Diagnosis and treatment of osteoporosis before and after admission to long-term care institutions.

SUMMARY: Bisphosphonate treatment rates were examined before and after admission to long-term residential care. Bisphosphonate treatment rates were low (16%) pre-admission but doubled after long-term residential care admission (30%). Men were very undertreated for osteoporosis, while a history of falls with injury was not associated with treatment. INTRODUCTION: To determine the rates and independent correlates of bisphosphonate treatment in elderly residents before and after admission to long-term care (LTC) institutions. METHODS: Information was collected from records of 421 residents of four LTC institutions in Edmonton, Alberta, Canada. Osteoporosis-related diagnoses, treatments, and risk factors including falls in LTC and any adulthood fractures were abstracted. Osteoporosis was defined by physician diagnosis or documented fractures of the hip, spine, or upper extremity. Multivariable analyses were undertaken to determine factors independently associated with bisphosphonate treatment. RESULTS: Mean age was 84 ± 8 years and 290 (70%) were female. Overall, 142 (34%) had previous fractures, 170 (41%) had physician-diagnosed osteoporosis, and 227 (54%) residents met the study's clinical definition of osteoporosis. Of those with osteoporosis, 44 (19%) were men. Before admission, 36 (16%) patients with osteoporosis were treated with bisphosphonates; after admission another 31 (14%) were started on bisphosphonates by LTC physicians. Women were far more likely than men to start bisphosphonate treatment [30 (97%) women vs. 1 (3%) man, adjusted odds ratio (aOR) = 9.20 (95% confidence intervals 1.2,70.5)]. Falls with injury were common [72/227 (31%)] but not associated with bisphosphonate treatment (adjusted p value > 0.5). CONCLUSION: Rates of pre-admission bisphosphonate treatment were low, but did double after LTC admission. Women were almost ten times more likely to start bisphosphonate treatment than men, although one fifth of those with documented osteoporosis were men. Although falls cause most fractures, a history of falls with injury was not associated with bisphosphonate treatment. Our findings suggest that targeting men and residents with falls for treatment with bisphosphonates might be warranted.

Oral bisphosphonates are associated with reduced mortality after hip fracture.

UNLABELLED: Intravenous bisphosphonates reduce mortality following hip fracture. We determined whether new use of oral bisphosphonates was also associated with reductions in mortality in 209 hip fracture patients. Oral bisphosphonate exposure led to relative reduction of 8% per month of use (p = 0.001) or about a 60% reduction in mortality per year of use. INTRODUCTION: Intravenous bisphosphonates reduce mortality following hip fracture. Using prospectively collected long-term data from a randomized trial of osteoporosis quality improvement for hip fracture, we determined whether new use of oral bisphosphonates was associated with reductions in mortality or the composite outcome of death or new fracture. METHODS: Originally, 220 hip fracture patients were randomized to case manager (n = 110) or usual care followed by facilitated bone mineral density (BMD) testing (n = 110) interventions. All were eligible for bisphosphonate treatment. Post-randomization, we followed patients for 3 years and ascertained bisphosphonate treatment, medication adherence and persistence, all-cause mortality, and new clinical fractures. Proportional hazards analyses with time-varying treatment status were undertaken. RESULTS: The final study cohort included 209 patients: 136 (65%) females, 104 (50%) older than 75 years, 90 (43%) with poor self-reported health, and 38 (18%) underweight. Of these, 76 (36%) had a previous fracture before hip fracture and 132 (81%) had low BMD. A total of 101 (46%) patients started oral bisphosphonates and 65 (64%) remained on treatment at the final evaluation. Overall, 24 (11%) patients died, 19 (9%) had new fractures, and 42 (20%) reached the composite outcome of death or fracture. Compared to no treatment, bisphosphonate exposure was independently associated with reduced mortality (17[16%] vs. 7[7%]; adjusted hazard ratio (aHR) = 0.92 per month treated; 95%CI, 0.88-0.97) and composite endpoints (28[26%] vs. 5[15%]; aHR = 0.94 per month treated; 95%CI, 0.91-0.97). CONCLUSION: Like intravenous bisphosphonates after hip fracture, our study suggests that oral bisphosphonates may be associated with reductions in all-cause mortality.

Cost-effectiveness of a multifaceted intervention to improve quality of osteoporosis care after wrist fracture.

UNLABELLED: In a randomized trial, a multifaceted intervention tripled rates of osteoporosis treatment in older patients with wrist fracture. An economic analysis of the trial now demonstrates that the intervention tested "dominates" usual care: over a lifetime horizon, it reduces fracture, increases quality-adjusted life years, and saves the healthcare system money. INTRODUCTION: In a randomized trial (N = 272), we reported a multifaceted quality improvement intervention directed at older patients and their physicians could triple rates of osteoporosis treatment within 6 months of a wrist fracture when compared with usual care (22% vs 7%). Alongside the trial, we conducted an economic evaluation. METHODS: Using 1-year outcome data from our trial and micro-costing time-motion studies, we constructed a Markov decision-analytic model to determine cost-effectiveness of the intervention compared with usual care over the patients' remaining lifetime. We took the perspective of third-party healthcare payers. In the base case, costs and benefits were discounted at 3% and expressed in 2006 Canadian dollars. One-way deterministic and probabilistic sensitivity analyses were conducted. RESULTS: Median age of patients was 60 years, 77% were women, and 72% had low bone mineral density (BMD). The intervention cost $12 per patient. Compared with usual care, the intervention strategy was dominant: for every 100 patients receiving the intervention, three fractures (one hip fracture) would be prevented, 1.1 quality-adjusted life year gained, and $26,800 saved by the healthcare system over their remaining lifetime. The intervention dominated usual care across numerous one-way sensitivity analyses: with respect to cost, the most influential parameter was drug price; in terms of effectiveness, the most influential parameter was rate of BMD testing. The intervention was cost saving in 80% of probabilistic model simulations. CONCLUSIONS: For outpatients with wrist fractures, our multifaceted osteoporosis intervention was cost-effective. Healthcare systems implementing similar interventions should expect to save money, reduce fractures, and gain quality-adjusted life expectancy.

Nurse case-manager vs multifaceted intervention to improve quality of osteoporosis care after wrist fracture: randomized controlled pilot study.

UNLABELLED: Few outpatients with fractures are treated for osteoporosis in the years following fracture. In a randomized pilot study, we found a nurse case-manager could double rates of osteoporosis testing and treatment compared with a proven efficacious quality improvement strategy directed at patients and physicians (57% vs 28% rates of appropriate care). INTRODUCTION: Few patients with fractures are treated for osteoporosis. An intervention directed at wrist fracture patients (education) and physicians (guidelines, reminders) tripled osteoporosis treatment rates compared to controls (22% vs 7% within 6 months of fracture). More effective strategies are needed. METHODS: We undertook a pilot study that compared a nurse case-manager to the multifaceted intervention using a randomized trial design. The case-manager counseled patients, arranged bone mineral density (BMD) tests, and prescribed treatments. We included controls from our first trial who remained untreated for osteoporosis 1-year post-fracture. Primary outcome was bisphosphonate treatment and secondary outcomes were BMD testing, appropriate care (BMD test-treatment if bone mass low), and costs. RESULTS: Forty six patients untreated 1-year after wrist fracture were randomized to case-manager (n = 21) or multifaceted intervention (n = 25). Median age was 60 years and 68% were female. Six months post-randomization, 9 (43%) case-managed patients were treated with bisphosphonates compared with 3 (12%) multifaceted intervention patients (relative risk [RR] 3.6, 95% confidence intervals [CI] 1.1-11.5, p = 0.019). Case-managed patients were more likely than multifaceted intervention patients to undergo BMD tests (81% vs 52%, RR 1.6, 95%CI 1.1-2.4, p = 0.042) and receive appropriate care (57% vs 28%, RR 2.0, 95%CI 1.0-4.2, p = 0.048). Case-management cost was $44 (CDN) per patient vs $12 for the multifaceted intervention. CONCLUSIONS: A nurse case-manager substantially increased rates of appropriate testing and treatment for osteoporosis in patients at high-risk of future fracture when compared with a multifaceted quality improvement intervention aimed at patients and physicians. Even with case-management, nearly half of patients did not receive appropriate care. TRIAL REGISTRY: clinicaltrials.gov identifier: NCT00152321.

Mechanisms of endocrine dysfunction in patients with testicular cancer.

To determine mechanisms of endocrine dysfunction in patients with testicular cancer, we performed static and dynamic testing of the hypothalamic-pituitary-testicular axis and testicular exocrine function in 13 patients and 11 normal control subjects, as well as in vitro studies of tumor tissue and remaining adjacent "normal" testicular tissue in the 13 patients. In tumor tissue, we demonstrated (a) elevated concentrations of total serum estradiol and serum estradiol not bound to sex hormone-binding globulin, (b) impaired spermatogenesis and sperm motility, and (c) blocking of multiple enzymes necessary for steroidogenesis. The data were consistent with a paracrine-endocrine mechanism in which tumor-produced human chorionic gonadotropin stimulates production of estradiol by "normal" testicular tissue but not tumor tissue, and the high estradiol levels then result in impaired spermatogenesis.

Gynecomastia after cytotoxic therapy for metastatic testicular cancer.

Four men with metastatic nonseminomatous testicular cancer, who had received combination chemotherapy, experienced transient gynecomastia. Serum hormone levels were measured during and after the resolution of the gynecomastia. These levels indicated toxic effects on Leydig's cells and germinal epithelium concurrent with raised serum estradiol levels in the absence of tumor. The gynecomastia may have been caused by Leydig's cell dysfunction or refeeding after substantial weight loss. This benign form of gynecomastia must be recognized to avoid unnecessary investigation and treatment.

Transforming growth factor beta 1 inhibits placental differentiation and human chorionic gonadotropin and human placental lactogen secretion.

Previously, no inhibitors of placental differentiation have been described. In this study, we determined the effect of transforming growth factor beta 1 (TGF beta 1) on cytotrophoblast differentiation. Monolayer cultures of pure cytotrophoblasts were exposed to 0.001-10 ng/ml TGF beta 1 with and without the presence of 10 ng/ml epidermal growth factor (EGF), an inducer of placental differentiation. Over 7 days of culture, in 11 separate experiments, phase contrast microscopy demonstrated marked inhibition of EGF-induced syncytial formation by TGF beta 1. Basal human (h)CG and h-placental lactogen (PL) release were reduced compared to control by fractions of 0.75 (TGF beta 1/control) and 0.54, respectively. EGF alone induced fractional (EGF/control) increases in hCG and hPL release of 2.46 and 2.68, respectively. However, this stimulation was significantly inhibited by 10 ng/ml TGF beta 1. Dose-response studies showed that maximal TGF beta 1 inhibition of EGF-stimulated hormone secretion occurred at 0.1 ng/ml or more TGF beta 1. Partial differentiation (syncytium formation) occurred despite the presence of TGF beta 1, suggesting a portion of cytotrophoblasts were committed to differentiation at the time of culture. We conclude that TGF beta 1 acts as a major inhibitor of trophoblast differentiation and concomitant peptide hormone secretion.

The dual effect of epidermal growth factor upon human chorionic gonadotropin secretion by the first trimester placenta in vitro.

Epidermal growth factor (EGF) is believed to play an important role in the regulation of placental function. We have examined the effect of EGF upon first trimester (7-10 gestational weeks) placental hCG secretion and cellular differentiation using both static (explants and isolated cells) and kinetic (superfusion of explants) culture methods. In superfused explants, short (1-4 min) pulses of EGF increased both the rate and amplitude of spontaneous pulsatility of hCG. The frequency increased from 3/h to 5/h, and the amplitude increased compared to the control channels as calculated by the area under the curve. This effect was dose dependent and the concentration of 50 ng/ml, which was the lowest dose tested, was the most effective. In explants cultured for 24 h, EGF caused a 2-fold increase in hCG secretion, compared to control, P less than 0.05. In two different dispersed trophoblastic cell cultures, EGF added daily for the first week caused a 180% increase in hCG secretion, P less than 0.05. However, according to morphological criteria, i.e. light microscopy and vital staining, no significant effect upon the rate of differentiation to syncytiotrophoblast was observed in long-term cultures of one of these preparations. In conclusion, EGF plays a dual trophic role in stimulating hCG secretion in the first trimester. However, this effect is not dependent on cellular differentiation.

Epidermal growth factor induces differentiation and secretion of human chorionic gonadotropin and placental lactogen in normal human placenta.

Human trophoblast differentiates by the fusion of cytotrophoblasts to form syncytiotrophoblast. To determine factors controlling this process, the effects of epidermal growth factor (EGF) on trophoblast differentiation were studied using long term serum-free culture of isolated trophoblast. Only trophoblast was present in the cultures, as demonstrated by positive immunoperoxidase staining with beta hCG, cytokeratin, and trophoblast-specific H315 monoclonal antisera and by the absence of contaminating endothelial cells, fibroblasts, and macrophages, as shown by negative staining with vimentin and OKM1 monoclonal antisera. EGF induced large sustained increases in hCG and human placental lactogen (hPL) secretion in a dose-dependent manner. The minimum effective dose was 0.1 ng/mL, and the maximum effective dose was 1 ng/mL. Light and electron microscopic studies showed EGF-induced differentiation of cytotrophoblast to form syncytiotrophoblast. DNA content and cell number did not change during the process. The formation of syncytia thus probably accounted for the increase in hCG and hPL secretion. We conclude that EGF causes morphological differentiation, but not cell proliferation, of trophoblasts, and the differentiation results in increased hCG and hPL secretion from the syncytia.

Life and death in the placenta: new peptides and genes regulating human syncytiotrophoblast and extravillous cytotrophoblast lineage formation and renewal.

Differential techniques have revealed several novel genes and peptides involved in trophoblast development including PL74/gdf15/MIC-1, a TGFbeta family cytokine that controls apoptosis and differentiation, PL48, a new serine-threonine protein kinase, serum and glucocorticoid-induced kinase, PBK-1, a tunicamycin-responsive gene, a cathepsin D-like gene (DAP-1) and hypoxia- regulated genes HRF-1,2,6,8 and HIF-1alpha, HIF-1beta, and hEPAS-1. Syncytin, a cell fusion- inducing gene, has been cloned from placenta where it regulates cell fusion. ERV-3 has also been demonstrated to promote cell fusion. These two genes represent the first demonstrated functions of endogenous retroviral sequences in human tissues. Endoglin, PlGF, TGFbeta3, IGF-II, IGFBP-1, and a placental IGFBP protease have found new roles in regulating cytotrophoblast proliferation and invasiveness. A specific placental p105 rasGAP protein has been identified. The homeobox genes DLX4, HB24, MSX2 and MOX2 also likely play a role in development at the epithelial-mesenchymal boundary. Transcription factors such as TEF-5, Hand1, HEB, HASH-2 and two genes represented by ESTs may have regulatory roles in placental development. Evidence suggests that the placenta has an unusual two-cell system for apoptosis regulation in which the cytotrophoblast may direct later apoptotic events in the syncytium, and with syncytialization possibly triggered by the "phosphatidylserine flip". Thus, the placenta is both a rich source of new growth-regulatory substances, and a model system for originating new paradigms of developmental biology.

PL48: a novel gene associated with cytotrophoblast and lineage-specific HL-60 cell differentiation.

The cDNA for a novel gene, PL48, isolated by subtractive hybridization between undifferentiated human term cytotrophoblast and differentiating cytotrophoblast, has been cloned and sequenced. PL48 contains an open reading frame coding for a 537-amino acid protein, has multiple potential PKC, casein kinase II, and cAMP/cGMP-dependent kinase phosphorylation sites, and N-linked glycosylation sites. It is not present in a wide variety of proliferating cancer cells, but PL48 mRNA shows marked expression during cytotrophoblast and granulocyte lineage-specific HL-60 promyelocytic cell differentiation induced by DMSO.

Demonstration of specific secretory granules for human chorionic gonadotropin in placenta.

Existence of secretory granules and exocytosis during secretion of human chorionic gonadotropin (hCG) in human placenta has been a point of controversy. Using two methods, the highly sensitive avidin-biotin complex (ABC) method and the protein A-gold technique, for immunochemical identification of beta-hCG on electron microscopic sections, we have examined placentas at 8-10 weeks gestation and at term for the presence of secretory granules. First-trimester placentas demonstrated plentiful syncytiotrophoblast cytoplasmic granules, some undergoing exocytosis, when stained using specific beta-hCG antiserum in the ABC and protein A-gold methods. Term placentas did not show positive reaction product. The data demonstrate that the classic secretory granule-exocytosis pathway mediates placental hCG secretion. However, clear morphological differences exist between placenta granules and hormone secretory granules observed in pituitary, consistent with known functional differences between these organs. This methodology will be useful for further studies of the secretory pathways for placental peptides.

Ultrastructural localization of human placental lactogen in distinctive granules in human term placenta: comparison with granules containing human chorionic gonadotropin.

Human placental lactogen (hPL) is known to originate in the syncytiotrophoblast, as demonstrated by light microscopic peroxidase and immunofluorescent staining. However, ultrastructural localization of hPL has not previously been performed. In these experiments, immunostaining of electron microscopic sections using protein A-gold and avidin-biotin complex techniques was used to study hPL and human chorionic gonadotropin (beta hCG) localization in first trimester and term placentae. HPL was localized in many small (0.12-0.25 micron) granules. In contrast, beta hCG was found in large (0.40-1.2 micron) granule complexes. The results therefore demonstrate that these two hormones are stored in two morphologically distinct types of cytoplasmic granules. Since hPL and hCG have different secretory mechanisms, this methodology will be useful in studying these differing mechanisms in human placenta.

EGF promotes development of a differentiated trophoblast phenotype having c-myc and junB proto-oncogene activation.

Human placental cytotrophoblast cells differentiate by a process of fusion into a syncytium. This process is stimulated by EGF but also occurs spontaneously at a slower rate in cultured cytotrophoblast cells. To determine nuclear proto-oncogene changes mediating these events, c-myc, c-fos, c-jun and junB were measured in spontaneously differentiating cells and in cells exposed to EGF. c-myc showed a transient rise in expression at 4-8 h with augmented expression by EGF, occurring even in the absence of serum or attachment. c-myc and c-jun declined during culture, but c-fos and particularly junB showed increased expression by day 3 with marked responses to EGF stimulation. Syncytia induced to form by EGF exposure for 48 h demonstrated marked junB expression after rechallenge with 40 min EGF exposure, but negligible responses of c-fos and c-jun. c-myc showed increased expression after 6 h EGF exposure throughout the culture period and in syncytia. The results indicate EGF promotes a syncytial phenotype characterized by c-fos and junB expression during syncytial formation. EGF continues to elicit junB and c-myc responsiveness in more mature syncytium, indicative of continued EGF actions which may include acting as a survival factor, as an hCG secretagogue, and as an inducer of continued development of the syncytium.

The role of Bcl-2 expression in EGF inhibition of TNF-alpha/IFN-gamma-induced villous trophoblast apoptosis.

The inflammatory cytokines tumour necrosis factor alpha (TNF-alpha) and immune interferon gamma (IFN-gamma) stimulate villous cytotrophoblast apoptosis while epidermal growth factor (EGF) protects. We hypothesize that TNF-alpha, IFN-gamma and EGF regulate apoptosis in part by modulating cellular expression levels of the anti-death gene bcl-2. While Bcl-2 is reported to be strongly expressed in villous syncytiotrophoblasts, it is not known whether the protein is expressed in cultured villous cytotrophoblasts (CT) and, if so, whether it is functional. We show by Northern blot analysis that bcl-2 mRNA is expressed in cultured CT and by immunoblot analysis that the protein is strongly expressed in highly purified first trimester and term villous cytotrophoblasts. The expression levels of Bcl-2 protein were the same in first trimester and term cytotrophoblasts. Culture with TNF-alpha/IFN-gamma and EGF did not alter expression of either Bcl-2 protein or of the pro-apoptotic Bcl-2 family member Bak. Double label flow cytometric analysis that measured apoptosis and Bcl-2 content simultaneously showed that cells expressing low levels of Bcl-2 underwent TNF-alpha/IFN-gamma-induced apoptosis at a higher frequency than cells expressing lower levels. We conclude that Bcl-2 is expressed in cytotrophoblasts, that its expression is constitutive and that modulation of its expression levels does not mediate cytokine and growth factor regulation of apoptosis in these cells.

Preparation and functional characterization of villous cytotrophoblasts free of syncytial fragments.

Recent studies suggest that purified villous cytotrophoblasts are largely contaminated by mononucleated syncytial fragments and therefore unsuitable for studies of trophoblast differentiation. We assessed highly purified (>99.99 per cent) populations of villous trophoblasts for fragment contamination using the syncytial markers placental alkaline phosphatase (PLAP, by immunohistochemistry) and exteriorized phosphatidyl serine (ePS, by flow cytometric analysis). The preparations contained from 4-46 per cent syncytial fragments. However, we find that PLAP negative cells preferentially adhere to tissue culture surfaces and that all preparations were <2 per cent PLAP positive after routine plating and washing procedures. A second purification procedure eliminated dead (propidium iodide permeable) cells and separated viable syncytial fragments (ePS-positive) from viable cytotrophoblasts (ePS-negative) by two colour fluorescence activated cell sorting (FACS). Viable ePS-positive cells were ultrastructurally apoptotic, adhered poorly in culture and those that adhered rapidly underwent apoptosis. Viable ePS-negative cells contained large heterochromic nuclei and cytoplasmic structures, adhered strongly in culture and remained viable. The latter population (putative true villous CT) differentiated into syncytialized cells when cultured with EGF. We conclude that villous CT can be routinely purified, are viable in culture and can undergo syncytial fusion without extensive preformed syncytium.

Functional, long-term cultures of human term trophoblasts purified by column-elimination of CD9 expressing cells.

We have extended previous observations of expression of the trypsin-resistant cell surface antigen CD9 on placental fibroblasts to virtually all cells in the villous stroma and developed a method for eliminating CD9 expressing cells from trypsinized placental preparations. Preparations incubated with the mouse anti-human CD9 monoclonal antibody 50H.19 were passed through a goat anti-mouse immunoglobulin column that captures CD9 expressing cells. Approximately 95 per cent of the eluted cells stained positive with the villous trophoblast specific antibody GB25 and could be cryopreserved and thawed with > 80 per cent recovery in culture. One week cultures contained fewer than 0.3 per cent vimentin positive (mesenchymal) cells and maintained secretion of hCG. Two week cultures remained free of fibroblasts and macrophages. Clusters of trophoblasts that formed spontaneously during the first week of culture were shown by microinjection of carboxyfluorescein and by staining with anti-desmoplakin antibody to be a patchwork of mononuclear cells and syncytial units. Although the DNA content of the culture decreased by 35 per cent during the 2 week culture, the metabolic capacity and protein content remained relatively constant. Thus, CD9 immuno-elimination gives a high yield of enriched and viable trophoblasts that can be cultured for at least 2 weeks with almost no contamination by stromal cells.

In vitro cultured human term cytotrophoblast: a model for normal primary epithelial cells demonstrating a spontaneous differentiation programme that requires EGF for extensive development of syncytium.

Normal human term cytotrophoblast cells prepared by trypsin-DNAse I digestion with and without secondary immunological purification with CD9 antibodies were investigated for the expression of morphological and genetic markers of proliferation and differentiation. After 24 h of culture, the cell preparations demonstrated spontaneous formation of microvilli and formation of small syncytial units as assessed by desmoplakin staining and FITC-dextran microinjection. EGF was required for mature syncytial formation. Compared to log-phase proliferating HeLa cells, uptake of [3H]thymidine incorporation was low and quickly decreased to negligible levels. Expression of the proto-oncogenes c-myc, c-fos, and c-jun and histone 2A decreased rapidly in the first 24 h of culture in both cell preparations, followed by an increase in expression of c-fos and junB over the next 3 days of culture. Proto-oncogene changes were similar in attached and suspension cells. Spontaneous increases in alpha hCG, pregnancy-specific beta(1)-glycoprotein and 3 beta-hydroxysteroid dehydrogenase (3 beta OHSD) occurred within 1 day in both cell preparations. EGF receptor blocking antibodies did not inhibit minor degrees of spontaneous syncytial formation nor inhibit spontaneous expression of alpha hCG or 3 beta OHSD mRNA, but did prevent extensive synctialization induced by EGF. The results demonstrate that term cytotrophoblast cells even in serum-free conditions or suspension culture rapidly commit to a non-proliferative differentiation program in culture which includes limited syncytialization and marked hormone mRNA expression. However, EGF is required for extensive syncytial development.

Identification by subtractive hybridization of a spectrum of novel and unexpected genes associated with in vitro differentiation of human cytotrophoblast cells.

We have previously demonstrated that epidermal growth factor (EGF), colony stimulating factor-1 (CSF-I), and granulocyte-monocyte colony stimulating factor (GMCSF) stimulate, while transforming growth factor beta 1 (TGF beta 1) inhibits, cytotrophoblast differentiation. To identify genes mediating EGF induced differentiation, we constructed a subtracted cDNA library between undifferentiated cytotrophoblast and differentiating cytotrophoblast. We identified six novel genes and four known syncytial products alpha-human chorionic gonadotrophin (alpha hCG) pregnancy-specific beta 1-glycoprotein, 3 beta-hydroxysteroid dehydrogenase, and plasminogen activator inhibitor type 1 whose mRNAs increased during differentiation. Ten other genes were identified whose mRNAs increased during differentiation. Five of these (keratin 19, calcreticulin, heat shock protein 27, serum and glucocorticoid-regulated kinase and adrenomedullin) were not previously reported to be expressed in placenta. Five other genes known to be expressed in placenta were identified. keratin 8, fibronectin, mitochondrial ATP synthase, 1119, and cytosolic copper-zinc superoxide dismutase (SOD-1). Several of these genes may have regulatory functions in trophoblast differentiation.

Cloning of PL33: a novel probable serpentine membrane receptor associated with human cytotrophoblast and lineage-specific HL-60 cell differentiation.

From a subtracted library of differentiating human cytotrophoblast cells, we have cloned a gene termed PL33, a novel probable member of the G-protein-linked seven transmembrane domain receptor family. The 496 amino acid open reading frame of PL33 codes for a protein of predicted molecular weight of 54.7 kDa. Northern blot analysis shows that the two mRNA transcripts of PL33 are expressed in testes, keratinocytes, differentiating cytotrophoblast and in monocyte/macrophage lineage-specific HL-60 cell differentiation.