Alternative low-cost approach to the synthesis of magnetic iron oxide nanoparticles by thermal decomposition of organic precursors.

A new approach to thermal decomposition of organic iron precursors is reported, which results in a simpler and more economical method to produce well crystallized γ-Fe2O3 nanoparticles (NPs) with average sizes within the 3-17 nm range. The NPs were characterized by TEM, SAED, XRD, DLS-QELS, Mössbauer spectroscopy at different temperatures, FT-IR and magnetic measurements. The obtained γ-Fe2O3 NPs are coated with oleic acid and, in a lower quantity, with oleylamine (about 1.5 nm in thickness). It was shown that changing operative variables allows us to tune the average particle diameters and obtain a very narrow or monodisperse distribution of sizes. The γ-Fe2O3 NPs behave superparamagnetically at room temperature and their magnetization saturation is reduced by about 34% in comparison with bulk maghemite. The results indicate that the distance between two neighbour NPs, generated by the coating, of about 3 nm is insufficient to inhibit interparticle magnetic interactions when the average diameter is 8.8 nm. The good quality of the NPs, obtained through the present low-cost and easy-handling process, open a new perspective for future technological applications.

Antiproliferative activity and interactions with cell-cycle related proteins of the organotin compound triethyltin(IV)lupinylsulfide hydrochloride.

Organotin compounds, particularly tri-organotin, have demonstrated cytotoxic properties against a number of tumor cell lines. On this basis, triethyltin(IV)lupinylsulfide hydrochloride (IST-FS 29), a quinolizidine derivative, was synthesized and developed as a potential antitumor agent. This tin-derived compound exhibited potent antiproliferative effects on three different human cancer cell lines: teratocarcinoma of the ovary (PA-1), colon carcinoma (HCT-8) and glioblastoma (A-172). Cytotoxic activity was assessed by MTT and cell count assays during time course experiments with cell recovery after compound withdrawal. Significant cell growth inhibition (up to 95% in HCT-8 after 72 h of exposure), which also persisted after drug-free medium change, was reported in all the cell lines by both assays. In addition, the cytocidal effects exerted by IST-FS 29 appeared more consistent with necrosis or delayed cell death, rather than apoptosis, as shown by morphologic observations under light microscope, DNA fragmentation analysis and flow cytometry. In the attempt to elucidate whether this compound might affect genes playing a role in G1/S phase transition, the expressions of p53, p21(WAF1), cyclin D1 and Rb, mainly involved in response to DNA-damaging stress, were analyzed by Western blot. Heterogeneous patterns of expression during exposure to IST-FS 29 were evidenced in the different cell lines suggesting that these cell-cycle-related genes are not likely the primary targets of this compound. Thus, the present data seem more indicative of a direct effect of IST-FS-29 on macromolecular synthesis and cellular homeostasis, as previously hypothesized for other organotin complexes.

Cytotoxicity in vitro and preliminary antitumor activity in vivo of a novel organotin compound.

The cytotoxic effect and antitumor activity induced by the novel organotin compound triethyltin(IV)lupinyisulfide hydrochloride, have been investigated. Different patterns of antiproliferative effects have been observed in a panel of human tumor cell lines in vitro. Toxicity studies in mice reported acute toxicity at the doses of 21 and 17.5 mg/kg which progressively disappeared at lower concentrations. On this basis, the doses of 3.5, 7 and 14 mg/kg were selected to assess the antitumor activity in vivo against the P388 leukemic cells xenografted in mice. This compound was able to induce a dose-dependent significant reduction of tumor volume, up to 46%, at the highest concentration (p = 0.0062) without important toxicity, as also confirmed by histological analysis of the main organ tissues. This preliminary study seems to hold interest for further investigations in different tumor models as well as for the evaluation of optimal drug route and schedule.

Increased cyclin D1 expression is associated with features of malignancy and disease recurrence in ovarian tumors.

Alterations in the expression of cyclin D1 have been reported frequently in several human cancers, but their significance in the multistep model of carcinogenesis has been scantly described. To define the pattern of cyclin D1 expression in the development of ovarian cancer and clinical outcome, 55 cases of benign ovarian tumors, 12 borderline cases, and 37 ovarian carcinomas (32 primary and 5 recurrent carcinomas) were studied. Analyses were carried out on fresh tumor specimens by Western blotting and reverse transcription-PCR and provided significant superimposable results (P = 0.00001). Cyclin D1 abundance was classed according to the densitometric values as undetectable, detectable, well detectable, and highly detectable. A significant increase (P < 0.000001) in median cyclin D1 values was observed from benign (0.038; range, 0.001-0.705) to borderline (0.226; range, 0.001-0.623) to malignant (0.347; range, 0.027-2.330) to recurrent (0.887; range, 0.309-2.2260) tumors. In addition, higher median cyclin D1 values were reported in serous carcinomas (P = 0.058) and advanced-stage diseases (P = 0.003). Survival analyses carried out in the 32 primary carcinomas showed no significant difference in overall survival between detectable versus well/highly detectable cyclin D1 neoplasms. Conversely, a significant relationship between cyclin D1 expression and progression-free survival was found (P = 0.031). These results may elucidate the function of altered cyclin D1 expression in ovarian tumorigenesis and provide a basis for additional studies on its prognostic role.

[Rotavirus laboratory network: results after one year of observation]. / Red de laboratorios de Rotavirus: resultados del primer año de vigilancia.

Rotavirus is the most common cause of severe diarrhea in children and it has been estimated that in Argentina Rotavirus is responsible for 21,000 hospitalizations, 85,000 medical attentions and an annual medical cost of US$ 27 millions. Given that a Rotavirus vaccine is about to be approved, a laboratory network based surveillance system was organized. Herein, we present the results after one year of study. Severe diarrhea was responsible for 9% of pediatric hospitalizations and rotavirus was detected in 42.1% of the diarrhea cases. We estimated that Rotavirus causes 3.8% of pediatric hospitalizations. The number of diarrhea and Rotavirus diarrhea hospitalizations was greater during the first year of life (62% and 71.3%, respectively). The number of diarrhea hospitalizations during the December-May semester was significantly higher than the rest of the year. A Rotavirus diarrhea peak was detected between April and June. These results indicate that Rotavirus is the most important etiological agent of severe diarrhea in Argentine children and show the importance of performing Rotavirus diagnosis in every pediatric hospital. The additional costs will be compensated by many benefits such as better use of antibiotics, improved nosocomial spread control, better handling of hospital beds and of laboratory resources and of the hospitalized patient.

Red de laboratorios de Rotavirus: resultados del primer año de vigilancia / Rotavirus laboratory network: results after one year experience

Rotavirus es el principal agente productor de diarrea infantil y se ha estimado que provoca en Argentina 21.000 hospitalizaciones, 85.000 atenciones ambulatorias, y un costo mayor a los 27 millones de dolares anuales. Ante la inminente aprobación de una vacuna contra este patógeno se organizó un Sistema de Vigilancia Epidemiológica en base a una Red de laboratorios. Se presentan los resultados obtenidos luego del primer año de funcionamiento de esta Red. Se encontró que el 9 por ciento de la internación pediátrica es debido a diarrea aguda, y rotavirus se halló en el 42,1 por ciento de los casos estudiados. Se estimó que rotavirus provoca el 3,8 por ciento de las internaciones pediátricas. La internación por diarrea y la internación asociada a diarrea por rotavirus fue mayor en el primer año de vida (62 por ciento y 71,3 por ciento respectivamente). En el semestre de diciembre a mayo el número de internaciones por diarrea fue significativamente mayor que en el semestre restante. Se detectó un pico de diarreas por rotavirus entre abril y junio en las distintas Unidades centinelas. Estos resultados señalan a los rotavirus como el principal agente etiológico de la gastroenteritis infantil aguda en nuestro país y avalan la necesidad de incorporar su diagnóstico en todos los hospitales pediátricos. Los costos adicionales serán ampliamente superados por los beneficios relacionados con elmejor manejo de las camas hospitalarias, los recursos del laboratorio, y el paciente internado por diarrea, el uso correcto de antibióticos, y el control de la diseminación intrahospitalaria de rotavirus

Red de laboratorios de Rotavirus: resultados del primer año de vigilancia / Rotavirus laboratory network: results after one year experience

Rotavirus es el principal agente productor de diarrea infantil y se ha estimado que provoca en Argentina 21.000 hospitalizaciones, 85.000 atenciones ambulatorias, y un costo mayor a los 27 millones de dolares anuales. Ante la inminente aprobación de una vacuna contra este patógeno se organizó un Sistema de Vigilancia Epidemiológica en base a una Red de laboratorios. Se presentan los resultados obtenidos luego del primer año de funcionamiento de esta Red. Se encontró que el 9 por ciento de la internación pediátrica es debido a diarrea aguda, y rotavirus se halló en el 42,1 por ciento de los casos estudiados. Se estimó que rotavirus provoca el 3,8 por ciento de las internaciones pediátricas. La internación por diarrea y la internación asociada a diarrea por rotavirus fue mayor en el primer año de vida (62 por ciento y 71,3 por ciento respectivamente). En el semestre de diciembre a mayo el número de internaciones por diarrea fue significativamente mayor que en el semestre restante. Se detectó un pico de diarreas por rotavirus entre abril y junio en las distintas Unidades centinelas. Estos resultados señalan a los rotavirus como el principal agente etiológico de la gastroenteritis infantil aguda en nuestro país y avalan la necesidad de incorporar su diagnóstico en todos los hospitales pediátricos. Los costos adicionales serán ampliamente superados por los beneficios relacionados con elmejor manejo de las camas hospitalarias, los recursos del laboratorio, y el paciente internado por diarrea, el uso correcto de antibióticos, y el control de la diseminación intrahospitalaria de rotavirus(AU)

Red de laboratorios de Rotavirus: resultados del primer año de vigilancia. / [Rotavirus laboratory network: results after one year of observation]

Rotavirus is the most common cause of severe diarrhea in children and it has been estimated that in Argentina Rotavirus is responsible for 21,000 hospitalizations, 85,000 medical attentions and an annual medical cost of US$ 27 millions. Given that a Rotavirus vaccine is about to be approved, a laboratory network based surveillance system was organized. Herein, we present the results after one year of study. Severe diarrhea was responsible for 9

of pediatric hospitalizations and rotavirus was detected in 42.1

of the diarrhea cases. We estimated that Rotavirus causes 3.8

of pediatric hospitalizations. The number of diarrhea and Rotavirus diarrhea hospitalizations was greater during the first year of life (62

and 71.3

, respectively). The number of diarrhea hospitalizations during the December-May semester was significantly higher than the rest of the year. A Rotavirus diarrhea peak was detected between April and June. These results indicate that Rotavirus is the most important etiological agent of severe diarrhea in Argentine children and show the importance of performing Rotavirus diagnosis in every pediatric hospital. The additional costs will be compensated by many benefits such as better use of antibiotics, improved nosocomial spread control, better handling of hospital beds and of laboratory resources and of the hospitalized patient.

Control of cyclin D1 expression by antisense oligonucleotides in three ovarian cancer cell lines.

Cyclin D1 is a critical gene controlling the G1 phase progression through the cell cycle. Alterations of cyclin D1 have been demonstrated in a variety of cancer types. We recently reported that increased cyclin D1 expression is associated with malignancy also in ovarian tumors. Three human ovarian cancer cell lines (SW626, OVCAR-3, IGROV1), expressing high levels of this gene, were used to investigate the effects induced by antisense oligonucleotides to cyclin D1 as antiproliferative compounds. Unmodified 18 mer oligomers, targeted to the translation start site of the cyclin D1 cDNA, were able to inhibit the growth of the three cell lines after a single administration of 40 microM. The pattern of cell number reduction ranged between 30 and 55% after 48 h of treatment. Moreover, by RT-PCR and Western blotting, a marked decrease of the cyclin D1 transcript and protein (up to 77% in the SW626) was detected after 24 and 48 h, respectively, from antisense exposure. Conversely, no relevant inhibition was reported in the sense-treated cells. The present data confirm the role of cyclin D1 expression in the proliferative behavior of ovarian cancer and provide additional information that might be helpful in the search for new therapeutic strategies of this disease.

Synthesis and biological activity of gold and tin compounds in ovarian cancer cells.

We have investigated the patterns of in vitro cytotoxicity, induced by six newly synthesized gold and tin compounds, in three human ovarian cancer cell lines (SW 626, IGROV 1 and OVCAR-3). Four gold compounds, i.e. gold(I)lupinylsulfide hydrochloride [1] (containing a naked gold atom), triethylphosphinogold(I)lupinylsulfide hydrochloride [2], triphenyl-phosphinogold(I)lupinylsulfide hydrochloride [3] and 1 ,2-bis(diphenylphosphino)ethane bis[gold(I)lupinylsulfide] dihydrochloride [4] (all containing a gold atom coordinated with different phosphines), were prepared. Moreover, the triethylphosphinogold(I)(2-diethylamino)ethylsulfide hydrochloride [5] in which the simple diethylaminoethylthiol replaced the bulky lupinylthiol was synthesized. The tin compound, triethyltin(IV)lupinylsulfide hydrochlorlde [6], was also studied. Comparative tests with cisplatin, the most widely used antitumor agent in ovarian cancer, were carried out in biological Investigations. In vitro cytotoxicity, by MTT assay, showed that compound [4] and compound [6] exhibited interesting antiproliferative activity in all the three cell lines (mean IC50=1.3 and 0.7 microM, respectively) compared to cisplatin (mean IC50=4.8 microM). In addition, the PA-1 cell line, more sensitive to cisplatin (IC50=0.6 microM), was included as a comparison in the study. Cell count assays confirmed the cytotoxic properties of compounds [4] and [6] against the four cell lines, reporting higher growth Inhibition potency than cisplatin, with IC50 values in the sub-micromolar range.

Comparison of total alkaline phosphatase and three assays for bone-specific alkaline phosphatase in childhood and adolescence.

We compared serum levels of total alkaline phosphatase (TAP) and bone-specific alkaline phosphatase (BAP) as determined by three different assays (lectin affinity electrophoresis, immunoradiometric assay, enzyme-linked immunosorbent assay) in subjects aged 5-20 years suffering from X-linked hypophosphatemic rickets (n = 14), chronic renal failure (n = 10) and chronic cholestatic liver disease (n = 16). Results were compared to controls of the same age and were expressed as standard deviation scores (SDS). TAP correlated significantly with BAP (r > 0.9 for each assay; p < 0.001) in controls. In children with cholestatic diseases, TAP (median SDS + 2.0) was elevated, but BAP, as measured by the electrophoretic assay, was within the reference range for most patients (median SDS: -0.4; p = 0.003 for the difference between the median SDS of TAP and BAP). In contrast, results for BAP as determined by the two immunoassays were not significantly different from TAP in any of the three patient groups (p > 0.05 in each group for both assays). In this study, the two immunoassays did not have a detectable advantage over lectin affinity electrophoresis in the determination of BAP.

Expression of cyclin D1 correlates with malignancy in human ovarian tumours.

Cyclin D1 is a cell cycle regulator of G1 progression that has been suggested to play a relevant role in the pathogenesis of several human cancer types. In the current study, the expression of cyclin D1 has been investigated in a series of 33 patients, with benign (10 patients), borderline (five patients) and malignant (18 patients) ovarian disease. Cyclin D1 protein and mRNA content were analysed by Western blotting and reverse transcriptase polymerase chain reaction respectively. The levels of cyclin D1 protein were undetectable in patients with benign disease, detectable in the majority of patients with borderline disease and elevated in those with ovarian carcinomas, being significantly related to the degree of malignancy (carcinoma vs benign, P = 0.0001; benign vs borderline, P = 0.0238). A significant relationship between cyclin D1 expression and tumour proliferative activity was also found (P = 0.000001). Moreover, eight benign lesions, two borderline tumours and 11 carcinomas proved to be suitable for the analysis of cyclin D1 transcript, and emerging data demonstrated significant agreement between protein abundance and mRNA expression. Results from the current study suggest that cyclin D1 expression is associated with the degree of transformation and most probably plays a role in the early development of ovarian malignancy.

Antisense oligonucleotides as therapeutic agents.

The potential for modulating gene expression by the use of antisense oligonucleotides has become increasingly interesting in recent years. Antisense oligonucleotides are complementary nucleic acid fragments that hybridize to target sequences within RNA to form a DNA-RNA duplex, resulting in the block of translation of messenger RNA into the protein. Advances in chemistry and molecular biology have provided the basis to develop antisense oligodeoxynucleotides and improve their selectivity, stability and specificity of action. The antisense technology has been extensively used in vitro and in vivo as a tool to study the regulatory mechanisms in biologic processes and as potential therapeutic agents in cancer, viral infections and genetic disorders. In the present review, the various approaches for the use of antisense molecules in oncology, virology, genetic and inflammatory diseases are described; several studies, supporting the in vitro and in vivo applications of this technology, are also presented. Moreover, the potential clinical use of antisense therapies is discussed.

Establishment and characterization of three new cell lines derived from the ascites of human ovarian carcinomas.

Three human cancer cell lines (OC 314, OC 315, and OC 316) were newly established in permanent culture from the ascites of patients with serous adenocarcinoma of the ovary. OC 314 was derived from an untreated tumor presenting with ascites at diagnosis; OC 315 was isolated from a neoplasm progressing after cisplatin-containing regimen; and OC 316 was collected from a patient with pleural metastasis at diagnosis, resistant to different chemotherapeutic treatments including Taxol. These cell lines were repetitively subcultured once to twice a week through 75-80 passage generations. Tumor cells grew as monolayers and displayed epithelial-like morphology, consistent with a feature of adenocarcinoma, which was then confirmed by the expressions of cytokeratins and vimentin. The cell lines proved highly tumorigenic when transplanted into nude mice, both subcutaneously and intraperitoneally. In addition, the mice inoculated with subcutaneous OC 316 developed extremely aggressive tumor, also invading the peritoneum, which correlated with the malignant behavior of the original tumor. Drug sensitivity, evaluated by the MTT assay, showed that the three cell lines expressed similar sensitivity to doxorubicin. Responses to cisplatin essentially reported low sensitivity of OC 314 and OC 315 and resistance of OC 316, thus reflecting the original sensitivity at the clinical level.

Inhibition of DNA polymerase alpha expression and cell growth, a possible triple helix mechanism.

The antiproliferative effects mediated by a 14-mer homopyrimidine oligonucleotide (5' CTTTCT-CTTTTCTC3'), designed to form DNA triplex with a purine region of the DNA polymerase alpha promoter, were evaluated on the human breast cancer cell line MDA-MB 231. In order to stabilize the triple complex under physiologic conditions, replacement of cytosines by methylcytosines in the oligomer sequence was carried out. Band-shift analyses demonstrated a complete triplex formation between the radiolabeled target duplex DNA and the methylcytosine-modified oligomer at the concentration of 0.1 microM under physiologic pH and temperature. A single exposure of MDA-MB 231 cells to 0.5 microM methylcytosine-modified oligonucleotide was able to markedly reduce the cell number and the percentage of cells in DNA synthesis up to 58% and 66%, respectively, compared with controls. Furthermore, a 48% reduction in the amount of the DNA polymerase alpha mRNA was reported after treatment with the oligomer. In conclusion, data from the present study demonstrate that an oligonucleotide to DNA polymerase alpha promoter, designed to form a triple helix with target double-stranded DNA, inhibits the expression of the reporter gene at the biologic and molecular levels, suggesting a possible triplex-mediated mechanism of action.

Feasibility of thymidine labelling index on fine needle aspirates from breast cancer: comparison with surgical samples.

Fine-needle aspiration (FNA) provides a suitable diagnostic tool in the management of patients with breast cancer lesions. The current study reports on tumor proliferative activity, by 3H-Thymidine Labelling Index (TLI), assessed on 59 FNA (TLI1) and 28 surgical specimens (TLI2) from the same breast cancer patients. Median TLI values from FNA and surgical material were 1.0% and 0.7%, respectively. In the 28 patients, evaluable for the comparison between TLI1 and TLI2, the association was found to be highly significant (p = 0.000). Moreover, no change in tumor proliferative activity was observed in the majority (79%) of cases when evaluated preoperatively and at surgery. This study confirms the feasibility of TLI analysis on FNA from breast cancer and provides results superimposable on those obtained in a tissue sample from the same patient.

Injectable gold dermatitis and proteinuria: retreatment with auranofin.

Seven female patients with classical rheumatoid arthritis (RA), treated successfully with injectable gold salts (Fosfocrisolo ICI, 0.10 g/week, with a serum gold concentration of 200-400 mcg/dl), experienced severe gold side-effects after 3 to 20 months of therapy, requiring their withdrawal from gold despite the good results in both clinical and laboratory findings. Four patients showed mucocutaneous side-effects (2 dermatitis and 2 stomatitis) and three a moderate or severe proteinuria. Renal biopsy was performed in these patients, with a histological picture of membranous glomerulonephritis referable to gold therapy. Remission inducing drug (R.I.D.) therapy being mandatory in patients with a chronic progressive disease, and in view of the previous efficacy of gold salts, the patients were put on oral gold, Auranofin being administered 3 mg b.i.d. Both the mucocutaneous side-effects and the proteinuria ameliorated within 2 to 6 months, and the remission of the disease was maintained. The chemical and pharmacokinetic differences between the above two gold compounds are discussed.