1990;58:289C296. received immune milk were lower than those in the control group. These results suggest that milk produced from immunized cow may be useful for controlling in the human oral cavity. Dental care caries is one of the most prevalent infectious diseases in humans, and has been implicated as a causative organism in human dental caries (5, 12, 22). Colonization on tooth surfaces by this microorganism is considered to be the first step in the induction Tacrine HCl Hydrate of dental caries. adheres to tooth surfaces by sucrose-independent and sucrose-dependent mechanisms (8, 10). The former mechanism is due to the binding of a 190-kDa surface protein antigen (PAc) of to human salivary components on tooth surfaces (9). The latter, sucrose-dependent binding, is due to the synthesis of water-insoluble glucan from sucrose catalyzed by glucosyltransferases (GTFs) (11). The important functions of PAc and water-insoluble-glucan-synthesizing GTF (GTF-I) in the cariogenicity of make them rational targets for the development of an anticaries vaccine and an adhesion inhibitor (7). Simultaneous inhibition of these colonization factors may result in the protection of teeth from dental caries. We previously constructed a fusion protein, PAcA-GB, that fuses the saliva-binding alanine-rich region (PAcA) of PAc with the glucan-binding (GB) domain name of GTF-I (27) and have immunized Holstein cows with the fusion protein (19). Moreover, we have shown that antibodies purified from your milk of these immunized Holstein cows inhibit both the adhesion of to saliva-coated hydroxyapatite beads and glucan synthesis by GTF-I (19). In this study, we investigated the effect of passive immunization with milk from an immunized cow on recolonization of the human oral cavity by TK18 by ammonium sulfate precipitation, chromatography on DEAE-cellulose, and subsequent gel filtration on Sepharose CL-6B (Pharmacia, Uppsala, Sweden) (9). For preparation of GTF-I, transformant UA130B+, which is usually defective in both the and gene products (27), was produced in brain heart infusion broth (Difco Laboratories, Detroit, Mich.) at 37C for 18 h. GFT-I was extracted from whole cells of the transformant by treatment with 8 M urea at 25C for 1 h. The extract was centrifuged at 5,000 for 20 min, and the supernatant was dialyzed against 10 mM potassium phosphate buffer (pH 6.0). The supernatant was used as the GTF-I preparation (27). ELISA. For ELISA, 96-well microtiter plates were coated with 100 l of rPAc or Tacrine HCl Hydrate GTF-I (5 g/ml) in 50 mM carbonate-bicarbonate Tacrine HCl Hydrate buffer (pH 9.6). After incubation at 37C for 90 min, the plates were washed with phosphate-buffered saline (PBS) made up of 0.05% (vol/vol) Tween 20 (PBST) and blocked with PBST containing 1% (wt/vol) chicken egg albumin at 37C for 90 min. After the plates were washed three times with PBST, twofold serial dilutions of pasteurized bovine milk were added (100 l per well) and the plates were incubated at Eno2 37C for 90 min. The bound antibodies were detected with alkaline phosphatase-conjugated rabbit anti-bovine immunoglobulin G (heavy and light chains) (Zymed Laboratories, South San Francisco, Calif.) followed by the addition of = 4; 3 female, 1 male) and control (= 4; 2 female, 2 male) groups. Written informed consent was obtained from all volunteers, and the protocol was approved by the ethics committee of the Faculty of Dental care Science, Kyushu University or college, Fukuoka, Tacrine HCl Hydrate Japan. None of the volunteers experienced any clinically detectable active carious lesions. For the baseline values of the mean count, the ratio of to total streptococci, and the mean dental caries experience (decayed, missing, packed teeth), there was no statistical difference between the groups. Experimental design. Three different samples of saliva and plaque were collected from each subject during a 2-week period. The baseline level in each subject was Tacrine HCl Hydrate determined from your mean value of the three samples. Before rinsing their mouths with milk, all of the subjects received cetylpyridinium chloride (CPC) treatment and professional mechanical tooth cleaning (PMTC) for 5 days to lower the level of in the oral cavity. After PMTC with a rubber cup and an abrasive made up of fluoride, 10 ml of 1 1.0% CPC answer was applied once a day for 5 min using a custom-made dentition tray. During this treatment period, the subjects also rinsed their mouths with 10 ml of 0.2% CPC answer for 1 min twice a day (morning and night) after brushing their teeth. On the day after the last CPC treatment, they started rinsing their mouths with milk. Mouth rinsing was performed with 10 ml of immune and control milk for 1 min twice a day (morning and night) after tooth brushing for a period of 14.