Clinical Pharmacogenetics Implementation Consortium (CPIC) Guidelines for CYP2C9 and HLA-B Genotype and Phenytoin Dosing

Phenytoin is a widely used antiepileptic drug with a narrow therapeutic index and large inter-patient variability partly due to genetic variations in CYP2C9. Furthermore, the variant allele HLA-B*15:02 is associated with an increased risk of Stevens-Johnson syndrome and toxic epidermal necrolysis in response to phenytoin treatment. We summarize evidence from the published literature supporting these associations and provide recommendations for the use of phenytoin based on CYP2C9 and/or HLA-B genotype (also available on PharmGKB: www.pharmgkb.org)

Clinical Pharmacogenetics Implementation Consortium (CPIC) Guidelines for Human Leukocyte Antigen B (HLA-B) Genotype and Allopurinol Dosing: 2015 update

The Clinical Pharmacogenetics Implementation Consortium (CPIC) Guidelines for HLA-B*58:01 Genotype and Allopurinol Dosing was originally published in February 2013. We reviewed the recent literature and concluded that none of the evidence would change the therapeutic recommendations in the original guideline; therefore, the original publication remains clinically current. However, we have updated the Supplemental Material and included additional resources for applying CPIC guidelines into the electronic health record. Up-to-date information can be found at PharmGKB (http://www.pharmgkb.org)

Oogenesis in cultures derived from adult human ovaries

Ten years ago, we reported that in adult human females the ovarian surface epithelium (OSE) is a source of germ cells. Recently, we also demonstrated that new primary follicles are formed by assembly of oocytes with nests of primitive granulosa cells in the ovarian cortex. The components of the new primary follicles, primitive granulosa and germ cells, differentiated sequentially from the OSE, which arises from cytokeratin positive mesenchymal progenitor cells residing in the ovarian tunica albuginea. In the present study, we investigated the possibility that the oocytes and granulosa cells may differentiate in cultures derived from adult human ovaries. Cells were scrapped from the surface of ovaries and cultured for 5 to 6 days, in the presence or absence of estrogenic stimuli [phenol red (PhR)]. The OSE cells cultured in the medium without PhR differentiated into small (15 micron) cells of granulosa phenotype, and epithelial, neural, and mesenchymal type cells. In contrast, OSE cells cultured in the presence of PhR differentiated directly into large (180 micron) cells of the oocyte phenotype. Such cells exhibited germinal vesicle breakdown, expulsion of the polar body, and surface expression of zona pellucida proteins, i.e. characteristics of secondary oocytes. These in vitro studies confirm our in vivo observations that in adult human ovaries, the OSE is a bipotent source of oocytes and granulosa cells. Development of numerous mature oocytes from adult ovarian stem cells in vitro offers new strategies for the egg preservation, IVF utilization, and treatment of female infertility. In addition, other clinical applications aiming to utilize stem cells, and basic stem cell research as well, may employ totipotent embryonic stem cells developing from fertilized oocytes

A comparative study of self-perceived leadership skills in coeducational, male-only, and female-only educational settings

The purpose of this quasi-experimental study was to examine the effect that a gender-specific classroom had on men’s and women’s self-perceived leadership abilities as compared to coeducational classrooms where the students were studying leadership together. The sample for the study comprised 81 junior and senior students enrolled in a survey leadership course (ALED 340) in the College of Agriculture and Life Sciences, Department of Agricultural Leadership, Education, and Communications during the Spring 2007 semester at Texas A&M University. The students were assigned to one of five leadership laboratory sections; three sections were traditional coeducational, one was all-male, and one was all-female. During the last week of the course, the students voluntarily participated in a Leadership Skills Inventory survey that asked them to rate their self-perceptions of their leadership skills. The instrument used the post-then design method that asked for their perceptions prior to beginning the course and their perceptions at the conclusion of the course. The data were analyzed using SPSS version 14.0. Results of the study showed statistically significantly higher self-perceptions of leadership skills abilities for those students who participated in the gender-specific laboratory sections. The all-male section’s self-perceptions were statistically significantly higher than both the males in the coeducational sections and the coeducational sections as a whole. The all-female section’s self-perceptions were statistically significantly higher than the coeducational sections as a whole. The study also revealed that leadership experience in organizations and activities in high school and college prior to enrolling in a college-level leadership course statistically significantly improves self-perceptions of leadership skills ability. Results of this study agree with many research studies that support single-sex schooling and education

VMAT2-Deficient Mice Display Nigral and Extranigral Pathology and Motor and Nonmotor Symptoms of Parkinson's Disease

Dopamine is transported into synaptic vesicles by the vesicular monoamine transporter (VMAT2; SLC18A2). Disruption of dopamine storage has been hypothesized to damage the dopamine neurons that are lost in Parkinson's disease. By disrupting vesicular storage of dopamine and other monoamines, we have created a progressive mouse model of PD that exhibits catecholamine neuron loss in the substantia nigra pars compacta and locus coeruleus and motor and nonmotor symptoms. With a 95% reduction in VMAT2 expression, VMAT2-deficient animals have decreased motor function, progressive deficits in olfactory discrimination, shorter latency to behavioral signs of sleep, delayed gastric emptying, anxiety-like behaviors at younger ages, and a progressive depressive-like phenotype. Pathologically, the VMAT2-deficient mice display progressive neurodegeneration in the substantia nigra (SNpc), locus coeruleus (LC), and dorsal raphe (DR) coupled with α-synuclein accumulation. Taken together, these studies demonstrate that reduced vesicular storage of monoamines and the resulting disruption of the cytosolic environment may play a role in the pathogenesis of parkinsonian symptoms and neurodegeneration. The multisystem nature of the VMAT2-deficient mice may be useful in developing therapeutic strategies that go beyond the dopamine system

Origin of germ cells and formation of new primary follicles in adult human ovaries

Recent reports indicate that functional mouse oocytes and sperm can be derived in vitro from somatic cell lines. We hypothesize that in adult human ovaries, mesenchymal cells in the tunica albuginea (TA) are bipotent progenitors with a commitment for both primitive granulosa and germ cells. We investigated ovaries of twelve adult women (mean age 32.8 ± 4.1 SD, range 27–38 years) by single, double, and triple color immunohistochemistry. We show that cytokeratin (CK)+ mesenchymal cells in ovarian TA differentiate into surface epithelium (SE) cells by a mesenchymal-epithelial transition. Segments of SE directly associated with ovarian cortex are overgrown by TA, forming solid epithelial cords, which fragment into small (20 micron) epithelial nests descending into the lower ovarian cortex, before assembling with zona pellucida (ZP)+ oocytes. Germ cells can originate from SE cells which cover the TA. Small (10 micron) germ-like cells showing PS1 meiotically expressed oocyte carbohydrate protein are derived from SE cells via asymmetric division. They show nuclear MAPK immunoexpression, subsequently divide symmetrically, and enter adjacent cortical vessels. During vascular transport, the putative germ cells increase to oocyte size, and are picked-up by epithelial nests associated with the vessels. During follicle formation, extensions of granulosa cells enter the oocyte cytoplasm, forming a single paranuclear CK+ Balbiani body supplying all the mitochondria of the oocyte. In the ovarian medulla, occasional vessels show an accumulation of ZP+ oocytes (25–30 microns) or their remnants, suggesting that some oocytes degenerate. In contrast to males, adult human female gonads do not preserve germline type stem cells. This study expands our previous observations on the formation of germ cells in adult human ovaries. Differentiation of primitive granulosa and germ cells from the bipotent mesenchymal cell precursors of TA in adult human ovaries represents a most sophisticated adaptive mechanism created during the evolution of female reproduction. Our data indicate that the pool of primary follicles in adult human ovaries does not represent a static but a dynamic population of differentiating and regressing structures. An essential mission of such follicular turnover might be elimination of spontaneous or environmentally induced genetic alterations of oocytes in resting primary follicles

Immunoregulation of follicular renewal, selection, POF, and menopause in vivo, vs. neo-oogenesis in vitro, POF and ovarian infertility treatment, and a clinical trial

Abstract The immune system plays an important role in the regulation of tissue homeostasis ("tissue immune physiology"). Function of distinct tissues during adulthood, including the ovary, requires (1) Renewal from stem cells, (2) Preservation of tissue-specific cells in a proper differentiated state, which differs among distinct tissues, and (3) Regulation of tissue quantity. Such morphostasis can be executed by the tissue control system, consisting of immune system-related components, vascular pericytes, and autonomic innervation. Morphostasis is established epigenetically, during morphogenetic (developmental) immune adaptation, i.e., during the critical developmental period. Subsequently, the tissues are maintained in a state of differentiation reached during the adaptation by a “stop effect” of resident and self renewing monocyte-derived cells. The later normal tissue is programmed to emerge (e.g., late emergence of ovarian granulosa cells), the earlier its function ceases. Alteration of certain tissue differentiation during the critical developmental period causes persistent alteration of that tissue function, including premature ovarian failure (POF) and primary amenorrhea. In fetal and adult human ovaries the ovarian surface epithelium cells called ovarian stem cells (OSC) are bipotent stem cells for the formation of ovarian germ and granulosa cells. Recently termed oogonial stem cells are, in reality, not stem but already germ cells which have the ability to divide. Immune system-related cells and molecules accompany asymmetric division of OSC resulting in the emergence of secondary germ cells, symmetric division, and migration of secondary germ cells, formation of new granulosa cells and fetal and adult primordial follicles (follicular renewal), and selection and growth of primary/preantral, and dominant follicles. The number of selected follicles during each ovarian cycle is determined by autonomic innervation. Morphostasis is altered with advancing age, due to degenerative changes of the immune system. This causes cessation of oocyte and follicular renewal at 38 +/-2 years of age due to the lack of formation of new granulosa cells. Oocytes in primordial follicles persisting after the end of the prime reproductive period accumulate genetic alterations resulting in an exponentially growing incidence of fetal trisomies and other genetic abnormalities with advanced maternal age. The secondary germ cells also develop in the OSC cultures derived from POF and aging ovaries. In vitro conditions are free of immune mechanisms, which prevent neo-oogenesis in vivo. Such germ cells are capable of differentiating in vitro into functional oocytes. This may provide fresh oocytes and genetically related children to women lacking the ability to produce their own follicular oocytes. Further study of "immune physiology" may help us to better understand ovarian physiology and pathology, including ovarian infertility caused by POF or by a lack of ovarian follicles with functional oocytes in aging ovaries. The observations indicating involvement of immunoregulation in physiological neo-oogenesis and follicular renewal from OSC during the fetal and prime reproductive periods are reviewed as well as immune system and age-independent neo-oogenesis and oocyte maturation in OSC cultures, perimenopausal alteration of homeostasis causing disorders of many tissues, and the first OSC culture clinical trial.</p

Abnormal expression of p27kip1 protein in levator ani muscle of aging women with pelvic floor disorders – a relationship to the cellular differentiation and degeneration

BACKGROUND: Pelvic floor disorders affect almost 50% of aging women. An important role in the pelvic floor support belongs to the levator ani muscle. The p27/kip1 (p27) protein, multifunctional cyclin-dependent kinase inhibitor, shows changing expression in differentiating skeletal muscle cells during development, and relatively high levels of p27 RNA were detected in the normal human skeletal muscles. METHODS: Biopsy samples of levator ani muscle were obtained from 22 symptomatic patients with stress urinary incontinence, pelvic organ prolapse, and overlaps (age range 38–74), and nine asymptomatic women (age 31–49). Cryostat sections were investigated for p27 protein expression and type I (slow twitch) and type II (fast twitch) fibers. RESULTS: All fibers exhibited strong plasma membrane (and nuclear) p27 protein expression. cytoplasmic p27 expression was virtually absent in asymptomatic women. In perimenopausal symptomatic patients (ages 38–55), muscle fibers showed hypertrophy and moderate cytoplasmic p27 staining accompanied by diminution of type II fibers. Older symptomatic patients (ages 57–74) showed cytoplasmic p27 overexpression accompanied by shrinking, cytoplasmic vacuolization and fragmentation of muscle cells. The plasma membrane and cytoplasmic p27 expression was not unique to the muscle cells. Under certain circumstances, it was also detected in other cell types (epithelium of ectocervix and luteal cells). CONCLUSIONS: This is the first report on the unusual (plasma membrane and cytoplasmic) expression of p27 protein in normal and abnormal human striated muscle cells in vivo. Our data indicate that pelvic floor disorders are in perimenopausal patients associated with an appearance of moderate cytoplasmic p27 expression, accompanying hypertrophy and transition of type II into type I fibers. The patients in advanced postmenopause show shrinking and fragmentation of muscle fibers associated with strong cytoplasmic p27 expression

Pharmacogenetic allele nomenclature: International workgroup recommendations for test result reporting

This manuscript provides nomenclature recommendations developed by an international workgroup to increase transparency and standardization of pharmacogenetic (PGx) result reporting. Presently, sequence variants identified by PGx tests are described using different nomenclature systems. In addition, PGx analysis may detect different sets of variants for each gene, which can affect interpretation of results. This practice has caused confusion and may thereby impede the adoption of clinical PGx testing. Standardization is critical to move PGx forward

Expression and localization of estrogen receptor-alpha protein in normal and abnormal term placentae and stimulation of trophoblast differentiation by estradiol

Estrogens play an important role in the regulation of placental function, and 17-beta-estradiol (E2) production rises eighty fold during human pregnancy. Although term placenta has been found to specifically bind estrogens, cellular localization of estrogen receptor alpha (ER-alpha) in trophoblast remains unclear. We used western blot analysis and immunohistochemistry with h-151 and ID5 monoclonal antibodies to determine the expression and cellular localization of ER-alpha protein in human placentae and cultured trophoblast cells. Western blot analysis revealed a ~65 kDa ER-alpha band in MCF-7 breast carcinoma cells (positive control). A similar band was detected in five normal term placentae exhibiting strong expression of Thy-1 differentiation protein in the villous core. However, five other term placentae, which exhibited low or no Thy-1 expression (abnormal placentae), exhibited virtually no ER-alpha expression. In normal placentae, nuclear ER-alpha expression was confined to villous cytotrophoblast cells (CT), but syncytiotrophoblast (ST) and extravillous trophoblast cells were unstained. In abnormal placentae no CT expressing ER-alpha were detected. Normal and abnormal placentae also showed ER-alpha expression in villous vascular pericytes and amniotic (but not villous) fibroblasts; no staining was detected in amniotic epithelial cells or decidual cells. All cultured trophoblast cells derived from the same normal and abnormal placentae showed distinct ER-alpha expression in western blots, and the ER-alpha expression was confined to the differentiating CT, but not to the mature ST. Trophoblast cells from six additional placentae were cultured in normal medium with phenol red (a weak estrogen) as above (PhR+), or plated in phenol red-free medium (PhR-) without or with mid-pregnancy levels of E2 (20 nM). Culture in PhR- medium without E2 caused retardation of syncytium formation and PhR-medium with E2 caused acceleration of syncytium formation compared to cultures in PhR+ medium. These data indicate that the considerable increase in estrogen production during pregnancy may play a role, via the ER-alpha, in the stimulation of CT differentiation and promote function in normal placentae. This mechanism, however, may not operate in abnormal placentae, which show a lack of ER-alpha expression