A simulated oral hygiene model to determine the efficacy of repeated exposure of amine oxide on the viability of Streptococcus mutans biofilms.

The use of biocide in oral care products is important for controlling microbial pathogens. However, the use of biofilm tests that investigate repeated exposure to biocide, to mimic in situ treatment, has rarely been reported in the literature. The present study describes the application of a biofilm-based efficacy protocol, for testing the effect of repeated exposure to antimicrobials on biofilm, in an attempt to mimic oral care regimens. The activity of different treatment regimens, including repeated exposure to amine oxide (AO; C(10)-C(16)-alkyldimethyl N-oxides; 1.1% v/v), was conducted against 16-h Streptococcus mutans biofilms grown on hydroxyapatite disks. Single exposure to AO alone produced a 3 log(10) reduction in microbial count, but when combined with mechanical removal, a 5 log(10) reduction in microbial count was observed. Treatments incorporating repeated exposure to AO reduced the microbial count below the level of detection, even when exposure to AO was interspersed with recovery periods. The presence of organic load produced an additional 2 log(10) reduction in the microbial count. This study showed that the application of a biofilm-based efficacy protocol to mimic oral care regimens allowed the reproducible testing of repeated antimicrobial exposures against bacterial biofilm. In addition, AO was confirmed to be an excellent biocide for eliminating S. mutans biofilms and could therefore be beneficial in oral care formulations.

Effects of progesterone and estradiol on uterine secretion of prostaglandin f(2alpha)in response to oxytocin in ovariectomized sows.

Thirty ovariectomized sows were used in an experiment designed to determine whether the ability of the porcine uterus to release prostaglandin (PG) F(2alpha) in response to oxytocin is regulated by progesterone (P(4)) and estradiol (E(2)). Sows were assigned to one of four treatment groups: 1) no steroids (ovariectomized controls; n = 8), 2) E(2) (n = 8), 3) P(4) (n = 7), or 4) E(2) + P(4) (n = 7). P(4) and E(2) were administered so as to mimic the normal temporal changes that occur in these hormones during the estrous cycle. A group of intact sows (n = 9) was included for comparison. All sows received an injection of oxytocin (30 IU, i.v.) on Days 12, 15, and 18 postestrus. Jugular venous blood samples were collected from 60 min before through 120 min after injection of oxytocin for quantification of 13,14-dihydro-15-keto-PGF(2alpha) (PGFM). Preinjection baseline concentrations of PGFM, the magnitude of the PGFM response above baseline, and area under the PGFM response curve (AUC) were calculated for each sow on each day and compared among treatment groups by ANOVA. Among the ovariectomized sows receiving steroid replacement, baseline concentrations of PGFM were low on Day 12 postestrus in all four groups. On Days 15 and 18, baseline concentrations remained low in the two groups that did not receive P(4) but increased in those that did. Both the magnitude of the response to oxytocin and AUC were small on Day 12 postestrus in all 4 groups. By Day 15, the magnitude of the response and AUC increased in the group that received both P(4) and E(2) but remained low in the other three groups. By Day 18, responses to oxytocin were greater in both groups that received P(4) than in those that did not. Baseline concentrations were similar in intact sows and in those that received both P(4) and E(2) on all three days examined. The magnitude of the response and the AUC were greater in the ovariectomized sows receiving P(4) and E(2) replacement than in the intact control sows on Days 15 and 18 postestrus. From these results, we conclude that P(4) and E(2) interact to control the time when the uterus begins to secrete PGF(2alpha) in response to oxytocin and the amount of PGF(2alpha) secreted.

Unique regulation of CYP17 expression in the trophectoderm of the preattachment porcine blastocyst.

Expression of the gene encoding cytochrome P450 17alpha-hydroxylase, CYP17, is necessary for adrenal and gonadal steroidogenesis in most species. However, some animals, such as the pig, express CYP17 in the trophectoderm of the preattachment blastocyst, an event associated with estrogen synthesis and the establishment of pregnancy. How trophoblastic expression of CYP17 is regulated in the porcine blastocyst remains unknown and forms the basis of the following studies. The porcine CYP17 gene, including the complete coding and several kilobases of 5'-flanking regions, was cloned and sequenced. Blastocysts were examined by Northern analysis to verify the level of CYP17 transcript, and tissue-specific expression in the trophectoderm was confirmed by in situ hybridization. Primer extension, S1 nuclease protection, and 5'-rapid amplification of cDNA ends confirmed a common proximal transcription start site in adrenals and gonads (-48 bp) but identified a unique distal start site used in porcine trophectoderm (-182 bp). Additionally, reporter analysis of the CYP17 regulatory region demonstrated that constructs (-27 to -718 bp) were unresponsive to forskolin when expressed in porcine trophoblast cells, suggesting that trophoblast may not be able to respond to cAMP induction of this gene. The identification of this distal, previously undescribed, transcriptional start site suggests that unique mechanisms control the expression of CYP17 in porcine trophectoderm and possibly other genes important in implantation and early placental development.

Development and differentiation of the interstitial and tubular compartments of fetal porcine testes.

Male differentiation is initiated by fetal testicular androgen synthesis, catalyzed by the enzyme 17alpha-hydroxylase/17,20 lyase cytochrome P450 (P450c17). This study was an investigation of testicular development and differentiation in porcine fetuses recovered on Days 30-42 of gestation. The expression of P450c17 was localized in fetal gonads by in situ hybridization and immunocytochemistry and related to cellular proliferation through expression of the proliferating cell nuclear antigen (PCNA). Gonadal P450c17 expression was quantified by Western immunoblot analysis and related to testosterone secretion by cultured explants of fetal gonads. P450c17 transcripts were detected in the interstitium surrounding testicular cords preceding the appearance of the enzyme protein. The intensity of both P450c17 hybridization and staining was greater in Yorkshire fetal gonads, which also exhibited more advanced tubular development. PCNA staining was prominent within tubular primordia and was higher in testes from Yorkshire than from Meishan fetuses on all days examined. P450c17 expression paralleled testosterone secretion, which decreased by Day 42, and was generally less in cultures of Meishan than of Yorkshire fetal gonads. These data demonstrate that the expression of P450c17 in porcine fetal testes coincides with differentiation of central medullary cells and androgen secretion during gonadal development between Days 30 and 42 of gestation. This occurs as medullary cords organize and is associated with changes in cellular proliferation within the tubular compartment.

Effects of progesterone withdrawal on uterine secretion of prostaglandin F2 alpha in response to oxytocin in ewes.

Two experiments were conducted to determine if withdrawal of progesterone during the luteal phase of the oestrous cycle affected the ability of the ovine uterus to secrete prostaglandin F2 alpha (PGF2 alpha) in response to oxytocin. In Experiment 1, 18 ewes were ovariectomized on Day 9 and Day 12 after oestrus. Ewes were subdivided into three treatment groups (n = 6 per group): Group-1 ewes underwent sham surgery; Group-2 ewes received oestradiol (OVX + O); and Group-3 ewes received oestradiol + progesterone (OVX + O,P). Oxytocin was administered to each ewe on Days 10, 13 and 15 after oestrus. Concentrations of 13, 14-dihydro-15-keto-PGF2 alpha (PGFM) were determined in samples of jugular venous blood for 2 h after oxytocin challenge. The magnitude of the PGFM response 24 h after ovariectomy was greater (P < 0.1) in ewes from which progesterone had been withdrawn (OVX + O) than in ewes in which progesterone was maintained (intact controls and OVX + O,P). Therefore, progesterone appears to exert an inhibitory effect on uterine secretory responsiveness to oxytocin which is removed by progesterone withdrawal. In Experiment 2, ewes were ovariectomized on Day 11 and assigned to 1 of 4 treatment groups (n = 6 per group): Group 1, no steroid replacement (OVX); Group 2, oestradiol replacement (OVX + O); Group 3, progesterone replacement (OVX + P); or Group 4, progesterone + oestradiol replacement (OVX + O,P). Ewes received oxytocin on Day 12 and Day 15. On Day 12, uterine secretory responsiveness to oxytocin was greatest in ewes in the OVX + O group (P < 0.1). Responsiveness was low in ewes in the OVX group, as it was in ewes in both groups that received progesterone replacement. Therefore, the increase in uterine secretory responsiveness to oxytocin following progesterone withdrawal is dependent on oestradiol replacement.

Differences in the expression of cytochromes P450 17 alpha-hydroxylase and aromatase in Meishan and Yorkshire conceptuses at days 10.5-14.0 of gestation.

The expression of cytochromes P450 17 alpha-hydroxylase (P450c17) and aromatase (P450arom) was compared between preimplantation Chinese Meishan and domestic Yorkshire conceptuses during the period encompassing maternal recognition of pregnancy. Individual conceptuses were recovered on days 10.5, 11.0, 11.5, 12.0, and 14.0 of gestation. Diameter (spherical blastocysts), length (elongated blastocysts), DNA, protein and oestradiol content, as well as the amounts of P450c17 and P450arom (western analysis) were determined in individual conceptuses. Comparisons were made only between conceptuses of similar diameters on each day which restricted analyses to blastocysts 6 mm or less in diameter on days 10.5-12.0. Nonetheless, both DNA and protein content were greater in Yorkshire than in Meishan conceptuses. Oestradiol content also tended to be greater in Yorkshire than in Meishan conceptuses across days. A significant effect of breed and breed by day interaction was detected for P450c17. Expression of P450c17 in Yorkshire conceptuses increased markedly above that in Meishan conceptuses by day 11, remained high until day 11.5 and returned to values similar to those of Meishans by day 12. The expression of P450arom was also greater in Yorkshire than in Meishan conceptuses, but no breed by day interaction was detected. These data suggest that differences in development between Meishan and Yorkshire conceptuses include trophoblastic differentiation during preattachment stages. The significance and impact of this divergence in development on subsequent growth and survival remains to be determined.

Changes in uterine secretion of prostaglandin F2 alpha in response to oxytocin during the estrous cycle, early pregnancy, and estrogen-induced pseudopregnancy in swine.

Thirty-one sows were used in an experiment designed to determine whether the ability of the porcine uterus to release prostaglandin (PG) F2 alpha in response to oxytocin was suppressed in pregnancy and pseudopregnancy. Sows were assigned to one of three treatment groups: nonbred (nonpregnant) controls (n = 8), pseudopregnant (5 mg estradiol benzoate, i.m., daily on Days 11-15 postestrus; n = 8), or bred (bred once daily throughout the estrous period; n = 15). Jugular venous blood samples were collected daily for quantification of progesterone. Pregnancy was determined by uterine examination at slaughter 51-72 days postmating. On the basis of progesterone and embryo recovery, bred sows were classified into three subgroups: confirmed pregnant (n = 4), suspected pregnant based on delayed luteal regression (n = 5), or bred/not pregnant (n = 6). All sows received an injection of oxytocin (30 IU, i.v.) on Days 12, 15, and 18 postestrus. Jugular venous blood samples were collected from 60 min prior to through 120 min after injection of oxytocin for quantification of 13, 14-dihydro-15-keto-PGF2 alpha (PGFM). Magnitude of response above baseline and area under the PGFM response curve (AUC) were calculated for each sow on each day and compared among treatment groups by analysis of variance. Responses in pregnant and suspected-pregnant sows were not different on any day examined; therefore the two groups were combined (n = 9) and considered pregnant for all subsequent analyses. Responses in the nonpregnant and bred/not pregnant sows were pooled and compared to the responses in the pregnant and pseudopregnant sows. Magnitudes of response were similar between these pooled groups on Day 12 (p > 0.5), but were less in pregnant and pseudopregnant sows on Days 15 and 18 (p < 0.01). When nonpregnant and bred/not pregnant sows were compared to each other, the magnitudes of the response were similar on Days 12, 15, and 18 (p > 0.3 on each day). In contrast, when pregnant and pseudopregnant sows were compared, pseudopregnant sows had a lower magnitude of response that was consistent across all 3 days (p < 0.02). Similar relationships were apparent for the AUC. These results demonstrate that uterine secretory responsiveness to oxytocin is suppressed during early pregnancy and that this effect may be mediated through estrogen secreted by conceptuses.

Lack of effect of sex on pig embryonic development in vivo.

The effect of sex on pig conceptus development to day 12 of gestation was investigated. On day 2 of gestation, reciprocal embryo transfers were performed resulting in four groups (Yorkshire-Yorkshire, Yorkshire-Meishan, Meishan-Yorkshire and Meishan-Meishan). Conceptuses at day 12 were recovered from each recipient and diameter, as well as DNA, protein and oestradiol content were determined for individual conceptuses. The sex of individual conceptuses at day 12 was determined by amplification of a fragment of the pig SRY gene, using the polymerase chain reaction. Embryos developed more rapidly to day 12 in Yorkshire recipients, but there was no detectable effect of sex on the diameter, DNA, protein or oestradiol content of conceptuses from any transfer group. Thus, no sex effect was apparent under conditions either promoting or retarding the rate of early pig blastocyst growth. These results provide strong evidence that pig embryonic development occurs at a rate determined by uterine environment and not by sex of the conceptus.

Immunohistochemical localization of 3 beta-hydroxysteroid dehydrogenase and P450 17 alpha-hydroxylase during follicular and luteal development in pigs, sheep, and cows.

Follicular and luteal morphology and steroidogenic function were investigated by immunohistochemistry for cytochrome P450 17 alpha-hydroxylase (P450c17) and 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) during the estrous cycle in pigs, sheep, and cows. The theca interna of all species expressed P450c17 during follicular development. In the pig, this constituted a continuous layer of cells around the follicle, but a sheath of cells lining the basement membrane appeared not to express P450c17. Neither was expression of P450c17 in ovine and bovine follicles uniform throughout the theca interna. In these two species, a beaded appearance was given by P450c17, since it was expressed in some regions but not in others. Therefore, staining for P450c17 defined functional sub-populations of cells within the theca interna of pigs, sheep, and cows. Ovulation was associated with a decrease in P450c17 in all species, but some expression persisted in theca-derived cells of developing and mature porcine CL. Expression of 3 beta-HSD in the preovulatory follicle was confined to the theca of the pig and sheep; in contrast, in the cow, it was highest in the granulosa. In general, 3 beta-HSD expression appeared to be greater in porcine than ovine or bovine follicles, the physiological relevance of which is discussed. Porcine and ovine theca continued to express 3 beta-HSD after ovulation, and granulosa-derived cells increased their 3 beta-HSD expression markedly as they luteinized in all three species. During early luteal development in pigs and sheep, theca-derived cells with high 3 beta-HSD encircled luteal lobules, but these cells appeared throughout the parenchyma of the mature CL. Luteal regression in sheep and cows was typified by the loss of many cells expressing 3 beta-HSD, whereas others, adjacent to them, appeared to be intact without loss of enzyme expression. These data further define differences in steroidogenesis during follicular and luteal development among the pig, sheep, and cow.

HIV testing: issues for the family physician.

Human immunodeficiency virus (HIV) infection affects all communities. To provide optimal patient care, physicians must understand the ramifications of HIV-antibody testing and provide appropriate counseling before and after testing. The seropositive patient requires knowledgeable surveillance. In addition, the family physician must be prepared for the psychologic and social stresses that HIV infection imposes on the patient and the family.