Interleukin-10 production by tumor infiltrating macrophages plays a role in Human Papillomavirus 16 tumor growth

Background: Human Papillomavirus, HPV, is the main etiological factor for cervical cancer. Different studies show that in women infected with HPV there is a positive correlation between lesion grade and number of infiltrating macrophages, as well as with IL-10 higher expression. Using a HPV16 associated tumor model in mice, TC-1, our laboratory has demonstrated that tumor infiltrating macrophages are M2-like, induce T cell regulatory phenotype and play an important role in tumor growth. M2 macrophages secrete several cytokines, among them IL-10, which has been shown to play a role in T cell suppression by tumor macrophages in other tumor models. In this work, we sought to establish if IL-10 is part of the mechanism by which HPV tumor associated macrophages induce T cell regulatory phenotype, inhibiting anti-tumor activity and facilitating tumor growth. Results: TC-1 tumor cells do not express or respond to IL-10, but recruit leukocytes which, within the tumor environment, produce this cytokine. Using IL-10 deficient mice or blocking IL-10 signaling with neutralizing antibodies, we observed a significant reduction in tumor growth, an increase in tumor infiltration by HPV16 E7 specific CD8 lymphocytes, including a population positive for Granzyme B and Perforin expression, and a decrease in the percentage of HPV specific regulatory T cells in the lymph nodes. Conclusions: Our data shows that in the HPV16 TC-1 tumor mouse model, IL-10 produced by tumor macrophages induce regulatory phenotype on T cells, an immune escape mechanism that facilitates tumor growth. Our results point to a possible mechanism behind the epidemiologic data that correlates higher IL-10 expression with risk of cervical cancer development in HPV infected women.

Influence of Caffeine and Chondroitin Sulfate on Swine Sperm Capacitation and In Vitro Embryo Production

Background: The establishment of an in vitro production (IVP) of embryo in swine allows the generation of embryos with the same quality as in vivo produced embryos with less costs and time. In order to achieve successful fertilization under normal circumstances in vivo, mammalian spermatozoa must first undergo capacitation and then acrosome reaction. The purpose of this study was compared the efficacious of IP/CFDA fluorescence and Coomassie Blue G (CB) staining to detect capacitated sperm cells in refrigerated and fresh semen. Morever, it was investigated the efficacious of caffeine and chondroitin sulphate to promote in vitro sperm capacitation and in vitro embryo produced (IVP) of swine embryos. Materials, Methods & Results: A sperm-rich fraction from ejaculate was obtained using the gloved-hand method and the gel-free fraction was separated using sterile gauze. The semen was diluted in BTS at a final concentration of 1.5 x 10(8) cells/mL. The sperm suspension was incubated for 2 h at 25 degrees C, refrigerated and maintained for 1 h at 15-18 degrees C (refrigerated group) or used immediately (fresh group). Sperm capacitation was assessed by IP/CFDA fluorescence and CB staining for both fresh and refrigerated semen. For PI/CFDA evaluation, a final solution containing 1.7 mM formaldehyde, 7.3 mM PI and 20 mM CFDA in 950 mu L saline was prepared. In the dark, 40 mu L PI/CFDA final solution was added to 10 mu L semen and after 8 min, slides were analyzed on epifluorescence microscopy. For CB evaluation, sperm cells were fixed in 4% paraformaldehyde for 10 min and centrifuged twice at 320 x g in ammonium acetate pH 9 for 8 min. A smear was made and stained with 2.75 mg/mL CB in solution containing 12.5% methanol, 25% glacial acetic acid and 62.5% water, for 2 min. The smear was washed in running water, air dried and sealed with Permount (R), diluted 2:1 in xilol to avoid staining oxidation. Our results showed that refrigeration did not affect sperm capacitation and comparing staining methods, the PI/CFDA combination was more efficient to detect capacitated sperm, when compared to CB staining. In experiment 2, we evaluated the effect of different incubation time (1 - 5 h) with chondroitin sulfate and caffeine on sperm capacitation. For in vitro fertilization, oocytes were obtained from slaughterhouse ovaries. Oocytes with a thick and intact cumulus oophurus layer and cytoplasm with homogenous granules were selected for in vitro maturation for 44 h. According to the results of experiment 2, it was used for in vitro fertilization refrigerated semen was capacitated with 50 mu g/mL chondroitin sulfate for 2 h or capacitated with 5 mu g/mL caffeine for 3 h. Six hours after insemination, cumulus oophorus cells were mechanically removed and oocytes were washed and incubated in microdrops of culture medium. Embryo development after fertilization with sperm capacitated with caffeine or chondroitin sulfate was evaluated on days 3, 5 and 7 of culture. No differences were observed in days 3 or 5 of in vitro culture. However, it was observed an increase on blastocyst rate on Day 7 of culture when caffeine was used as the capacitor agent. Discussion: Molecular basis of sperm capacitation is still poor understood. Sperm capacitation can occur in vitro spontaneously in defined media without addition of biological fluids. We observed that sperm capacitation increased as incubation period enlarged and it was observed using Coomassie blue G and PI/CFDA for fresh semen and for refrigerated semen. It can be concluded that the cooling of semen did not change their pattern of sperm capacitation and this is best assessed by IP/CFDA than by CB. In addition to the use of caffeine in sperm capacitation produces more blastocysts than the chondroitin sulfate after in vitro fertilization.

A Paradigm Shift in Predoctoral Dental Curricula in Brazil: Evaluating the Process of Change

In 2002, the Brazilian Ministry of Education approved the official curricular guidelines for undergraduate courses in Brazil to be adopted by the nation's 188 dental schools. In 2005-06, the Brazilian Dental Education Association (BDEA) promoted workshops in forty-eight of the schools to verify the degree of transformation of the curriculum based on these guidelines. Among the areas analyzed were course philosophy (variables were v1: knowledge production based on the needs of the Brazilian Public Health System [BPHS]; v2: health determinants; and v3: postgraduate studies and permanent education); pedagogical skills (v4: curricular structure; v5: changes in pedagogic and didactic skills; and v6: course program orientation); and dental practice scenarios (v7: diversity of the scenarios for training/learning; v8: academic health care centers opened to the BPHS; and v9: participation of students in health care delivery for the population). The subjects consisted of faculty members (n=711), students (n=228), and employees (n=14). The results showed an incipient degree of curriculum transformation. The degree of innovation was statistically different depending on the type of university (public or private) for variables I, 2, 4, 5, 6, and 7. Private schools reported a higher level of innovation than public institutions. Resistance to transforming the dental curriculum according to the official guidelines may be linked to an ideological conception that supports the private practice model, continues to have faculty members direct all classroom activities, and prevents students from developing an understanding of professional practice as targeted towards the oral health needs of all segments of society.

PRACTICAL USE OF MOLECULAR MARKERS IN BEEF AND DAIRY ZEBU CATTLE IN BRAZIL

Selection of zebu (Bos indicus) beef and dairy cattle in Brazil and the validation process of genetic markers for growth, carcass and meat quality traits and also for milk production, fat and protein milk content are discussed as concerned to the concepts and details of their use as auxiliary tools in selection processes. It is highlighted, also, the importance of right selection of ova donor cows for production of embryos to be transferred.

Additive genetic relationship of longevity with fertility and production traits in Nellore cattle based on bivariate models

Survival or longevity is an economically important trait in beef cattle. The main inconvenience for its inclusion in selection criteria is delayed recording of phenotypic data and the high computational demand for including survival in proportional hazard models. Thus, identification of a longevity-correlated trait that could be recorded early in life would be very useful for selection purposes. We estimated the genetic relationship of survival with productive and reproductive traits in Nellore cattle, including weaning weight (WW), post-weaning growth (PWG), muscularity (MUSC), scrotal circumference at 18 months (SC18), and heifer pregnancy (HP). Survival was measured in discrete time intervals and modeled through a sequential threshold model. Five independent bivariate Bayesian analyses were performed, accounting for cow survival and the five productive and reproductive traits. Posterior mean estimates for heritability (standard deviation in parentheses) were 0.55 (0.01) for WW, 0.25 (0.01) for PWG, 0.23 (0.01) for MUSC, and 0.48 (0.01) for SC18. The posterior mean estimates (95% confidence interval in parentheses) for the genetic correlation with survival were 0.16 (0.13-0.19), 0.30 (0.25-0.34), 0.31 (0.25-0.36), 0.07 (0.02-0.12), and 0.82 (0.78-0.86) for WW, PWG, MUSC, SC18, and HP, respectively. Based on the high genetic correlation and heritability (0.54) posterior mean estimates for HP, the expected progeny difference for HP can be used to select bulls for longevity, as well as for post-weaning gain and muscle score.

Serum-Starved Apoptotic Fibroblasts Reduce Blastocyst Production but Enable Development to Term after SCNT in Cattle

Cell cycle synchronization by serum starvation (SS) induces apoptosis in somatic cells. This side effect of SS is hypothesized to negatively affect the outcome of somatic cell nuclear transfer (SCNT). We determined whether apoptotic fibroblasts affect SCNT yields. Serum-starved, adult, bovine fibroblasts were stained with annexin V-FITC/propidium iodide to allow apoptosis detection by flow cytometry. Positive and negative cells sorted by fluorescence activated cell sorting (FACS) and an unsorted control group were used as nuclear donors for SCNT. Reconstructed embryos were cultured in vitro and transferred to synchronized recipients. Apoptosis had no effect on fusion and cleavage rates; however, it resulted in reductions in blastocyst production and quality measured by apoptotic index. However, reconstructed embryos with apoptotic cells resulted in pregnancy rates similar to that of the control on day 30, and generated one live female calf. In conclusion, we showed that apoptotic cells present in serum-starved cultures negatively affect embryo production after SCNT without compromising full-term development. Further studies will evaluate the ability of the oocyte to reprogram cells in specific phases of apoptosis.

Effect of eucalyptus (Eucalyptus citriodora) fresh or residue leaves on methane emission in vitro

Rumen fermentation and methane emission for eucalyptus (Eucalyptus citriodora) fresh leaves (FL) or residue leaves (RL), after essential oil extraction from eucalyptus leaves in comparison with alfalfa (Medicago sativa) hay, were investigated in vitro. Eucalyptus FL and RL were obtained from the Distillery Trees Barras Company, Torrinha City, Sao Paulo, Brazil. The semi-automatic system of gas production was used to measure gas production, methane emission and rumen fermentation after 24 h incubation in vitro. The results showed that the crude protein (CP) contents were 76.4, 78.1 and 181.9 g kg(-1) DM for eucalyptus FL, RL and alfalfa hay, respectively. The neutral-detergent fibre (NDF) and acid-detergent fibre (ADF) were significantly lower in eucalyptus FL and RL than alfalfa hay. The Eucalyptus fresh and residue leaves were rich in total phenols (TP) and total tannins (TT) but had negligible content of condensed tannins (CT). There was significant reduction in cumulative gas production about 54 and 51% with eucalyptus FL and RL, respectively, compared with alfalfa hay. The methane emission (mL/g DM) was reduced (P<0.05) by 53 and 57% with eucalyptus FL and RL, respectively, but the reduction was 21 and 16% when expressed on truly digested organic matter basis. There were a decline (P<0.05) in true dry and organic matter degradation in vitro in eucalyptus FL and RL compared with alfalfa hay substrate. The partitioning factor values were higher (P<0.05) in eucalyptus FL and RL than alfalfa hay. There was no significant difference observed between eucalyptus FL, RL and alfalfa hay in protozoa count. It is concluded that the eucalyptus leaves have potential effect to mitigate CH4 production in vitro, which may be attributed to a decrease in fermentable substrate rather than to a direct effect on methanogenesis.

The metal content of bulge field stars from FLAMES-GIRAFFE spectra - I. Stellar parameters and iron abundances

Aims. We determine the iron distribution function (IDF) for bulge field stars, in three different fields along the Galactic minor axis and at latitudes b = -4 degrees, b = -6 degrees, and b = -12 degrees. A fourth field including NGC 6553 is also included in the discussion. Methods. About 800 bulge field K giants were observed with the GIRAFFE spectrograph of FLAMES@VLT at spectral resolution R similar to 20 000. Several of them were observed again with UVES at R similar to 45 000 to insure the accuracy of the measurements. The LTE abundance analysis yielded stellar parameters and iron abundances that allowed us to construct an IDF for the bulge that, for the first time, is based on high-resolution spectroscopy for each individual star. Results. The IDF derived here is centered on solar metallicity, and extends from [Fe/H] similar to -1.5 to [Fe/H] similar to + 0.5. The distribution is asymmetric, with a sharper cutoff on the high-metallicity side, and it is narrower than previously measured. A variation in the mean metallicity along the bulge minor axis is clearly between b = -4 degrees and b = -6 degrees ([Fe/H] decreasing similar to by 0.6 dex per kpc). The field at b = -12 degrees. is consistent with the presence of a gradient, but its quantification is complicated by the higher disk/bulge fraction in this field. Conclusions. Our findings support a scenario in which both infall and outflow were important during the bulge formation, and then suggest the presence of a radial gradient, which poses some challenges to the scenario in which the bulge would result solely from the vertical heating of the bar.

The stellar content of obscured Galactic giant H II regions. VI. W51A

We present K-band spectra of newly born OB stars in the obscured Galactic giant H II region W51A and approximate to 0.8 '' angular resolution images in the J, H, and K(S)-bands. Four objects have been spectroscopically classified as O-type stars. The mean spectroscopic parallax of the four stars gives a distance of 2.0 +/- 0.3 kpc (error in the mean), significantly smaller than the radio recombination line kinematic value of 5.5 kpc or the values derived from maser proper motion observations (6-8 kpc). The number of Lyman continuum photons from the contribution of all massive stars (NLyc approximate to 1.5 x 10(50) s(-1)) is in good agreement with that inferred from radio recombination lines (NLyc = 1.3 x 10(50) s(-1)) after accounting for the smaller distance derived here. We present analysis of archival high angular resolution images (NAOS CONICA at VLT and T-ReCS at Gemini) of the compact region W51 IRS 2. The K(S)-band images resolve the infrared source IRS 2 indicating that it is a very young compact H II region. Sources IRS 2E was resolved into compact cluster (within 660 AU of projected distance) of three objects, but one of them is just bright extended emission. W51d1 and W51d2 were identified with compact clusters of three objects (maybe four in the case of W51d1) each one. Although IRS 2E is the brightest source in the K-band and at 12.6 mu m, it is not clearly associated with a radio continuum source. Our spectrum of IRS 2E shows, similar to previous work, strong emission in Br gamma and He I, as well as three forbidden emission lines of Fe III and emission lines of molecular hydrogen (H(2)) marking it as a massive young stellar object.

Transiting exoplanets from the CoRoT space mission XIII. CoRoT-13b: a dense hot Jupiter in transit around a star with solar metallicity and super-solar lithium content

We announce the discovery of the transiting planet CoRoT-13b. Ground-based follow-up in CFHT and IAC80 confirmed CoRoT's observations. The mass of the planet was measured with the HARPS spectrograph and the properties of the host star were obtained analyzing HIRES spectra from the Keck telescope. It is a hot Jupiter-like planet with an orbital period of 4.04 days, 1.3 Jupiter masses, 0.9 Jupiter radii, and a density of 2.34 g cm(-3). It orbits a G0V star with T(eff) = 5 945 K, M(*) = 1.09 M(circle dot), R(*) = 1.01 R(circle dot), solar metallicity, a lithium content of +1.45 dex, and an estimated age of between 0.12 and 3.15 Gyr. The lithium abundance of the star is consistent with its effective temperature, activity level, and age range derived from the stellar analysis. The density of the planet is extreme for its mass, implies that heavy elements are present with a mass of between about 140 and 300 M(circle plus).

THE STELLAR CONTENT OF OBSCURED GALACTIC GIANT H II REGIONS. VII. W3

Spectrophotometric distances in the K band have been reported by different authors for a number of obscured Galactic H II regions. Almost 50% of them show large discrepancies compared to the classical method using radial velocities measured in the radio spectral region. In order to provide a crucial test of both methods, we selected a target that does not present particular difficulty for any method and which has been measured by as many techniques as possible. The W3 star-forming complex, located in the Perseus arm, offers a splendid opportunity for such a task. We used the Near-Infrared Integral Field Spectrograph on the Frederick C. Gillett Gemini North telescope to classify candidate ""naked photosphere"" OB stars based on Two Micron All Sky Survey photometry. Two of the targets are revealed to be mid-O-type main-sequence stars leading to a distance of d = 2.20 kpc. This is in excellent agreement with the spectrophotometric distance derived in the optical band (d = 2.18 pc) and with a measurement of the W3 trigonometric parallax (d = 1.95 kpc). Such results confirm that the spectrophotometric distances in the K band are reliable. The radio-derived kinematic distance, on the contrary, gives a distance twice as large (d = 4.2 kpc). This indicates that this region of the Perseus arm does not follow the Galactic rotation curve, and this may also be the case for other H II regions for which discrepancies have been found.

Production of workers, queens and males in Plebeia remota colonies (Hymenoptera, Apidae, Meliponini), a stingless bee with reproductive diapause

Queen, male and worker production was studied during one year in three Plebeia remota colonies from Atlantic Rainforest in Cunha, Sao Paulo State, and two from a subtropical Araucaria forest in Prudentopolis, Parana State. All the colonies were kept in Sao Paulo city during our study. Plebeia remota has reproductive diapause during autumn and winter, which makes its biology of special interest. Brood production begins before spring, renewing the colony cycle. We sampled brood combs monthly in these five colonies. The number of cells in each comb varied significantly with time of the year; the smallest brood combs appear to be a consequence of reduced food availability. However, worker, queen and male frequencies did not differ significantly in time, and this presumably is due to the fact that they all are necessary for the growth, maintenance and reproduction of the colony. Although some molecular, morphological and behavioral differences have been detected in several studies comparing populations from Cunha and from Prudentopolis, we did not find significant differences between the colonies from these two localities in number of brood cells and worker, queen and male production.

Control of the Intracellular Redox State by Glucose Participates in the Insulin Secretion Mechanism

Background: Production of reactive oxygen species (ROS) due to chronic exposure to glucose has been associated with impaired beta cell function and diabetes. However, physiologically, beta cells are well equipped to deal with episodic glucose loads, to which they respond with a fine tuned glucose-stimulated insulin secretion (GSIS). In the present study, a systematic investigation in rat pancreatic islets about the changes in the redox environment induced by acute exposure to glucose was carried out. Methodology/Principal Findings: Short term incubations were performed in isolated rat pancreatic islets. Glucose dose- and time-dependently reduced the intracellular ROS content in pancreatic islets as assayed by fluorescence in a confocal microscope. This decrease was due to activation of pentose-phosphate pathway (PPP). Inhibition of PPP blunted the redox control as well as GSIS in a dose-dependent manner. The addition of low doses of ROS scavengers at high glucose concentration acutely improved beta cell function. The ROS scavenger N-acetyl-L-cysteine increased the intracellular calcium response to glucose that was associated with a small decrease in ROS content. Additionally, the presence of the hydrogen peroxide-specific scavenger catalase, in its membrane-permeable form, nearly doubled glucose metabolism. Interestingly, though an increase in GSIS was also observed, this did not match the effect on glucose metabolism. Conclusions: The control of ROS content via PPP activation by glucose importantly contributes to the mechanisms that couple the glucose stimulus to insulin secretion. Moreover, we identified intracellular hydrogen peroxide as an inhibitor of glucose metabolism intrinsic to rat pancreatic islets. These findings suggest that the intracellular adjustment of the redox environment by glucose plays an important role in the mechanism of GSIS.

Comparative Analysis of Activation Phenotype, Proliferation, and IFN-gamma Production by Spleen NK1.1(+) and NK1.1(-) T Cells During Plasmodium chabaudi AS Malaria

The NK1.1 molecule participates in NK, NKT, and T-cell activation, contributing to IFN-gamma production and cytotoxicity. To characterize the early immune response to Plasmodium chabaudi AS, spleen NK1.1(+) and NK1.1(-) T cells were compared in acutely infected C57BL/6 mice. The first parasitemia peak in C57BL/6 mice correlated with increase in CD4(+)NK1.1(+)TCR-alpha beta(+), CD8(+)NK1.1(+)TCR-alpha beta(+), and CD4(+)NK1.1(-)TCR-alpha beta(+) cell numbers per spleen, where a higher increment was observed for NK1.1(+) T cells compared to NK1.1(-) T cells. According to the ability to recognize the CD1d-alpha-GalCer tetramer, CD4(+)NK1.1(+) cells in 7-day infected mice were not predominantly invariant NKT cells. At that time, nearly all NK1.1(+) T cells and around 30% of NK1.1(-) T cells showed an experienced/activated (CD44(HI)CD69(HI)CD122(HI)) cell phenotype, with high expression of Fas and PD-L1 correlating with their low proliferative capacity. Moreover, whereas IFN-gamma production by CD4(+)NK1.1(+) cells peaked at day 4 p.i., the IFN-gamma response of CD4(+)NK1.1(-) cells continued to increase at day 5 of infection. We also observed, at day 7 p.i., 2-fold higher percentages of perforin(+) cells in CD8(+)NK1.1(+) cells compared to CD8(+)NK1.1(-) cells. These results indicate that spleen NK1.1(+) and NK1.1(-) T cells respond to acute P. chabaudi malaria with different kinetics in terms of activation, proliferation, and IFN-gamma production.

Production of the refolded oligopeptide-binding protein (OppA) encoded by the citrus pathogen Xanthomonas axonopodis pv. citri

The oligopeptide-binding protein, OppA, binds and ushers oligopeptide substrates to the membrane-associated oligopeptide permease (Opp), a multi-component ABC-type transporter involved in the uptake of oligopeptides expressed by several bacterial species. In the present study, we report the cloning, purification, refolding and conformational analysis of a recombinant OppA protein derived from Xanthomonas axonopodis pv. citri (X. citri), the etiological agent of citrus canker. The oppA gene was expressed in Escherichia coli BL21 (DE3) strain under optimized inducing conditions and the recombinant protein remained largely insoluble. Solubilization was achieved following refolding of the denatured protein. Circular dichroism analysis indicated that the recombinant OppA protein preserved conformational features of orthologs expressed by other bacterial species. The refolded recombinant OppA represents a useful tool for structural and functional analyses of the X. citri protein.