TRAF6-IRF5 kinetics, TRIF, and biophysical factors drive synergistic innate responses to particle-mediated MPLA-CpG co-presentation.

Innate immune responses to pathogens are driven by co-presentation of multiple pathogen-associated molecular patterns (PAMPs). Combinations of PAMPs can trigger synergistic immune responses, but the underlying molecular mechanisms of synergy are poorly understood. Here, we used synthetic particulate carriers co-loaded with monophosphoryl lipid A (MPLA) and CpG as pathogen-like particles (PLPs) to dissect the signaling pathways responsible for dual adjuvant immune responses. PLP-based co-delivery of MPLA and CpG to GM-CSF-driven mouse bone marrow-derived antigen-presenting cells (BM-APCs) elicited synergistic interferon-ß (IFN-ß) and interleukin-12p70 (IL-12p70) responses, which were strongly influenced by the biophysical properties of PLPs. Mechanistically, we found that MyD88 and interferon regulatory factor 5 (IRF5) were necessary for IFN-ß and IL-12p70 production, while TRIF signaling was required for the synergistic response. Both the kinetics and magnitude of downstream TRAF6 and IRF5 signaling drove the synergy. These results identify the key mechanisms of synergistic Toll-like receptor 4 (TLR4)-TLR9 co-signaling in mouse BM-APCs and underscore the critical role of signaling kinetics and biophysical properties on the integrated response to combination adjuvants.

Medieval women's early involvement in manuscript production suggested by lapis lazuli identification in dental calculus.

During the European Middle Ages, the opening of long-distance Asian trade routes introduced exotic goods, including ultramarine, a brilliant blue pigment produced from lapis lazuli stone mined only in Afghanistan. Rare and as expensive as gold, this pigment transformed the European color palette, but little is known about its early trade or use. Here, we report the discovery of lapis lazuli pigment preserved in the dental calculus of a religious woman in Germany radiocarbon-dated to the 11th or early 12th century. The early use of this pigment by a religious woman challenges widespread assumptions about its limited availability in medieval Europe and the gendered production of illuminated texts.

Ring-substituted analogs of 3,3'-diindolylmethane (DIM) induce apoptosis and necrosis in androgen-dependent and -independent prostate cancer cells.

We recently reported that novel ring-substituted analogs of 3,3'-diindolylmethane (ring-DIMs) have anti-androgenic and growth inhibitory effects in androgen-dependent prostate cancer cells. The objectives of this study were to confirm the ability of 4,4'- and 7,7'-dibromo- and dichloro-substituted ring-DIMs to inhibit androgen-stimulated proliferation of androgen-dependent LNCaP human prostate cancer cells using a non-invasive, real-time monitoring technique. In addition, their ability to induce apoptotic and necrotic cell death in androgen-dependent as well as -independent (PC-3) prostate cancer cells was studied. Prostate cancer cells were treated with increasing concentrations of DIM and ring-DIMs (0.3-30 µM) and effects on cell proliferation were measured in real-time using an xCELLigence cellular analysis system. Chromatin condensation and loss of membrane integrity were determined by Hoechst and propidium iodide staining, respectively. Apoptotic protein markers were measured by immunoblotting and activation of caspases determined using selective fluorogenic substrates. Intra- and extracellular concentrations of DIM and ring-DIMs were assessed by electrospray ionization tandem mass spectrometry. Ring-DIMs inhibited androgen-stimulated LNCaP cell proliferation and induced apoptosis and necrosis in LNCaP and PC-3 cells with 2-4 fold greater potencies than DIM. DIM and the ring-DIMs increased caspases -3, -8 and -9 activity, elevated expression of Fas, FasL, DR4 and DR5 protein, and induced PARP cleavage in both cell lines. The cytotoxicity of the most potent ring-DIM, 4,4'-dibromoDIM, but not the other compounds was decreased by an inhibitor of caspase -3. The 4,4'-dibromoDIM was primarily found in the extracellular medium, whereas all other compounds were present to a much larger extent in the cell. In conclusion, ring-DIMs inhibited prostate cancer cell growth and induced cell death in LNCaP and PC-3 cells with greater potencies than DIM; they also structure-dependently activated different cell death pathways suggesting that these compounds have clinical potential as chemopreventive and chemotherapeutic agents in prostate cancer, regardless of hormone-dependency.

Measurement of the cosmic-ray antiproton-to-proton abundance ratio between 4 and 50 GeV.

We present a new measurement of the antiproton-to-proton abundance ratio, pbar/p, in the cosmic radiation. The HEAT-pbar instrument, a balloon borne magnet spectrometer with precise rigidity and multiple energy loss measurement capability, was flown successfully in Spring 2000, at an average atmospheric depth of 7.2 g/cm(2). A total of 71 antiprotons were identified above the vertical geomagnetic cutoff rigidity of 4.2 GV. The highest measured proton energy was 81 GeV. We find that the pbar/p abundance ratio agrees with that expected from a purely secondary origin of antiprotons produced by primary protons with a standard soft energy spectrum.

High-resolution anion-exchange and partition thin-layer chromatography for complex mixtures of 32P-postlabeled DNA adducts.

32P-Postlabeling has emerged as a major tool for detecting DNA adducts resulting from exposure to complex carcinogen mixtures. An integral component of this assay is multi-directional PEI-cellulose TLC in which lipophilic 32P-adducts are resolved in high-salt, high-urea solvents following removal of the bulk of non-adduct radioactivity. This TLC system is very effective for adducts formed following exposure to individual carcinogens; however, adducts resulting from exposure to complex mixtures (e.g. cigarette smoke) generally appear in the form of the so-called diagonal radioactive zones. By using mixtures of polycyclic aromatic hydrocarbon- and aromatic amine-DNA adducts as well as adducts in mouse skin treated with cigarette smoke condensate, we have demonstrated that a combination of 0.3-0.4 M NH4OH and isopropanol-4 M NH4OH (1-1.4:1) solvents can provide more sharply defined adduct spots than the commonly used urea solvents. The non-urea solvents also result in excellent resolution of many adducts which otherwise may remain buried in diagonal radioactive zones when using the urea solvents. In addition, the signal-to-noise ratio is increased 2- to 5-fold over the urea solvents enabling detection of discrete adducts at < or = 3 adducts per 10(10) nucleotides. These partition TLC solvents also involve fewer manipulations (e.g. no water washes to remove salt and urea), and are likely to be more informative with regards to the type of individual adducts detected in the biomonitoring of humans than has hitherto been possible.

DNA adducts of the ubiquitous environmental contaminant cyclopenta[cd]pyrene.

The bioactivation of cyclopenta[cd]pyrene (CPP) was investigated to determine the major DNA adduct-forming metabolite(s) of this widespread environmental contaminant and suspect carcinogen. DNA adducts were analyzed by 32P-postlabeling. Four major and at least seven minor adducts formed when CPP was incubated with calf thymus DNA in the presence of rat liver microsomal systems. P450 subfamilies IA and IIB both activated CPP as microsomes from either phenobarbital- or beta-naphthoflavone-treated rats produced quantitatively similar and qualitatively identical adducts. When the epoxide hydrolase inhibitors, 1,1,1-trichloropropene-2,3-oxide or cyclohexene oxide were added to the incubations, binding increased 2.5- to 4-fold, suggesting epoxidation as a mechanism of adduct formation in vitro. Sprague-Dawley rats were killed 1, 3, 7, 18, 45 and 80 days postdosing i.p. with 50 mg/kg CPP. In all tissues analyzed, four major and several minor qualitatively identical adducts were produced. Binding was highest and most persistent in lung followed by heart, white blood cells (WBCs) and liver. CPP adducts were detectable at doses from 1 microgram/kg to 50 mg/kg. Rat lung DNA adducts were cochromatographed with standardized deoxyguanosine and deoxyadenosine adducts produced by reaction of CPP-3,4-epoxide in vitro. All rat lung adducts comigrated with the deoxyguanosine adducts but one was clearly deoxyadenosine derived. Mouse skin DNA adducts from NIH Swiss mice and mouse lung DNA adducts from B6C3F1 mice were also analyzed. All adducts from either mouse tissue comigrated with rat lung DNA adducts, suggesting CPP-3,4-epoxide was also the major DNA adduct-forming species in the mouse. CPP-3,4-epoxide has been suggested to be the key mediator of the biological activities of CPP. Evidence presented here strongly suggests CPP-3,4-epoxide as the major adduct-forming species of CPP as catalyzed in vitro by rat liver preparations known to mediate the mutagenic activation of CPP, in the rat in vivo, and in mouse skin and lung, two tissues with known sensitivity to CPP tumorigenicity.

Photon transmission tomography techniques for screening organs in biological specimen banking.

Biological specimen banking methodology dictates that when a number of specimens of a particular type are available then the selected specimen be representative of the population in question. It is apparent though that this sorting process may present a formidable task which can be facilitated by employing tomographic transmission techniques using photons. Thus it is possible to discriminate between and discern abnormalities in specimens by obtaining the distribution of the photon linear attenuation coefficient, a function of atomic number and physical density, in sections through the specimen whilst maintaining its integrity. Fresh and freeze-dried specimens of porcine kidney were initially examined under differing temperature conditions using a single point photon transmission method, which was not successful due to the heterogeneity of the tissues. Whereas photon transmission tomography is shown to be well suited in testing for homogeneity, highlights the importance of scanning before cryogenic treatment and provides the possibility of monitoring tissue status over storage period by observing small changes in linear attenuation coefficient.

Reaction of cyclopenta[c,d]pyrene-3,4-epoxide with DNA and deoxynucleotides.

Cyclopenta[c,d]pyrene (CPP) is a widespread polycyclic aromatic hydrocarbon with potent mutagenic and carcinogenic activity. The trans isomer of 3,4-dihydro-3,4-dihydroxy-cyclopenta[c,d]pyrene has been shown to be the major metabolic product of CPP in rat, mouse or human microsomal systems, as well as in peroxyl radical-generating systems, indicating the preferential formation of its obligatory precursor, CPP-3,4-epoxide. The direct mutagenicity of CPP-3,4-epoxide, the inactivity of 3,4-dihydro-CPP and the DNA adduct forming capacity of CPP in vivo has prompted analysis of the DNA adducts produced by CPP-3,4-epoxide to provide information pertaining to: (i) the role this postulated major ultimate mutagenic metabolite may play in the formation of DNA adducts in vivo; (ii) the base selectivity of CPP-3,4-epoxide DNA adducts; and (iii) the role of CPP-3,4-epoxide in the mutagenicity/carcinogenicity of CPP. CPP-3,4-epoxide was reacted with calf thymus DNA, dGp, dAp, dTp, dCp, poly dG-dC, poly dA-dT and poly dG. Adducts were analyzed by the butanol-enhanced version of 32P-postlabeling. Four major and at least three minor adducts formed with DNA in vitro, which were further analyzed for their base selectivity. A similar spectrum of adducts was exhibited by dGp, poly dG-dC and poly dG. dCp, dTp, and dAp formed one, two, and four adducts respectively. The relative binding in adducts per 10(7) nucleotides was in the following descending order: dGp (6000), poly dG-dC (5800), dTp (5300), dAp (4800), calf thymus DNA (3800), poly dA-dT (2300), poly dG (2600) and dCp (20). Adducts derived from either dGp, poly dG-dC or poly dG co-migrated with the DNA adducts in three solvent systems, indicating that CPP-3,4-epoxide forms DNA adducts almost exclusively with deoxyguanosine.

Improved thin-layer chromatographic separation of 32P-postlabeled DNA adducts.

DNA adducts represent the putative initiating event in the chemical carcinogenesis process. 32P-Postlabeling is one of several assays which have been developed for the sensitive detection of DNA adducts. An integral part of the 32P-postlabeling assay is the separation of adducted nucleotides by multidirectional, multisolvent, anion-exchange polyethyleneimine-cellulose thin-layer chromatography. Standard since the introduction of this assay has been the use of high-salt, high-urea solvents for the resolution of adducts during the D3 and D4 phases of the chromatography. Urea solvents are able to separate adducts resulting from a number of chemicals, however, they are time-consuming, retain a lot of background noise, may push adducts into inadequately resolved diagonal radioactive zones, and may not separate adducts of similar structure. In this study we introduce the use of a dilute ammonium hydroxide solvent for D4 chromatography and compare it to other standard solvents such as lithium chloride-Tris.HCl-urea, sodium phosphate-Tris.HCl-urea, and isopropanol-4 M ammonium hydroxide for adduct separation, resolution, recovery, retention of background noise, and chromatography development time. We found that 0.2 M ammonium hydroxide worked well for the recovery, separation, and resolution of a wide array of adducts derived from highly lipophilic polycyclic aromatic hydrocarbons and aromatic amines. In addition, this solvent required much less time (< 1/4) as compared to the other solvents and more importantly allowed the separation of adducts which otherwise comigrated and were not visible when using the other three D4 solvents.(ABSTRACT TRUNCATED AT 250 WORDS)

Interception of reactive, DNA adduct-forming metabolites present in rodent serum following carcinogen exposure: implications for use of body fluids in biomonitoring.

The detection of adduct-forming metabolites in the serum of carcinogen treated animals by 32P-postlabeling was evaluated as a novel approach to overcome the stringent requirement of obtaining DNA from tissues in human biomonitoring assessments. Benzo[a]pyrene (BP) was given i.p. to B6C3F1, C57B1/6, ICR, and DBA/2 mouse strains as well as Sprague-Dawley rats. Three adducts related to BP were detected in the liver and/or lung of Sprague-Dawley rats or B6C3F1, C57B1/6, and ICR mice; a single adduct was detected in the liver and lung of the DBA/2 mouse strain. Adducts chromatographically similar to those found in these tissues were also detected when salmon sperm DNA was incubated with the serum of BP-treated animals. Benzidine treatment induced the formation of one adduct in the liver of B6C3F1 mice, which was chromatographically similar to dG-C8-N'-acetylbenzidine. An identical adduct was detected in the salmon sperm DNA incubated with the serum of these mice. Cyclopenta[cd]pyrene treatment produced four major and three minor adducts in the liver or lung of B6C3F1 mice, all but two of which were detected in DNA incubated with serum of cyclopenta[cd]pyrene-treated animals. Large interstrain differences in the serum level of BP adduct-forming metabolites as well as tissue DNA adducts were found which correlated with previously observed strain-specific trends in sensitivity to PAH-mediated carcinogenesis. Thus, levels of BP adduct-forming metabolites were found in the following descending order: B6C3F1, C57B1/6, ICR, and DBA/2. BP-derived adduct-forming metabolites were detectable as late as 2 d and 5 d post-treatment in the serum of C57B1/6 mice or Sprague-Dawley rats, respectively, which seems to coincide well with the reported species-specific turnover of serum albumin; a protein know to be involved in the transport of reactive metabolites throughout the systemic circulation. The results obtained clearly indicate the presence of adduct-forming carcinogen metabolites in the serum of treated animals, which seemingly irrespective of their chemical nature, can be intercepted with exogenous DNA and detected by 32P-postlabeling. Successful application of a serum-based approach coupled with the use of the generally applicable, ultrasensitive 32P-postlabeling assay could evade the need for obtaining DNA from tissues, currently the major impediment in human biomonitoring studies.

DNA adducts and induction of sister chromatid exchanges in the rat following benzo[b]fluoranthene administration.

Benzo[b]fluoranthene (B[b]F) was administered (100 mg/kg by i.p. injection) to male Sprague--Dawley rats. Lungs, livers and peripheral blood lymphocytes (PBLs) were harvested 1, 3, 5, 7, 14, 28 and 56 days after treatment. Several DNA adducts were observed in each tissue, with maximal levels occurring at approximately 7 days after treatment. Lung DNA exhibited consistently higher adduct levels than liver or PBL DNA. At 56 days after B[b]F administration, the adducts in liver and PBL DNA were present at < 10 amol/microgram DNA, while in lung there were 100 amoles/microgram DNA. No significant differences were observed between tissues in the types of adducts produced. Co-chromatography with synthetic standards showed that only a minor adduct produced in vivo is derived from trans-9,10-dihydro-9,10-dihydroxybenzo[b]fluoranthene-11,12-oxide. Sister chromatid exchanges (SCEs) from whole blood cultures were significantly increased relative to concurrent controls between 1 and 14 days after B[b]F administration, with maximum levels at 14 days. By 28 days after treatment, SCEs had essentially returned to control levels. SCE induction did not correlate with the amount of B[b]F--DNA adducts remaining in the PBLs at harvest time.

Additive effects and potential inhibitory mechanism of some common aromatic pollutants on in vitro mitochondrial respiration.

The in vitro toxicity of multiple hydrophobic compounds was the focus of this study. A mitochondrial respiratory assay, sensitive to perturbations caused by hydrophobic chemicals, was utilized to measure the effects of individual aromatic hydrocarbon pollutants and their mixtures on mitochondrial respiratory function. Benzene, naphthalene, acenaphthene, and 1-chloronaphthalene, common industrial solvents shown to interact additively in vivo, were evaluated using this in vitro assay system. Mitochondrial respiration was inhibited 50% (EC50) by 525 ppm (6.7 mM) benzene, 15 ppm (117 microM) naphthalene, 3.9 ppm (25.5 microM) acenaphthene, or 3.8 ppm (23.4 microM) 1-chloronaphthalene. NADH:O2 oxidoreductase (NADH-->O2), NADH:ubiquinone oxidoreductase, and ubiquinol:O2 oxidoreductase activities were inhibited by all four compounds, whereas succinate:O2 oxidoreductase, cytochrome c oxidase, and duroquinol:O2 oxidoreductase activities were not inhibited. Inhibition of mitochondrial respiration occurred at the level of ubiquinone (coenzyme Q10) for all four aromatic hydrocarbons. The ultraviolet absorbance spectrum of isolated Q10 was also altered by naphthalene, acenaphthene, or 1-chloronaphthalene, suggesting a specific interaction between that component of the respiratory chain and these aromatic hydrocarbons. Inhibition by a mixture of 2, 3, or 4 of the compounds tested was additive, reflecting a summation effect of each compound present in the mixture. This additive nature is consistent with previously reported effects of these compounds in vivo and with compounds having similar modes of action. The similar mode of action in vitro is a specific interaction with coenzyme Q10, not a generalized membrane perturbation as speculated to occur in vivo, and is the likely mechanism for the observed additive toxicity.

Ultrasonic equipment for the measurement of backfat on unshorn live sheep.

Because of the ease of application to the animal's exposed skin, the measurement by A-scan ultrasonics of backfat on pigs is an established technique; but difficulties are experienced with unshorn sheep because the fleece presents an obstacle, as a parting of the wool offers only a limited aperture for insonification of the subcutaneous tissues. Also, movement of the typical nervous sheep usually provides somewhat intermittent return echo signals, rendering difficult an otherwise simple measurement. The present instrument has overcome these problems by an accumulator-averaging technique, implemented by a microprocessor, allowing estimation of live backfat thickness to the nearest 0.5 mm. This paper describes the instrument function, and presents results of a series of experiments which examined the correlation with the carcass backfat thickness.