Hepcidin, soluble transferrin receptor and IL-6 levels in obese children and adolescents with and without type 2 diabetes mellitus/impaired glucose tolerance and their association with obstructive sleep apnea.

PURPOSE: Obesity, type 2 diabetes mellitus (T2DM), and obstructive sleep apnea (OSA) are associated with chronic low-grade inflammation. Iron metabolism is linked with insulin-resistant states and with OSA in adults. The association of body iron status with T2DM in children remains undefined. We aimed to evaluate plasma interleukin-6 (IL-6), hepcidin, and soluble transferrin receptor (sTfR) levels in obese patients with T2DM or impaired glucose tolerance (IGT) and in those without, and the contribution of OSA to their levels. METHODS: In this cross-sectional study, obese children and adolescents with and without T2DM/IGT underwent overnight polysomnography. Fasting plasma concentrations of IL-6, hepcidin, and sTfR were measured and evaluated according to glycemic status (T2DM/IGT and normal glucose tolerance) and the presence of OSA. RESULTS: Ten patients with T2DM (age 15.9 ± 3.6 years), 8 with IGT (age 13.1 ± 2.5 years) and 20 subjects with normal glucose tolerance matched for body mass index standard deviation score (age 12.6 ± 3.3 years) were studied. Sleep measures or IL-6, hepcidin, and sTfR levels were not significantly different between the group with T2DM/IGT vs. the control group. No significant differences were found in hepcidin or sTfR levels between patients with OSA and those without. However, patients with OSA showed higher plasma IL-6 values compared with those without (4.56 ± 2.92 vs. 2.83 ± 1.54 pg/ml, P = 0.025), and the highest values were evident in patients affected by both T2DM/IGT and OSA. CONCLUSIONS: Higher IL-6 levels were associated with both glycemic status and OSA. No differences in body iron regulator levels were found in obese patients with T2DM/IGT compared to those without or in those with OSA compared to those without. Further longitudinal studies in larger population samples are warranted.

Differential regulation of the apoptotic machinery during megakaryocyte differentiation and platelet production by inhibitor of apoptosis protein Livin.

Livin is a member of the inhibitor of apoptosis proteins (IAP) family of intracellular antiapoptotic proteins that act by binding and inhibiting caspases. Upon strong apoptotic stimuli, it is then specifically cleaved by caspases to produce a truncated protein (tLivin) with a paradoxical proapoptotic activity. Intriguingly, we have detected robust protein levels of Livin in normal mature bone marrow megakaryocyte (MK) and platelets. To evaluate the potential role of Livin in thrombopoiesis, we used the human BCR-ABL+ cell line, LAMA-84, and cord blood CD34+ cells to induce differentiation toward MKs. Upon differentiation, induced by phorbol myristate acetate and concurrent with increase in Livin protein expression, LAMA-84 cells formed functional platelet-like particles. Livin overexpression in CD34+ progenitor cells induced higher endoreplication in the MKs generated. Furthermore, overexpression of Livin increased the ability of both primary MKs and differentiated LAMA-84 cells to produce functional platelets. In the differentiated LAMA-84 cells, we observed accumulation of proapoptotic tLivin concomitant with increased caspase-3 activity. Downregulation of Livin with small interfering RNA in both leukemic and primary MK cells decreased their ability to produce functional platelets. We suggest that Livin has a role in thrombopoiesis by regulating the apoptotic and antiapoptotic balance in MK endoreplication and platelet production.

Rapid in vivo testing of drug response in multiple myeloma made possible by xenograft to turkey embryos.

BACKGROUND: The best current xenograft model of multiple myeloma (MM) in immune-deficient non-obese diabetic/severe-combined immunodeficient mice is costly, animal maintenance is complex and several weeks are required to establish engraftment and study drug efficacy. More practical in vivo models may reduce time and drug development cost. We recently described a rapid low-cost xenograft model of human blood malignancies in pre-immune turkey. Here, we report application of this system for studying MM growth and the preclinical assessment of anticancer therapies. METHODS: Cell lines and MM patient cells were injected intravenously into embryonic veins on embryonic day 11 (E11). Engraftment of human cells in haematopoietic organs was detected by quantitative real-time polymerase chain reaction, immunohistochemistry, flow cytometry and circulating free light chain. RESULTS: Engraftment was detected after 1 week in all embryos injected with cell lines and in 50% of those injected with patient cells. Injection of bortezomib or lenalinomide 48 h after cell injection at therapeutic levels that were not toxic to the bone marrow dramatically reduced MM engraftment. CONCLUSION: The turkey embryo provides a practical, xenograft system to study MM and demonstrates the utility of this model for rapid and affordable testing therapeutics in vivo. With further development, this model may enable rapid, inexpensive personalised drug screening.

Increased cord serum inflammatory markers in small-for-gestational-age neonates.

OBJECTIVE: The pathological picture in ischemic tissue injury shares features with the inflammatory response. Hypoxia-mediated induction of interleukin-6 (IL-6) could set in motion the mechanisms limiting inflammation in ischemia. Intrauterine growth restriction (IUGR) represents a human model of chronic fetal hypoxia. The purpose of this study was a first-time exploration to determine whether cord blood obtained at the delivery of small-for-gestational-age (SGA) infants has increased concentrations of inflammatory markers. STUDY DESIGN: Cord blood was collected from 20 SGA (term and near-term) infants and 20 appropriate-for-gestational-age (AGA) controls. Infants exposed to maternal smoking, diabetes, maternal chronic diseases, or alcohol or drug use were excluded. Both groups had Apgar score ≥7 at 1 min with a normal cord pH (>7.25). Cord-serum cytokines and thrombopoietin (TPO) levels were measured by enzyme linked immunosorbent assay. C-reactive protein (CRP) was measured using a turbidometric immunoassay. RESULT: SGA infants had a significantly smaller birth weight than AGA controls, with a smaller gestation age by 1 week. There were significant elevations in IL-6, tumor necrosis factor (TNF-α), CRP and TPO in the SGA compared with the AGA group, which persisted in multiple regression analysis even after gestational age was taken into account. CONCLUSION: As hypothesized, significant increases in the cord blood concentrations of known inflammatory markers were found in SGA infants compared with the controls.

Prohepcidin concentrations and erythroid progenitors in cord blood of appropriate versus small for gestational age neonates.

OBJECTIVE: Prohepcidin (Pro-Hep), synthesized in the liver, is the prohormone of hepcidin (Hep), which reduces iron absorption in the gut; its synthesis is enhanced by inflammation and is reduced during hypoxia. We aimed to study the hypothesis that infants born small for gestational age (SGA) have reduced cord blood concentrations of Pro-Hep. STUDY DESIGN: Cord blood was collected from 20 SGA (term and near term >35 week gestation) infants and 20 appropriate for gestational age (AGA) controls. We excluded infants exposed to maternal chronic diseases, smoking, diabetes, alcohol or drug use. Both groups had a 1 min Apgar score above or equal to 7 and had normal cord blood pH (above 7.25). ELISA was used to determine serum concentrations of Pro-Hep and erythropoietin (EPO). Circulating CD71(+)/CD45(-)/SSC(low) cells were measured by flow cytometry as an index of erythroid progenitors. RESULT: There were no significant differences between groups in terms of hemoglobin concentrations, and Pro-Hep. In contrast, EPO levels and circulating CD71(+)/CD45(-)/SSC(low) erythroid progenitors were significantly higher in the SGA group. These differences remained significant even after controlling for gestational age and gravidity. CONCLUSION: Contrary to EPO upregulation during intrauterine growth restriction (IUGR), and higher concentrations of circulating erythroid progenitors, Pro-Hep concentration is not affected by IUGR.

The CXCR4 antagonist AMD3100 impairs survival of human AML cells and induces their differentiation.

The chemokine stromal cell-derived factor-1 (SDF-1) and its receptor, CXCR4, participate in the retention of acute myeloblastic leukemia (AML) cells within the bone marrow microenvironment and their release into the circulation. AML cells also constitutively express SDF-1-dependent elastase, which regulates their migration and proliferation. To study the molecular events and genes regulated by the SDF-1/CXCR4 axis and elastase in AML cells, we examined gene expression profiles of the AML cell line, U937, under treatment with a neutralizing anti-CXCR4 antibody or elastase inhibitor, as compared with non-treated cells, using DNA microarray technology. Unsupervised hierarchical clustering analysis demonstrated similar gene expression profiles of anti-CXCR4 antibody or elastase inhibitor-treated cells, as compared with control. Pathway and functional analysis showed a greater tendency toward differentiation in cells under either one of both treatment modalities. Thus given, we further analyzed the effects of CXCR4 inhibition on AML cell growth and differentiation using the antagonist AMD3100. AMD3100 arrested proliferation in AML cell lines and triggered changes that mimicked differentiation, including morphological changes and the expression of myeloid differentiation antigens. Inhibition of elastase also triggered the differentiation of AML cells. Our study defines a new role for the SDF-1/CXCR4 axis in the regulation of leukemic cell survival and differentiation.

[Stress urinary incontinence and suburetral slings. Implications for sexuality]. / Incontinence urinaire d'effort et bandelette sous-urétrale: implications sur la sexualité

The impact of stress urinary incontinence (SUI) is not limited to physical and psychological consequences classically evaluated for this disease. In fact, some studies emphasize the indisputable existence of sexual disorders directly imputable to SUI. Sexual function is an important evaluation element before and after surgical treatment of SUI. Many validated questionnaires about sexual disorders exist, the most frequently utilized in present literature being the female sexual function index (FSFI). Suburethral slings, in spite of discordant results, do not seem to impair global sexuality of patients, some authors reporting a benefit effect of surgery, related to a decrease of coital incontinence after surgery and a decrease of apprehension before sexual intercourse.

[Obstetric management after ectopic pregnancy in the caesarean section scar: a case report and review of literature]. / Prise en charge obstétricale après grossesse ectopique intracicatricielle: à propos d'un cas et revue de la littérature.

We report the case of a patient who came out pregnancy twelve months after medical and surgical treatment of an ectopic pregnancy in a previous caesarean section scar. The preconceptional management consisted in a saline infusion sonohysterography and a pelvic magnetic resonance imaging. Judging the risks of abnormal placental insertion to be higher in this case compared to a simple caesarean section, a careful ultrasonography with color doppler imaging was carried out. The myometrium fragility caused by the ectopic pregnancy in the caesarean section brought us to recommend a prophylactic caesarean section around 37. The high risks of hemorrhage required a medical center with embolization possibilities. A review of literature in order to define the medical care adapted in this case was come out.

[Are there any factors predicting failure or complications rates of trans-obturator surgery for stress urinary incontinence?]. / Existe-t-il des facteurs prédictifs d'échec ou de complications dans la chirurgie de l'incontinence urinaire d'effort par voie transobturatrice?

OBJECTIVE: Since 2001 and the publication by Delorme of the trans-obturator route in the stress urinary incontinence (SUI), this technique has known an increasing development in France. The aim of this study is to evaluate the impact of different predicting factors on results and complications of trans-obturator surgery. PATIENTS AND METHODS: It is a retrospective, multicentric study, including 4 centers, 14 surgeons and 196 patients operated between February 2003 and August 2005. We have realized a univariate (Chi2 test) and multivariate (logistic regression test) statistic analysis concerning 7 sub-groups defined according to the literature on the TVT. RESULTS: Age>55 years (P=0,044) and SUI grade>2 (P=0,028) are statistically associated with a decrease of surgical success, age>55 years is also associated with an increase of complications rate in univariate (P=0,033) and multivariate (P=0,048) analysis. DISCUSSION AND CONCLUSION: Age>55 years should be considered, according to us, as a risk factor of surgical failure and complications in the trans-obturator surgery for SUI, none of the others risk factors found in the literature on the TVT seems to have an influence, in this study, on the results of trans-obturator surgery for SUI.

The prothrombin 20209 C-->T mutation in Jewish-Moroccan Caucasians: molecular analysis of gain-of-function of 3' end processing.

BACKGROUND: Mutations of the 3' end mRNA-processing signal of the prothrombin (F2) gene have been reported to cause elevated F2 plasma concentrations, thrombosis, and complications of pregnancy. Whereas the common F2 20210*A mutation is almost exclusively found in Caucasians, the F2 20209*T mutation has been reported in Afro-Americans and Afro-Caribbeans only. PATIENTS AND METHODS: Using LightCycler technology, three unrelated Jewish-Moroccan patients tested for obstetric complications were found to be carriers of the F2 20209*T allele. A detailed molecular analysis was performed to identify the functional impact of this mutation. RESULTS: We report three unrelated women of Jewish-Moroccan origin with a F2 20209*T mutation and fetal loss or infertility. The functional analysis revealed that the F2 20209*T mutation stimulates 3' end processing and up-regulates prothrombin protein expression as assessed by a highly sensitive luminescence-based reporter system. CONCLUSIONS: This is the first report of 20209*T in Caucasians, and functional analysis demonstrates that F2 20209*T falls into a general category of mutations of the F2 gene, which may possibly contribute to thrombophilia and complications of pregnancy by interfering with a tightly balanced architecture of non-canonical F2 3' end formation signals.

Time dependent protection of amifostine from renal and hematopoietic cisplatin induced toxicity.

Efficacy of chemotherapy may be maximized and its toxicity can be minimized if drugs would be administered at specified daily times. The present study was aimed to examine if the protection of amifostine against cisplatin toxicity is time dependent. Amifostine is an organic thiophosphate that protects selectively normal tissues, but not tumors, against the cytotoxicity of DNA binding chemotherapeutic agents such as cisplatin. ICR male mice which were entrained to Light:Dark (L:D) 14:10 were injected (intrapritoneal bolus) for 5 consecutive days with either: cisplatin, cisplatin plus amifostine (administered 30 minutes prior to cisplatin). Injections were given at either 08:00, 13:00, 20:00 or 01:00. Five days later, on day 10, each set of mice was sacrificed (at the same hour corresponds to the injection hour), blood count, blood creatinine and blood urea nitrogen (BUN) were assayed. Cisplatin treated mice exhibited nephrotoxicity, as indicated by increased blood urea nitrogen values and by high blood urea nitrogen to creatinine ratios, as well as myelotoxicity that was indicated by low levels of hemoglobin and platelets. Co-administration of amifostine-cisplatin reversed both, the nephrotoxicity of cisplatin, and its myelosuppressive effects. For BUN, hemoglobin and platelets, maximal protections were observed at 08:00, (p <0.05, p <0.01 and p <0.01 respectively). For BUN/Cr ratio (p <0.05), maximal protections was observed at 13:00. These findings show that amifostine exhibits time dependent protection against cisplatin toxicity and thus it is recommended to use the protector when treatments are given during morning hours. The results also further validate the notion that chronochemotherapy is advantageous at least in reducing drug toxicity and thus should be integrated in the design of clinical protocols.

[Kell alloimmunization in pregnancy]. / Allo-immunisation anti-Kell et grossesse.

INTRODUCTION: Kell alloimmunization is a rare disease, although its incidence is the highest after after anti-D alloimmunization. METHODS: We report two recent cases and a review of the literature to describe practical management of Kell alloimmunization in pregnancy. DISCUSSION: When an immunization against the Kell antigen was diagnosed, amniocentesis was performed at 14 weeks gestation to determine the fetal blood group. If the fetus was Kell positive, a first fetal blood sample was drawn at 17 weeks gestation in case of fetal hydrops, and at 20 weeks without fetal hydrops. The diagnosis of anemia led to in utero transfusion. A second fetal blood sample was taken at 8 to 10 days, every two weeks during the second trimester and every three or four weeks during the third trimester. Fetal well-being was assessed with weekly sonography and rates of hemoglobin decline. These measures enable adapting the frequency of fetal blood sampling.

Flow-resistant red blood cell aggregation in morbid obesity.

OBJECTIVE: Enhanced red blood cell (RBC) aggregation has an adverse effect on microcirculatory blood flow and tissue oxygenation. It has been previously shown that obesity is associated with increased RBC aggregation. The objectives of the present study were to further characterize obesity-related RBC aggregation and to examine whether the enhanced aggregation is a plasma- or cellular-dependent process. METHODS: Obese (body mass index (BMI)=40+/-6.3 kg/m2, n=22) and nonobese (BMI=24+/-3.4 kg/m2, n=18) individuals were evaluated for inflammation markers and aggregation parameters. Aggregation parameters were derived from the distribution of RBC population into aggregate sizes, and from the variation of the distribution as a function of flow-derived shear stress, using a cell flow properties analyzer. To differentiate plasmatic from cellular factors, we determined the aggregation in the presence of autologous plasma or dextran-500 kDa and calculated the plasma factor (PF) in the obese group. PF ranges from 0 to 1. When the PF=1, the aggregation is all due to plasmatic factors, when PF=0, the altered aggregation depends entirely on cellular factors, whereas 0

ARP, a peptide derived from the stress-associated acetylcholinesterase variant, has hematopoietic growth promoting activities.

BACKGROUND: Psychological stress induces rapid and long-lasting changes in blood cell composition, implying the existence of stress-induced factors that modulate hematopoiesis. Here we report the involvement of the stress-associated "readthrough" acetylcholinesterase (AChE-R) variant, and its 26 amino acid C-terminal domain (ARP) in hematopoietic stress responses. MATERIALS AND METHODS: We studied the effects of stress, cortisol, antisense oligonucleotides to AChE, and synthetic ARP on peripheral blood cell composition and clonogenic progenitor status in mice under normal and stress conditions, and on purified CD34 cells of human origin. We employed in situ hybridization and immunocytochemical staining to monitor gene expression, and 5-bromo-2-deoxyuridine (BrdU), primary liquid cultures, and clonogenic progenitor assays to correlate AChE-R and ARP with proliferation and differentiation of hematopoietic progenitors. RESULTS: We identified two putative glucocorticoid response elements in the human ACHE gene encoding AChE. In human CD34+ hematopoietic progenitor cells, cortisol elevated AChE-R mRNA levels and promoted hematopoietic expansion. In mice, a small peptide crossreacting with anti-ARP antiserum appeared in serum following forced swim stress. Ex vivo, ARP was more effective than cortisol and equally as effective as stem cell factor in promoting expansion and differentiation of early hematopoietic progenitor cells into myeloid and megakaryocyte lineages. CONCLUSIONS: Our findings attribute a role to AChE-R and ARP in hematopoietic homeostasis following stress, and suggest the use of ARP in clinical settings where ex vivo expansion of progenitor cells is required.

The effect of i.v. iron alone or in combination with low-dose erythropoietin in the rapid correction of anemia of chronic renal failure in the predialysis period.

BACKGROUND: It is now more and more evident that anemia of predialysis chronic renal failure (CRF) should be actively treated, since long-standing anemia may cause irremediable damage to the heart. The most common form of treatment of this anemia is subcutaneous erythropoietin (EPO). iron (Fe) deficiency can also contribute to anemia in predialysis CRF, and intravenous iron (i.v. Fe) can frequently improve it. It is possible, therefore, that the combination of EPO and i.v. Fe may have an additive effect, and cause a rapid improvement in anemia with relatively small doses of EPO. PURPOSE: The purpose of this study was an initial study: to assess the ability of a combination of low-dose EPO and i.v. Fe, given weekly for 5 doses, to correct the anemia of predialysis CRF patients compared to the use of i.v. Fe alone in a randomized study. In the follow-up study: to assess the ability of the maintenance of adequate iron stores for one year to achieve and maintain the target Hct of 35% with the minimum dose of EPO. Initial study: METHOD: Ninety predialysis CRF patients (creatinine clearance 10-40 ml/min/1.73 m2 received either: Group A (45 patients): 200 mg i.v. Fe as Fe sucrose (Venofer, Vifor Int.) once per week for 5 doses in combination with 2,000 international units (IU) EPO (Eprex, Cilag-Janssen), subcutaneously given simultaneously also for 5 doses. Group B (45 patients): the same dose of i.v. Fe as in Group A but without EPO. RESULTS: The mean increase in hematocrit (Hct) and hemoglobin (Hb) by one week after the last dose was greater in group A, 4.54 +/- 2.64% (p < 0.01) and 1.37 +/- 0.84 g% (p < 0.01), respectively, than in Group B, 2.74 +/- 2.72% (p < 0.05) and 0.91 +/- 0.78 g% (p < 0.05), respectively. 80% of those in Group A had an increase in Hct of 3 vol% or more compared to 48.9% in Group B (p < 0.01). 40% of those in Group A reached the target Hct of 35% compared to 28.9% in Group B (p > 0.05). Follow-up study: During a 12-month follow-up period, enough i.v. iron was given to maintain the Hct at 35%, while keeping the serum ferritin at < 400 ug/l and % Fe Sat at < 40%. If the i.v. Fe alone was not capable of maintaining the target Hct, EPO was given in increasing doses. Eighteen patients required dialysis. Of the 72 patients who did not require dialysis, 24 (33.3%) maintained the target Hct with i.v. Fe alone, without EPO. All the remaining 48 patients (66.7%) continued to receive EPO in addition to the i.v. Fe, and 47 achieved and maintained the target Hct with a mean EPO dose of 2,979 +/- 1,326 IU/week. CONCLUSION: The combination of low-dose EPO and i.v. Fe had a rapid and additive effect on the correction of anemia in CRF predialysis patients. Maintaining adequate iron stores with i.v. Fe during a subsequent maintenance phase allowed the target Hct of 35% to be reached and maintained with low-dose EPO in two-thirds of the predialysis patients and with no EPO at all in one-third.

The inflammation meter: novel technology to detect the presence of infection/inflammation in patients without leukocytosis but with increased leukocyte adhesiveness/aggregation.

OBJECTIVES: To reveal the presence of infection/inflammation in patients with relatively normal white blood cell count (WBCC) by using the leukocyte adhesiveness/aggregation test (LAAT). METHODS: The LAAT was performed by using a simple slide test and image analysis (Inflamet), the WBCC, by an electronic cell analyzer, C-reactive protein, by Laser nephelometry and CD11b/CD18 by whole blood flow cytometry. RESULTS: Forty out of a cohort of 121 patients with nonviral acute febrile illness had a WBCC within normal limits. The intensity of the inflammatory response in these individuals as judged by either C-reactive protein, or fibrinogen concentrations, erythrocyte sedimentation or polymorphonuclear leukocyte CD11b/CD18 expression was similar to that observed in patients with a leukocytic response. Our present finding that 63% out of the group with documented infection/inflammation and no leukocytosis had a significantly increased LAAT suggest that the lack of leukocytosis is in part a pseudoleukopenia, or is associated with some degree of uncompensated tissue leukostasis. CONCLUSIONS: The lack of a leukocytic response in a patient with nonviral infection/inflammation is by no means a sign of a less inflammatory response. The increased state of leukocyte adhesiveness/aggregation might help to disclose the presence of inflammation in these individuals.

Megakaryocyte maturation is associated with expression of the CXC chemokine connective tissue-activating peptide CTAP III.

Connective tissue-activating peptide III (CTAP III), a CXC chemokine derived from the chemokine precursor platelet basic protein (PBP) by proteolytic cleavage, has been identified in platelets, activated macrophages, neutrophils and T lymphocytes. CTAP III can support stem cell-derived haematopoiesis yet inhibits the proliferation of committed megakaryocyte (MK) progenitors. This investigation was aimed at characterizing CTAP III expression in human MKs and determining it's role in MK differentiation. We report high expression of CTAP III in mature human bone marrow (BM) MKs and megakaryoblast cell lines following differentiation induction with phorbol ester 12-myristate 13-acetate (PMA). Immunostaining with anti-CTAP III antibodies demonstrated its prominent presence in platelet producing zones in the cytoplasm and intense staining around the periphery of the large BM MKs. In cultures of logarithmically growing megakaryoblast cell lines DAMI, CHRF-288 or MEG01, which contain primarily 2N cells, only 15% of the cells expressed CTAP III. The addition of PMA stimulated high levels of CTAP III after 24 h in more than 75% of the cells, being expressed in both the 2N and large polyploid MKs. Reverse transcription polymerase chain reaction (RT-PCR) revealed upregulation of CTAP III mRNA after only 1 h of exposure to PMA that was sustained for 24 h. In the bone marrow of idiopathic thrombocytopenic pupura (ITP) patients undergoing accelerated MK maturation and thrombopoiesis, 99% of large MKs and 95% of small MKs expressed high levels of CTAP III. While the biological function of this chemokine in MKs is not known, these studies demonstrate that molecular upregulation of CTAP III in MKs is associated with maturation and, as with other chemokines, may be involved in proliferation arrest and cellular interactions with extracellular matrix and platelet production.

Expression and purification of the hormone binding domain of the Drosophila ecdysone and ultraspiracle receptors.

Escherichia coli vectors were constructed for the production of a protein complex that mimics the native ecdysone receptor (EcR) isolated from Drosophila. The two steroid receptors, ultraspiracle (USP) and EcR, were expressed as truncations, retaining primarily the hormone binding domains. The recombinant receptor complex was able to mimic the pharmacology of the native receptor with respect to both synthetic and natural agonists. USP and EcR fusion proteins could be expressed in separate cell lines and then recombined following isolation to yield a ligand binding preparation with a dissociation constant (K(D)) for Ponasterone A of 1.5 nM and a total yield of 1.9 pmol ligand binding sites/mg protein. Alternatively, the simultaneous coexpression of both receptors increased yields by several orders of magnitude to 6 nmol ligand binding sites/mg protein with a K(D) of 0.6 nM. Chromatographic analysis under native conditions showed that EcR, when expressed alone, migrated as a variety of complexes, mostly coming out in the void volume as denatured, insoluble, aggregate. In contrast, purified extracts of coexpressed EcR and USP eluted as a single peak with a mobility indicating a heterodimer. The majority of the coexpressed fusion receptors, following purification, formed functional steroid binding sites. A detailed scheme is provided for the expression and isolation of milligram quantities of highly purified receptor dimer.

Placing the newborn on the maternal abdomen after delivery increases the volume and CD34 cell content in the umbilical cord blood collected: an old maneuver with new applications.

OBJECTIVE: Our purpose was to increase the number of the progenitor cells in umbilical cord blood collected for transplantation. STUDY DESIGN: We randomly assessed the effect of "upper" and "lower" positions of the newborn on the volume and progenitor cell (CD34(+)) content of the umbilical cord blood collected from 49 healthy, vaginally delivered, term neonates. RESULTS: Twenty-two collections were performed in the "upper" and 27 in the "lower" position. The volume of umbilical cord blood obtained in the "upper" position was 108.1 +/- 19.1 mL compared with 42.6 +/- 19.5 mL in the "lower" position (P <.0001). Mononuclear cell separation revealed significantly higher numbers of cells in umbilical cord blood obtained in the "upper" group (P <.01). Although the percentage of CD34(+) cells was comparable, the absolute number of CD34(+) cells was significantly higher in the "upper" group because of the larger volume collected (P <.02). At 24 hours after delivery the hemoglobin levels were not significantly different between newborns of the 2 groups. CONCLUSIONS: Placing the newborn on the maternal abdomen after delivery and before cord clamping may significantly increase the volume of umbilical cord blood collected and therefore the CD34(+) counts that improve transplantation success without placing the mother or the newborn at risk.