Cisplatin and Etoposide in Childhood Germ Cell Tumor: Brazilian Pediatric Oncology Society Protocol GCT-91

Purpose In 1988, we formed a consortium of Brazilian institutions to develop uniform standards for the diagnostic assessment and multidisciplinary treatment of children and adolescents with germ cell tumors. We also implemented the first childhood Brazilian germ cell tumor protocol, GCT-91, evaluating two-agent chemotherapy with cisplatin and etoposide (PE). We now report on the clinical characteristics and survival of children and adolescents with germ cell tumors treated on this protocol. Patients and Methods From May 1991 to April 2000, 115 patients (106 assessable patients) were enrolled onto the Brazilian protocol with a diagnosis of germ cell tumor. Results Patients were treated with surgery only (n = 35) and chemotherapy (n = 71). Important prognostic factors included stage (P = .025), surgical procedure at diagnosis according to resectability (P = .032), and abnormal lactate dehydrogenase value at diagnosis (P = .001). Conclusion The improvement in survival by the introduction of a standard protocol is an important achievement. This is of particular importance for smaller institutions with previous limited experience in the treatment of childhood germ cell tumors. In addition, the results of a two-agent regimen with PE were favorable (5-year overall survival rate is 83.3% for patients in the high-risk group [n = 36] who received PE v 58.8% for patients in the high-risk patients group who received PE plus ifosfamide, vinblastine, and bleomycin [n = 17; P = .017]). Thus for selected patients, complex three-agent regimens may not be necessary to achieve long-term survival, even for some patients with advanced disease.

Molecular Characterization of Strains of Respiratory Syncytial Virus Identified in a Hematopoietic Stem Cell Transplant Outpatient Unit Over 2 Years: Community or Nosocomial Infection?

Respiratory syncytial virus (RSV) is recognized as the leading cause of nosocomial respiratory infection among hematopoietic stem cell transplant (HSCT) recipients, causing considerable morbidity and mortality. RSV is easily transmitted by contact with contaminated surfaces, and in HSCT units, more than 50% of RSV infections have been characterized as of nosocomial origin. From April 2001 to October 2002, RSV was identified by direct immunofluorescent assay in 42 symptomatic HSCT recipients. Seven RSV strains from 2001 and 12 RSV strains from 2002 were sequenced. RNA extraction, cDNA synthesis, and seminested polymerase chain reaction (PCR) with primers complementary to RSV genes G and F were pet-formed. PCR products were analyzed by nucleotide sequencing of the C-terminal region of gene G for typing (in group A or B). Of the 7 strains analyzed in 2001, only 2 belonged to group B; the other 5 belonged to group A. Of these 7 strains, 3 were identical and were from recipients receiving outpatient care. In 2002, of the 12 strains analyzed, 3 belonged to group A and the other 9 belonged to group B. Of these 9 strains, 7 were genetically identical and were also from recipients receiving outpatient care. Therefore, multiple strains of RSV cocirculated in the hematopoietic stem cell transplant units (ward and outpatient units) between 2001 and 2002. Nosocomial transmission was more likely to occur at the HSCT outpatient unit than in the HSCT ward. Infection control practices should also be implemented in the outpatient setting.

Comparison of the efficacy of two commercially available media for culturing one-cell embryos in the in vitro fertilization mouse model

Objective: To examine the effects of two commercial media on the development of mouse ova fertilized in vitro to the blastocyst stage. Design: Animal model. Setting: Academic institution. Animal(s): Eight-week old, superovulated mice. Intervention(s): One-cell embryos cultured in vitro up to the blastocyst stage in potassium-enriched simplex optimized medium (KSOM) or G1/G2 medium. Main Outcome Measure(s): Blastocyst and hatching rates, total cell number count, and proportion of allocation of cells to the inner cell mass (ICM) and trophectoderm (TE). Result(s): The percentage of zygotes that developed to the blastocyst stage 96 and 120 hours after insemination was statistically significantly higher in the KSOM group. The percentage of blastocysts that partially or completely hatched by day 5 of culture was 84% and 71% for the KSOM and G1/G2 groups, respectively, showing a statistically significant difference between the groups. The mean number of ICM cells was 11.7 +/- 4.0 and 9.2 +/- 5.2 for the zygotes cultured in KSOM and G1/G2 media, respectively, revealing a statistically significantly higher cell number in the ICM of blastocysts derived from culture in KSOM medium. The ICM/TE ratio in the blastocysts cultured in KSOM or G1/G2 media was similar in both groups. Conclusion(s): Commercially available KSOM medium is superior to sequential G1/G2 media for culturing one-cell embryos up to the blastocyst stage in the mouse IVF model.

Presence of residual basal cell carcinoma in re-excised specimens is more probable when deep and lateral margins were positive

In order to quantify presence of residual BCC in patients with histologic positive margins after the first excison and to correlate the presence of residual tumor in re-excised lesions with the location of the positive margin on the first excision, a retrospective evaluation of 2053 surgically treated BCC was performed. Only 38.3% of the re-excised lesions showed residual tumor. In the group of re-excised lesions where residual BCC was found, 13% had lateral positive margin in the first excision, 39% had deep positive margin and 48% had both lateral and deep positive margins. In the group of re-excised lesions where no residual BCC was found, 49% of the primary excised lesions had lateral positive margin, 32% had deep positive margin and 19% had both deep and lateral positive margins. The association between residual tumor and positive margins was statistically significant (p = 0.01). Our findings confirm that presence of residual tumour is more likely when both lateral and deep margins are compromised.

Topical interleukin-1 receptor antagonist inhibits inflammatory cell infiltration into the cornea

Interleukin (IL)-1 alpha and beta are important modulators of many functions of corneal epithelial and stromal cells that occur following injury to the cornea, including the influx of bone marrow-derived inflammatory cells into the stroma attracted by chemokines released from the stroma and epithelium. In this study, we examined the effect of topical soluble IL-1 receptor antagonist on bone marrow-derived cell influx following corneal epithelial scrape injury in a mouse model. C57BL/6 mice underwent corneal epithelial scrape followed by application of IL-1 receptor antagonist (Amgen, Thousand Oaks, CA) at a concentration of 20 mg/ml or vehicle for 24 h prior to immunocytochemical detection of marker CD11b-positive cells into the stroma. In two experiments, topical IL-1 receptor antagonist had a marked effect in blocking cell influx. For example, in experiment 1, topical IL-1 receptor antagonist markedly reduced detectible CD11b-positive cells into the corneal stroma at 24 It after epithelial injury compared with the vehicle control (3.5 +/- 0.5 (standard error of the mean) cells/400x field and 13.9 +/- 1.2 cells/400x field, respectively, p < 0.01). A second experiment with a different observer performing cell counting had the same result. Thus, the data demonstrate conclusively that topical IL-1 receptor antagonist markedly down-regulates CD-11b-positive monocytic cell appearance in the corneal stroma. Topical IL-1 receptor antagonist could be an effective adjuvant for clinical treatment of corneal conditions in which unwanted inflammation has a role in the pathophysiology of the disorder. (c) 2008 Elsevier Ltd. All rights reserved.

Antigenotoxicity and antimutagenicity of lycopene in HepG2 cell line evaluated by the comet assay and micronucleus test

Epidemiological studies have provided evidence that high consumption of tomatoes effectively reduces the risk of reactive oxygen species (ROS)-mediated diseases such as cancer. Tomatoes are rich sources of lycopene, a potent singlet oxygen-quenching carotenoid. In addition to its antioxidant properties, lycopene shows an array of biological effects including antimutagenic and anticarcinogenic activities. In the present study, the chemopreventive action of lycopene was examined on DNA damage and clastogenic or aneugenic effects of H2O2 and n-nitrosodiethylamine (DEN) in the metabolically competent human hepatoma cell line (HepG2 cells). Lycopene at concentrations of 10. 25, and 50 mu M, was tested under three protocols: before, simultaneously, and after treatment with the mutagen, using the comet and micronucleus assays. Lycopene significantly reduced the genotoxicity and mutagenicity of H2O2 in all of the conditions tested. For DEN, significant reductions of primary DNA damage (comet assay) were detected when the carotenoid (all of the doses) was added in the cell culture medium before or simultaneously with the mutagen. In the micronucleus test, the protective effect of lycopene was observed only when added prior to DEN treatment. In conclusion, our results suggest that lycopene is a suitable agent for preventing chemically-induced DNA and chromosome damage. (C) 2007 Elsevier Ltd. All rights reserved.

Evidences of a Role for Eukaryotic Translation Initiation Factor 5A (eIF5A) in Mouse Embryogenesis and Cell Differentiation

Eukaryotic translation initiation factor 5A (eIF5A) has a unique character: the presence of an unusual amino acid, hypusine, which is formed by post-translational modifications. Even before the identification of hypusination in eIF5A, the correlation between hypusine formation and protein synthesis, shifting cell proliferation rates, had already been observed. Embryogenesis is a complex process in which cellular proliferation and differentiation are intense. In spite of the fact that many studies have described possible functions for eIF5A, its precise role is under investigation, and to date nothing has been reported about its participation in embryonic development. In this study we show that eIF5A is expressed at all mouse embryonic post-implantation stages with increase in eIF5A mRNA and protein expression levels between embryonic days E10.5 and E13.5. Immunohistochemistry revealed the ubiquitous presence of eIF5A in embryonic tissues and organs at E13.5 day. Interestingly, stronger immunoreactivity to eIF5A was observed in the stomodeum, liver, ectoderm, heart, and eye, and the central nervous system; regions which are known to undergo active differentiation at this stage, suggesting a role of eIF5A in differentiation events. Expression analyses of MyoD, a myogenic transcription factor, revealed a significantly higher expression from day E12.5 on, both at the mRNA and the protein levels suggesting a possible correlation to eIF5A. Accordingly, we next evidenced that inhibiting eIF5A hypusination in mouse myoblast C2C12 cells impairs their differentiation into myotubes and decreases MyoD transcript levels. Those results point to a new functional role for eIF5A, relating it to embryogenesis, development, and cell differentiation. J. Cell. Physiol. 225: 500-505, 2010. (C) 2010 Wiley-Liss, Inc.

Mesenchymal Stem Cell Properties of Periodontal Ligament Cells From Deciduous and Permanent Teeth

Background: Human postnatal stem cells have been identified in periodontal ligaments (PDLs). In this study, the in vitro biologic properties of CD105(+) enriched cell subsets from PDLs harvested from deciduous (DePDL) and permanent (PePDL) teeth are comparatively assessed. Methods: PDL tissue was obtained from 12 teeth (six primary and six permanent) from which CD105(+) CD34(-) CD45(-) cells were isolated by magnetic cell sorting. To identify and quantitatively compare the stem cell markers, DePDL and PePDL cells were assessed for CD166 surface antigen expression by flow cytometry, real-time polymerase chain reaction, and immunostaining for Stro-1 and Oct-4, osteogenic and adipogenic differentiation, and proliferation rate by trypan blue method. Results: Magnetic cell sorting isolated cell populations containing 23.87% (+/- 11.98%) and 11.68% (+/- 6.27%) of CD105(+) expressing cells from PePDL and DePDL, respectively. Flow cytometric analysis demonstrated a higher proportion of CD105(+) cells coexpressing CD166 surface antigen in PePDL, whereas immunostaining and real-time polymerase chain reaction analysis demonstrated that both cell subsets expressed Stro-1 and Oct-4. DePDL-CD105(+) subsets were more proliferative compared to PePDL subsets, and both cell populations showed multipotential capabilities to differentiate in vitro to osteoblast/cementoblast- and adipocyte-like cells. However, a higher expression of adipogenic-related genes was observed in DePDL cells, whereas PePDL-CD105(+) cell subset presented a more homogeneous osteoblast/cementoblast response. Conclusion: These findings demonstrate that highly purified mesenchymal progenitor cell subsets can be obtained from the PDLs of both deciduous and permanent teeth, and further indicate phenotype dissimilarities that may have an impact on their clinical applications. J Periodontol 2010;81:1207-1215.

Characterization and in vitro activities of cell-free antigens from Histoplasma capsulatum-loaded biodegradable microspheres

In the last decades, the incidence of histoplasmosis, a pulmonary fungal disease caused by Histoplasma capsulatum, has increased worldwide. In this context, vaccines for the prevention of this infection or therapies are necessary. Cell-free antigens (CFAgs) from H. capsulatum when administered for murine immunization purposes are able to confer protection and control of the infection, since they activate cellular immunity. However the most of vaccination procedures need several anti, gens administrations and immunoadjuvants, which are not approved for use in humans. The aim of this study was to develop and characterize a vaccination approach using biodegradable PLGA microspheres (MS) that could allow the controlled and/or sustained release of the encapsulated antigens from H. capsulatum. CFAgs-loaded MS presented a size less than 10 mu m, were marked engulfed by bone marrow-derived macrophages (BMDM phi) and induced the nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha) production by these cells. Our data show that CFAgs-loaded MS induce cell activation, suggesting an immunostimulant effect to be further investigated during immunization procedures. CFAgs-loaded MS present potential to be used as vaccine in order to confer protection against H. capsulatum infection. (C) 2009 Elsevier B.V. All rights reserved.

Synthesis, spectroscopic characterization, DFT studies and biological assays of a novel gold(I) complex with 2-mercaptothiazoline

A new gold(I) complex with 2-mercaptothiazoline (MTZ) with the coordination formula [AuCN(C(3)H(5)NS(2))] was synthesized and characterized by chemical and spectroscopic measurements, OFT studies and biological assays. Infrared (IR) and (1)H, (13)C and (15)N nuclear magnetic resonance (NMR) spectroscopic measurements indicate coordination of the ligand to gold(I) through the nitrogen atom. Studies based on OFT confirmed nitrogen coordination to gold(I) as a minimum of the potential energy surface with calculations of the hessians showing no imaginary frequencies. Thermal decomposition starts at temperatures near 160 degrees C, leading to the formation of Au as the final residue at 1000 degrees C. The gold(I) complex with 2-mercaptothiazoline (Au-MTZ) is soluble in dimethyl sulfoxide (DMSO), and is insoluble in water, methanol, ethanol, acetonitrile and hexane. The antibacterial activities of the Au-MTZ complex were evaluated by an antibiogram assay using the disc diffusion method. The compound showed an effective antibacterial activity against Staphylococcus aureus (Gram-positive) and Escherichia coli and Pseudomonas aeruginosa (Gram-negative) bacterial cells. Biological analysis for evaluation of the cytotoxic effect of the Au-MTZ complex was performed using HeLa cells derived from human cervical adenocarcinoma. The complex presented a potent cytotoxic activity, inducing 85% of cell death at a concentration of 2.0 mu mol L(-1). (C) 2011 Elsevier Ltd. All rights reserved.

Application of recombinant antibody library for screening specific antigens in a bovine sperm cell subpopulation

Antibody phage display libraries are a useful tool in proteomic analyses. This study evaluated an antibody recombinant library for identification of sex-specific proteins on the sperm cell surface. The Griffin.1 library was used to produce phage antibodies capable of recognizing membrane proteins from Nelore sperm cells. After producing soluble monoclonal scFv, clones were screened on Simental sperm cells by flow cytometry and those that bound to 40-60% of cells were selected. These clones were re-analyzed using Nelore sperm cells and all clones bound to 40-60% of cells. Positive clones were submitted to a binding assay against male and female bovine leukocytes by flow cytometry and one clone preferentially bound to male cells. The results indicate that phage display antibodies are an alternative method for identification of molecules markers on sperm cells. (C) 2007 Elsevier B.V. All rights reserved.

Phenotypic and functional characterization of pulmonary macrophages subpopulations after intratracheal injection of Paracoccidioides brasiliensis cell wall components

A shift in the activation of pulmonary macrophages characterized by an increase of IL-1, INF-alpha and IL-6 production has been induced in mice infected with Paracoccidioides brasiliensis. It is still unclear whether a functional shift in the resident alveolar macrophage population would be responsible for these observations due to the expression of cell surface molecules. We investigated pulmonary macrophages by flow cytometry from mice treated with P. brasiliensis derivatives by intratracheal route. In vivo labeling with the dye PKH26GL was applied to characterize newly recruited pulmonary macrophages from the bloodstream. Pulmonary macrophages from mice inflamed with P. brasiliensis derivatives showed a high expression of the surface antigens CD11b/CD18 and CD23 among several cellular markers. The expression of these markers indicated a pattern of activation of a subpopulation characterized as CD11b(+) or CD23(+), which was modulated in vitro by IFN-gamma and IL-4. Analysis of monocytes labelled with PKH26GL demonstrated that CD11b(+) cells did infiltrate the lung exhibiting a proinflammatoni pattern of activation, whereas CD23(+) cells were considered to be resident in the lung. These findings may contribute to better understand the pathology of lung inflammation caused by P. brasiliensis infection. (C) 2010 Elsevier GmbH. All rights reserved.

Host genetic background affects regulatory T-cell activity that influences the magnitude of cellular immune response against Mycobacterium tuberculosis

Using two mouse strains with different abilities to generate interferon (IFN)-gamma production after Mycobacterium tuberculosis infection, we tested the hypothesis that the frequency and activity of regulatory T (Treg) cells are influenced by genetic background. Our results demonstrated that the suppressive activity of spleen Treg cells from infected or uninfected BALB/c mice was enhanced, inhibiting IFN-gamma and interleukin (IL)-2 production. Infected C57BL/6 mice exhibited a decrease in the frequency of lung Treg cells and an increased ratio CD4(+):CD4(+)Foxp3(+) cells compared with infected BALB/c mice and uninfected C57BL/6 mice. Moreover, infected C57BL/6 mice also had a decrease in the immunosuppressive capacity of spleen Treg cells, higher lung IFN-gamma and IL-17 production, and restricted the infection better than BALB/c mice. Adoptive transfer of BALB/c Treg cells into BALB/c mice induced an increase in bacterial colony-forming unit (CFU) counts. Furthermore, BALB/c mice treated with anti-CD25 antibody exhibited lung CFU counts significantly lower than mice treated with irrelevant antibody. Our results show that in BALB/c mice, the Treg cells have a stronger influence than that in C57BL/6 mice. These data suggest that BALB/c and C57BL/6 mice may use some different mechanisms to control M. tuberculosis infection. Therefore, the role of Treg cells should be explored during the development of immune modulators, both from the perspective of the pathogen and the host. Immunology and Cell Biology (2011) 89, 526-534; doi:10.1038/icb.2010.116; published online 19 October 2010