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1.  Concurrent use of amphetamine stimulants and antidepressants by undergraduate students 
Undergraduate students were recruited to participate in an online survey to report their use of amphetamine stimulants and other drugs. Significant differences were found between students reporting (n=79; 4.0%) and not reporting (n=1,897; 96%) amphetamine-stimulant use in the past month – in terms of race/ethnicity, class standing, residence, health symptoms, self-health report – in addition to alcohol, tobacco, pain-reliever, and antidepressant use. Health symptoms reported more often by stimulant users included depression, diarrhea, difficulty sleeping, fatigue, dizziness, difficulty concentrating, and nicotine craving. Health care providers of college students should query these patients about symptoms that could be related to depression and amphetamine use. In particular, they should provide education at the point of care around the risks of amphetamine use in general and the specific risks in those students who have symptoms of depression and/or are taking antidepressant medication. Prevention programs should also target the risks of concurrent use of amphetamines, antidepressants, and other drugs among college students.
doi:10.2147/PPA.S74602
PMCID: PMC4309786  PMID: 25653508
stimulant use; depression; college students; self-medication
2.  Diversity of the Vaginal Microbiome Correlates With Preterm Birth 
Reproductive Sciences  2014;21(1):32-40.
Reproductive tract infection is a major initiator of preterm birth (PTB). The objective of this prospective cohort study of 88 participants was to determine whether PTB correlates with the vaginal microbiome during pregnancy. Total DNA was purified from posterior vaginal fornix swabs during gestation. The 16S ribosomal RNA gene was amplified using polymerase chain reaction primers, followed by chain-termination sequencing. Bacteria were identified by comparing contig consensus sequences with the Ribosomal Database Project. Dichotomous responses were summarized via proportions and continuous variables via means ± standard deviation. Mean Shannon Diversity index differed by Welch t test (P = .00016) between caucasians with PTB and term gestation. Species diversity was greatest among African Americans (P = .0045). Change in microbiome/Lactobacillus content and presence of putative novel/noxious bacteria did not correlate with PTB. We conclude that uncultured vaginal bacteria play an important role in PTB and race/ethnicity and sampling location are important determinants of the vaginal microbiome.
doi:10.1177/1933719113488838
PMCID: PMC3857766  PMID: 23715799
PTB; vaginal microbiome; race/ethnicity
3.  Coculturing human endometrial epithelial cells and stromal fibroblasts alters cell-specific gene expression and cytokine production 
Fertility and sterility  2013;100(4):10.1016/j.fertnstert.2013.06.007.
Objective
To determine the effects of coculturing endometrial epithelial cells (eEC) with paired endometrial stromal fibroblasts (eSF) on cell-specific gene expression and cytokine secretion patterns.
Design
In vitro study.
Setting
University research laboratory.
Patient(s)
Endometrial biopsies were obtained from premenopausal women.
Intervention(s)
Polarized eEC and subject-paired eSF were cultured for 12.5 hours alone (monoculture) or combined in a two-chamber coculture system without cell-cell contact. Cells and conditioned media were analyzed for global gene expression and cytokine secretion, respectively. Purified, endometrial tissue-derived eEC and eSF isolated by fluorescent activated cell sorting (FACS) were used as noncultured controls.
Main Outcome Measure(s)
Cell-specific global gene expression profiling and analysis of secreted cytokines in eEC/eSF cocultures and respective monocultures.
Result(s)
Transepithelial resistance, diffusible tracer exclusion, expression of tight junction proteins, and apical/basolateral vectorial secretion confirmed eEC structural and functional polarization. Distinct transcriptomes of eEC and eSF were consistent with their respective lineages and their endometrial origin. Coculture of eEC with eSF resulted in altered cell-specific gene expression and cytokine secretion.
Conclusion(s)
This coculture model provides evidence that interactions between endometrial functionally polarized epithelium and stromal fibroblasts affect cell-specific gene expression and cytokine secretion underscoring their relevance when modeling endometrium in vitro.
doi:10.1016/j.fertnstert.2013.06.007
PMCID: PMC3820417  PMID: 23849844
Endometrium; coculture; polarized epithelium; stroma; microarray; cytokines
4.  The dynamics of the vaginal microbiome during infertility therapy with in vitro fertilization-embryo transfer 
Purpose
To determine the vaginal microbiome in women undergoing IVF-ET and investigate correlations with clinical outcomes.
Methods
Thirty patients had blood drawn for estradiol (E2) and progesterone (P4) at four time points during the IVF-ET cycle and at 4–6 weeks of gestation, if pregnant. Vaginal swabs were obtained in different hormonal milieu, and the vaginal microbiome determined by deep sequencing of the 16S ribosomal RNA gene.
Results
The vaginal microbiome underwent a transition during therapy in some but not all patients. Novel bacteria were found in 33% of women tested during the treatment cycle, but not at 6–8 weeks of gestation. Diversity of species varied across different hormonal milieu, and on the day of embryo transfer correlated with outcome (live birth/no live birth). The species diversity index distinguished women who had a live birth from those who did not.
Conclusions
This metagenomics approach has enabled discovery of novel, previously unidentified bacterial species in the human vagina in different hormonal milieu and supports a shift in the vaginal microbiome during IVF-ET therapy using standard protocols. Furthermore, the data suggest that the vaginal microbiome on the day of embryo transfer affects pregnancy outcome.
Electronic supplementary material
The online version of this article (doi:10.1007/s10815-011-9694-6) contains supplementary material, which is available to authorized users.
doi:10.1007/s10815-011-9694-6
PMCID: PMC3270134  PMID: 22222853
Metagenomics; Vagina; Microbiome; Infertility; IVF; Pregnancy
5.  Biobanking Human Endometrial Tissue and Subject Blood Specimens: Standard Operating Procedure and Importance to Reproductive Biology Research and Diagnostic Development 
Fertility and sterility  2011;95(6):2120-2122.e12.
Objective
To develop a standard operating procedure (SOP) for collection, transport, storage of human endometrial tissue and blood samples, subject and specimen annotation, and establishing sample priorities.
Design
The SOP synthesizes sound scientific procedures, the literature on ischemia research, sample collection and gene expression profiling, good laboratory practices, and the authors’ experience of workflow and sample quality.
Setting
The NIH University of California San Francisco Human Endometrial Tissue and DNA Bank.
Patients
Women undergoing endometrial biopsy or hysterectomy for non-malignant indications.
Intervention
Collecting, processing, storing, distributing endometrial tissue and blood samples under approved institutional review board (IRB) protocols and written informed consent from participating subjects.
Main outcome measure
SOP.
Results
The SOP addresses rigorous and consistent subject annotation, specimen processing and characterization, strict regulatory compliance, and a reference for researchers to track collection and storage times that may influence their research.
Conclusion
The comprehensive and systematic approach to the procurement of human blood and endometrial tissue in this SOP ensures the high quality, reliability, and scientific usefulness of biospecimens made available to investigators by the NIH University of California San Francisco Human Endometrial Tissue and DNA Bank. The detail and perspective in this SOP also provides a blueprint for implementation of similar collection programs at other institutions.
doi:10.1016/j.fertnstert.2011.01.164
PMCID: PMC3080464  PMID: 21371706
endometrium; biobanking; standard operating procedure
6.  Glycoengineered Pichia produced anti-HER2 is comparable to trastuzumab in preclinical study 
mAbs  2011;3(3):289-298.
Mammalian cell culture systems are used predominantly for the production of therapeutic monoclonal antibody (mAb) products. A number of alternative platforms, such as Pichia engineered with a humanized N-linked glycosylation pathway, have recently been developed for the production of mAbs. The glycosylation profiles of mAbs produced in glycoengineered Pichia are similar to those of mAbs produced in mammalian systems. This report presents for the first time the comprehensive characterization of an anti-human epidermal growth factor receptor 2 (HER2) mAb produced in glycoengineered Pichia, and a study comparing the anti-HER2 from Pichia, which had an amino acid sequence identical to trastuzumab, with trastuzumab. The comparative study covered a full spectrum of preclinical evaluation, including bioanalytical characterization, in vitro biological functions, in vivo anti-tumor efficacy and pharmacokinetics in both mice and non-human primates. Cell signaling and proliferation assays showed that anti-HER2 from Pichia had antagonist activities comparable to trastuzumab. However, Pichia-produced material showed a 5-fold increase in binding affinity to FcγIIIA and significantly enhanced antibody dependent cell-mediated cytotoxicity (ADCC) activity, presumably due to the lack of fucose on N-glycans. In a breast cancer xenograft mouse model, anti-HER2 was comparable to trastuzumab in tumor growth inhibition. Furthermore, comparable pharmacokinetic profiles were observed for anti-HER2 and trastuzumab in both mice and cynomolgus monkeys. We conclude that glycoengineered Pichia provides an alternative production platform for therapeutic mAbs and may be of particular interest for production of antibodies for which ADCC is part of the clinical mechanism of action.
doi:10.4161/mabs.3.3.15532
PMCID: PMC3149709  PMID: 21487242
glycoengineered Pichia; anti-HER2; trastuzumab; xenograft; PK; ADCC
7.  Characterization of Notch1 Antibodies That Inhibit Signaling of Both Normal and Mutated Notch1 Receptors 
PLoS ONE  2010;5(2):e9094.
Background
Notch receptors normally play a key role in guiding a variety of cell fate decisions during development and differentiation of metazoan organisms. On the other hand, dysregulation of Notch1 signaling is associated with many different types of cancer as well as tumor angiogenesis, making Notch1 a potential therapeutic target.
Principal Findings
Here we report the in vitro activities of inhibitory Notch1 monoclonal antibodies derived from cell-based and solid-phase screening of a phage display library. Two classes of antibodies were found, one directed against the EGF-repeat region that encompasses the ligand-binding domain (LBD), and the second directed against the activation switch of the receptor, the Notch negative regulatory region (NRR). The antibodies are selective for Notch1, inhibiting Jag2-dependent signaling by Notch1 but not by Notch 2 and 3 in reporter gene assays, with EC50 values as low as 5±3 nM and 0.13±0.09 nM for the LBD and NRR antibodies, respectively, and fail to recognize Notch4. While more potent, NRR antibodies are incomplete antagonists of Notch1 signaling. The antagonistic activity of LBD, but not NRR, antibodies is strongly dependent on the activating ligand. Both LBD and NRR antibodies bind to Notch1 on human tumor cell lines and inhibit the expression of sentinel Notch target genes, including HES1, HES5, and DTX1. NRR antibodies also strongly inhibit ligand-independent signaling in heterologous cells transiently expressing Notch1 receptors with diverse NRR “class I” point mutations, the most common type of mutation found in human T-cell acute lymphoblastic leukemia (T-ALL). In contrast, NRR antibodies failed to antagonize Notch1 receptors bearing rare “class II” or “class III” mutations, in which amino acid insertions generate a duplicated or constitutively sensitive metalloprotease cleavage site. Signaling in T-ALL cell lines bearing class I mutations is partially refractory to inhibitory antibodies as compared to cell-penetrating gamma-secretase inhibitors.
Conclusions/Significance
Antibodies that compete with Notch1 ligand binding or that bind to the negative regulatory region can act as potent inhibitors of Notch1 signaling. These antibodies may have clinical utility for conditions in which inhibition of signaling by wild-type Notch1 is desired, but are likely to be of limited value for treatment of T-ALLs associated with aberrant Notch1 activation.
doi:10.1371/journal.pone.0009094
PMCID: PMC2817004  PMID: 20161710
8.  Cytotrophoblast induction of arterial apoptosis and lymphangiogenesis in an in vivo model of human placentation 
Journal of Clinical Investigation  2006;116(10):2643-2652.
We studied the vascular effects of invasive human cytotrophoblasts in vivo by transplanting placental villi to the fifth mammary fat pads or beneath the kidney capsules of Scid mice. Over 3 weeks, robust cytotrophoblast invasion was observed in both locations. The architecture of the mammary fat pad allowed for detailed analysis of the cells’ interactions with resident murine blood vessels, which revealed specific induction of apoptosis in the endothelial cells and smooth muscle walls of the arterioles. This finding, and confirmation of the results in an in vitro coculture model, suggests that a parallel process is important for enabling cytotrophoblast endovascular invasion during human pregnancy. Cytotrophoblast invasion of the kidney parenchyma was accompanied by a robust lymphangiogenic response, while in vitro, the cells stimulated lymphatic endothelial cell migration via the actions of VEGF family members, FGF, and TNF-α. Immunolocalization analyses revealed that human pregnancy is associated with lymphangiogenesis in the decidua since lymphatic vessels were not a prominent feature of the nonpregnant endometrium. Thus, the placenta triggers the development of a decidual lymphatic circulation, which we theorize plays an important role in maintaining fluid balance during pregnancy, with possible implications for maternal-fetal immune cell trafficking.
doi:10.1172/JCI27306
PMCID: PMC1570373  PMID: 16998586

Results 1-8 (8)