Allele specificity of structural requirement for peptides bound to HLA-DRB1*0405 and -DRB1*0406 complexes: implication for the HLA-associated susceptibility to methimazole-induced insulin autoimmune syndrome.

Self-peptides bound to HLA-DR4 (DRA-DRB1*0405 complex) were eluted from the purified DR4 complex, fractionated on reverse-phase HPLC, and subjected to NH2-terminal sequencing. Seven independent sequences were obtained, and all putative peptides synthesized bound to DRB1*0405 as well as DRB1*0406 complex, which differ only at DR beta residues 37, 57, 74, and 86. Binding assay using analogue peptides of a DR4 binder GSTVFDNLPNPE revealed that FxxLxN is an important anchor motif necessary for binding (where x is any amino acid), which was common to DRB1*0405 and 0406. Determination of the binding affinity of 60 synthetic AAFAALANAA-based analogue peptides showed that substituting F to W or C; L to F, W, or Y; and N to Q or S on AAFAALANAA changed the affinity substantially between DRB1*0405 and DRB1*0406. It is noteworthy that all patients with methimazole-induced insulin autoimmune syndrome are positive for DRB1*0406 and negative for DRB1*0405. Interestingly, the quantitative structural motif identified in this study predicted that 8TSICSLYQLE17 of human insulin alpha chain may bind specifically to DRB1*0406 using its 10IxxLxQ15 motif. Indeed, DRB1*0406 complex bound 8TSICSLYQLE17 with a high affinity, and in striking contrast, DRB1*0405 complex did not. Furthermore, a short-term T cell line specific to human insulin established from a DRB1*0406-bearing individual did show reactivity with a peptide fragment containing the 10IxxLxQ15 motif. Although this fragment probably exists at a very low level under normal physiological conditions due to the disulfide bond between flanking cysteine residues (6Cys-11Cys), a reducing compound such as methimazole may cleave the disulfide bond in vivo and allow DR alpha-DRB1*0406 complex on antigen-presenting cells to bind much of the linear fragment of insulin alpha chain, which may lead to the activation of self-insulin-specific T-helper cells.

Length of the flagellar hook and the capacity of the type III export apparatus.

Length determination in biology generally uses molecular rulers. The hook, a part of the flagellum of motile bacteria, has an invariant length. Here, we examined hook length and found that it was determined not by molecular rulers but probably by the amount of subunit protein secreted by the flagellar export apparatus. The export apparatus shares common features with the type III virulence-factor secretion machinery and thus may be used more widely in length determination of structures other than flagella.

Differential production of MCP-1 and cytokine-induced neutrophil chemoattractant in the ischemic brain after transient focal ischemia in rats.

Chemokines have been shown to play an important role in leukocyte infiltration into ischemic lesions. Recently, the increased expression of monocyte chemoattractant protein-1 (MCP-1) and cytokine-induced neutrophil chemoattractant (CINC) was observed in experimental stroke models where infiltrated leukocytes were supposed to induce tissue injury, however, the protein level and time course of these chemokines have not been fully elucidated. Therefore, we analyzed the time-dependent production of MCP-1 and CINC in the rat brain after transient middle cerebral artery occlusion (MCAO) by means of specific enzyme-linked immunosorbent assay systems. The MCP-1 levels in the ipsilateral hemispheres increased from 6 h, peaked at 2 days, and thereafter gradually decreased. The peak MCP-1 concentration was 89.2+/-28.2 ng/g tissue wet weight (mean +/- SEM, n = 5, 49.3-fold greater than the contralateral value at the same time, P < 0.05), which is supposed to be high enough to exert its biological effects. In contrast, the maximum CINC concentration that corresponded to 2.9+/-0.7 ng/g tissue wet weight (mean +/- SEM, n = 5, 55.0-fold greater than the contralateral value at the same time, P < 0.05), was observed at 6 h. In addition, we confirmed the temporal profile of leukocyte subtypes that infiltrated into the ischemic brain, thus, neutrophil infiltration occurred at early stages (1-3 days), followed by massive infiltration of macrophages at later stages (2-7 days). These studies suggest that MCP-1 in cerebral ischemia actually plays a significant role in the migration of macrophages into the lesion and that the differential temporal production of these chemokines contributes to the regulation of infiltrated leukocyte subtypes.

Peroxisome proliferator-activated receptors are expressed in mouse bone marrow-derived mast cells.

We examined the expression of peroxisome proliferator-activated receptors (PPARs) and the role of PPARs in cytokine production in mouse bone marrow-derived mast cells (mBMMCs). mBMMCs expressed PPARbeta strongly and gamma slightly, but not alpha. Activation of mBMMCs with antigen or calcium ionophore resulted in the increased expression of PPARgamma mRNA specifically. 15-Deoxy-Delta(12, 14)-prostaglandin J(2) (15d-PGJ(2)) and troglitazone, all PPARgamma ligands, attenuated the antigen-induced cytokine production by mBMMCs. Carbaprostacyclin, a PPARbeta ligand, also inhibited cytokine production, whereas PPARalpha ligands did not. These results suggest that PPARbeta and gamma might be included in the negative regulation of mast cell activation.

Preventive effects of sequential treatment with alendronate and 1 alpha-hydroxyvitamin D3 on bone mass and strength in ovariectomized rats.

Because accumulating evidence has shown that bisphosphonates are unable to maintain their bone-sparing effects after the withdrawal of the drug, a replacement treatment is needed when bisphosphonate treatment cannot be continued for some reason. The present study investigated the preventive effects of alendronate followed by 1alpha(OH)D3 on the mass and mechanical strength of trabecular and cortical bones in ovariectomized rats. Sprague-Dawley rats were ovariectomized or sham-operated at 48 weeks of age. Ovariectomized rats treated with vehicle alone (OVX group) showed significant decreases in bone mineral density (BMD) and mechanical strength of the lumbar vertebra and the midfemur during a 20-week period after the operation as compared with sham-operated rats. These decreases were prevented by continuous treatment with alendronate (0.5 mg/kg/day, po) for 20 weeks (ALN-C group), whereas the values reverted to those of the OVX group when alendronate was withdrawn at 10 weeks (ALN-W group). The sequential treatment with alendronate and 1alpha(OH)D3 (0.05 microg/kg/day, po) for 10 weeks each (ALN --> 1alpha group) resulted in higher BMD and mechanical strength of the lumbar vertebra and the midfemur in this group than in the OVX and ALN-W groups. The increase in mechanical strength was proportional to that in BMD at both sites, suggesting that the stimulatory effects of these treatments on bone strength were due to those on bone mass. Analyses of histology, computed tomography, and biochemical markers confirmed the preventive effects of the sequential treatment. Therefore, we propose that 1alpha(OH)D3 may be a good choice to replace alendronate when alendronate treatment cannot be continued for some reason.

Novel naphthalene derivatives as inhibitors of human immunoglobulin E antibody production.

A series of naphthalene derivatives with a variety of substituents at the 2-position was prepared in order to evaluate their suppressive effect on immunoglobulin E (IgE) antibody production by human peripheral blood mononuclear cells provoked with anti-CD40 antibody (alpha-CD40), interleukin-4 (IL-4), and interleukin-10 (IL-10). Compounds having a 1,4-phenylene spacer moiety tethered between the 2-naphthyl nucleus and anthranilic acid suppressed IgE antibody production in vitro in preference to that of IgG antibody without affecting cell viability. Deletion of the anthranilic acid moiety diminished the inhibitory activities. Changing the 2-naphthyl to a 1-naphthyl or phenyl nucleus led to no change in the potency, indicating that the aromatic group at this position is indispensable for the inhibitory activities. On the other hand, changing the 1,4-phenylene spacer to a 1,3-phenylene one resulted in reduced potency. Similarly, inhibitory activities were lost when the CO2H moiety at the 2-position was moved to the 3- or 4-position on the terminal benzene. These observations suggest that the conformation around the anthranilic acid moiety affects the inhibitory activities toward IgE biosynthesis. 2-(4-(2-Naphthyloxy)benzamido)benzoic acid (29) seemed to be a more potent inhibitor of IgE production than of IgG production. Insertion of a methylene between the inter-phenylene and the amide moiety resulted in 2-((4-(2-naphthyloxy)phenyl)acetamido)benzoic acid (31), which provided a stronger inhibition of both IgE and IgG production, although the selectivity toward IgE was lower than that of 29. Introduction of a benzyl group at the 6-position on the naphthalene ring considerably increased the inhibitory activity toward IgE production with an IC50 of 8.3 nM (36). The potency of 31 and 36 was retained when hydrocortisone or lipopolysaccharide was used instead of alpha-CD40 and IL-10 as costimulatory factors with IL-4, implying that these compounds may interfere with signal transduction between IL-4/IL-4 receptor cognition and genetic transcription that induce class-switching of immunoglobulin in B cells. These novel naphthalene derivatives are thus excellent candidates for further investigation with a view toward a therapeutic remedy against IgE-mediated allergic diseases.

Fc portion of intravenous immunoglobulin suppresses the induction of experimental allergic neuritis.

To clarify how intravenous immunoglobulin (IVIg) acts on Guillain-Barré syndrome, we investigated the effects of intact-type IVIg treatment on experimental allergic neuritis (EAN) induced by immunizing with synthetic peptide from bovine P2 protein. Treatment with intact-type IVIg (400 mg/kg/day) on days 0, 7, 14, 15 and 16 after immunization prevented the paralysis, whereas treatment with F(ab')2 failed to alter the clinical course. Intact-type IVIg treatment given on days 0 and 1 showed almost the same efficacy. These results suggest that intact-type IVIg is superior to F(ab')2 in ameliorating the clinical course of EAN and that the Fc portion might affect the immune system.

Regulation of RANTES and IL-8 production in normal human dermal fibroblasts by active vitamin D3 (tacalcitol).

1. The production of chemokines, RANTES and IL-8 in cultured human dermal fibroblasts and the effects of tacalcitol (1alpha,24(R)-dihydroxyvitamin D3) were studied using an enzyme-linked immunosorbent assay. 2. In the unstimulated condition, RANTES and IL-8 were at a trace level in the culture supernatant. On stimulation with TNF-alpha alone for 24 h, RANTES and IL-8 production were induced. Tacalcitol suppressed RANTES and IL-8 production dose-dependently at concentrations between 10(-12) M and 10(-7) M. 3. When the cells were treated with TNF-alpha and IFN-gamma in combination, RANTES production was enhanced, but IL-8 production was not changed, compared to TNF-alpha-treated cells. Tacalcitol decreased IL-8 production dose-dependently as observed in the TNF-alpha-treated cells. On the other hand, RANTES production was enhanced by 10(-11) M and 10(-10) M of tacalcitol, and dose-dependently suppressed by tacalcitol concentrations higher than 10(-9) M. 4. Active vitamin D3 compounds, betamethasone valerate and cyclosporin A were compared with respect to their effects on chemokine production. Three active vitamin D3 compounds, tacalcitol, 1alpha,25-dihydroxyvitamin D3 and MC903 (calcipotriol), inhibited the production of RANTES and IL-8, with very similar potencies. Betamethasone valerate also inhibited these chemokine productions, but with greater potency than active vitamin D3 compounds. Cyclosporin A significantly stimulated RANTES production at 10(-6) M and IL-8 production at 10(-7) M and 10(-6) M. 5. The results of this study suggest that active vitamin D3 compounds exert some beneficial effects in the treatment of inflammatory skin diseases via regulation of the production of chemokines by dermal fibroblasts.

Hypouricemic effect of allopurinol and the novel xanthine oxidase inhibitor TEI-6720 in chimpanzees.

The hypouricemic effect of a newly synthesized xanthine oxidase/xanthine dehydrogenase inhibitor, TEI-6720, 2-(3-cyano-4-isobutoxyphenyl)-4-methyl-5-thiazolecarboxylic acid, was investigated and compared with that of allopurinol in male chimpanzees (n = 3). When allopurinol (10 mg/kg) was administered orally once a day for three consecutive days, it cumulatively reduced serum urate levels by 29.7, 50.1 and 60.2%, 24, 48 and 72 h, respectively, after the initial dose. This effect was dose dependent at doses of 3 and 10 mg/kg. At 3 mg/kg, the mean serum urate levels were 3.1, 2.4, 2.5 and 2.3 mg/dl before and 24, 48 and 72 h, respectively, after the initial dose. Animals treated with 10 mg/kg of allopurinol showed serum urate levels of 3.3, 2.3, 1.6 and 1.3 mg/dl, respectively. The urate-lowering effect of TEI-6720 was then compared with that of allopurinol at a daily dose of 5 mg/kg (n = 3). Both compounds caused striking reductions in serum and urinary uric acid levels accompanied by an increase in urinary xanthine levels. These effects of TEI-6720 were more potent than those of allopurinol. TEI-6720 reduced serum urate levels by 55.9, 69.6 and 73.6%, 24, 48 and 72 h, respectively, after the first dose, whereas the corresponding values after allopurinol were 28.1, 41.6 and 45.1%. These results suggest that the hypouricemic effect of TEI-6720 may be more potent than that of allopurinol in the treatment of hyperuricemia and gout, and that TEI-6720 may become an effective alternative drug.

Hypouricemic effect of the novel xanthine oxidase inhibitor, TEI-6720, in rodents.

We investigated the xanthine oxidase/xanthine dehydrogenase inhibitory activity and hypouricemic effect of a newly synthesized xanthine oxidase/xanthine dehydrogenase inhibitor, TEI-6720, 2-(3-cyano-4-isobutoxyphenyl)-4-methyl-5-thiazole-carboxylic acid, and compared its effects with those of allopurinol in rodents. TEI-6720 was found to inhibit bovine milk xanthine oxidase, and mouse liver and rat liver xanthine oxidase/xanthine dehydrogenase with IC50 values of 1.4, 1.8 and 2.2 nM, respectively. On bovine milk xanthine oxidase, TEI-6720 exhibited mixed-type inhibition and the Ki value was 0.7 nM. TEI-6720 displayed prolonged urate-lowering activity in normal mice and rats. We evaluated the hypouricemic effect of TEI-6720 on hyperuricemia induced by the uricase inhibitor, potassium oxonate (250 mg/kg s.c., 1 h before the test drugs), and measured the total molarity of both serum allantoin and urate in rats. Oral TEI-6720 and allopurinol had a hypouricemic effect 2 h after their administration to oxonate-pretreated rats with ED50 values of 1.5 and 5.0 mg/kg, respectively. Both compounds also reduced the combined molarity of uric acid and allantoin in rats. The ED50 values of TEI-6720 and allopurinol were 2.1 and 6.9 mg/kg p.o., respectively. These results suggest that TEI-6720 may be useful for the treatment of hyperuricemia.

Beneficial effects of a novel anti-hypoxemic agent, TEI-7322, on bleomycin-induced experimental hypoxemia in rats.

Almitrine bismesylate is known to be an anti-hypoxemic agent that acts via the enhancement of hypoxic pulmonary vasoconstriction. However, screening for this class of compounds has been minimal, owing, in part, to a lack of convenient hypoxemic models in small animals. The present study was designed to establish a convenient model of hypoxemia induced by bleomycin and to evaluate anti-hypoxemic agents including a newly synthesized compound. TEI-7322, 2-allylamino-4-tert-butyl-amino-7-methyl-7H-pyrrolo[2,3- d]pyrimidine hydrochloride by using this model. Bleomycin was intratracheally instilled into rats. After 3 weeks, the arterial blood gas pressures were monitored in the animals in the conscious state. Then, prednisolone, doxapram, almitrine or TEI-7322 was administered to the bleomycin-treated rats to monitor changes in arterial blood gas pressures. Bleomycin-treated rats showed a decrease in the arterial blood O2 pressure (PaO2). The blood CO2 pressure (PaCO2) increased, along with an increase in the alveolar-arterial oxygen difference (AaDO2). These blood gas pressures in bleomycin-treated rats were not affected by treatment with prednisolone. Doxapram decreased the PaCO2 but did not change the PaO2. However, administration of almitrine or TEI-7322 significantly improved the PaO2 of bleomycin-treated rats with a decrease in the PaCO2. In conclusion, (1) bleomycin-induced lung injury causes hypoxemia in rats, probably resulting from ventilation-perfusion inequality; thus this model may be useful for evaluating anti-hypoxemic agents; and (2) TEI-7322, as well as almitrine, showed anti-hypoxemic effects in this model with different properties from those of doxapram, possibly due to improvement of ventilation-perfusion inequality, indicating that TEI-7322 may be a potent candidate for the treatment of hypoxemia.

Inhibitory effect of activated protein C on platelet aggregation induced by the prothrombin-converting reaction.

The present study was undertaken to elucidate the effect on platelet aggregation of the prothrombin-converting reaction on platelets with or without activated protein C (APC). A reaction mixture of washed platelets from human individuals, Factor Xa and prothrombin markedly induced platelet aggregation; maximum aggregation rates, 31.3-92.5%, and times to reach to maximum aggregation, 11.6 to 20.1 min. This aggregation was inhibited by the addition of APC with 50% inhibition concentration (IC50) value of 14.4 U/ml. APC also inhibited thrombin generation in the reaction mixture in a dose-dependent manner with IC50 value of 0.96 U/ml. However, APC did not inhibit the thrombin (0.1 CU/ml)-induced platelet aggregation at concentrations of up to 30 U/ml. These findings suggest that APC has no direct inhibitory effect on platelet aggregation and that APC inhibits platelet aggregation through inhibition of thrombin generation.

Allopurinol induces renal toxicity by impairing pyrimidine metabolism in mice.

We investigated the relationship between the toxic effect of allopurinol and pyrimidine metabolism in mice. Allopurinol-induced increases in plasma transaminase levels in dinitrofluorobenzene (DNFB)-sensitized mice were not affected by uridine. In contrast, plasma creatinine and BUN tended to decrease 18 hr after the last injection of uridine. Both plasma and urinary orotidine (OD) were detected in DNFB-sensitized mice after administration of a single dose of allopurinol. In contrast, TEI-6720, a newly synthesized xanthine oxidase/xanthine dehydrogenase inhibitor, caused neither pyrimidine metabolism abnormality nor renal impairment in DNFB-sensitized mice. Also, normal mice administered high doses of allopurinol showed abnormal pyrimidine metabolism together with renal toxicity which could be ameliorated by uridine, indicating that allopurinol essentially causes pyrimidine metabolism abnormality leading to renal impairment. In DNFB-sensitized mice, allopurinol increased urinary OD excretion to an extent similar to that in normal mice administered the same dose of allopurinol. However, renal impairment by allopurinol was more striking in DNFB-sensitized mice than in normal mice. Histopathological observations showed that allopurinol induced calculus formation in the collecting tubules and papillary duct. Calculus formation was increased by DNFB and decreased by uridine. These observations indicate that the enhancement of the renal toxicity of allopurinol by DNFB-sensitization may be due to some biological interactions between DNFB and allopurinol. In humans, it is possible that there are some biological interactions which serve to enhance the toxicity of allopurinol, resulting in the development of allopurinol hypersensitivity syndrome (AHS). In contrast, TEI-6720, had no effect on pyrimidine metabolism and showed no toxic effect.

Effects of ultrasound and 1,25-dihydroxyvitamin D3 on growth factor secretion in co-cultures of osteoblasts and endothelial cells.

It has been shown that low-intensity pulsed ultrasound (US) accelerates fracture healing in animal models and in clinical studies. However, the mechanism by which US accelerates fracture healing remains unclear. Systemic factors and several growth factors, such as platelet-derived growth factor (PDGF), are thought to be involved in the process of fracture healing. In the present study, we examined the effects of US and 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] on growth factor secretion in a co-culture system of human osteoblastic cells (SaOS-2) and endothelial cells (HUVEC). US was applied to cultured cells for 20 min daily for four consecutive days. US treatment increased the PDGF-AB level in the conditioned media. 1,25-(OH)2D3 (1 x 10(-8) M) also enhanced PDGF-AB secretion. The secretion of PDGF-AB was synergistically increased by the combination of US and 1,25-(OH)2D3. These results suggest that the stimulation of growth factor secretion from cells by US and 1,25-(OH)2D3 treatment may be involved in the acceleration of fracture healing.

Effects of progesterone on the elevation of tail skin temperature in ovariectomized rats.

We examined the effects of progesterone on the elevation of tail skin temperature (TST) in ovariectomized rats and compared them with those of estradiol. Progesterone showed only insignificant effects on the TST elevation, whereas estradiol showed complete inhibition. The TST elevation induced by ovariectomy is caused by estradiol deficiency, but progesterone plays little or no role.

Effect of addition of Orvus ES paste to frozen canine semen extender on sperm acrosomes.

Using the triple-stain technique, we investigated whether sperm acrosomes in frozen canine semen were protected during freezing and thawing by addition of a surfactant, Orvus ES Paste (OEP), to the extender. Acrosomes were clearly shown to be protected by the addition of OEP to the entender when compared with those in sperm frozen without OEP addition (p<0.05).

PK/PD and safety of a single dose of TMX-67 (febuxostat) in subjects with mild and moderate renal impairment.

A single oral dose of 20 mg febuxostat was administered to subjects with normal, mild or moderate impairment in renal function. There was less than a 2-fold difference in AUC of plasma unchanged febuxostat among the renal function groups, and changes in plasma urate levels from pre-dose levels were not significant. A total of five adverse events were reported with all mild in severity. The results indicate that renal impairment will have little clinical impact on the pharmacokinetics (PK), pharmacodynamics (PD) and safety of the study drug.

Pharmacokinetics and pharmacodynamics of febuxostat (TMX-67), a non-purine selective inhibitor of xanthine oxidase/xanthine dehydrogenase (NPSIXO) in patients with gout and/or hyperuricemia.

The diurnal change of sUA and the effect of febuxostat on this change were investigated in 10 patients with gout and/or hyperuricemia. The diurnal sUA change after the last dose during the 4-week treatment phase (20 mg, QD) was almost the same as the pre-treatment value. Considering the dose, the AUC(obs) and Cmax of unchanged drug in patients with gout and/or hyperuricemia were estimated to be similar to those of healthy male adults. The results show that a 6-week treatment with febuxostat is safe and well-tolerated in the target patient population for this drug.