Unusual magnetization process and magnetocaloric effect in α-CoV2O6driven by pulsed magnetic fields.

In low-dimensional Ising spin systems, an interesting observation is the presence of step magnetization at low temperatures. Here we combine both DC and pulsed magnetic fields to study the 1/3 magnetization plateau and multiple steps in the Ising spin-chain material α-CoV2O6. Magnetization in pulsed fields is quite different from that in DC fields, showing multiple steps in an intermediate range of 4.2-6 K, inverted hysteresis below 4.2 K and asymmetric magnetization in negative fields below 11 K. We demonstrate that these unusual behaviors in magnetization are caused by the spin dynamics and the anomalous magnetocaloric effect (MCE) in α-CoV2O6, i.e., abrupt changes of sample temperature in adiabatic conditions. We successfully separate the influence between the intrinsic slow spin dynamics and the quasi-extrinsic temperature change. From the MCE, we find that some irreversible behavior is originated from the slow spin dynamics. Two different slow dynamics associated with the metastable steps are observed: one is sensitive to the slow field sweep rate at the order of ∼mT s-1and weakly depends on temperature, while the other responds to the rapid field sweep rate of ∼kT s-1and dominates at lowest temperature. We also distinguish that the metastable transition atH4is the first order and crucial for the ferrimagnetic to ferromagnetic transition. This study is useful to the understanding of multistep magnetization in α-CoV2O6and sheds light on recent experimental findings of related compounds.

Monoclonal antibody against macrophage colony-stimulating factor suppresses circulating monocytes and tissue macrophage function but does not alter cell infiltration/activation in cutaneous lesions or clinical outcomes in patients with cutaneous lupus erythematosus.

This study's objective was to assess the effects of PD-0360324, a fully human immunoglobulin G2 monoclonal antibody against macrophage colony-stimulating factor in cutaneous lupus erythematosus (CLE). Patients with active subacute CLE or discoid lupus erythematosus were randomized to receive 100 or 150 mg PD-0360324 or placebo via intravenous infusion every 2 weeks for 3 months. Blood and urine samples were obtained pre- and post-treatment to analyse pharmacokinetics and pharmacodynamic changes in CD14(+) CD16(+) monocytes, urinary N-terminal telopeptide (uNTX), alanine/aspartate aminotransferases (ALT/AST) and creatine kinase (CK); tissue biopsy samples were taken to evaluate macrophage populations and T cells using immunohistochemistry. Clinical efficacy assessments included the Cutaneous Lupus Erythematosus Disease Area and Severity Index (CLASI). Among 28 randomized/analysed patients, peak/trough plasma concentrations increased in a greater-than-dose-proportional manner with dose increases from 100 to 150 mg. Statistically significant differences were observed between active treatment and placebo groups in changes from baseline in CD14(+) CD16(+) cells, uNTX, ALT, AST and CK levels at most time-points. The numbers, density and activation states of tissue macrophages and T cells did not change from baseline to treatment end. No between-group differences were seen in CLASI. Patients receiving PD-0360324 reported significantly more adverse events than those receiving placebo, but no serious adverse events. In patients with CLE, 100 and 150 mg PD-0360324 every 2 weeks for 3 months suppressed a subset of circulating monocytes and altered activity of some tissue macrophages without affecting cell populations in CLE skin lesions or improving clinical end-points.

Effects of rumen-protected γ-aminobutyric acid on performance and nutrient digestibility in heat-stressed dairy cows.

This experiment was conducted to investigate the effects of rumen-protected γ-aminobutyric acid (GABA) on performance and nutrient digestibility in heat-stressed dairy cows. Sixty Holstein dairy cows (141±15 d in milk, 35.9±4.3kg of milk/d, and parity 2.0±1.1) were randomly assigned to 1 of 4 treatments according to a completely randomized block design. Treatments consisted of 0 (control), 40, 80, or 120mg of true GABA/kg of dry matter (DM). The trial lasted 10wk. The average temperature-humidity indices at 0700, 1400, and 2200h were 78.4, 80.2, and 78.7, respectively. Rectal temperatures decreased linearly at 0700, 1400, and 2200h with increasing GABA concentration. Supplementation of GABA had no effect on respiration rates at any time point. Dry matter intake, energy-corrected milk, 4% fat-corrected milk, and milk fat yield tended to increase linearly with increasing GABA concentration. Supplementation of GABA affected, in a quadratic manner, milk protein and lactose concentrations, and milk protein yield, and the peak values were reached at a dose of 40mg of GABA/kg. Milk urea nitrogen concentration responded quadratically. Total solids content increased linearly with increasing GABA concentration. Supplementation of GABA had no effect on milk yield, lactose production, total solids, milk fat concentration, somatic cell score, or feed efficiency. Apparent total-tract digestibilities of DM, organic matter, crude protein, neutral detergent fiber, and acid detergent fiber were similar among treatments. These results indicate that rumen-protected GABA supplementation to dairy cows can alleviate heat stress by reducing rectal temperature, increase DM intake and milk production, and improve milk composition. The appropriate supplemental GABA level for heat-stressed dairy cows is 40mg/kg of DM.

Simultaneous determination of aflatoxin M1, ochratoxin A, zearalenone and α-zearalenol in milk by UHPLC-MS/MS.

In this study, a sensitive and rapid method has been developed for the simultaneous determination of aflatoxin M1, ochratoxin A, zearalenone and α-zearalenol in milk by ultra high performance liquid chromatography combined with electrospray ionisation triple quadrupole tandem mass spectrometry (UHPLC-ESI-MS/MS). The milk samples were purified using Oasis HLB cartridge. The matrix effects were evaluated by determining the signal suppression-enhancement (SSE) and corrected by external matrix-matched calibration. The limits of quantity (LOQ) of the mycotoxins were in the range of 0.003-0.015µgkg(-1). The high correlation coefficients (R(2)⩾0.996) were obtained in the range of 0.01-1.00µgkg(-1) of the mycotoxins, along with good recovery (87.0-109%), repeatability (3.4-9.9%) and intra-laboratory reproducibility (4.0-9.9%) at the concentrations of 0.025, 0.1 and 0.5µgkg(-1). The detected rates of the mycotoxins were from 16.7% to 96.7% in raw milk, liquid milk and milk powder samples collected from the dairy farms and supermarkets in Beijing. The method proposed is suitable for the simultaneous determination of aflatoxin M1, ochratoxin A, zearalenone, and α-zearalenol, and could be performed for analysing the mycotoxins in milk.

The innate immune response, clinical outcomes, and ex vivo HCV antiviral efficacy of a TLR7 agonist (PF-4878691).

Hepatitis C virus (HCV) infection is an issue of global concern, and studies are ongoing to identify new therapies that are both effective and safe. PF-4878691 is a Toll-like receptor 7 (TLR7) agonist modeled so as to dissociate its antiviral activities from its inflammatory activities. In a proof-of-mechanism study in healthy volunteers who received doses of 3, 6, and 9 mg of PF-4878691 twice a week for 2 weeks, PF-4878691 induced biomarkers of the immune and interferon (IFN) responses in a dose-dependent and dose-frequency-related manner. A novel finding was induction of TLR7 expression in vivo in response to PF-4878691, leading to an amplified biomarker response. A nonresponder at the 9-mg dose had a polymorphism in the IFN-α receptor 1 subunit (Val168Leu). Two subjects who had received 9-mg doses experienced serious adverse events (SAEs), characterized by flu-like symptoms, hypotension, and lymphopenia, leading to early termination of the study. TLR7 stimulation results in a pharmacologic response at levels commensurate with predicted antiviral efficacy, but these doses are associated with SAEs, raising concerns about the therapeutic window of this class of compounds for the treatment of HCV infection.

Specific immune milk production of cows implanted with antigen-release devices.

A new antigen-release device (ARD) that can be implanted to enhance the titer of specific IgG and concentration of total IgG in milk of lactating cows was evaluated. An immunostimulating complex-based vaccine in the core of the ARD was made from the adjuvant Quil A and type XIII lipase from Pseudomonas spp. with a polylactide acid capsule that was used to control antigen release. Forty lactating Holstein dairy cows were divided into 2 groups (n = 20). All cows in the test group were implanted in the right iliac lymph node with 3 types of ARD at the same time, which were designed to release antigens on different days. The other group was used as a control with no implantation. The 3 ARD were designed to release the antigen on d 0, 14, and 28 after implantation. Specific IgG titers in whey and serum were measured by indirect ELISA, and total IgG concentrations were measured using sandwich ELISA. The results indicated that ARD implantation brought no negative effects on the health status, production performance of cows, and caused neither subclinical nor acute mastitis. The levels of specific IgG in serum (200,000 +/- 45,000 vs. 1,200 +/- 360) and whey (41,000 +/- 6,000 vs. 820 +/- 210) increased in the cows implanted with ARD. Specific IgG in whey was increased after 9 d. The dynamics of specific IgG titer demonstrated a pattern with the release of the antigen from 3 types of ARD. The average ELISA titer of test group in whey was 41,000 +/- 6,000, which suggested high efficiency of immune milk production caused by the ARD implantation. For total IgG in milk, greater concentration in the test compared with the control cows occurred from 11 to 20 d following implantation. The IgG mass was consistent with the dynamics of specific IgG titer and was higher from 15 to 30 d between test and control group (7.89 +/- 1.34 vs. 6.48 +/- 1.17 g). In conclusion, ARD implantation was effective in improving specific antibody concentration in serum and whey. Furthermore, the whey:serum ratio of specific IgG titer, the milk:serum ratio of total IgG concentration and total IgG mass in milk suggested that a transiently upregulated IgG transfer occurred after ARD implantation.

Factors affecting the transfer of immunoglobulin G1 into the milk of Holstein cows.

Immunoglobulin (Ig) G1 concentrations in milk from Holstein cows was measured to determine if transfer and concentration was influenced by production factors (lactation number, stage of lactation, daily milk production), milk composition (milk fat, protein, lactose, and total solids content) or by serum IgG1 concentration. Two hundred and ninety-nine Chinese Holstein cows were randomly selected from four herds containing a total of more than 1600 lactating animals. The concentration of IgG1 in the milk and serum was determined by ELISA. Milk IgG1 concentrations varied between 0.030 and 0.614 mg/mL and significantly correlated with lactation number, stage of lactation, daily milk production and somatic cell count. The IgG1 mass was found to highly correlate with lactation number, stage of lactation, daily milk production and milk protein content. Lactation number had the highest positive direct relationship with IgG1 concentration.

The effect of implanting an antigen release device on lactoferrin concentration in serum and milk.

The objective of this study was to evaluate the effect of implanting an Antigen Release Devices (ARD) into dairy cows during the lactation cycle to induce an immune response. Subsequently, the concentrations of lactoferrin in serum and milk were measured. Forty healthy adult Chinese Holstein cows were divided into two equal groups: a test group and a control group. Animals in the test group received ARD implants, whereas the control group animals were not treated. An even spread across the two groups was maintained with animal selection based on parity, the lactation days and milk yields. The concentrations of lactoferrin in the serum and milk of all forty animals were measured using an Enzyme-Linked Immunosorbent Assay (ELISA). The results show that the implantation of an ARD did not significantly increase the concentration of lactoferrin in the serum and milk throughout the whole experiment period except on two occasions. The levels of lactoferrin in the milk and serum significantly increased on day 7 and on day 11 after implantation (p<0.05). There was a strong correlation between milk lactoferrin and serum lactoferrin (r=0.564, P<0.01). Three separate ARDs were used releasing its antigen load on day 0, 14 and 28 to induce a primary, secondary and tertiary response respectively. As the significant increases in the lactoferrin levels were only observed after the first ARD release, the effects of lactoferrin appears to be associated with the early phase of the immune response, consistent with its role in the host's innate defense system.

Factors affecting the lactoferrin concentration in bovine milk.

Lactoferrin (LF) concentrations in the milk with different levels of the somatic cell count score were examined using an ELISA to determine whether milk LF concentration is influenced by parity of the cow, stage of lactation, and the somatic cell count. The study animals were 198 Chinese Holstein cows randomly chosen from more than 1,600 cows in 4 dairy farms in the Beijing area. The cows had shown no sign of mastitis for 2 mo. Daily milk production was recorded, and milk samples were taken from individual cow samples. The LF concentration varied between 31.78 and 485.63 microg/mL in milk from normal animals. Lactoferrin was significantly associated with stage of lactation (r = 0.557) and daily milk production (r = -0.472). Nevertheless, there was no significant relationship with parity. Moreover, milk LF concentration tended to be correlated with the somatic cell count score (r = 0.375). This finding suggests that milk LF may be helpful as an indicator for intramammary infection in dairy cows.

Characterization of chemokine CCR3 agonist-mediated eosinophil recruitment in the Brown-Norway rat.

1. The ability of various C-C chemokines to elicit tissue eosinophil infiltration following intradermal injection or peripheral blood eosinophilia following intravenous injection were compared in the Brown-Norway rat. 2. Eotaxin (0.1 - 3 microg site-1) of human and murine origin produced equivalent, dose-dependent increases in eosinophil peroxidase activity in rat dermis 4 h post-injection. 3. Human eotaxin-2 was equipotent with human eotaxin in terms of dermal eosinophil recruitment. Other human CCR3 agonists, such as MCP-3, RANTES and MCP-4 failed to increase dermal eosinophil peroxidase activity at doses up to 1 microg site-1 whereas the latter did produce a small effect at 3 microg site-1. 4. Consistent with observations in vivo, human eotaxin displaced [125I]-eotaxin from rat spleen membranes more potently (IC50=2 nM) than did MCP-4 (IC50=500 nM). RANTES did not compete with the radiolabelled chemokine at concentrations up to 1 microM. 5. Human eotaxin (5 microg) administered intravenously increased circulating eosinophils approximately 3 fold whereas MCP-4 (5 microg i.v.) increased circulating monocytes approximately 3 fold without affecting eosinophil numbers. 6. Dexamethasone pretreatment inhibited eotaxin-induced dermal eosinophil influx only at a steroid dose (0.1 mg kg-1, s.c.) which significantly reduced circulating eosinophil numbers. The steroid also reduced eosinophilia in peripheral blood resulting from systemic eotaxin administration (5 microg, i.v.). 7. These data suggest differences in rat CCR3 relative to other species as surmised from a distinctive rank order of chemokine potency. In addition to its chemotactic effects eotaxin, but not MCP-4, promotes eosinophil recruitment into the circulation. One of the mechanisms by which glucocorticoids, such as dexamethasone, acutely inhibits eotaxin-induced dermal eosinophil influx is to diminish the circulating numbers of these cells available for tissue recruitment.

Discovery of CP-199,330 and CP-199,331: two potent and orally efficacious cysteinyl LT1 receptor antagonists devoid of liver toxicity.

CP-199,330 (3) and CP-199,331 (4) are cysLT1 receptor antagonists that are equipotent to marketed cysLT1 receptor antagonists zafirlukast and pranlukast, show good pharmacokinetics in rats and monkeys, and are devoid of liver toxicity in monkeys as seen in CP-85,958 (1).

Isolation and characterization of PDE9A, a novel human cGMP-specific phosphodiesterase.

We have cloned and characterized the first human isozyme in a new family of cyclic nucleotide phosphodiesterases, PDE9A. By sequence homology in the catalytic domain, PDE9A is almost equidistant from all eight known mammalian PDE families but is most similar to PDE8A (34% amino acid identity) and least like PDE5A (28% amino acid identity). We report the cloning of human cDNA encoding a full-length protein of 593 amino acids, including a 261-amino acid region located near the C terminus that is homologous to the approximately 270-amino acid catalytic domain of other PDEs. PDE9A is expressed in all eight tissues examined as a approximately 2. 0-kilobase mRNA, with highest levels in spleen, small intestine, and brain. The full-length PDE9A was expressed in baculovirus fused to an N-terminal 9-amino acid FLAG tag. Kinetic analysis of the baculovirus-expressed enzyme shows it to be a very high affinity cGMP-specific PDE with a Km of 170 nM for cGMP and 230 microM for cAMP. The Km for cGMP makes PDE9A one of the highest affinity PDEs known. The Vmax for cGMP (4.9 nmol/min/microg recombinant enzyme) is about twice as fast as that of PDE4 for cAMP. The enzyme is about twice as active in vitro in 1-10 mM Mn2+ than in the same concentration of Mg2+ or Ca2+. PDE9A is insensitive (up to 100 microM) to a variety of PDE inhibitors including rolipram, vinpocetine, SKF-94120, dipyridamole, and 3-isobutyl-1-methyl-xanthine but is inhibited (IC50 = 35 microM) by zaprinast, a PDE5 inhibitor. PDE9A lacks a region homologous to the allosteric cGMP-binding regulatory regions found in the cGMP-binding PDEs: PDE2, PDE5, and PDE6.

Isolation and characterization of PDE8A, a novel human cAMP-specific phosphodiesterase.

As part of our efforts to understand the regulation of intracellular cAMP and to generate new targets for pharmacological intervention, we have cloned and characterized the first isozyme in a new family of cyclic nucleotide phosphodiesterases, PDE8A. PDE8A is most similar to PDE4 (38.5% amino acid identity in the catalytic domain), but is clearly not a member of any of the seven known PDE families. We report the cloning of human cDNA encoding the C-terminal 713 amino acids of the protein, including a 283 amino acid region located near the C-terminus that is homologous to the approximately 270 amino acid catalytic domain of other PDEs. In addition, we found cDNA sequences consistent with alternative 5' mRNA splicing analogous to that seen in other PDE genes. PDE8A is expressed in a wide variety of tissues as a approximately 4.5 kb mRNA, with highest levels in testis, ovary, small intestine, and colon. The C-terminal 545 amino acids of PDE8A (the region shared among all splice variants) were expressed in baculovirus. Kinetic analysis of the baculovirus expressed enzyme shows it to be a very high affinity cAMP specific PDE with a Km of 55 nM for cAMP and 124 microM for cGMP. The Vmax (150 pmol/min/microgram recombinant enzyme) is about 10 times slower than that of PDE4. The cAMP hydrolytic activity of PDE8A is not modulated by cGMP at concentrations up to 100 microM. The enzyme requires the presence of at least 1 mM Mn2+ or Mg2+ for maximal activity in vitro, while 100 mM Ca2+ restores only about 20% activity. PDE8A is insensitive (up to 100 microM) to a variety of PDE inhibitors including rolipram, zaprinast, vinpocetine, SKF-94120, and IBMX, but is inhibited (IC50 = 9 microM) by the PDE inhibitor dipyridimole. To give PDE8A a descriptive name that distinguishes it from the other two known high affinity cAMP-specific phosphodiesterases (PDE4 and PDE7), we denote PDE8A as the high affinity cAMP-specific and IBMX-insensitive PDE.

7-Oxo-4,5,6,7-tetrahydro-1H-pyrazolo[3,4-c]pyridines as novel inhibitors of human eosinophil phosphodiesterase.

High-throughput file screening against inhibition of human lung PDE4 led to the discovery of 3-ethyl-1-(4-fluorophenyl)-6-phenyl-7-oxo-4, 5,6,7-tetrahydro-1H-pyrazolo[3,4-c]pyridine (11) as a novel PDE4 inhibitor. Subsequent SAR development, using an eosinophil PDE assay, led to analogues up to 50-fold more potent than 11 with IC50 values of 0.03-1.6 microM. One such compound, CP-220,629 (22) (IC50 = 0.44 microM), was efficacious in the guinea pig aerosolized antigen induced airway obstruction assay (ED50 2.0 mg/kg, po) and demonstrated a significant reduction in eosinophil (55%), neutrophil (65%), and IL-1beta (82%) responses to antigen challenge in atopic monkeys (10 mg/kg, po).

Development of new chromanol antagonists of leukotriene D4.

By addressing the issues of potency and metabolism in 3, a new series of LTD4 antagonists represented by (+)-26 was developed which is equipotent to clinical LTD4 antagonists Zafirlukast (1) and Pranlukast (2).

N-carbamoyl analogs of Zafirlukast: potent receptor antagonists of leukotriene D4.

Exploration of the indole nitrogen region of Zafirlukast (1) has uncovered a potent series of cysteinyl leukotriene D4 (LTD4) antagonists. These studies showed that a variety of functionality could be incorporated in this region of the molecule without sacrificing potency. Efforts to exploit this site in order to improve oral efficacy are discussed.

Development of 2,2-dimethylchromanol cysteinyl LT1 receptor antagonists.

A new series of cysLT1 receptor antagonists represented by CP-288,886 (7) and CP-265,298 (8) were developed which are equipotent to clinical cysLT1 receptor antagonists Zafirlukast (1) and Pranlukast (2).

Differential in vivo and in vitro bronchorelaxant activities of CP-80,633, a selective phosphodiesterase 4 inhibitor.

CP-80,633, a selective phosphodiesterase (PDE) 4 inhibitor, potently reverses histamine bronchoconstriction in anesthetized guinea pigs (ED50 10 micrograms/kg) but only weakly relaxes histamine-constricted guinea pig trachea (EC50 137 microM). Using CP-80,633 as a prototype PDE4 inhibitor, we evaluated the hypothesis that bronchodilation induced by PDE4 inhibitors is not mediated by direct relaxation of airway smooth muscle. In anesthetized guinea pigs, a bronchodilatory dose of CP-80,633 did not increase plasma catecholamines, nor did propranolol pretreatment significantly alter the ability of CP-80,633 to reverse histamine bronchoconstriction. In an isolated organ system, the activity of bronchorelaxants may be attenuated by the lack of endogenous activators of adenylyl cyclase or by decreased levels of intracellular cyclic nucleotides. Pretreatment with the beta-adrenoceptor agonist, salbutamol, or the PDE3 inhibitor imazodan did not potentiate the bronchorelaxant ability of CP-80,633. Milrinone pretreatment increased the potency of CP-80,633 to relax carbachol-constricted tracheal rings, but only at concentrations where nonspecific effects have been reported. By comparing the bronchorelaxant abilities of PDE inhibitors in tracheal rings with or without epithelium, we determined that the epithelium did not serve as a barrier to drug penetration. In conclusion, CP-80,633 is a potent bronchodilator in vivo, whose activity is neither mediated by direct airway smooth muscle relaxation nor dependent upon endogenous catecholamines.

The phosphodiesterase type 4 (PDE4) inhibitor CP-80,633 elevates plasma cyclic AMP levels and decreases tumor necrosis factor-alpha (TNFalpha) production in mice: effect of adrenalectomy.

Rolipram was previously reported to elevate plasma cyclic adenosine 3',5'-monophosphate (cAMP) and inhibit serum tumor necrosis factor-alpha (TNF-alpha) production in mice. CP-80,633, a new cyclic nucleotide phosphodiesterase (PDE4) inhibitor, has been shown to augment intracellular cAMP levels and to inhibit TNFalpha release from human monocytes in vitro. This study was undertaken to determine the effect of p.o. CP-80,633 on plasma cAMP levels and lipopolysaccharide-induced TNFalpha production in mice with and without adrenal glands. CP-80,633 dose-dependently (3-32 mg/kg p.o.) elevated plasma cAMP levels and decreased systemic TNFalpha production in response to i.p. injection of lipopolysaccharide. Elevated plasma cAMP levels can be detected for up to 4 hr. CP-80,633 (10 mg/kg p.o.) caused a 6-fold increase in the plasma cAMP level, a 2-fold increase in the plasma epinephrine level and a greater than 95% reduction in TNFalpha production. Unlike CP-80,633, neither vinpocetine, dipyridamole, SKB-94,120 nor zaprinast, at 100 mg/kg p.o., modified the cAMP response, which suggests that this response is mediated by inhibition of PDE4. Adrenalectomy reduced the cAMP response and completely blocked the epinephrine response; however, the levels of plasma cAMP in the CP-80,633-treated mice (10 mg/kg p.o.) remained elevated (vehicle: 47.3 +/- 6.8 vs. CP-80,633: 98.4 +/- 10.3 pmol/ml, n = 7, P < .05). This effect is mimicked by treatment of control mice with propranolol, which demonstrates that beta adrenoreceptors contribute to the cAMP response. Removal of adrenal glands significantly increased the LPS-induced elevation of serum TNFalpha. The ability of CP-80,633 to block the TNFalpha response was only slightly affected by adrenalectomy (ED50 = 1.2 mg/kg in controls vs. 3.9 mg/kg in adrenalectomized mice). Taken together, these results show that CP-80,633, when given p.o. to mice, is capable of elevating plasma cAMP and inhibiting TNFalpha production and that adrenal catecholamines contribute significantly to the effect of CP-80,633 on the cAMP response but only slightly to its effect on the systemic TNFalpha response.