High level IL-12 production by murine dendritic cells: upregulation via MHC class II and CD40 molecules and downregulation by IL-4 and IL-10.

We have shown previously that dendritic cells (DC) produce IL-12 upon interaction with CD4+ T cells. Here we ask how this IL-12 production is induced and regulated. Quantitative PCR and in situ hybridization for IL-12 p40 and an ELISA specific for the p70 heterodimer were used to determine IL-12 production. We demonstrate that ligation of either CD40 or MHC class II molecules independently trigger IL-12 production in DC, and that IL-12 production is downregulated by IL-4 and IL-10. The levels of bioactive IL-12 that can be released by triggering with an anti-CD40 mAb or with a T cell hybridoma are high (range 260-4700 pg/ml from 1 X 10(6) DC in 72 h). The CD40-mediated pathway indicates that IL-12 production is induced in DC upon interaction with activated, CD40 ligand-expressing helper T cells, even in the absence of cognate antigen recognition. Side-by-side comparison of IL-12 production, and blocking experiments employing an anti-CD40 ligand mAb, suggest that the CD40-mediated pathway is quantitatively more significant than induction via the MHC class II molecule. The importance of the CD40/CD40 ligand interaction for IL-12 induction in DC likely contributes to the recent finding that mice lacking the CD40 ligand are impaired in mounting Th1 type cell-mediated immune responses.

A mammary cell-specific enhancer in mouse mammary tumor virus DNA is composed of multiple regulatory elements including binding sites for CTF/NFI and a novel transcription factor, mammary cell-activating factor.

Mouse mammary tumor virus (MMTV) is a milk-transmitted retrovirus involved in the neoplastic transformation of mouse mammary gland cells. The expression of this virus is regulated by mammary cell type-specific factors, steroid hormones, and polypeptide growth factors. Sequences for mammary cell-specific expression are located in an enhancer element in the extreme 5' end of the long terminal repeat region of this virus. This enhancer, when cloned in front of the herpes simplex thymidine kinase promoter, endows the promoter with mammary cell-specific response. Using functional and DNA-protein-binding studies with constructs mutated in the MMTV long terminal repeat enhancer, we have identified two main regulatory elements necessary for the mammary cell-specific response. These elements consist of binding sites for a transcription factor in the family of CTF/NFI proteins and the transcription factor mammary cell-activating factor (MAF) that recognizes the sequence G Pu Pu G C/G A A G G/T. Combinations of CTF/NFI- and MAF-binding sites or multiple copies of either one of these binding sites but not solitary binding sites mediate mammary cell-specific expression. The functional activities of these two regulatory elements are enhanced by another factor that binds to the core sequence ACAAAG. Interdigitated binding sites for CTF/NFI, MAF, and/or the ACAAAG factor are also found in the 5' upstream regions of genes encoding whey milk proteins from different species. These findings suggest that mammary cell-specific regulation is achieved by a concerted action of factors binding to multiple regulatory sites.

Lactogenic hormone and cell type-specific control of the whey acidic protein gene promoter in transfected mouse cells.

The whey acidic protein (WAP) is a major milk protein. It is abundantly expressed in mammary epithelial cells, and its gene is controlled by lactogenic hormones. The identification of regulatory cis-acting sequences of the mouse WAP gene was so far dependent on the analysis of transgenic animals. We report here the possibility of analyzing regulatory sequences by gene transfer experiments using the lactogenic hormone-dependent mammary epithelial cell line HC11. A WAP-chloramphenicol acetyltransferase construct containing 2.5 kilobases of the 5'-flanking sequence of the WAP gene was stably transfected into HC11 cells. High chloramphenicol acetyltransferase activity was induced in pools of transfected cells cultured in the presence of the lactogenic hormones glucocorticoid, PRL, and insulin. A lower induction was observed by glucocorticoid hormone alone. PRL by itself was not able to induce the WAP gene promoter above the level observed in the absence of lactogenic hormones. A time course of hormone induction showed a weak initial response with a steady increase over at least 4 days of hormone treatment. Induction was not observable in the mammary epithelial cell line NOG-8 and NIH3T3 fibroblasts, despite the presence of functional glucocorticoid receptor in these cells. This indicates the requirement for a cell type-specific transcription factor present in the mammary epithelial cell line HC11, but not in NOG-8 epithelial cells or NIH3T3 fibroblasts.(ABSTRACT TRUNCATED AT 250 WORDS)

A distal enhancer region in the human beta-casein gene mediates the response to prolactin and glucocorticoid hormones.

The 5' flanking region of the human beta-casein gene was investigated for the presence of regulatory sequences mediating the action of the lactogenic hormones prolactin and dexamethasone. DNA encompassing 9389 base pairs of the flanking region was isolated and a sequence comparison performed with regulatory regions previously identified in the beta-casein gene of rodents and ruminants. The analysis revealed the presence of a distal region between -4700 and -4550 with a high percentage of identity to the bovine beta-casein enhancer region, and a proximal region between -1 and -200 similar to the proximal promoter regions found in rodents and ruminants. Reporter gene constructs under the control of the distal or the proximal region of the human beta-casein gene were tested for their responsiveness to prolactin and dexamethasone. In transfection experiments, the distal region functioned as a lactogenic hormone inducible enhancer, whereas the proximal region exhibited low activity. In electromobility shift assays, multiple binding sites for Stat5, CCAAT/enhancer-binding proteins, and Ets domain proteins were identified in the distal human enhancer. These transcription factors have already been demonstrated as important regulators of the transcription of milk protein genes in rodents. Thus, a common set of transcription factors appears to be required for the expression of the human beta-casein gene and of milk protein genes in other species.

First determination of generalized polarizabilities of the proton by a virtual compton scattering experiment

Absolute differential cross sections for the reaction ep-->epgamma have been measured at a four-momentum transfer with virtuality Q2 = 0.33 GeV2 and polarization epsilon = 0.62 in the range 33.6 to 111.5 MeV/c for the momentum of the outgoing photon in the photon-proton center of mass frame. The experiment has been performed with the high-resolution spectrometers at the Mainz Microtron MAMI. From the photon angular distributions, two structure functions which are a linear combination of the generalized polarizabilities have been determined for the first time.

Measurement of the Gerasimov-Drell-Hearn Integrand for 2H from 200 to 800 MeV.

A measurement of the helicity dependence of the total inclusive photoabsorption cross section on the deuteron was carried out at MAMI (Mainz) in the energy range 200

Measurement of helicity-dependent photoabsorption cross sections on the neutron from 815 to 1825 MeV.

Helicity-dependent total photoabsorption cross sections on the deuteron have been measured for the first time at ELSA (Bonn) in the photon energy range from 815 to 1825 MeV. Circularly polarized tagged photons impinging on a longitudinally polarized LiD target have been used together with a highly efficient 4pi detector system. The data around 1 GeV are not compatible with predictions from existing multipole analyses. From the measured energy range an experimental contribution to the GDH integral on the neutron of [33.9 +/- 5.5(stat) +/- 4.5(syst)] microb is extracted.

Involvement of Ets-related proteins in hormone-independent mammary cell-specific gene expression.

Regulatory regions have been located in the 5' flanking sequence of the mouse whey acidic protein gene which contribute to its tissue- and stage-specific expression in the mammary gland. They can be functionally separated into elements which mediate the action of lactogenic hormones prolactin and glucocorticoids and elements which control mammary cell-specific transcription in the absence of hormones. By mutational analysis, we have located a site in the whey acidic protein promoter between -120 and -100 which is important for hormone-independent promoter function. In stably transfected HC11 mammary epithelial cells, the hormone-independent activity of the mutated promoter was reduced 40-fold, whereas the capability to respond to lactogenic hormones was retained. The site was specifically recognised by two nuclear factors contained in extracts of cultivated mammary epithelial cells or mammary glands. Electrophoretic mobility shift assay, DNase I footprinting and methylation interference experiments indicated a relation of both factors to the Ets family of DNA-binding proteins. One of these factors also recognised a functionally important site in the mammary cell-specific enhancer of the mouse mammary tumor virus long terminal repeat. The results suggest that factors related to the Ets family are important determinants in mammary cell-specific gene expression.

Measurement of the beam-helicity asymmetry in the p((-->)e,e'p)pi(0) reaction at the energy of the Delta(1232) resonance.

In a p((-->)e,e'p)pi(0) out-of-plane coincidence experiment at the three-spectrometer setup of the Mainz Microtron MAMI, the beam-helicity asymmetry has been precisely measured around the energy of the Delta(1232) resonance and Q(2) = 0.2(GeV/c)(2). The results are in disagreement with three up-to-date model calculations. This is interpreted as a lack of understanding of the nonresonant background, which in dynamical models is related to the pion cloud.

Neutron polarizabilities investigated by quasifree Compton scattering from the deuteron.

Measuring Compton scattered photons and recoil neutrons in coincidence, quasifree Compton scattering by the neutron has been investigated at MAMI (Mainz) at theta(lab)(gamma) = 136 degrees in an energy range from 200 to 400 MeV. From the data a polarizability difference of alpha(n)-beta(n) = 9.8+/-3.6(stat)+2.1-1.1(syst)+/-2.2(model) in units of 10(-4) fm(3) has been determined. In combination with the polarizability sum alpha(n)+beta(n) = 15.2+/-0.5 deduced from photoabsorption data, the first precise results for the neutron electric and magnetic polarizabilities, alpha(n) = 12.5+/-1.8(stat)+1.1-0.6(syst)+/-1.1(model) and beta(n) = 2.7-/+1.8(stat)+0.6-1.1(syst)-/+1.1(model), are obtained.

Measurement of the recoil polarization in the p(e-->, e'p-->)pi(0) reaction at the Delta(1232) resonance.

The recoil proton polarization has been measured in the p(e-->,e'p-->)pi(0) reaction in parallel kinematics around W = 1232 MeV, Q2 = 0.121 (GeV/c)2, and epsilon = 0.718 using the polarized cw electron beam of the Mainz Microtron. All three proton polarization components, Px/P(e) = (-11.4+/-1.3+/-1.4)%, P(y) = (-43.1+/-1.3+/-2.2)%, and P(z)/P(e) = (56.2+/-1.5+/-2.6)%, could be measured simultaneously. The Coulomb quadrupole to magnetic dipole ratio, CMR = (-6.4+/-0.7(stat)+/-0.8(syst))%, was determined from Px in the framework of the Mainz Unitary Isobar Model. The consistency among the reduced polarizations and the extraction of the ratio of longitudinal-to-transverse response is discussed.

Neutral pion threshold production at Q(2) = 0.05 GeV(2)/c(2) and chiral perturbation theory.

New data are presented on the p(e,e'p)pi(0) reaction at threshold at a four-momentum transfer of Q(2) = 0.05 GeV(2)/c(2). The data were taken with the three-spectrometer setup of the A1 Collaboration at the Mainz Microtron MAMI. The complete center of mass solid angle was covered up to a center of mass energy of 4 MeV above threshold. Combined with measurements at three different values of the virtual photon polarization epsilon, the structure functions sigma(T), sigma(L), sigma(TT), and sigma(TL) are determined. The results are compared with calculations in heavy baryon chiral perturbation theory and with a phenomenological model. The measured cross section is significantly smaller than both predictions.

Helicity dependence of gammap --> Npi below 450 MeV and contribution to the Gerasimov-Drell-Hearn sum rule.

The helicity dependence of the single pion photoproduction on the proton has been measured in the energy range from 200 to 450 MeV for the first time. The experiment, performed at the Mainz microtron MAMI, used a 4pi-detector system, a circularly polarized, tagged photon beam, and a frozen-spin target. The data obtained provide new information for multipole analyses of pion photoproduction and determine the main contributions to the Gerasimov-Drell-Hearn sum rule and the forward spin polarizability gamma(0).

Measurement of the exclusive 3He(e,e'p) reaction below the quasielastic peak.

New, high-precision measurements of the 3He(e,e(')p) reaction using the A1 Collaboration spectrometers at the Mainz microtron MAMI are presented. These were performed in antiparallel kinematics at energy transfers below the quasielastic peak, and at a central momentum transfer of 685 MeV/c. Cross sections and distorted momentum distributions were extracted and compared to theoretical predictions and existing data. The longitudinal and transverse behavior of the cross section was also studied. Sizable differences in the cross-section behavior from theoretical predictions based on the plane wave impulse approximation were observed in both the two- and three-body breakup channels. Full Faddeev-type calculations account for some of the observed excess cross-section, but significant differences remain.

Experimental check of the Gerasimov-Drell-Hearn sum rule for 1H.

For the first time we checked the fundamental Gerasimov-Drell-Hearn (GDH) sum rule for the proton experimentally in the photon energy range from 0.2-2.9 GeV with the tagged photon facilities at MAMI (Mainz) and ELSA (Bonn). New data of the doubly polarized total cross section difference are presented in the energy range from 1.6 to 2.9 GeV. The contribution to the GDH integral from 0.2-2.9 GeV yields [254+/-5(stat)+/-12(syst)] microb with negative contributions in the Regge regime at photon energies above 2.1 GeV. This trend supports the validity of the GDH sum rule.

Helicity amplitudes A1/2 and A3/2 for the D13(1520) resonance obtained from the gamma-->p-->-->ppi(0) Reaction.

The helicity dependence of the gamma-->p-->-->ppi(0) reaction has been measured for the first time in the photon-energy range from 550 to 790 MeV. The experiment, performed at the Mainz microtron MAMI, used a 4pi-detector system, a circularly polarized, tagged photon beam, and a longitudinally polarized frozen-spin target. These data are predominantly sensitive to the D13(1520) resonance and are used to determine its helicity amplitudes.

First Measurement of the Gerasimov-Drell-Hearn Sum Rule for 1H from 0.7 to 1.8 GeV at ELSA.

To verify the fundamental Gerasimov-Drell-Hearn (GDH) sum rule for the first time experimentally, we measured the helicity dependent total photoabsorption cross section with circularly polarized real photons and longitudinally polarized nucleons in the photon energy range 0.68-1.82 GeV with the tagged photon facility at ELSA. The experiment was carried out with a 4pi detection system, a circularly polarized tagged photon beam, and a frozen spin polarized proton target. The contribution to the GDH sum rule in this photon energy range is [49.9+/-2.4(stat)+/-2.2(syst)] microb.