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.

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.

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.

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.

Impact of Manaus City on the Amazon Green Ocean atmosphere: ozone production, precursor sensitivity and aerosol load

As a contribution to the Large-Scale Biosphere-Atmosphere Experiment in Amazonia - Cooperative LBA Airborne Regional Experiment (LBA-CLAIRE-2001) field campaign in the heart of the Amazon Basin, we analyzed the temporal and spatial dynamics of the urban plume of Manaus City during the wet-to-dry season transition period in July 2001. During the flights, we performed vertical stacks of crosswind transects in the urban outflow downwind of Manaus City, measuring a comprehensive set of trace constituents including O(3), NO, NO(2), CO, VOC, CO(2), and H(2)O. Aerosol loads were characterized by concentrations of total aerosol number (CN) and cloud condensation nuclei (CCN), and by light scattering properties. Measurements over pristine rainforest areas during the campaign showed low levels of pollution from biomass burning or industrial emissions, representative of wet season background conditions. The urban plume of Manaus City was found to be joined by plumes from power plants south of the city, all showing evidence of very strong photochemical ozone formation. One episode is discussed in detail, where a threefold increase in ozone mixing ratios within the atmospheric boundary layer occurred within a 100 km travel distance downwind of Manaus. Observation-based estimates of the ozone production rates in the plume reached 15 ppb h(-1). Within the plume core, aerosol concentrations were strongly enhanced, with Delta CN/Delta CO ratios about one order of magnitude higher than observed in Amazon biomass burning plumes. Delta CN/Delta CO ratios tended to decrease with increasing transport time, indicative of a significant reduction in particle number by coagulation, and without substantial new particle nucleation occurring within the time/space observed. While in the background atmosphere a large fraction of the total particle number served as CCN (about 60-80% at 0.6% supersaturation), the CCN/CN ratios within the plume indicated that only a small fraction (16 +/- 12 %) of the plume particles were CCN. The fresh plume aerosols showed relatively weak light scattering efficiency. The CO-normalized CCN concentrations and light scattering coefficients increased with plume age in most cases, suggesting particle growth by condensation of soluble organic or inorganic species. We used a Single Column Chemistry and Transport Model (SCM) to infer the urban pollution emission fluxes of Manaus City, implying observed mixing ratios of CO, NO(x) and VOC. The model can reproduce the temporal/spatial distribution of ozone enhancements in the Manaus plume, both with and without accounting for the distinct (high NO(x)) contribution by the power plants; this way examining the sensitivity of ozone production to changes in the emission rates of NO(x). The VOC reactivity in the Manaus region was dominated by a high burden of biogenic isoprene from the background rainforest atmosphere, and therefore NO(x) control is assumed to be the most effective ozone abatement strategy. Both observations and models show that the agglomeration of NO(x) emission sources, like power plants, in a well-arranged area can decrease the ozone production efficiency in the near field of the urban populated cores. But on the other hand remote areas downwind of the city then bear the brunt, being exposed to increased ozone production and N-deposition. The simulated maximum stomatal ozone uptake fluxes were 4 nmol m(-2) s(-1) close to Manaus, and decreased only to about 2 nmol m(-2) s(-1) within a travel distance >1500 km downwind from Manaus, clearly exceeding the critical threshold level for broadleaf trees. Likewise, the simulated N deposition close to Manaus was similar to 70 kg N ha(-1) a(-1) decreasing only to about 30 kg N ha(-1) a(-1) after three days of simulation.

Heavy quark production in deep inelastic electron-nucleus scattering

Heavy quark production has been very well studied over the last years both theoretically and experimentally. Theory has been used to study heavy quark production in ep collisions at HERA, in pp collisions at Tevatron and RHIC, in pA and dA collisions at RHIC, and in AA collisions at CERN-SPS and RHIC. However, to the best of our knowledge, heavy quark production in eA has received almost no attention. With the possible construction of a high energy electron-ion collider, updated estimates of heavy quark production are needed. We address the subject from the perspective of saturation physics and compute the heavy quark production cross section with the dipole model. We isolate shadowing and nonlinear effects, showing their impact on the charm structure function and on the transverse momentum spectrum.

Exclusive vector meson production in electron-ion collisions

We calculate the nuclear cross section for coherent and incoherent vector meson production within the QCD color dipole picture, including saturation effects. Theoretical estimates for scattering on both light and heavy nuclei are given over a wide range of energy.

Observation of double electron-positron pair production by gamma rays reexamined

An experiment was conducted to observe triple- and quadruple-escape peaks, at a photon energy equal to 6.128 MeV, in the spectra recorded with a high-purity Ge detector working in coincidence with six bismuth germanate detectors. The peak intensities may be explained having recourse to only the bremsstrahlung cascade process of consecutive electron-positron pair creation; i.e., the contribution of simultaneous double pair formation (and other cascade effects) is much smaller. The experimental peak areas are in reasonably good agreement with those predicted by Monte Carlo simulations done with the general-purpose radiation-tran sport code PENELOPE.

Measurement of neutral mesons in p plus p collisions at root s=200 GeV and scaling properties of hadron production

The PHENIX experiment at the Relativistic Heavy Ion Collider has measured the invariant differential cross section for production of K(S)(0), omega, eta', and phi mesons in p + p collisions at root s 200 GeV. Measurements of omega and phi production in different decay channels give consistent results. New results for the omega are in agreement with previously published data and extend the measured p(T) coverage. The spectral shapes of all hadron transverse momentum distributions measured by PHENIX are well described by a Tsallis distribution functional form with only two parameters, n and T, determining the high-p(T) and characterizing the low-p(T) regions of the spectra, respectively. The values of these parameters are very similar for all analyzed meson spectra, but with a lower parameter T extracted for protons. The integrated invariant cross sections calculated from the fitted distributions are found to be consistent with existing measurements and with statistical model predictions.

Cross Section and Parity-Violating Spin Asymmetries of W(+/-) Boson Production in Polarized p plus p Collisions at root s=500 Gev

Large parity-violating longitudinal single-spin asymmetries A(L)(e+) = 0.86(-0.14)(+0.30) and Ae(L)(e-) = 0.88(-0.71)(+0.12) are observed for inclusive high transverse momentum electrons and positrons in polarized p + p collisions at a center-of-mass energy of root s = 500 GeV with the PHENIX detector at RHIC. These e(+/-) come mainly from the decay of W(+/-) and Z(0) bosons, and their asymmetries directly demonstrate parity violation in the couplings of the W(+/-) to the light quarks. The observed electron and positron yields were used to estimate W(+/-) boson production cross sections for the e(+/-) channels of sigma(pp -> W(+)X) X BR(W(+) -> e(+) nu(e)) = 144.1 +/- 21.2(stat)(-10.3)(+3.4)(syst) +/- 21.6(norm) pb, and sigma(pp -> W(-)X) X BR(W(-) -> e(-) (nu) over bar (e)) = 3.17 +/- 12.1(stat)(-8.2)(+10.1)(syst) +/- 4.8(norm) pb.

Cross section and double helicity asymmetry for eta mesons and their comparison to pi(0) production in p plus p collisions at root s=200 GeV

Measurements of double-helicity asymmetries in inclusive hadron production in polarized p + p collisions are sensitive to helicity-dependent parton distribution functions, in particular, to the gluon helicity distribution, Delta g. This study focuses on the extraction of the double-helicity asymmetry in eta production ((p) over right arrow + (p) over right arrow -> eta + X), the eta cross section, and the eta/pi(0) cross section ratio. The cross section and ratio measurements provide essential input for the extraction of fragmentation functions that are needed to access the helicity-dependent parton distribution functions.

Measurement of transverse single-spin asymmetries for J/psi production in polarized p plus p collisions at root s=200 GeV

We report the first measurement of transverse single-spin asymmetries in J/psi production from transversely polarized p + p collisions at root s = 200 GeV with data taken by the PHENIX experiment in 2006 and 2008. The measurement was performed over the rapidity ranges 1.2 < vertical bar y vertical bar < 2.2 and vertical bar y vertical bar < 0.35 for transverse momenta up to 6 GeV/c. J/psi production at the Relativistic Heavy Ion Collider is dominated by processes involving initial-state gluons, and transverse single-spin asymmetries of the J/psi can provide access to gluon dynamics within the nucleon. Such asymmetries may also shed light on the long-standing question in QCD of the J/psi production mechanism. Asymmetries were obtained as a function of J/psi transverse momentum and Feynman-x, with a value of -0.086 +/- 0.026(stat) +/- 0.003(syst) in the forward region. This result suggests possible nonzero trigluon correlation functions in transversely polarized protons and, if well defined in this reaction, a nonzero gluon Sivers distribution function.