the numbers of patients studied were small, and many of the changes were trends that did not reach statistical significance. The availability of a non-pharmacological treatment that improves exercise capacity and quality-of-life would be a major advance. However, further studies will need to address the question of mortality and morbidity benefits of such intervention.