Identification of human saliva by antisera to alpha-amylase in human salivary glands

Amylase activities were detected significantly in saliva from human, macaques and rodents and slightly in the vegetable and fruit extracts. Dried stains on filter paper prepared from human and mammalian saliva, and the vegetable and fruit extracts were subjected to starch-iodine test and blue starch polymer-agar plate test. Both tests showed strong positive reactions with the macaque and rodent saliva stains as well as human, but the vegetable and fruit stains showed clear positive reactions only in the starch-iodine test. The results suggest that these tests are not specific for human saliva and that for screening test of saliva stains, blue starch polymer-agar plate test is more suitable than starch-iodine test. Rabbit antisera against alpha-amylase isolated from the human submaxillary glands were prepared. In double immunodiffusion test with the human and mammalian saliva, human and macaque saliva produced precipitation lines. Human saliva gave patterns of partial identity with saliva from Japanese monkey and crab-eating monkey and these two macaque saliva gave total identity with each other. Anti-alpha-amylase sera absorbed with Japanese monkey saliva reacted only with human saliva. This suggested that the anti-alpha-amylase sera absorbed with macaque saliva make it possible to identify human saliva stain.     

Antiarrhythmic effects of pilsicainide hydrochloride and effects on cardiac function and ECG in dogs: comparison with disopyramide

Antiarrhythmic effects and cardiovascular effects of pilsicainide hydrochloride were compared with those of disopyramide in a canine model of coronary ligation-induced ventricular arrhythmias and anesthetized dogs. Pilsicainide (1.25, 2.5 and 5 mg/kg) and disopyramide (2.5 and 5 mg/kg) decreased the arrhythmic ratio ?(ventricular arrhythmias/total heart rate) x 100? dose-dependently. Pilsicainide at 2.5 and 5 mg/kg and disopyramide at 5 mg/kg suppressed ventricular arrhythmias more than 50%. The effective dose of pilsicainide was lower than that of disopyramide, but the effective plasma concentration of pilsicainide was between 3 and 8 micrograms/ml, which was almost the same as that of disopyramide. In anesthetized dogs, both drugs decreased LV dP/dt max in almost the same concentration-dependent manner. PQ-interval was prolonged by pilsicainide, but not by disopyramide. QRS and QTc were prolonged by both drugs in a concentration-dependent manner. However, the prolongation of QTc by disopyramide was provoked at lower plasma concentrations than by pilsicainide. Because the excessive prolongation of QTc lead to the lethal arrhythmias such as torsades de pointes, pilsicainide may be useful as an injectable antiarrhythmic agent superior to disopyramide.     

Curative effect of combined treatment with alendronate and 1 alpha-hydroxyvitamin D3 on bone loss by ovariectomy in aged rats

We investigated the combined effects of alendronate and 1alpha-hydroxyvitamin D3 (1alpha(OH)D3) on the bone mass and strength in aged ovariectomized rats and compared them with those of single treatments. Forty-week-old female rats underwent ovariectomy or sham operation, and after 15 weeks, ovariectomized rats were daily administered vehicle alone, alendronate (0.2 or 1.0 mg/kg,p.o.), 1alpha(OH)D3 (0.02 microg/kg, p.o.), or the combinations of 0.2 or 1.0 mg/kg of alendronate and 1alpha(OH)D3. After 12 weeks, the groups receiving combined treatments had significantly increased bone density and mechanical strength of the 4th lumbar vertebral body and the midfemur compared to the vehicle-treated group, and the effects were almost equal to or slightly less than the addition of those of the respective single treatments. The increase in mechanical strength was proportional to that in bone mineral density, suggesting that the stimulatory effects of these treatments on bone strength are ascribable primarily to those on bone mass. Analyses of histology, computed tomography, and biochemical markers confirmed the strong effect of the combined treatment on trabecular bone in particular, which was associated with increased trabecular number and decreased bone turnover. We propose that the combination of daily alendronate and 1alpha(OH)D3 is clinically promising as a curative treatment of established postmenopausal osteoporosis.     

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 (1α,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-α 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-α and IFN-γ in combination, RANTES production was enhanced, but IL-8 production was not changed, compared to TNF-α-treated cells. Tacalcitol decreased IL-8 production dose-dependently as observed in the TNF-α-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, 1α,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.

     

A comparative study on the hypouricemic activity and potency in renal xanthine calculus formation of two xanthine oxidase/xanthine dehydrogenase inhibitors: TEI-6720 and allopurinol in rats

In this study, the hypouricemic efficacy of a novel xanthine oxidase/xanthine dehydrogenase inhibitor, TEI-6720, was compared with that of allopurinol in a hyperuricemic rat model established by feeding the animals oxonate, a uricase inhibitor. In addition, using normal rats, the changes in xanthine concentration in plasma and the concentrations and absolute quantities of uric acid, allantoin and xanthine in urine were analyzed during a 28-day period of repeated administration of TEI-6720 to determine the changes occurring during this period and the conditions required for the formation of xanthine crystals and calculi in comparison with allopurinol. TEI-6720 and allopurinol caused a significant dose-dependent decrease in plasma uric acid levels in the hyperuricemic rat model and the ED50 of TEI-6720 was lower than that of allopurinol, indicating that in terms of hypouricemic efficacy TEI-6720 is more potent than allopurinol. TEI-6720 also showed more potent activity than allopurinol in decreasing urinary uric acid and allantoin levels in normal rats. In addition, TEI-6720 and allopurinol showed similar dose-response curves for the decrease in uric acid or allantoin concentration, and the associated increase in xanthine concentration, indicating that TEI-6720 and allopurinol have similar pharmacological characteristics although the dosage required differs. The efficacy of TEI-6720 in increasing plasma and urinary xanthine levels in normal rats was approximately 10- to 30-fold greater than that of allopurinol. However, with respect to renal xanthine calculus formation, there was only about a 3-fold difference in dosage comparing TEI-6720 and allopurinol. This difference suggests that there may be another factor independent of xanthine, and dependent on the drug itself, involved in renal calculus formation caused by allopurinol. The daily excretion of purine metabolites per body weight was about 20-fold higher in rats than in humans. From these results, it is concluded that TEI-6720 has potent hypouricemic activity and that, compared to allopurinol, administration of TEI-6720 is not likely to result in a higher incidence of calculus formation.     

Nephrotoxic effects of allopurinol in dinitrofluorobenzene-sensitized mice: comparative studies on TEI-6720

Allopurinol is widely used and generally well-tolerated. However, when used in patients with renal insufficiency it may have life-threatening toxic effects known as allopurinol hypersensitivity syndrome (AHS). We previously found that allopurinol increased ear swelling and mortality in a DNFB-induced contact hypersensitivity mouse model. In the present study, we investigated the toxic effect of allopurinol on DNFB-sensitized mice in order to clarify the mechanism responsible for the lethal effect of allopurinol. Allopurinol increased plasma GPT and GOT in DNFB-sensitized mice and markedly increased plasma creatinine and BUN. The increase in plasma GPT and GOT was moderate and declined time-dependently. In contrast, the increase in plasma creatinine and BUN was striking and continued until 18 hr after administration of allopurinol at 100 mg/kg/day. Although allopurinol increased GOT and GPT in DNFB-sensitized mice, no effect was observed in non-sensitized mice even at 100 mg/kg/day, indicating that allopurinol essentially has no toxic effect on the liver. A high dose of allopurinol induced renal impairment even in non-sensitized mice. These observations indicate that there is some biological interaction between allopurinol and DNFB, and suggest that allopurinol may modulate or enhance the inflammatory reactions induced by DNFB, and/or that DNFB may cause metabolic changes via inflammation, leading to the enhanced toxicity of allopurinol. In contrast, TEI-6720, a newly synthesized XOD/XDH inhibitor, had almost no effect on DNFB-sensitized mice. TEI-6720 at 1 mg/kg, in terms of hypouricemic effect, appeared to be more potent than allopurinol at 3 mg/kg. Therefore, the nephrotoxic effect of allopurinol observed in the present study may not be related to XOD/XDH inhibitory activity.     

1 alpha,25-dihydroxyvitamin D3 suppresses interleukin-1beta-induced interleukin-8 production in human whole blood: an involvement of erythrocytes in the inhibition

Interleukin (IL)-8, which is involved in inflammatory responses, is produced by a variety of cell types, monocytes/macrophages and neutrophils, in response to inflammatory stimuli including lipopolysaccharide, IL-1, and tumor necrosis factor alpha. Here we report the inhibitory effects of 1alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3) on IL-8 production in human whole blood culture. 1,25(OH)2D3 inhibited only the late phase of the biphasic IL-8 production in lipopolysaccharide-stimulated human whole blood. It also effectively inhibited IL-8 production induced by IL-lbeta compared with that induced by tumor necrosis factor alpha. IL-8 mRNA expression in IL-lbeta-stimulated whole blood was found to require de novo protein synthesis. Although monocytes were found to be mainly responsible for IL-1beta-induced IL-8 production in whole blood, 1,25(OH)2D3 inhibited IL-8 production by isolated mononuclear cells only marginally. The inhibitory effect of 1,25(OH)2D3 on mononuclear cells was restored by adding erythrocytes. These results suggest that erythrocytes play a role in mediating the inhibitory effects of 1,25(OH)2D3 on IL-8 production in IL-1beta-stimulated whole blood.     

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.