Establishment of a CYP2C19 genotyping assay for clinical use.

Conversion of clopidogrel (Plavix) to its active metabolite is catalyzed largely by the P450 enzyme 2C19 (CYP2C19). Numerous allelic variants of CYP2C19 exist. The *1 allele is considered wild type, whereas the *2 and *3 alleles have no in vivo enzymatic activity. Conversely, the *17 allele has increased expression, resulting in increased clopidogrel activation. Poor metabolizers (*2/*2 and *2/*3 genotypes) experience higher rates of therapeutic failure. For this reason, we have validated a CYP2C19 genotyping assay for the *1, *2, *3, and *17 alleles. Genomic DNA extracted from 30 deidentified EDTA whole-blood samples from patients was analyzed at 2 independent facilities using specific TaqMan realtime polymerase chain reaction primers and probes. Concordant genotypes were generated on all samples tested. Of the 30 samples, 15 were CYP2C19*1/*1, 8 were CYP2C19*1/*17, 5 were CYP2C19*1/*2, and 2 were CYP2C19*2/*17. There were no CYP2C19*3 alleles or *2/*2 homozygous genotypes detected. This CYP2C19 genotyping assay is appropriate for clinical testing, demonstrating excellent interlaboratory concordance, enabling the selection of the most effective clopidogrel treatment regimen for patients undergoing percutaneous coronary intervention.

Using interphase fluorescence in situ hybridization (I-FISH) to detect the transfer of infant cells during breastfeeding.

Using fluorochrome-labeled probes for the X and Y chromosomes, fluorescence in situ hybridization (FISH) was used to test for the presence of male cells in the milk of women breastfeeding male babies. After breastfeeding, the women wiped one breast and collected a test sample. Wiping efficiency was evaluated in 2 mothers by running a solution over the nipple after wiping. Cells were collected from both the milk and the rinse samples by centrifugation. The cells were fixed to glass slides for hybridization with the X and Y probes. Two cell populations were observed: small female (XX) cells from the mother, and large epithelial cells that were shown to be male (XY), presumably from the baby's mouth. Results varied from 0 to 175 male cells detected per milliliter of breast milk. This finding suggests that small molecules, including possible pathogens, in a baby's mouth, might also be passed directly into the breast.

Histological characteristics of singleton placentas delivered before the 28th week of gestation.

AIMS: The placenta is a record of the fetal environment and its examination may provide information about the baby's subsequent growth and development. We describe the histological characteristics of 947 singleton placentas from infants born between 23 and 27 weeks gestation. METHODS: Consent was obtained from mothers who delivered before 28 weeks (clinical estimate). We evaluated the gross and histopathological features of the placenta and assessed pair-wise correlations between variables. RESULTS: Lesions of uteroplacental circulation (abruption, extensive infarction or thrombosis, marked basal or perivillous fibrin deposition, increased syncytial knots) were inversely related to those associated with inflammation of the membranes and cord. Earlier age favoured inflammatory variables, while older age favoured characteristics attributed to impaired blood flow. We observed inflammation of the chorionic plate in 43%, the cord in 19%, and of chorionic plate vessels in 30%. Of the placentas with umbilical cord inflammation, 8% had no inflammation of the chorionic plate. CONCLUSIONS: This study population is unique in its size and recruitment by gestational age rather than birth weight. Inflammation occurred frequently, but not in placentas that had characteristics of vasculopathy. The prevalence of inflammation decreased with increasing gestational age, while vasculopathy increased. Funisitis need not be accompanied by chorionic inflammation.

Triplet placentas: reference values for weights.

The occurrence of twins, triplets, and other multiple births increased significantly between 1970 and 2000 in the United States and other industrialized countries. The number of triplet placentas submitted for examination as pathologic specimens has also markedly increased, but no reference values are published for triplet weights. We examined 196 normal triplet placentas. Specimens with associated conditions known to affect the weights of the placentas were excluded. The gestational ages ranged between 20 and 38 weeks. Mean weights for different gestational ages are summarized as follows: 253 g for 20 weeks, 319 g for 22 weeks, 406 g for 24 weeks, 509 g for 26 weeks, 621 g for 28 weeks, 738 g for 30 weeks, 855 g for 32 weeks, 965 g for 34 weeks, 1,065 g for 36 weeks, and 1,147 g for 38 weeks. Weight gain of triplet placentas appears to parallel that of twin placentas. The mean values of placental weights for triplets at each gestational age are less than triple those of singleton weights for the same duration of gestation. The placental weights in multiple gestations do not increase proportionately with the number of fetuses.

Expression of estrogen receptor beta in the fetal, neonatal, and prepubertal human prostate.

BACKGROUND: Although androgens have long been implicated in the development, regulation, and pathophysiology of the prostate, evidence suggests that estrogens may also affect these processes. Specifically, estrogens have been shown to influence the development of the fetal and neonatal rodent prostate and to induce a pathognomonic change, termed squamous metaplasia, in the developing and adult prostate. Studies have been inconclusive, however, as to whether estrogens enhance or restrain the growth of the gland. Although the fetal rodent prostate has been reported to contain both estrogen receptor alpha (ER-alpha) and beta (ER-beta), there have been no reports as to whether either of the ER subtypes is expressed in the developing human prostate. METHODS: In the present study, we used a novel antibody, directed against a unique sequence in the F domain of ER-beta, and laser capture microdissection/reverse transcriptase-polymerase chain reaction to study the expression of the receptor in the fetal, neonatal, and prepubertal human prostate. Results were compared with the expression of ER-alpha, androgen receptor (AR), prostatic acid phosphatase (PAP), prostate specific antigen (PSA), high molecular weight cytokeratin (HMCK), and the proliferative marker Ki67. RESULTS: For the first time, we report that ER-beta is the only estrogen receptor detected at the protein level in the morphologically normal developing human fetal prostate. By midgestation, strong immunostaining for ER-beta was detected in the nuclei of nearly 100% of epithelial and in the majority of stromal cells. This pattern of expression was evident in the fetal, neonatal, and early prepubertal prostate. However, by 11 years postnatal, staining for the receptor became restricted primarily to the basal epithelial and stromal compartments, a pattern analogous to that observed in the normal adult gland. ER-alpha mRNA was present in microdissected stroma of the fetal gland. Although ER-alpha was not immunodetected in any morphologically normal fetal epithelial or stromal cells, weak staining for the receptor, however, was found in some examples of squamous metaplasia, suggesting the role of alpha-subtype in this lesion. ER-alpha was clearly visualized immunohistochemically at 1 month of postnatal development where it was then localized exclusively in periacinar stromal nuclei, which suggests that it may exert paracrine influences on further prostatic glandular development. Interestingly, the expression of ER-beta early in prostatic development occurred coincident with both the increasing rate of epithelial cell proliferation, observed in the first half of gestation, and the reported high levels of estrogen in the gland from midgestation until term. Paradoxically, however, staining for the receptor remained intense, despite the dramatic decrease in Ki67 labeling observed in the second half of gestation. CONCLUSION: Our results indicate that the effects of estrogens on the growth of the human fetal prostate are mediated primarily by ER-beta but that ER-alpha contributes to postnatal glandular development. Furthermore, these results suggest that ER-beta, possibly in concert with androgens, may mediate diverse effects on prostate epithelial proliferation by first promoting cell expansion early in gestation, and then acting to limit growth later in prostatic development.