Examination of Genetic Susceptibility in Radiation-Associated Meningioma.

Previous epidemiological studies have demonstrated elevated susceptibility to ionizing radiation in some families, thus suggesting the presence of genetic components that conferred increased rate of radiation-associated meningioma (RAM). In this study, we exome-sequenced and investigated the segregation pattern of rare deleterious variants in 11 RAM pedigrees. In addition, we performed a rare-variant association analysis in 92 unrelated familial cases of RAM that were ancestry-matched with 88 meningioma-free controls. In the pedigree analysis, we found that each family carried mostly a unique set of rare deleterious variants. A follow-up pathway analysis of the union of the genes that segregated within each of the 11 pedigrees identified a single statistically significant (q value = 7.90E-04) "ECM receptor interaction" set. In the case-control association analysis, we observed no statistically significant variants or genes after multiple testing correction; however, examination of ontological categories of the genes that associated with RAM at nominal P values <0.01 identified biologically relevant pathways such as DNA repair, cell cycle and apoptosis. These results suggest that it is unlikely that a small number of highly penetrant genes are involved in the pathogenesis of RAM. Substantially larger studies are needed to identify genetic risk variants and genes in RAM.

The primacy of NF1 loss as the driver of tumorigenesis in neurofibromatosis type 1-associated plexiform neurofibromas.

Neurofibromatosis type 1 (NF1) is a common tumor-predisposition disorder due to germline mutations in the tumor suppressor gene NF1. A virtually pathognomonic finding of NF1 is the plexiform neurofibroma (PN), a benign, likely congenital tumor that arises from bi-allelic inactivation of NF1. PN can undergo transformation to a malignant peripheral nerve sheath tumor, an aggressive soft-tissue sarcoma. To better understand the non-NF1 genetic contributions to PN pathogenesis, we performed whole-exome sequencing, RNASeq profiling and genome-wide copy-number determination for 23 low-passage Schwann cell cultures established from surgical PN material with matching germline DNA. All resected tumors were derived from routine debulking surgeries. None of the tumors were considered at risk for malignant transformation at the time; for example, there was no pain or rapid growth. Deep (~500X) NF1 exon sequencing was also conducted on tumor DNA. Non-NF1 somatic mutation verification was performed using the Ampliseq/IonTorrent platform. We identified 100% of the germline NF1 mutations and found somatic NF1 inactivation in 74% of the PN. One individual with three PNs had different NF1 somatic mutations in each tumor. The median number of somatic mutations per sample, including NF1, was one (range 0-8). NF1 was the only gene that was recurrently somatically inactivated in multiple tumors. Gene Set Enrichment Analysis of transcriptome-wide tumor RNA sequencing identified five significant (FDR<0.01) and seven trending (0.01⩽FDR<0.02) gene sets related to DNA replication, telomere maintenance and elongation, cell cycle progression, signal transduction and cell proliferation. We found no recurrent non-NF1 locus copy-number variation in PN. This is the first multi-sample whole-exome and whole-transcriptome sequencing study of NF1-associated PN. Taken together with concurrent copy-number data, our comprehensive genetic analysis reveals the primacy of NF1 loss as the driver of PN tumorigenesis.

Fine mapping of genetic susceptibility to polycystic ovary syndrome on chromosome 19p13.2 and tests for regulatory activity.

CONTEXT: Little is known about genes that contribute to polycystic ovary syndrome (PCOS). We previously found linkage and association of PCOS with the dinucleotide marker D19S884 in two independent sets of families; allele 8 of D19S884 confers increased risk. OBJECTIVE/DESIGN: The objectives of the study were: 1) use the transmission/disequilibrium test (TDT) to assess linkage and association between PCOS and D19S884 (and nearby markers) in a third set of families; and 2) test D19S884 and surrounding DNA sequence for in vitro regulatory activity in lymphoblastoid cell lines (LCLs) and granulosa cells. SETTING/SUBJECTS: We studied 98 new families with a PCOS proband, father, mother, and other available offspring. We analyzed data from these families separately and in combination with data obtained previously. INTERVENTIONS: Interventions were venipuncture. MAIN OUTCOME MEASURES: Measures were transmission frequencies and in vitro functional studies. RESULTS: The first result we found was that in the 98 new families, the TDT was significant for allele 8 of D19S884 (P = 0.043). In the total collection of 465 families, the TDT evidence is very strong (nominal P < 7 x 10(-5)). Results for all other genetic markers near D19S884 were nonsignificant after correction for multiple testing. The second result was that an approximately 800-bp fragment containing various alleles of D19S884 showed modest but reproducible promoter activity in LCLs. However, no allelic differences were detected. No activity of this fragment was detected in granulosa cells. CONCLUSIONS: This is the second independent confirmation of linkage and association of D19S884 with PCOS. We found in addition that some sequence in the region of D19S884 confers in vitro promoter activity in LCLs.

Pharmaceutical development, quality control, stability and compatibility of a parenteral lyophilized formulation of the investigational polymer-conjugated platinum antineoplastic agent AP5346.

AP5346 is a low molecular weight polymer-conjugated platinum antineoplastic agent. The lyophilized drug product has completed a phase I clinical trial. In order to guarantee a constant quality of AP5346 pharmaceutical products, quality control and analysis of the drug substance and final product were performed. The identity of AP5346 was confirmed using 1H NMR, 195Pt NMR and IR spectroscopy. Furthermore, the free platinum content, platinum release characteristics, molecular size and size distribution were established. With the selected analytical techniques, AP5346 could be distinguished very well from its polymeric analogues, such as AP5280 and AP5279. Stability experiments revealed that AP5346 final product is stable for 12 months at 5 degrees C, in the dark. For administration to patients, AP5346 final product is reconstituted with 5% w/v dextrose and diluted in infusion containers. To investigate the influence of container materials, the stability of AP5346 after reconstitution and dilution in infusion containers was determined. The infusion containers investigated were composed of glass, polyvinyl chloride (PVC, intraflex) and low density polyethylene (LD-PE, Ecoflac). AP5346 was shown to be stable after reconstitution and dilution with 5% w/v dextrose in these infusion containers for at least 96 h at 2-8 degrees C in the dark and at room temperature with ambient light conditions.

Enhanced serelaxin signalling in co-cultures of human primary endothelial and smooth muscle cells.

BACKGROUND AND PURPOSE: In the phase III clinical trial, RELAX-AHF, serelaxin caused rapid and long-lasting haemodynamic changes. However, the cellular mechanisms involved are unclear in humans. EXPERIMENTAL APPROACH: This study examined the effects of serelaxin in co-cultures of human primary endothelial cells (ECs) and smooth muscle cells (SMCs) on cAMP and cGMP signalling. KEY RESULTS: Stimulation of HUVECs or human coronary artery endothelial cells (HCAECs) with serelaxin, concentration-dependently increased cGMP accumulation in co-cultured SMCs to a greater extent than in monocultures of either cell type. This was not observed in human umbilical artery endothelial cells (HUAECs) that do not express the relaxin receptor, RXFP1. Treatment of ECs with l-N(G) -nitro arginine (NOARG; 30 µM, 30 min) inhibited serelaxin-mediated (30 nM) cGMP accumulation in HUVECs, HCAECs and co-cultured SMCs. In HCAECs, but not HUVECs, pre-incubation with indomethacin (30 µM, 30 min) also inhibited cGMP accumulation in SMCs. Pre-incubation of SMCs with the guanylate cyclase inhibitor ODQ (1 µM, 30 min) had no effect on serelaxin-mediated (30 nM) cGMP accumulation in HUVECs and HCAECs but inhibited cGMP accumulation in SMCs. Serelaxin stimulation of HCAECs, but not HUVECs, increased cAMP accumulation concentration-dependently in SMCs. Pre-incubation of HCAECs with indomethacin, but not l-NOARG, abolished cAMP accumulation in co-cultured SMCs, suggesting involvement of prostanoids. CONCLUSIONS AND IMPLICATIONS: In co-cultures, treatment of ECs with serelaxin caused marked cGMP accumulation in SMCs and with HCAEC also cAMP accumulation. Responses involved EC-derived NO and with HCAEC prostanoid production. Thus, serelaxin differentially modulates vascular tone in different vascular beds.

Serelaxin-mediated signal transduction in human vascular cells: bell-shaped concentration-response curves reflect differential coupling to G proteins.

BACKGROUND AND PURPOSE: In a recently conducted phase III clinical trial, RELAX-AHF, serelaxin infusion over 48 h improved short- and long-term clinical outcomes in patients with acute heart failure. In this study we used human primary cells from the umbilical vasculature to better understand the signalling mechanisms activated by serelaxin. EXPERIMENTAL APPROACH: We examined the acute effects of serelaxin on signal transduction mechanisms in primary human umbilical vascular cells and its chronic actions on markers of cardiovascular function and disease. KEY RESULTS: The RXFP1 receptor, the cognate serelaxin receptor, was expressed at the cell surface in HUVECs and human umbilical vein smooth muscle cells (HUVSMCs), human umbilical artery smooth muscle cells (HUASMCs) and human cardiac fibroblasts (HCFs), but not human umbilical artery endothelial cells. In HUVECs and HUVSMCs, serelaxin increased cAMP, cGMP accumulation and pERK1/2, and the concentration-response curves (CRCs) were bell-shaped. Similar bell-shaped CRCs for cGMP and pERK1/2 were observed in HCFs, whereas in HUASMCs, serelaxin increased cAMP, cGMP and pERK1/2 with sigmoidal CRCs. Gαi/o and lipid raft disruption, but not Gαs inhibition, altered the serelaxin CRC for cAMP and cGMP accumulation in HUVSMC but not HUASMC. Longer term serelaxin exposure increased the expression of neuronal NOS, VEGF, ETß receptors and MMPs (gelatinases) in RXFP1 receptor-expressing cells. CONCLUSIONS AND IMPLICATIONS: Serelaxin caused acute and chronic changes in human umbilical vascular cells that were cell background dependent. Bell-shaped CRCs that were observed only in venous cells and fibroblasts involved Gαi/o located within membrane lipid rafts.

Familial testicular germ cell tumors (FTGCT) - overview of a multidisciplinary etiologic study.

This Review summarizes the cumulative results of the National Cancer Institute Clinical Genetics Branch Multidisciplinary Etiologic Study of Familial Testicular Germ Cell Tumors (FTGCT). Initiated 12 years ago, this protocol enrolled 724 subjects from 147 unrelated families with either ≥2 affected men (n = 90) with TGCT or a proband with bilateral TGCT and a negative family history for this cancer (n = 57). Data were collected directly from 162 subjects evaluated at the NIH Clinical Center, and 562 subjects provided information from their home communities (Field Cohort). The primary study aims included (i) ascertaining, enrolling eligible FTGCT kindred, (ii) characterizing the clinical phenotype of multiple-case families, (iii) identifying the underlying genetic mechanism for TGCT susceptibility in families, (iv) evaluating counseling, psychosocial, and behavioral issues resulting from membership in an FTGCT family, and (v) creating an annotated biospecimen repository to permit subsequent translational research studies. Noteworthy findings include (i) documenting the epidemiologic similarities between familial and sporadic TGCT, (ii) demonstrating significantly younger age-at-diagnosis for familial vs. sporadic TGCT, (iii) absence of a dysmorphic phenotype in affected family members, (iv) shifting the focus of gene discovery from a search for rare, highly penetrant susceptibility variants to the hypothesis that multiple, more common, lower penetrance genes underlie TGCT genetic risk, (v) implicating testicular microlithiasis in FTGCT risk, and (vi) observing that aberrant methylation may contribute to FTGCT risk. A clinically based, biospecimen-intensive, multidisciplinary research strategy has provided novel, valuable insights into the etiology of FTGCT, and created a research resource which will support FTGCT clinical and laboratory studies for years to come.

Development of a homologous equine relaxin radioimmunoassay.

Equine relaxin (eRlx) immunoactivity has previously been measured in the mare during pregnancy using the porcine relaxin (pRlx) RIA (pRlx-RIA). This was not the optimal system for measurement of eRlx because the dose-response curve obtained with equine plasma was not parallel to the pRlx standard curve. A homologous eRlx-RIA has been developed and used to measure relaxin immunoactivity during pregnancy and parturition in the mare. Highly purified eRlx was used for the generation of antiserum in rabbits, preparation of tracer, and as assay standards. A double antibody eRlx RIA (eRlx-RIA) was developed which was highly specific and sensitive (0.023 ng relaxin/tube). The dose-response curve obtained with eRlx plasma was parallel to the equine standard curve while there was no parallelism noted between the pRlx and eRlx standard curves. This assay was utilized to measure eRlx in samples collected during pregnancy which were previously measured for relaxin content in the pRlx-RIA. It was found that the two assay systems gave almost identical patterns of secretion throughout pregnancy. The two assays differed in the amount of relaxin measured, with the eRlx-RIA measuring considerably higher amounts during gestation than the pRlx assay. In oxytocin-induced foalings, the differences became greater with the equine assay measuring as much as 10-fold greater concentrations.

Purification and characterization of equine relaxin.

It has been previously determined that the equine placenta is the sole significant source of relaxin during pregnancy and that relaxin immunoactivity is also present in term placentas. Therefore, placentas obtained at the time of foaling were selected for starting material for purification of equine relaxin. Frozen whole placentas were ground and then extracted with 0.5 N HCl-85% acetone. Relaxin was precipitated by raising the acetone concentration to 97%. Equine relaxin was further purified by stepwise elution ion exchange, gel filtration, and gradient elution ion exchange chromatographies and trichloroacetic acid precipitation. Equine relaxin purified in this manner was shown to be heterogeneous with one major form (R-1) and several minor forms (two of which are identified as R-2 and R-3). About 1.5 mg R-1 were obtained per kg placenta. Purity was assessed by slab and disc gel electrophoresis and dansyl end group analysis. R-1 had a potency of 28 U/mg, as measured in the mouse inter-pubic ligament bioassay and displayed a dose-response curve parallel with porcine relaxin. Amino acid analysis indicated the presence of tyrosine, histidine, and proline, amino acids absent in porcine relaxin. Dansyl end group analysis indicated the blockage of N-terminal groups on R-1 and the presence of Lys on R-3. A lower molecular weight was indicated by the electrophoretic migration of relaxin reduced with 2-mercaptoethanol suggesting that equine relaxin consists of two chains.

Affinity purification and sequence determination of equine relaxin.

Relaxin, a polypeptide hormone normally associated with pregnancy, has been purified from many species, and the sequence determined for a growing number. Equine relaxin has been previously purified by acetone extraction, gel filtration, and ion exchange chromatographies. In an attempt to develop a more rapid and efficient method for relaxin purification, the use of affinity chromatography coupled with HPLC was explored. Monoclonal antibodies were raised against highly purified equine relaxin; large quantities of antibody were obtained by ascites production and attached to a solid phase support. An extract of term equine placentas was passed through the affinity column and washed, and relaxin was eluted by a change in pH. The isoforms of equine relaxin were separated by HPLC using a C18 (ODS) reverse phase column and a linear gradient of 25-30% acetonitrile. Four major and several minor isoforms of equine relaxin were obtained. The isoforms share similar mol wt by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), are composed of subunits (SDS-PAGE under reducing conditions), and have similar charges (native PAGE). Five isoforms tested positive for biological activity in the mouse interpubic ligament bioassay. Equine relaxin was sequenced by Edman degradation, and the sequence was confirmed by fast atom bombardment mass spectrometry. The isoforms of equine relaxin were found to be due to heterogeneity of the C-terminus of the B-chain. Equine relaxin appears to be the smallest relaxin sequenced. The A-chain consists of 20 residues, and the B-chain has 28 residues, with a total mol wt of 5253. Equine relaxin shares the greatest sequence homology with porcine relaxin (67% identity).

Measurement of periimplantational relaxin concentrations in the macaque using a homologous assay.

Circulating relaxin concentrations in the human rise in the late luteal phase and increase further in response to increasing circulating CG concentrations immediately after implantation. Similar events have not been documented in the laboratory macaque because of the lack of sensitivity of heterologous assay systems. A homologous enzyme-linked immunosorbent assay for authentic macaque relaxin was developed and validated. Using this enzyme-linked immunosorbent assay, relaxin concentrations were measured in peripheral and ovarian venous blood collected from cynomolgus and rhesus macaques. Relaxin concentrations rose in the late luteal phase of nonconceptive menstrual cycles in cynomolgus macaques, but it was not detected at other times in the cycle. In conceptive cycles, relaxin concentrations rose rapidly in close association with the appearance of mCG 13-14 days after mating. Pregnant rhesus macaques also had elevated relaxin concentrations in blood samples collected on days 15-17 postbreeding. Relaxin concentrations disappeared immediately after luteectomy or ablation of the trophoblast by either surgery or administration of methotrexate. The rise of relaxin paralleled the rise of mCG until 20-25 days postbreeding, while progesterone concentrations declined during this same time period. The lack of correlation between relaxin and progesterone secretion profiles suggests that either the cellular origins or the intracellular mechanisms promoting the secretion of these hormones are different. The periimplantational profile of serum relaxin in macaques was similar to the profile of relaxin observed during early human pregnancy.

Simulation of human luteal endocrine function with granulosa lutein cell culture.

Human granulosa cells collected from in vitro fertilization have previously been cultured to provide a system to simulate the granulosa lutein cells of the corpus luteum. In most of these systems, the cultures have been relatively short term, and attempts to simulate the normal pattern of hormone production observed during the luteal phase of the cycle have not been reported. Additionally, the hormone relaxin has generally been absent from the endocrine analysis of these systems. In this report, methods were used that supported secretion of ovarian steroids and relaxin that mimics the profiles of these hormones in vivo. This system was used to observe the endocrine responses of the granulosa lutein cells to three different protocols of CG administration designed to mimic the normal luteal phase, early pregnancy, and early pregnancy followed by pregnancy loss. The normal luteal phase was simulated by a constant baseline (0.02 IU/mL) CG model to simulate a nonconceptive cycle (baseline). The second model was baseline CG until day 8 of culture, followed by daily doubling from days 9-17 to simulate an early pregnancy (rescue-plateau). CG concentrations were then held constant from days 17-20 (5.12 IU/mL). A third model (rescue-drop) was used that was identical to the early pregnancy model except that on day 17 CG was returned to baseline concentrations (0.02 IU/mL) to simulate an early pregnancy loss. Baseline CG stimulation resulted in profiles of estrogen, progesterone, and relaxin secretion in culture that were closely related to secretory profiles previously reported in serum during the nonconceptive luteal phase. The timing of appearance of relaxin secretion and later declines in steroid and relaxin secretion paralleled that observed in serum. In the CG rescue protocols, ovarian steroids rose in response to daily doubling of CG and fell when CG either plateaued or fell. Relaxin did not show an increase in response to increasing CG, but its secretion did not drop when CG concentrations plateaued or dropped. This cell culture system model mimics the profile of ovarian steroids and relaxin seen in serum during the nonconceptive luteal phase, although the relative magnitude of the hormones was not the same as seen in vivo. It was also used to investigate responses to luteal rescue protocols designed to simulate early pregnancy and pregnancy loss. This culture system may be useful to study differences in endocrine response in granulosa cells collected from different patients and to provide information of clinical relevance. This culture system provides a model to study luteal function and its response to different protocols of luteal rescue and thus may provide insight into early pregnancy and pregnancy loss.

Titrating luteinizing hormone replacement to sustain the structure and function of the corpus luteum after gonadotropin-releasing hormone antagonist treatment in rhesus monkeys.

These studies were designed to identify 1) a regimen of a third generation GnRH antagonist that abolishes primate luteal function, and 2) the amount of LH replacement required to maintain the structure and functional life span of the corpus luteum of the menstrual cycle after GnRH antagonist treatment. A single injection of antide at 3 or 5 mg/kg BW on day 6 of the luteal phase suppressed serum progesterone levels within 1 day of treatment, but levels recovered within 4 days. Administration of antide (3 mg/kg) for 3 days (luteal days 6-8) reduced (P < 0.05) serum progesterone below 1 ng/mL and maintained these low levels for the entire sampling period; in subsequent experiments, all monkeys received this antide regimen. Fixed doses (5, 10, or 20 IU) of recombinant human LH administered at 8-h intervals during and after antide treatment stimulated progesterone production in a dose-dependent manner; these monkeys menstruated earlier than controls regardless of treatment group. Replacement with an escalating dose regimen (5-20 IU) of LH resulted in typical serum progesterone and relaxin levels throughout a luteal phase of normal length. Corpora lutea removed on day 10 from monkeys treated with antide alone had decreased wet weight (P < 0.05) and few large luteal cells; coadministration of the escalating dose regimen of LH maintained luteal structure similar to that seen in time-matched controls. Antide-only treatment increased progesterone receptor (PR) messenger ribonucleic acid, but decreased PR immunostaining in luteal tissue; the escalating dose regimen of LH maintained PR messenger ribonucleic acid and immunostaining similar to those in controls. This study indicates that during GnRH antagonist administration, an escalating dose regimen of LH replacement is optimal for maintenance of the structure and functional life span of the primate corpus luteum.

Stimulation of primate luteal function by recombinant human chorionic gonadotropin and modulation of steroid, but not relaxin, production by an inhibitor of 3 beta-hydroxysteroid dehydrogenase during simulated early pregnancy.

CG produced by fetal tissues extends the functional lifespan of the primate corpus luteum during early pregnancy. Previous studies showed that urinary hCG administered to monkeys to simulate the rising CG levels associated with early pregnancy enhanced both progesterone (P) and relaxin (RLX) production by the corpus luteum. The current study was designed: 1) to compare the ability of recombinant (r) and urinary (u) hCG to stimulate luteal function, and 2) to assess the role of P in the regulation of luteal RLX secretion during simulated early pregnancy by concomitant administration of hCG and the 3 beta-hydroxysteroid dehydrogenase inhibitor trilostane to reduce P production. Rhesus monkeys received injections of either r-hCG or u-hCG (Ares Serono) in increasing doses (15-2880 IU/dose, twice daily) for 9 days beginning on day 9 of the luteal phase (n = 5/group). An additional group (n = 4) received r-hCG as described above, with concomitant oral administration of trilostane (500 mg/dose twice daily; Sanofi Winthrop). Daily serum samples were assayed for hCG by immunoradiometric assay, steroid hormones by RIA, and RLX by enzyme-linked immunosorbent assay. Serum hCG levels typically were not different between the r-HCG and u-hCG groups during or after treatment. Concentrations of hCG peaked 1 day after the final injection in monkeys receiving r-hCG (mean +/- SEM. 2759 +/- 120 mIU/mL) and u-hCG (2120 +/- 60 mIU/mL) and dropped below 5 mIU/mL by 10 days after the final treatment in all groups. Both r-hCG and u-hCG stimulated luteal P and RLX production. Progesterone levels rose rapidly after the initiation of hCG treatment and peaked in animals receiving r-hCG (14.4 +/- 2.8 ng/mL) and u-hCG (11.9 +/- 1.4 ng/mL) 4 days after initial administration. RLX levels peaked in the r-hCG (400 +/- pg/mL) and u-hCG (323 +/- 85 pg/mL) groups within 4 days of the final hCG treatment. Trilostane with r-hCG reduced P concentrations to very low levels (< 0.5 ng/mL; P < 0.01) within 1 day of administration compared to those in animals receiving r-hCG only and maintained these low levels for the entire treatment interval. Nevertheless, trilostane administration did not alter luteal RLX production, with serum levels peaking at 377 +/- 76 pg/mL. These data indicate that r-hCG and u-hCG were equally efficacious in stimulating the steroidogenic and peptidergic activities of the corpus luteum during simulated early pregnancy. In addition, P deprivation during r-hCG administration did not alter circulating RLX levels, suggesting that P is not a major regulator of RLX production by the primate corpus luteum during early pregnancy.

Enhanced ovarian steroid secretion before implantation in early human pregnancy.

Previous studies have compared ovarian steroid production in the luteal phase of nonconceptive and conceptive cycles. Some investigators reported higher preimplantational levels of progesterone in conceptive cycles vs. nonconceptive cycles, but other studies have found no differences. Many of these results were difficult to interpret because the studies included infertile women and/or women who received exogenous hormones. In this study we have characterized the profiles of gonadotropin secretion and ovarian steroid response during early pregnancy in a population of spontaneously ovulating women and compared them to those in nonconceptive cycles of recently fertile women. Blood samples were collected daily during the luteal phase from 24 women during 51 cycles of artificial insemination with donor semen. Cycles were segregated to those from women who had a successful term pregnancy (normal group) and those having an early spontaneous abortion (SAB group) and were also classified as nonconceptive or conceptive based on measurements of hCG. Serum LH and FSH did not show marked differences between nonconceptive and conceptive cycles in the periimplantation period in either the normal or SAB group. In the normal group, estradiol concentrations were significantly higher in conceptive cycles than in nonconceptive cycles beginning 6 days after the LH peak and continuing through the end of the cycle, while differences in progesterone concentrations bordered on or exceeded significance during the same time period. In the SAB group, preimplantation differences in pituitary gonadotropin and ovarian steroid secretion were not observed, whereas the postimplantation hCG concentrations in the SAB group were significantly lower than those in the normal group. It is reasoned that embryos with defective post-implantation hCG secretion may have had this defect before detection of hCG in serum, thus accounting for the lack of stimulation of steroid secretion in these pregnancies. These findings suggest that the enhanced ovarian steroid secretion in conceptive cycles may be due to a gonadotropic stimulus from the preimplantation embryo.

The role of relaxin in glycodelin secretion.

Glycodelin is a glycoprotein named for its unique carbohydrate structure. Glycodelin is produced by the secretory endometrium during the late luteal phase and returns to baseline during menses of the ensuing cycle, whereas in conceptive cycles it rapidly increases. Although progesterone and possibly estradiol are required for glycodelin production, they are not directly involved in the synthesis and release of this protein. Their role may be development of the endometrial secretory glandular elements, whereas other factors are required to initiate and maintain glycodelin secretion. The pattern of relaxin secretion during the luteal phase and early pregnancy is similar to that of glycodelin, but their profiles have not been determined simultaneously. To investigate the relationship of relaxin and glycodelin, two studies were conducted. In the first study, relaxin, glycodelin, and ovarian steroids were measured in daily serum samples from nonconceptive and conceptive natural cycles. Profiles of relaxin and glycodelin were closely associated, with the onset of relaxin preceding glycodelin secretion by 1-2 days in nonconceptive cycles, and the pregnancy-associated increases in each hormone differing by about 2 days. The second study tested the hypothesis that relaxin stimulates glycodelin secretion. Samples were obtained from patients injected with human relaxin for 28 days. In subjects demonstrating ovarian cyclicity, glycodelin secretion was elevated, but it was not detected in subjects without ovarian cyclicity or in placebo-treated control subjects. This study reveals a close temporal and quantitative relationship between relaxin and glycodelin profiles in the late luteal phase and early pregnancy. It also demonstrates that relaxin administration can stimulate glycodelin production from a developed endometrium. This is the first report of a nonsteroidal ovarian factor that controls glycodelin secretion, and these results suggest a function for relaxin during early pregnancy. Glycodelin is a potent inhibitor of sperm zona pellucida binding by virtue of its extensive carbohydrate structure, but it is normally at a nadir in the periovulatory period. The data demonstrate that relaxin can stimulate glycodelin secretion throughout the menstrual cycle, including the periovulatory period, when relaxin-induced glycodelin secretion could have a contraceptive effect.

Relaxin in the peri-implantation period.

The time of appearance of relaxin in peripheral blood was determined in conceptive and non-conceptive cycles using a sensitive and specific double-antibody enzyme-linked immunoassay for human relaxin. For study of relaxin in early pregnancy, daily plasma samples were collected from women receiving artificial insemination of donor semen. The day of ovulation was determined by daily LH monitoring and ultrasound observation. In three conceptive cycles, relaxin was significantly elevated over baseline 9-10 days following the LH peak. Relaxin concentrations quickly rose over the next 15 days of observation to over 800 pg/ml. Relaxin was observed to increase 1 to 2 days prior to the first detectable increase in plasma hCG as measured by enzyme-linked immunosorbent assay. To compare the relaxin profile in conceptive cycles with normal luteal phase concentrations, relaxin was also measured in daily plasma samples collected from women contracepting with barrier methods, bilateral tubal ligation, or abstinence. A small but consistent rise in relaxin in the late luteal phase was observed in nine of eleven women, which began 6-9 days after the LH peak, averaged approximately 50 pg/ml, and was declining by the next menses. It is concluded that a small but measurable rise in plasma relaxin is associated with the normal luteal phase and that relaxin secretion is accelerated around the time that hCG is first detected in conceptive cycles. This acceleration of relaxin secretion which is associated with the onset of hCG may provide additional evidence for identification of transient early pregnancy.

Differences between young and aged mice in susceptibility to Friend virus.

Friend virus (FV) is a murine leukemia virus that infects progenitor red blood cells and causes an erythroleukemia in susceptible mouse strains, resulting in splenomegaly. Several genetic loci of the host have been identified that affect erythroleukemia development, differentiation status of target cells and virus replication. Since age may change expression of these loci, age may affect FV disease. To explore this possibility, FV expression in four genetically diverse strains of mice of different ages was examined. Extent of viral replication and of disease were evaluated by measuring spleen focus forming units (SFFU), spleen weight and reverse transcriptase (RT) activity in target organs. Young DBA/2 and (C57BL/6 x DBA/2)F1 mice exhibited a greater level of virus expression than their aged counterparts in all parameters investigated. Young CBA/Ca mice had slightly higher spleen weights and SFFU values than aged CBA/Ca mice, but a definitive age-related change was not observed in the RT activity of the target organs. C57BL/6 mice, which are genetically resistant to the development of FV-induced erythroleukemia, exhibited a limited degree of virus replication that was not effected by the age of the animal. Our results indicate that the age of the mouse, as well as the genetic background, can contribute to the level of susceptibility to FV.

Improved targeting of platinum chemotherapeutics. the antitumour activity of the HPMA copolymer platinum agent AP5280 in murine tumour models.

AP5280 is a novel N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-bound platinum (Pt) therapeutic designed to increase the therapeutic index relative to conventional, small-molecule platinum agents. The platinum-polymer construct accumulates in solid tumours on the basis of increased capillary permeability. The bound platinum moiety is present as an N,O-Pt chelate at the distal end of a tetrapeptide linker, glycine-phenylalanine-leucine-glycine, and the weight-average molecular weight (Mw) of the construct is 22 kDa. The antitumour activity and toxicity of AP5280 were assessed in the syngeneic murine B16F10 and Lewis lung tumour models, and in the human ovarian carcinoma 2008 and head and neck squamous carcinoma UMSCC10b xenograft models. The maximum tolerated dose (MTD) of AP5280 was 6-fold greater than that of carboplatin (CBDCA) in vivo. AP5280 was active in all four tumour models, and it displayed a higher therapeutic index than CBDCA in each of these tumour models. The antitumour effect of AP5280 given at 16% of its MTD was equivalent to that produced by a MTD of CBDCA. Thus, consistent with the design goal for this drug, and despite being less potent than CBDCA, AP5280 produced less systemic toxicity relative to its antitumour activity and thus has a greater therapeutic index. On the basis of the improved therapeutic index evidenced in these models, AP5280 has been advanced into clinical trials.

Evaluation of gastroesophageal reflux surgery in children.

Fifty-five infants and children with complications of gastroesophageal reflux required operative management for control of symptoms. All patients, except those with severe esophageal stricture, received a six-week trial with 60-degree constant elevation before an operation was considered necessary. The operation was performed to control (1) persistent vomiting, (2) vomiting with growth retardation, (3) esophagitis, (4) esophagitis with stricture, and (5) recurrent aspiration pneumonia. Preoperative and postoperative evaluation involved both X-ray fluoroscopy and esophageal manometry with pH studies. A good surgical result was not dependent upon an increase in the lower esophageal pressure following operation. The Boerema anterior gastropexy is simple and effective for controlling gastroesophageal reflux for cases uncomplicated by esophagitis, stricture, or previous operation. Complex cases with inflammatory or operative changes in the lower esophagus are more effectively treated by Nissen fundoplication.