Characterization of ASKP1240, a fully human antibody targeting human CD40 with potent immunosuppressive effects.

Blocking the CD40-CD154 interaction is reported to be effective for transplantation management and autoimmune disease models in rodents and nonhuman primates. However, clinical trials with anti-CD154 mAbs were halted because of high incidence of thromboembolic complications. Thus, we generated and characterized a fully human anti-CD40 mAb ASKP1240, as an alternative to anti-CD154 mAb. In vitro ASKP1240 concentration-dependently inhibited human peripheral blood mononuclear cell proliferation induced by soluble CD154. In addition, ASKP1240 did not destabilize platelet thrombi under physiological high shear conditions while mouse anti-human CD154 mAb (mu5C8) did. And ASKP1240 itself did not activate platelet and endothelial cells. In vivo administration of ASKP1240 (1 or 10 mg/kg, intravenously) to cynomolgus monkeys, weekly for 3 weeks, significantly attenuated both delayed-type hypersensitivity and specific antibody formation evoked by tetanus toxoid. The immunosuppressive effect was well correlated with the CD40 receptor saturation. Thus, these results suggest that ASKP1240 is immunosuppressive but not prothromboembolic, and as such appears to be a promising therapeutic candidate for the management of solid organ transplant rejection and autoimmune diseases therapy.

Breakthrough in purification of fossil pollen for dating of sediments by a new large-particle on-chip sorter.

Particle sorting is a fundamental method in various fields of medical and biological research. However, existing sorting applications are not capable for high-throughput sorting of large-size (>100 micrometers) particles. Here, we present a novel on-chip sorting method using traveling vortices generated by on-demand microjet flows, which locally exceed laminar flow condition, allowing for high-throughput sorting (5 kilohertz) with a record-wide sorting area of 520 micrometers. Using an activation system based on fluorescence detection, the method successfully sorted 160-micrometer microbeads and purified fossil pollen (maximum dimension around 170 micrometers) from lake sediments. Radiocarbon dates of sorting-derived fossil pollen concentrates proved accurate, demonstrating the method's ability to enhance building chronologies for paleoenvironmental records from sedimentary archives. The method is capable to cover urgent needs for high-throughput large-particle sorting in genomics, metabolomics, and regenerative medicine and opens up new opportunities for the use of pollen and other microfossils in geochronology, paleoecology, and paleoclimatology.

Characterization of a 2,4-dinitrochlorobenzene-induced chronic dermatitis model in rats.

Although many mouse models of atopic dermatitis have been reported, few rat models have been studied. In this study, a rat chronic allergic dermatitis model was developed and evaluated as a pharmacological model of atopic dermatitis. Prominent ear thickening and scratching were induced after the application of 2,4-dinitrochlorobenzene to the right ear of Brown Norway rats 3 times per week for 3 weeks. Histopathologically, infiltration of T cells in the ear was observed on day 7, and eosinophils and mast cells were found in addition to T cells on day 21. The expression of interferon-gamma and interleukin-4 was increased on day 7 when compared with normal rats. However, interferon-gamma expression had disappeared by day 21. Tacrolimus ointment applied after ear tissue thickening fully developed, suppressed chronic dermatitis in a dose-dependent manner. This model has some symptomatic and histopathological similarities to atopic dermatitis and might be useful in pharmacological studies.

Use of sonographic elastography of the masseter muscles for optimizing massage pressure: a preliminary study.

To examine the stiffness of the masseter muscle using sonographic elastography and to investigate its relationship with the most comfortable massage pressure in the healthy volunteers. In 16 healthy volunteers (10 men and 6 women), the Masseter Stiffness Index (MSI) was measured using EUB-7000 real-time tissue elastography. They underwent massages at three kinds of pressures using the Oral Rehabilitation Robot (WAO-1). A subjective evaluation regarding the comfort of each massage was recorded on the visual analogue scale. Elastography was also performed in two patients with temporomandibular joint dysfunction with the myofascial pain. The mean MSI of the right and left muscles in the healthy volunteers were 0.85 +/- 0.44 and 0.74 +/- 0.35 respectively. There was no significant difference between the right and left MSI in the healthy volunteers. The MSI was related to massage pressure at which the healthy men felt most comfortable. The two temporomandibular disorder patients had a large laterality in the MSI. The MSI was related to the most comfortable massage pressure in the healthy men. The MSI can be one index for determining the massage pressure.

Neointima formation in a restenosis model is suppressed in midkine-deficient mice.

Neointima formation is a common feature of atherosclerosis and restenosis after balloon angioplasty. To find a new target to suppress neointima formation, we investigated the possible role of midkine (MK), a heparin-binding growth factor with neurotrophic and chemotactic activities, in neointima formation. MK expression increased during neointima formation caused by intraluminal balloon injury of the rat carotid artery. Neointima formation in a restenosis model was strongly suppressed in MK-deficient mice. Continuous administration of MK protein to MK-deficient mice restored neointima formation. Leukocyte recruitment to the vascular walls after injury was markedly decreased in MK-deficient mice. Soluble MK as well as that bound to the substratum induced migration of macrophages in vitro. These results indicate that MK plays a critical role in neointima formation at least in part owing to its ability to mediate leukocyte recruitment.

Changes in electrical resistance of sound fissure enamel in first molars for 66 months from eruption.

The purposes of this study were to investigate the enamel maturation process in the occlusal pit of sound first molars by measuring electrical resistance. Ninety-nine sound first molars in 34 children (mean age of 6.47 +/- 0.51 years) were measured electrically once every 6 months and were monitored for a maximum of 66 months. Electrical resistance increased during the posteruptive period. However, the results suggest that posteruptive enamel maturation in the occlusal pits may not be completed even 66 months after tooth eruption.

Estrogen and progesterone receptors in human breast cancer with concomitant assay of plasma 17beta-estradiol, progesterone, and prolactin levels.

The specific estrogen receptor in the cytosol of human breast cancer tissue was assayed in 217 primary cases. The specific progesterone receptor was also assayed in 48 cases as evidence of estrogen action on the tissue. Both receptors were positive in 45.8% of all cases. Plasma 17beta-estradiol and progesterone were assayed concomitantly with these steroid receptors. The higher hormone levels were found in the cases with fewer receptor binding sites. The relationship between 17beta-estradiol levels in tumor cytosol and the number of binding sites was more clearly observed. Plasma prolactin levels, however, showed no correlation with the number of receptor binding sites or the plasma levels of sex steroids. None of these assayed substances had a clear correlation with the histological type of tumor or the clinical stage of the disease.

Modulations of glucocorticoid-induced apoptosis linked to the p53 deletion and to the apoptosis susceptibility gene Rapop1 (Radiation-induced apoptosis 1).

We have analysed the effects of p53 and of the apoptosis susceptibility gene Rapop1 (Radiation-induced apoptosis 1) located on chromosome 16 on glucocorticoid- and radiation-induced in vivo apoptosis of thymocytes. For those analyses, we used Rapop1 semicongenic mice heterozygous for the STS and BALB/cHeA alleles in the chromosomal segment containing Rapop1 in the BALB/cHeA background, mice bearing a p53 deficient allele in the BALB/cHeA background and the genetic crosses between these mice. The p53 wild type mice with a STS/A allele at the Rapop1 locus were less susceptible to both radiation- and glucocorticoid-induced apoptosis than those with homozygous BALB/cHeA alleles at this locus. Surprisingly, glucocorticoid-induced apoptosis was enhanced in the p53 hemizygous mice and considerably increased in the p53 nullizygous mice. In contrast, a sizable reduction of radiation-induced apoptosis was seen in the p53 hemizygous mice. The low susceptiblity to glucocortocoid-induced apoptosis linked to the STS allele of Rapop1 was less pronounced in the p53 hemizygous mice and a diminished effect of Rapop1 on radiation-induced apoptosis was seen in these mice. Although it remains to be established whether the genes modulating glucocortocoid-induced apoptosis are identical to p53 and Rapop1, our data suggest that p53 and Rapop1 may participate in glucocorticoid-induced apoptosis of thymocytes.

15-Hydroperoxyeicosapentaenoic acid inhibits arachidonic acid metabolism in rabbit platelets more potently than eicosapentaenoic acid.

The effect of 15-hydroperoxy-5,8,11,13,15-eicosapentaenoic acid (15-HPEPE), a hydroperoxy adduct of eicosapentaenoic acid (EPA), on the formation of 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE), thromboxane (TX) B2 and 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT) from exogenous arachidonic acid in washed rabbit platelets was examined. 15-HPEPE inhibited 12-HETE, TXB2 and HHT formation at concentrations ranging from 2 to 8 microM. The inhibitory effect of 15-HPEPE was dose-dependent (12-HETE, 16.0-82.9% inhibition; TXB2, 16.7-57.2% inhibition; HHT, 4.6-52.0% inhibition). EPA inhibited the production of these three metabolites, but the inhibitory effect was kept low (20-100 microM: 12-HETE, 8.3-31.1% inhibition; TXB2, 18.9-49.5% inhibition; HHT, 12.5-41.7% inhibition) as compared with 15-HPEPE. Experiments utilizing 15-hydroxy-5,8,11,13,15-eicosapentaenoic acid and hydroxyl radical scavengers (dimethyl sulfoxide and mannitol) revealed that 15-HPEPE exerted its effect in the form of the hydroperoxy adduct. These results suggest that 15-HPEPE has the potential to modulate the activities of the cyclo-oxygenase and 12-lipoxygenase in platelets. This may also be one convincing mechanism for the anti-thrombotic and anti-atherosclerotic actions of EPA.

Effective use of rabbit gastric antral mucosal slices in prostaglandin synthesis and metabolism studies.

Intact slice preparations of rabbit stomach (antral mucosa, corporal mucosa, antral muscle and corporal muscle) were incubated and the released prostaglandins (PGs) were measured by reverse-phase high-performance liquid chromatography using 9-anthryldiazomethane for derivatization. With respect to total PG production, the highest amounts were generated by antral mucosal slices. Antral mucosal slices produced PGE2, 6-keto PGF1 alpha, thromboxane B2, PGF2 alpha and PGD2 (in descending order of magnitude) and possessed a high capacity for producing 13,14-dihydro-15-keto derivatives of both PGE2 and PGF2 alpha. Studies utilizing aspirin, EGTA or Ca2+ revealed that PG release by antral mucosal slices in the present in vitro system reflects a composite of the activities of phospholipase A3, PG cyclooxygenase and PG-metabolizing enzymes. These results show that antral mucosal slices will be useful in physiological and pharmacological studies on PG synthesis and metabolism of the stomach.

Thymic stroma-derived T cell growth factor (TSTGF): III. Its ability to promote T cell proliferation without stimulating interleukin 2- or 4-dependent autocrine mechanism.

A thymic stroma-derived cell clone, MRL104.8a produced a T cell growth factor designated as thymic stroma-derived T cell growth factor (TSTGF). This factor that is distinct from previously described T cell growth factors such as interleukin (IL) 2 or 4 was capable of promoting the growth of IL2-dependent, antigen-specific helper T cell clones. While such growth promotion was induced without requirement of the relevant antigen and exogenous IL2, we further investigated whether it depended on activation of an IL2- or IL4-dependent autocrine mechanism. Helper T cell clones, 8-E and 8-5, were able to proliferate in response to stimulation with either antigen or TSTGF. 8-E and 8-5 produced IL2 and IL4, respectively, in cultures following antigenic stimulation, whereas neither IL2 nor IL4 activity was detected in cultures during TSTGF-induced proliferation. The proliferation of these helper T cell clones by antigenic stimulation was almost completely inhibited when anti-IL2 receptor or anti-IL4 antibody was added to the cultures. The addition of cyclosporin A (CsA) to cultures of 8-E and 8-5 clones together with antigen also resulted in the complete inhibition of cellular proliferation in association with the suppression of IL2 and IL4 production. In contrast, TSTGF-induced proliferation was not affected by addition of either type of antibody or CsA. These results indicate that TSTGF is a novel T cell growth factor that can exert its own growth-promoting effect without depending on an IL2- or IL4-operating autocrine mechanism.

Balanitis caused by Streptococcus pyogenes: a report of two cases.

Streptococcus pyogenes (the Lancefield group A streptococcus) is a cause of pharyngitis and impetigo. However, it has rarely been implicated as a sexually transmitted pathogen. We herein report two cases of severe balanitis due to S. pyogenes in sexually active men. It is postulated that penile cellulitis developed following the invasion of S. pyogenes through a traumatic abrasion acquired during fellatio performed by commercial sex workers. Both patients were treated successfully with oral administration of penicillin.

Design of nanoparticles composed of graft copolymers for oral peptide delivery.

The development of a dosage form that improves the absorption of peptide and protein drugs via the gastrointestinal tract is one of the greatest challenges in the pharmaceutical field. Many researchers have taken up the challenge, using approaches including mucoadhesive drug delivery, colon delivery, particulate drug delivery such as nanoparticles, microcapsules, liposomes, emulsions, micelles, and so on. The objective of this article is to provide the reader with outlines of novel nanoparticle technologies for oral peptide delivery based on polymer chemistry. The physicochemical properties of nanoparticles and their behavior on exposure to physiological media are greatly dominated by their chemical structures and surface characteristics. We will especially focus on the design of nanoparticles composed of novel graft copolymers having a hydrophobic backbone and hydrophilic branches as drug carriers.

The stimulatory and inhibitory guanine nucleotide-binding proteins of adenylate cyclase in erythrocytes from patients with pseudohypoparathyroidism type I.

Both the inhibitory and stimulatory guanine nucleotide-binding proteins of the adenylate cyclase complex were measured in erythrocyte membranes from patients with pseudohypoparathyroidism (PHP). The inhibitory guanine nucleotide-binding protein (Ni) of adenylate cyclase was measured by incorporation of [32P]ADP-ribose from [32P]NAD into the 39K subunit of Ni catalyzed by pertussis toxin. The ADP-ribosyltransferase activity of the toxin was expressed through incubation with dithiothreitol and erythrocyte membranes. Erythrocytes from 12 patients with PHP type I (PHP-I) had Ni values similar to those of 9 normal subjects and 2 patients with pseudopseudohypoparathyroidism. In 6 PHP-I patients, decreased activity of the stimulatory guanine nucleotide-binding protein (Ns) of adenylate cyclase, as determined by reconstitution of adenylate cyclase in the Ns-deficient membranes of cyc-S49 cells, corresponded with the reduced degree of ADP-ribosylation of the 42K subunit of Ns catalyzed by cholera toxin. These data suggest that the defect of Ns results in reduced stimulation of adenylate cyclase in some PHP-I patients, and that enhanced inhibition of the enzyme due to an increase in the 39K subunit of Ni does not account for the biochemical lesion in PHP-I patients.

Site-specific stable insertion into the human cytomegalovirus genome of a foreign gene under control of the SV40 promoter.

On the basis of a previous finding that the 7.8-kb HindIII-O fragment of the human cytomegalovirus strain Towne genome is nonessential for viral replication, we constructed a vector, pKM, that directs introduction of foreign genes by homologous recombination precisely replacing the O fragment. Using this vector, we constructed Towne-strain-derived recombinant virus in which a chimeric lacZ gene fused to the simian virus 40 promoter and a poly(A) signal were inserted in place of the O fragment. Two types of recombinants were obtained which carried the chimeric gene in opposite directions, beta-Galactosidase (beta Gal) was produced throughout the infection cycle in human embryonic lung cells infected with these recombinants, and the rate of its synthesis in the early stages of infection was comparable to that of synthesis of a 65-kDa viral glycoprotein, one of the abundantly produced viral proteins. The chimeric lacZ gene introduced was stable and no lacZ- revertants have been observed so far.

Effects of fatty acids and fatty acyl CoA esters on Cu(2+)-induced conversion of xanthine dehydrogenase to oxidase in rabbit liver.

Effects of various fatty acids and fatty acyl CoA esters on Cu(2+)-induced conversion of xanthine dehydrogenase to oxidase in rabbit liver were examined. Cu2+ (2-10 microM) brought about the conversion of xanthine dehydrogenase to oxidase in a dose-dependent manner. Oleic, arachidonic, eicosapentaenoic, and docosahexaenoic acids (50-200 microM) prevented the conversion of xanthine dehydrogenase to oxidase catalyzed by 6 microM-Cu2+. The effect of these four fatty acids was concentration-dependent, whereas palmitic, stearic, and linoleic acids had no effect on the conversion of xanthine dehydrogenase to oxidase at the same concentration range. On the other hand, palmitoyl, linoleoyl, and arachidonoyl CoAs elicited the inhibition of 6 microM-Cu(2+)-induced conversion of xanthine dehydrogenase to oxidase at concentrations of 50, 100, and 200 microM. These results suggest that oleic, arachidonic, eicosapentaenoic and docosahexaenoic acids, and fatty acyl CoAs have the potential to inhibit the conversion of xanthine dehydrogenase to oxidase in rabbit liver.

Effects of reactive oxygen species on arachidonic acid metabolism in rabbit platelets.

Rabbit platelets were exposed to a reactive oxygen species (ROS) generating system (xanthine plus xanthine oxidase) to explore the effect of ROS on the formation of 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE), thromboxane (TX) B2, and 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT) from exogenous arachidonic acid. Xanthine plus xanthine oxidase suppressed the production of 12-HETE, TXB2, and HHT by 65-69%. This effect was reversed by addition of catalase to the ROS generating system but not by superoxide dismutase, mannitol, or dimethylsulfoxide, indicating that H2O2 is the responsible metabolite. These results suggest that H2O2 plays an important role in the regulation of platelet cyclo-oxygenase and lipoxygenase activities.

Receptor protein tyrosine kinase DDR is up-regulated by p53 protein.

We have previously reported on radiation-induction of ptk-3 in rat astrocyte culture [Sakuma et al. (1995) Radiat. Res. 143, 1-7]. Ptk-3 was considered to be a rat version of human DDR (discoidin domain receptor). We cloned and analyzed genomic DNA of the DDR and its promoter region. We discovered that the promoter region contained a consensus sequence of the p53 tumor suppressor binding site. Adenovirus-mediated p53 transfection induced a high level of DDR mRNA in SAOS2 human osteosarcoma cells. These results indicate that DDR is up-regulated by the p53 protein.

Distribution of nociceptin/orphanin FQ precursor protein and receptor in brain and spinal cord: a study using in situ hybridization and X-gal histochemistry in receptor-deficient mice.

Nociceptin/orphanin FQ (N/OFQ) is an opioid-like heptadecapeptide agonist for the opioid receptor homolog, N/OFQ receptor. To explore the precise distribution of the peptide-receptor system, the authors examined the brain and spinal cord from receptor-deficient mice bearing the targeted mutation (morc(m1)), a lacZ insertional mutation in the N/OFQ receptor gene. Precursor protein N/OFQ (preproN/OFQ) mRNA was detected by using in situ hybridization, and the N/OFQ receptor was detected by using X-gal histochemistry. The N/OFQ receptor reflected by lacZ expression was observed at high levels in the dentate gyrus, lateral septum, subparafascicular thalamic nucleus, medial preoptic area, median preoptic nucleus, ventromedial preoptic nucleus, anterior hypothalamic area, paraventricular hypothalamic nucleus, ventromedial hypothalamic nucleus, auditory brainstem nuclei, pontine dorsal tegmentum, and nucleus of the solitary tract. In situ detection of the N/OFQ receptor mRNA by digoxigenin-labeled riboprobes coupled with tyramide signal amplification in normal and wild-type mice resulted in the regional distribution paralleling the lacZ expression in these regions. PreproN/OFQ mRNA was expressed at high levels in the subparafascicular thalamic nucleus, central gray, central tegmental field, auditory brainstem nuclei, caudal spinal trigeminal nucleus, and spinal dorsal horn. Furthermore, variable levels of expression of the peptide and receptor were seen in distinct sites of the brain and spinal cord. These data indicate a correspondence of the peptide and the receptor in local distribution at limbic, hypothalamic, and brainstem sites. Together with concurrent physiologic and behavioral studies in mutant mice, the results suggest functional roles for the N/OFQ system, including the central regulation of learning and memory, hearing ability, water balance, food intake, and blood pressure.

Computer-assisted conformation radiotherapy with a variable thickness multi-leaf filter.

A new computer-assisted conformation radiotherapy with a variable thickness multi-leaf filter system was developed. Each wedgeshaped compensating filter (aluminum, tan theta = 0.1) travels against the movement of the ipsilateral leaf and according to defined parameters. By using our filter, a homogenous dose distribution in the target area was obtained and high-dose levels outside the target area decreased significantly.