Interference with tissue factor prolongs intrahepatic islet allograft survival in a nonhuman primate marginal mass model

BACKGROUND: Tissue factor (TF) expression on islets can result in an instant blood-mediated inflammatory reaction (IBMIR) that contributes to early islet loss. We tested whether peritransplant protection of islets from IBMIR with a monoclonal anti-TF antibody (CNTO859) would enhance engraftment in our nonhuman primate marginal mass model. METHODS: Each of six pairs of cynomolgus monkeys (CM) with streptozotocin-induced diabetes was closely matched for metabolic control and was transplanted with 5,000 IEQ/kg allogeneic, ABO-compatible islets from the same donor under the cover of steroid-free immunosuppression. For each pair, experimental animals received islets cultured with 20 microg/mL anti-TF and were dosed with 6 mg/kg anti-TF intravenously, 10-25 min before islet infusion; control monkeys received an equal number of islets from the same preparation cultured without anti-TF and no in vivo treatment. RESULTS: Early fasting C-peptide (CP) values were different between (P<0.01), but not within, pairs and correlated with in vitro functional capacity of islets as assessed by perifusion (r=0.60; P=0.022). Compared to their matched controls, experimental animals had decreased posttransplant markers of coagulation, higher fasting CP levels (1 month posttransplant and end of study) and prolonged graft function. CONCLUSIONS: These data suggest that pretreatment of islets and the recipient with anti-TF may limit the effects of IBMIR, thereby enhancing islet engraftment and survival.     

Enhanced angiogenic capacity of human umbilical vein endothelial cells from women with preeclampsia

Maternal and placental angiogenic abnormalities are a common feature of preeclampsia. The aim of this study was to determine if endothelial cells from women with preeclampsia exhibit different angiogenic responses compared to healthy cells. Using the endothelial tube formation assay, we have shown that primary human umbilical vein endothelial cells (HUVECs) isolated from women with preeclampsia display greater levels of in vitro angiogenic branching compared to cells from healthy women. A comparable increase in tube formation was observed in healthy cells cultured at 0.5% O(2). Vascular endothelial growth factor (VEGF) receptor inhibition resulted in a decrease in angiogenesis in both healthy hypoxic cells and cells from women with preeclampsia. These findings demonstrate that HUVECs from women with preeclampsia exhibit inherent differences in their angiogenic capacity which are apparent in the absence of placental or maternal factors.     

DNA ligands as radiomodifiers: studies with minor-groove binding bibenzimidazoles.

An iodinated bibenzimidazole, iodoHoechst 33258, was previously reported to markedly sensitize DNA and cells to UV-A, exemplifying the potential of iodinated DNA ligands as radiosensitizers, a rational extension of sensitization by halogenated pyrimidines. However, unlike the latter sensitizers, iodoHoechst 33258 is not a sensitizer of ionizing radiation, presumably due to the innate radioprotective properties of the uniodinated ligand. Experiments with purified DNA show that both Hoechst 33258 and Hoechst 33342 decrease the yield the radiation-induced DNA strand breakage. The ligands bind at discrete sites in the minor groove of DNA, and analysis on DNA sequencing gels show pronounced protection at the ligand binding sites, as well as more generalized protection. The extent of protection of strand breakage on plasmid DNA and the fact that it persists in the presence of 0.5 M NaCl (which prevents low affinity ionic binding between the high affinity sites) suggests that the protective effects of bound ligand are not confined to the high affinity binding sites in the minor groove. The mechanisms of this generalized protection is unknown, but there is some evidence indicating that the H-atom donation from the ligand may account for the site-specific protection. The extent of protection is much diminished, but still evident, in the presence of 100 mM mannitol, a known hydroxyl radical scavenger, indicating that some of the protective effects might relate to DNA damage mediated by direct action. Further evaluation of the mechanisms of protection should enable development of both more active radioprotectors and, by elimination of the radioprotective features from halogenated DNA ligands, more effective radiosensitizers.     

Osteoblast response to fluid induced shear depends on substrate microarchitecture and varies with time

Osteoblasts are exposed to fluid shear in vivo but the effects are not well understood, particularly how substrate properties or length of exposure modify the response. Short exposure (1 h) to shear reduces the stimulatory effect of micron-scale surface structure on osteoblast differentiation, but the effects of longer term exposures are not known. To test the hypothesis that substrate-dependent responses of osteoblasts to shear depend on the length of exposure to fluid flow, MG63 osteoblasts were grown on tissue culture glass, which has an average roughness (Ra) < 0.2 microm; machined Ti disks (PT, Ra < 0.6 microm); Ti disks with a complex microarchitecture [sand blasted acid etched (SLA), Ra = 4-5 microm); and Ti plasma-sprayed surfaces [Ti via plasma spray (TPS), Ra = 7 microm]. Confluent cultures were exposed to pulsatile flow at shear forces of 0, 1, and 14 dynes/cm(2) for 0, 6, 12, and 24 h. Shear reduced cell number on all surfaces, with greatest effects on TPS. Shear had no effect on alkaline phosphatase on smooth surfaces but increased enzyme activity on SLA and TPS in a time-dependent manner. Its effects on osteocalcin, TGF-beta1, and PGE(2) in the conditioned media were greatest on these surfaces as well. Responses to fluid-induced shear were blocked by the general Cox inhibitor indomethacin and the Cox-2 inhibitor meloxicam, indicating that response to shear is mediated by prostaglandin produced via a Cox-2 dependent mechanism. These results show that the effects of fluid induced shear change with time and are substrate dependent, suggesting that substrate microarchitecture regulates the osteoblast phenotype and effects of shear are determined by the maturation state of the responding population.     

Analysis of dioxins in contaminated soils with the calux and caflux bioassays, an immunoassay, and gas chromatography/high-resolution mass spectrometry

The chemically activated luciferase expression assay, the chemically activated fluorescence expression assay, and the enzyme-linked immunosorbent assay (ELISA) are all bioanalytical methods that have been used for the detection and quantification of polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans (PCDD/Fs). However, no comparisons of the results obtained by these three methods have been published analyzing identical replicates of purified sample extracts. Therefore, we have evaluated the performance of each of these methods for analyzing PCDD/Fs in aliquots of extracts from aged-contaminated soil samples and compared the results with those obtained by gas chromatography/high-resolution mass spectrometry (GC/HRMS). The quantitative performance was assessed and the effects of sample purification and data interpretation on the quality of the bioassay results were investigated. Results from the bioanalytical techniques were, in principle, not significantly different from each other or from the GC/HRMS data (p = 0.05). Furthermore, properly used, all of the bioanalytical techniques examined were found to be sufficiently sensitive, selective, and accurate to be used in connection with soil remediation activities when aiming at the remediation goal recommended by the U.S. Environmental Protection Agency (i.e., <1000 pg toxic equivalency/g). However, a site-specific correction factor should be applied with the use of the ELISA to account for differences between the toxic equivalency factors and the ELISA cross-reactivities of the various PCDD/F congeners, which otherwise might significantly underestimate the PCDD/F content.     

Synthesis of 3-aryl-2-phosphinoimidazo[1,2-a]pyridine ligands for use in palladium-catalyzed cross-coupling reactions

3-Aryl-2-phosphinoimidazo[1,2-a]pyridine ligands were synthesized from 2-aminopyridine via two complementary routes. The first synthetic route involves the copper-catalyzed iodine-mediated cyclizations of 2-aminopyridine with arylacetylenes followed by palladium-catalyzed cross-coupling reactions with phosphines. The second synthetic route requires the preparation of 2,3-diiodoimidazo[1,2-a]pyridine or 2-iodo-3-bromoimidazo[1,2-a]pyridine from 2-aminopyridine followed by palladium-catalyzed Suzuki/phosphination or a phosphination/Suzuki cross-coupling reactions sequence, respectively. Preliminary model studies on the Suzuki synthesis of sterically-hindered biaryl and Buchwald–Hartwig amination compounds are presented with these ligands.

3-Aryl-2-phosphoimidazo[1,2-a]pyridine ligands were prepared via two complimentary synthetic routes and were evaluated in the Suzuki–Miyaura and Buchwald–Hartwig amination cross-coupling reactions.

Palladium-catalyzed cross-coupling reactions have revolutionized the formation of C–C and C–X bond formation in the academic and industrial synthetic organic chemistry sectors.^1,2 Applications such as synthesis of natural products,³ active pharmaceutical ingredients (API),⁴ agrochemicals,⁵ and materials for electronic applications⁶ are showcased. Snieckus described in his 2010 Nobel Prize review that privileged ligand scaffolds represented the “third wave” in the cross-coupling reactions where the “first wave” was the investigation of the metal catalyst-the rise of palladium and the “second wave” was the exploration of the organometallic coupling partner.¹ In the last twenty years, it was recognized that the choice of ligand facilitated the oxidative addition and reductive-elimination steps of the catalytic cycle of transition metal-catalyzed cross-coupling reactions, increasing the overall rate of the reaction. For example, bulky trialkylphosphines facilitated the oxidative addition processes of electron-rich, unactivated substrates such as aryl chlorides.^7,8 Sterically demanding ligands also provided enhanced rates of reductive elimination from [(L)_nPd(aryl)(R), R = aryl, amido, phenoxo, etc.] species by alleviation of steric congestion.⁹ Privileged ligands such as Buchwald''s biarylphosphines,^10,11 Fu''s trialkylphosphines,^7,8,12 Nolan–Hermann''s N-heterocyclic carbenes (NHC),^13–15 Hartwig''s ferrocenes,^16,17 Beller''s bis(adamantyl)phosphines^18,19 and N-aryl(benz)imidazolyl or N-pyrrolylphosphines,^20,21 Zhang''s ClickPhos ligands,^22,23 and Stradiotto''s biaryl P–N phosphines,^24,25 to mention a few, have found wide-spread use in Suzuki–Miyaura, Corriu–Kumada, Heck, Negishi, Sonogashira, C–X (X = S, O, P) cross-coupling and Buchwald–Hartwig amination reactions (Fig. 1). Preformed catalysts with these ligands attached to the palladium metal center are also recognized as well-defined entities in cross-coupling reactions.²⁶Open in a separate windowFig. 1Privileged ligands for palladium-catalyzed cross-coupling reactions.The term privileged structure was first coined by Evans et al. in 1988 and was defined as “a single molecular framework able to provide ligands for diverse receptors”.²⁷ In the last three decades, it is clear that privileged structures are exploited as opportunities in drug discovery programs.^28–31 For example, imidazo[1,2-a]pyridines are privileged structures in medicinal chemistry programs (Fig. 2).³² Imidazo[1,2-a]pyridines are a represented motif in several drugs on the market such as zolpidem, marketed as Ambien™ for the treatment of insomnia,³³ minodronic acid, marketed as Bonoteo™ for oral treatment of osteoporosis,³⁴ and olprinone, sold as Coretec™ as a cardiotonic agent.³⁵Open in a separate windowFig. 2Imidazo[1,2-a]pyridines as privileged structures in medicinal chemistry and in our cross-coupling reactions approach.Our group is interested in a long-term research program directed at the use of key privileged structures that are employed in drug discovery programs as potential phosphorus ligands for cross-coupling reactions. In our entry into the use of privileged structures from the medicinal chemistry literature for our investigation into new phosphorus ligands, we have developed two complementary synthetic routes for the preparation of 3-aryl-2-phosphinoimidazo[1,2-a]pyridine ligands from 2-aminopyridine as our initial substrate.Our first synthetic route for the preparation of 3-aryl-2-phosphinoimidazo[1,2-a]pyridine ligands 3a–3l required the copper(ii) acetate iodine-mediated double oxidative C–H amination of 2-aminopyridine (1) with arylacetylenes under an oxygen atmosphere to give 3-aryl-2-iodoimidazo[1,2-a]pyridines 2a–2d (Scheme 1).^36,37Open in a separate windowScheme 1Preparation of 3-aryl-2-phosphinoimidazo[1,2-a]pyridine ligands 3a–3l from 2-aminopyridine via copper-catalyzed arylacetylene cyclizations/palladium-catalyzed phosphination reactions sequences.Phenylacetylene and 2-/3-/4-methoxyphenylacetylenes were commercially available reagents. With intermediates 2a–d in hand, we explored several cross-coupling phosphination reactions and we found that palladium-catalyzed phosphination with DIPPF ligand in the presence of cesium carbonate as the base in 1,4-dioxane under reflux provided twelve new ligands 3a–3l as shown in 38 Moderate to good yields were obtained under these cross-coupling conditions. There are few commercially available dimethoxyphenylacetylenes, and most are prohibitively expensive, and so an alternative synthetic strategy was explored.Palladium-catalyzed phosphination of 3-aryl-2-iodoimidazo[1,2-a]pyridines 2a–2d^a

Birth weight and muscle strength: A systematic review and meta-analysis

Objective: Lower muscle strength is associated with a range of adverse health outcomes in later life. The variation in muscle strength between individuals is only partly accounted for by factors in adult life such as body size and physical activity. The aim of this review was to assess the strength of the association between intrauterine development (indicated by birth weight) and subsequent muscle strength. Design: Systematic review and meta-analysis of studies that assessed the association between birth weight and subsequent muscle strength. Results: Nineteen studies met inclusion criteria with 17 studies showing that higher birth weight was associated with greater muscle strength. Grip strength was used as a single measure of muscle strength in 15 studies. Meta-analysis (13 studies, 20 481 participants, mean ages 9.3 to 67.5) showed a 0.86 kg (95% CI 0.58, 1.15) increase in muscle strength per additional kilogram of birth weight, after adjustment for age, gender and height at the time of strength measurement. Conclusion: This review has found consistent evidence of a positive association between birth weight and muscle strength which is maintained across the lifecourse. Future work will be needed to elucidate the biological mechanisms underlying this association, but it suggests the potential benefit of an early intervention to help people maintain muscle strength in later life.     

Palpebral fissure responses to topical adrenergic drugs

We measured the magnitude and time course of the increase in palpebral fissure width in ten normal volunteers in response to direct-acting (2.5% phenylephrine, 1% apraclonidine) and indirect-acting (10% cocaine, 1% hydroxyamphetamine) topical adrenergic drugs given in one eye. The increase in the palpebral fissure width of the treated eye was compared to the width of the untreated, control eye during a period of 60 minutes after each drug was administered. The difference in fissure width (asymmetry) between the treated and untreated eyes increased significantly for all drugs during the three- to 60-minute time period after treatment. There was no significant difference in the maximum eyelid effect among the adrenergic drugs tested. The drugs exerted their maximum effect by 30 minutes in 39 of the 40 trials (97.5%). The direct-acting drugs tended to exert their effect more quickly than the indirect-acting drugs. Our results demonstrate the expected increase in palpebral fissure width in response to topical adrenergic drugs in normal eyes. This information will provide a basis for evaluating Müller's muscle and its sympathetic innervation in patients with blepharoptosis.