Is there a role for salvage radiotherapy in locally advanced breast cancer refractory to neoadjuvant chemotherapy?

INTRODUCTION: Locally advanced breast cancer (LABC) is a major problem, especially in developing countries. The standard treatment for LABC is neoadjuvant chemotherapy, with or without anti-Her2 therapy, followed by surgery, radiotherapy, and adjuvant systemic treatment if appropriate. However, there are few data in the literature addressing alternatives when neoadjuvant chemotherapy fails to reduce the tumour for surgery. MATERIALS AND METHODS: We conducted a retrospective study including all patients who had non-metastatic LABC treated with neoadjuvant chemotherapy and who were not eligible for surgical resection; these patients were submitted to salvage radiotherapy (RTX) between January 2000 and December 2012 at the Brazilian National Cancer Institute. RESULTS: Fifty-seven patients were included, with a median age of 51 (23-72) years. The most frequent clinical stages were IIIA and IIIB, corresponding to 19.3% and 70.2%, respectively; mean tumour size was 8.74 (3-18) cm, and 44 patients (77.2%) had nodal involvement. Chemotherapeutic regimens containing anthracyclines were prescribed to 98.2% of the patients. Fifteen patients (26.3%) received taxanes and anthracyclines. Radiation dose was 50 Gy divided into 25 fractions; 43 patients (75.4%) had their tumours downsized by RTX and underwent mastectomy. Overall survival (OS) was 38 (23-52) months. Patients who were submitted to surgery had an OS of 49 (28-70) months and those who were not eligible for mastectomy after radiotherapy had an OS of 18 (9-27) months. CONCLUSION: This retrospective study confirms that RTX is an effective treatment to downsize LABC tumours with low or no response to chemotherapy, thereby enabling surgical resection which may improve overall patient outcome.

Pointing to potential reference areas to assess soil mutagenicity.

Several have been performed to evaluate the mutagenicity of soil samples in urban and industrial areas. The use of uncontaminated reference areas has been an obstacle to the study of environmental mutagenesis. The study aimed to indicate a methodology to define reference areas in studies of environmental contamination based on "Ambient Background Concentration" of metallic elements associated with the Salmonella/microsome assay. We looked at three potential reference areas, two of them close by the industrial sources of contamination (São Jerônimo reference, near the coal-fired power plant, and Triunfo reference, near the wood preservative plant), but not directly influenced by them and an area located inside a protected area (Itapuã reference). We also carried out chemical analyses of some metals to plot the metal profile of these potential reference areas and define basal levels of these metals in the soils. After examining the mutagenicity of the inorganic extracts using strains TA98, TA97a, and TA100, in the presence and absence of S9 mix, we indicated the São Jerônimo reference and the Itapuã reference as two sites that could be used in future studies of mutagenicity of soils in southern Brazil. The association between a mutagenicity bioassay and the "Ambient Background Concentration" seems to be a useful method to indicate the reference areas in studies of contamination by environmental mutagens, where these results were corroborated by canonical correspondence analysis.

Generation and characterization of embryonic stem-like cell lines derived from in vitro fertilization Buffalo (Bubalus bubalis) embryos.

In the present study, buffalo embryonic stem-like (ES-like) cell lines were successfully isolated, cultured and characterized. From a total of 92 normal buffalo embryos obtained by in vitro fertilization, 18 were morulae, 33 were blastocyst and 41 were hatched blastocyst, the inside of morulae or inner cell masses of blastocysts were isolated mechanically and cultured onto mitomocin-C-inactivated buffalo embryonic fibroblasts as feeder layers. Alkaline phosphatase (AP) of ES-like cells, as well as the specific stage embryonic antigen SSEA-1, SSEA-3, SSEA-4 and transcription factor OCT-4, was used to evaluate the characterization of the cells. The spontaneous differentiation of ES-like cells was induced by culturing on leukaemia inhibitory factor-free medium for more than 2 weeks without passage. To evaluate mark gene expression, total RNA was extracted from cells, and specific primers were used for reverse transcriptase-polymerase chain reaction (RT-PCR). After 8-10 days of culture, primary ES-like cell colonies were formed in 0% (0/18) of morulae, 24.24% (8/33) of blastocysts and 60.98% (25/41) of hatched blastocysts, respectively. The forming rate of primary ES-like cells colonies in hatched blastocyst group was significantly (p < 0.05) higher than the obtained for other groups. Two ES-like cell lines could survive to eight passages at least by using the method of mechanical dissociation, but just three passages by using the method of enzymatic dissociation. The cells formed large, multicellular colonies with distinct boundaries, exhibited many important features of ES/ES-like cells, including positive AP, SSEA-1, SSEA-3 and SSEA-4 activity. Undifferentiated buffalo ES-like cells expressed Oct-4, Nanog, Sox2 gene mRNA. In vitro differentiation experiments had demonstrated that those cells were pluripotent.

Generation of buffalo (Bubalus bubalis) transgenic chimeric and nuclear transfer embryos using embryonic germ-like cells expressing enhanced green fluorescent protein.

The possibility of producing transgenic buffalo embryos by chimera and nuclear transfer (NT) using buffalo embryonic germ (EG)-like cells expressing enhanced green fluorescent protein (EGFP) has been explored in this study. Buffalo EG-like cells and fibroblasts with two to eight passages were transfected with the lined plasmid (pCE-EGFP-IRES-Neo-dNdB) using Lipofectamine 2000 and selected by culturing in 200 microg/ml G418 for 6-8 days. G418 resistant fibroblasts and EG-like cells were used for embryo chimera and NT. To produce blastocysts by chimera, 8-16 cells embryos were injected with EG-like and fibroblast cells. Then, to produce blastocysts by NT, in vitro maturated oocytes were enucleated and afterwards EG-like/fibroblast cells transferred into the perivitelline space. No statistical differences were observed for the total blastocyst produced by the chimeric method, using EG-like and fibroblasts as donor cells, resulting on an accomplishment of 35.6% vs 33.3%, respectively. Nevertheless, besides from the 37 blastocysts produced, 23 (62.2%) from EG-like cells expressed EGFP, none of blastocysts from foetal fibroblasts expressed this protein. When the NT method was used, no statistical difference among different generations was observed in the percentage of oocytes fused, cleaved, and developed to blastocysts after NT for EG-like cells. On average, the percentage of oocytes fused, cleaved, and developed to blastocysts after NT was respectively 81.8%, 67.7% and 10.7%. For the expression of EGFP, from the 12 blastocysts produced by NT, 7 of them were positive, while none of NT embryos from EGFP positive fibroblasts developed to blastocysts. Results of the present study clearly demonstrated that gene transfected buffalo EG-like cells have the ability to form chimeric embryos after injecting into buffalo early embryos and reprogramming ability after NT, which can be employed to produce transgenic buffalos through either embryo chimera or NT.

Combined analysis of EGF+61G>A and TGFB1+869T>C functional polymorphisms in the time to androgen independence and prostate cancer susceptibility.

Proliferative mechanisms involving the epidermal growth factor (EGF) and transforming growth factor beta (TGF-beta(1)) ligands are potential alternative pathways for prostate cancer (PC) progression to androgen independence (AI). Thus, the combined effect of EGF and TGFB1 functional polymorphisms might modulate tumor microenvironment and consequently its development. We studied EGF+61G>A and TGFB1+869T>C functional polymorphisms in 234 patients with PC and 243 healthy individuals. Intermediate- and high-proliferation genetic profile carriers have increased risk for PC (odds ratio (OR)=3.76, P=0.007 and OR=3.98, P=0.004, respectively), when compared with low proliferation individuals. Multivariate analysis showed a significantly lower time to AI in the high proliferation group, compared with the low/intermediate proliferation genetic profile carriers (HR=2.67, P=0.039), after adjustment for age, metastasis and stage. Results suggest that combined analysis of target genetic polymorphisms may contribute to the definition of cancer susceptibility and pharmacogenomic profiles. Combined blockage of key molecules in proliferation signaling pathways could be one of the most promising strategies for androgen-independent prostate cancer.

Trypanosoma (Megatrypanum) saloboense n. sp. (Kinetoplastida: Trypanosomatidae) parasite of Monodelphis emiliae (Marsupiala: Didelphidae) from Amazonian Brazil.

Trypanosoma (Megatrypanum) soloboense n. sp., is described in the Brazilian opossum Monodelphis emiliae (Thomas, 1912) from primary forest in the Salobo area of the Serra dos Carajás (6 degrees S, 50 degrees 18' W) Pará State, North Brazil. Two morphologically different trypomastigotes were noted. Slender forms, regarded as immature parasites, have a poorly developed undulating membrane adhering closely to the body: large, broad forms with a well developed membrane are considered to be the mature trypomastigotes and have a mean total length of 71.2 microm (62.4-76.2) and a width of 6.1 (5.0-8.0). Infections studied in two opossums were of very low parasitaemia. The large size of T. (M.) saloboense readily distinguishes it from the two previously described members of the subgenus Megatrypanum of neotropical marsupials, T. (M.) freitasi Régo et al., 1957 of Didelphis ozarae and D. marsupialis, and T. (M.) samueli Mello, 1977 of Monodelphis domesticus, which measure only 49.0-51.5 microm and 42.4 microm respectively. No infections were obtained in hamsters inoculated with triturated liver and spleen from one infected M. emiliae, or in laboratory mice inoculated with epimastigotes from a blood-agar culture. No division stages could be detected in the internal organs or the peripheral blood.

Association between FAS polymorphism and prostate cancer development.

The role of FAS polymorphisms in prostate cancer has not been studied. Using the PCR-based restriction fragment-length polymorphism methodology, we evaluated FAS gene locus -670 genotypes in DNA from 904 men: 657 prostate cancer patients and 247 healthy controls. We found that carriers of AG or GG genotypes have a statistically significant protection (odds ratio (OR)=0.30; confidence interval (CI): 0.20-0.44 and OR=0.22; CI: 0.12-0.74, respectively) for disease with extra-capsular invasion. Taken together, a 72% protection was found for G allele carriers (OR=0.28; CI: 0.19-0.41). Fas exist as membrane-bound and soluble forms and with opposite roles. They derive from the same gene by alternative splicing. Membrane Fas receptors trigger apoptosis whereas, on the other hand, soluble Fas (sFas) bind to Fas ligand antagonizing Fas-Fas ligand apoptotic pathway. Our results suggest that G allele may reduce sFas levels preventing the apoptotic inhibition caused by the soluble form.

New species of Eimeria and Isospora (Protozoa: Eimeriidae) in Geochelone spp. (Chelonia: Testudinidae) from Amazonian Brazil.

Tetrasporocystic, dizoic oocysts of reptiles have been separated by some authors into the genera Eimeria, Choleoeimeria and Acroeimeria (Protozoa: Eimeriidae), based on the site and mode of development of their endogenous stages. The majority of Eimeria species have been, and still are, however, described on oocyst morphology alone. Four different oocysts with this basic morphology were encountered in the faeces of Brazilian tortoises, Geochelone carbonaria Spix, 1824 and are assigned to the genus Eimeria, with the view that they can readily be transferred to the genus Choleoeimeria or Acroeimeria if this is indicated by a future examination of their endogenous development. A morphological comparison distinguishes the oocysts from those of Eimeria spp., previously described in chelonids of the family Testudinidae, and the names E. amazonensis, E. carbonaria, E. carajasensis and E. wellcomei n. spp. are proposed. Coccidial infection appears to be common in G. carbonaria, with three of seven animals examined passing oocysts. Oocysts of Isospora rodriguesae n. sp. (Protozoa: Eimeriidae) are described in the faeces of Geochelone denticulata Linnaeus, 1766. They are morphologically very different from those of Isospora testudae, Davronov, 1985 in Testudo horsfieldi. Eimeria motelo Hurková et al., 2000, previously described in Geochelone denticulata from Peru, is here recorded in the some chelonid from Amazonian Brazil.

Assessment of sperm apoptosis in cryopreserved bull semen after swim-up treatment: a flow cytometric study.

The techniques used to prepare bovine spermatozoa for in vitro fertilization, to enhance the percentage of motile sperm cells include the swim-up (SU) method, among others. The objective of the present study was to evaluate the phosphatidylserine (PS) translocation and plasma membrane integrity as the indicator of apoptosis and necrosis in post-thaw bull sperm after SU treatment using annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) assay. A flow cytometric method was employed to measure apoptosis levels on frozen-thawed bull spermatozoa. The assay detects PS translocation across the plasma membrane using a fluorescein-labelled annexin-V and PI. By using the annexin V/PI assay four different subpopulations of sperm were observed: (i) a population of apoptotic sperm, labelled with annexin V-FITC but not with PI; (ii) a population of early necrotic spermatozoa, sperm labelled with annexin-FITC and PI; (iii) a population of necrotic sperm, labelled with PI but not with annexin-FITC; and (iv) a population of fully viable sperm cells, sperm not labelled with annexin V-FITC and without PI. Results clearly indicated that SU technique itself could have an adverse effect on the spermatozoa membrane stability. It has also been found, significant differences between bulls in the levels of apoptotic sperm, after SU treatment.

Analysis of forces developed during mechanical preparation of extracted teeth using RaCe rotary instruments and ProFiles.

AIM: To compare the torque and load during instrumentation with ProFile and RaCe nickel-titanium rotary instruments. METHODOLOGY: Thirty human incisor roots 12 mm in length were embedded in epoxy resin, and divided into three groups of 10 specimens each. Instruments employed in each group were as follows: RaCe .02 Step Back Files in group 1; RaCe .04 Step Back Files in group 2; and .04 ProFiles in group 3. Two load cells, whose outputs were connected to a digital oscilloscope, recorded torque and vertical load. Data were analysed by one-way anova. RESULTS: Torque values were statistically different between groups 1 and 3, and between groups 2 and 3 (torque was higher in group 3 than in groups 1 and 2). Vertical loads were statistically higher in group 3 than in groups 1 and 2. CONCLUSIONS: Torsional and vertical forces can be evaluated during instrumentation of straight root canals using the device and methods described in this study. When the step-back technique was employed, torque and vertical load obtained with RaCe instruments were lower than that obtained with ProFiles.

Bacterial population of a two-phase anaerobic digestion process treating effluent of cassava starch factory.

Different groups of microorganisms in a two-phase anaerobic system were enumerated to evaluate the prevalence of specific groups and species. Total and fecal coliforms showed similar values both in acidogenic and methanogenic reactors. The fecal streptococci were 4-fold higher in the acidogenic reactor, when compared with those of the methanogenic reactor. As expected, no methane forming or sulfate reducing bacteria were found in the acidogenic reactor. The populations of methanogenic bacteria were dominated by a mixed population of straight to curved rods and multicellular filaments which strongly resembled members of the genus Methanosaeta. Seven prevalent species of facultative anaerobic gram-negative bacteria were identified as Previdencia alcalifacienciens, Providencia rettgeri, Enterobacter cloaceae, Citrobacter freundii, Klebsiella oxytoca, Proteus pennri and Yersinia enterocolitica. The species Pseudomonas aeruginosa, Stentophomonas maltophila and Acinotobacter iwoffi, were the most frequently isolated glucose non-fermenting gram-negative bacilli. Among these species, only P. aeruginosa was present in high number in each sample.

Rotavirus strains bearing genotype G9 or P[9] recovered from Brazilian children with diarrhea from 1997 to 1999.

Human rotavirus strains belonging to genotype G9 or P[9] were detected in a collection of stool specimens from children with diarrhea in two cities of the state of Rio de Janeiro, Brazil, between March 1997 and December 1999. G9 strains were first detected in April 1997 and remained prevalent until the end of the study, at a frequency of 15.9% (n = 157). A high percentage of VP7 nucleotide (99.0 to 99.5%) and deduced amino acid identity (98.6 to 99.1%) was found between three randomly selected Brazilian G9 strains and the American G9 strain US1205. A novel G9:P[4] genotype combination was detected in addition to G9:P[8] and G9:P[6], demonstrating that this G genotype may undergo constant genetic reassortment in nature. The P[9] rotavirus strains constituted 10.2%, the majority of which were detected between April and July 1997. The RNA electrophoretic migration pattern of the G3:P[9] strains resembled that of AU-1 virus (G3:P3[9]), suggesting a genetic similarity between the Brazilian G3:P[9] strains and the Japanese virus, which is similar to a feline rotavirus genetically.

Respiratory burst activity in activated and unstimulated isolated bovine blood neutrophils during experimentally induced Escherichia coli mastitis.

The respiratory burst activity, measured as H2O2 production, of isolated bovine polymorphonuclear leucocytes (PMN) was evaluated during experimentally induced Escherichia coli mastitis by means of flow cytometry in cells activated by phorbol 12-myristate 13-acetate (PMA) and in unstimulated cells. As expected, a significantly reduced respiratory burst activity was observed in PMA-activated PMN 18 h after intramammary inoculation with Escherichia coli. At this time only 75% of the PMA-activated PMN showed a respiratory burst, but with a higher intensity than that measured before and later after infection with Esch. coli. In addition, an increase in the respiratory burst activity was observed in unstimulated blood PMN during a short period at 18 h after infection, when up to 30% of the unstimulated PMN had a respiratory burst activity. The increase in the respiratory burst intensity of PMA-activated PMN and the spontaneously augmented production of reduced oxygen species by the unstimulated PMN during infection with Esch. coli might indicate the production of a natural stimulator of burst activity in circulation, most probably originating from the inflamed udder.

Development and maturation of neutrophils.

Inflammatory processes require the activation of immunocompetent cells. In mammals, neutrophil polymorphonuclear leukocytes (PMN) constitute one of the essential body defences against diseases. In this article knowledge of the development and maturation of neutrophils, the control of haematopoiesis and of the factors that regulate neutrophil production is reviewed. As it has recently become apparent that neutrophils can be primed by cytokines to have enhanced functional activity, the future utilization of these growth factors in bovine immunotherapy is briefly discussed.