Recombinant human luteinizing hormone: a partial physicochemical, biological and immunological characterization

The aim of this study was to partially characterize the glycoform composition of a recombinant human luteinizing hormone preparation (rhLH; Serono), an early version of the material (LHadi) which is currently being assessed for clinical application. Specifically, the charge (pl) and internal carbohydrate complexity of this rhLH was examined and compared with that of an alternative commercially available form of recombinant LH (Crystal Chem) and a pituitary International Reference Preparation (IRP). All preparations were separated by charge by chromatofocusing them on a pH gradient (7-4) using a 4 ml mono-P column is conjunction with a fast performance liquid chromatography system and by complexity of the oligosaccharide structures using concanavalin A (con-A) lectin affinity chromatography. LH in both the unfractionated and fractionated material was assessed by immunoradiometric assay (IRMA, I-LH) and by the in-vitro Leydig cell bioassay (B-LH). Both assays were calibrated against IRP 80/552. The in-vitro biopotency of the preparations was 18187 (Serono rhLH), 12063 (Crystal Chem rhLH) and 6658 (80/552) IU/mg; biological:immunological ratios were 1.14 (80/552), 1.90 (Crystal Chem rhLH) and 1.99 (Serono rhLH). However, similar qualitative data were obtained by both bioassay and immunoradiometric assay following fractionation, with the median pl of the bioactive LH in the preparations being 5.5 (24% > pH 6), 5.52 (18% > pH 6) and 4.97 (0% > pH 6) for the Serono, Crystal Chem and pituitary preparations respectively. Further all three contain < 1% of the complex carbohydrate structures and between 36-44% and 56-63% of the intermediate and simple forms of bioactive LH. In conclusion, the Serono recombinant LH preparation has a higher in-vitro bioactivity and is more basic than the other two preparations although the complexity of its carbohydrate moities appears to be similar.     

Luteinizing hormone response to gonadotrophin-releasing hormone in normal women undergoing ovulation induction with urinary or recombinant follicle stimulating hormone

Oestradiol enhances pituitary sensitivity to gonadotrophin-releasing hormone (GnRH) in normal women, while in women undergoing ovulation induction the putative factor gonadotrophin surge attenuating factor (GnSAF) attenuates the response of luteinizing hormone (LH) to GnRH. To study the relationships between oestradiol and GnSAF during ovulation induction, 15 normally ovulating women were investigated in an untreated spontaneous cycle (control, first cycle), in a cycle treated with daily i.m. injections of 225 IU urinary follicle-stimulating hormone (FSH) (Metrodin HP, uFSH cycle) and in a cycle treated with daily s.c. injections of 225 IU recombinant FSH (Gonal-F, rFSH cycle). Treatment with FSH started on cycle day 2. The women during the second and third cycle were allocated to the two treatments in an alternate way. One woman who became pregnant during the first treatment cycle (rFSH) was excluded from the study. In all cycles, an i.v. injection of 10 microg GnRH was given to the women (n = 14) daily from days 2-7 as well as from the day on which the leading follicle was 14 mm in diameter (day V) until mid-cycle (n = 7). The response of LH to GnRH at 30 min (deltaLH), representing pituitary sensitivity, was calculated. In the spontaneous (control) cycles, deltaLH values increased significantly only during the late follicular phase, i.e. from day V to mid-cycle, at which time they were correlated significantly with serum oestradiol values (r = 0.554, P < 0.01). Initially during the early follicular phase in the uFSH and the rFSH cycles, deltaLH values showed a significant decline which was not related to oestradiol (increased GnSAF bioactivity). Then, deltaLH values increased significantly on cycle day 7 and further on day v with no change thereafter up to mid-cycle. On these two days, deltaLH values were correlated significantly with serum oestradiol values (r = 0.587 and r = 0.652 respectively, P < 0.05). During the pre-ovulatory period, deltaLH values in the FSH cycles were significantly lower than in the spontaneous cycles. Significantly higher serum FSH values were achieved during treatment with uFSH than rFSH. However, serum values of oestradiol, immunoreactive inhibin, and deltaLH as well as the number of follicles > or = 12 mm in diameter did not differ significantly between the two FSH preparations. These results suggest that in women undergoing ovulation induction with FSH, oestradiol enhances pituitary sensitivity to GnRH, while GnSAF exerts antagonistic effects. The rFSH used in this study (Gonal-F) was at least as effective as the uFSH preparation (Metrodin-HP) in inducing multiple follicular maturation in normally cycling women.     

Structure-function relationship of recombinant follicle stimulating hormone (Puregon)

After separation by means of preparative isoelectrofocusing, the isohormones of a Chinese hamster ovary (CHO)-derived recombinant follicle stimulating hormone (rFSH, Puregon) were characterized with respect to structural and functional features. A carbohydrate analysis revealed that rFSH isohormones with a low isoelectric point (pl) have a high sialic acid/galactose ratio and are rich in tri- and tetra-antennary N-linked carbohydrate chains in comparison with the high pl isohormones. The relative basic isohormones exhibit receptor binding activity and intrinsic bioactivity 2-3-fold higher than the relative acidic isohormones. However, due to their lower clearance rate these acidic isohormones displayed a 20-fold higher in-vivo bioactivity in the rat. A comparison of the isohormone profile of rFSH and urinary FSH (Metrodin) revealed that rFSH contains about 2-fold more basic isohormones with pl > or = 4.7 and 2-fold less acidic isohormones with pl < 4.1. In-vitro studies showed that the receptor binding affinity and intrinsic bioactivity of both FSH preparations are similar. Also the in-vivo efficacy and the pharmacokinetic behaviour of rFSH and urinary FSH in the rat were similar, which is not surprising since both preparations were compared in terms of in-vivo bioactivity calibrated in the rat Steelman-Pohley assay. However, in dogs the bioavailability of rFSH was lower than that of urinary FSH, which is in agreement with the higher percentage of relative basic isohormones in rFSH. This suggests that the pharmacokinetic behaviour of FSH in rats and dogs is different, which is supported by the much longer elimination half-life of rFSH and urinary FSH in dogs (27.9 and 30.4 h respectively) compared with rats (11.4 and 10.4 h respectively) for rFSH and urinary FSH respectively. The observed differences in pharmacokinetic behaviour in dogs and rats indicate that the rat Steelman-Pohley assay might not be a valid model for the prediction of the FSH bioactivity in species other than rat.     

A less acidic human follicle-stimulating hormone preparation induces tissue-type plasminogen activator enzyme activity earlier than a predominantly acidic analogue in phenobarbital-blocked pro-oestrous rats

Follicle-stimulating hormone (FSH) exists in multiple molecular forms. In both experimental animals and in humans the production and secretion of less acidic, short-lived FSH glycoforms significantly increase during the peri-ovulatory period. To gain further insights on the physiological role of these FSH variants, we analysed the ability of two FSH compounds, recombinant FSH (rFSH) and purified FSH from urinary origin (uFSH), (less acidic and acidic pattern of FSH charge isoform distributors respectively) to induce ovarian tissue-type plasminogen activator (tPA) enzyme activity in vivo. FSH produced by Chinese hamster ovary cells and highly purified uFSH were injected at 15:00 h on the pro-oestrous day into phenobarbital-blocked rats and the ovaries were analysed for tPA enzyme activity and tPA mRNA concentrations at different times after FSH injection. Induction of tPA enzyme activity by uFSH and rFSH showed distinct dynamics depending on the particular preparation administered. In animals treated with uFSH, maximum tPA enzyme activity was detected at 20:00 h, and maximum tPA mRNA concentrations were detected at 17:00 h. tPA enzyme activity induction by rFSH was at the maximum at 17:00 h, and maximum tPA mRNA concentration was at 16:00 h (P< 0.05 for uFSH versus rFSH). All animals in the uFSH- and rFSH-treated groups and none in phenobarbital-blocked, saline-treated controls ovulated. No significant differences were present in the number of ova shed by rats treated with uFSH or rFSH and spontaneously ovulating rats (10.7+/-1.7, 10.0+/-2.6 and 11.3+/-1.6 respectively). These data indicate that the increased biological activity exhibited by less acidic FSH glycovariants at the target cell level may compensate for the drawback imposed by their relatively short plasma half-life.     

Binding effects in proton-nucleus elastic scattering

Recombinant human FSH (rhFSH) was obtained by expressing the human FSH alpha- and beta-subunit complementary DNAs in the chinese hamster ovary cell line. Isoforms of rhFSH were resolved into specific isoelectric (pI) fractions by chromatofocusing. rhFSH isoforms ranged from pI 3.0-5.5 with a modal value of pI 4.2. Analysis of the biological activity of specific pI isoforms of rhFSH was undertaken using both the rat granulosa cell aromatase (in vitro) bioassay and a RRA. More acidic isoforms (e.g. pI 3.5) showed significantly lower affinity (P < 0.05) for rat testicular FSH receptors than did the less acidic isoforms (e.g. pI 4.8). Consistent with the receptor binding affinity data, the more acidic fractions resulted in significantly less activation (P < 0.05) of rat granulosa cell aromatase activity, as measured by estrogen production, than did the less acidic isoforms. The observed bioactivities and their correlation with the pI values of the rhFSH isoforms are consistent with observations of differing bioactivities seen in both pituitary and urinary FSH isoforms. These results demonstrate that rhFSH, made in the chinese hamster ovary cell line, is both biologically active and has isoform profiles, and presumably carbohydrate structures, that closely resemble those seen in natural hFSH.     

Treatment of carbon monoxide poisoning with hyperbaric oxygen

This report describes differences in humoral immune response of acute and chronic phases of human Chagas disease. The reactivities of IgG, IgM, and IgA anti-Trypanosoma cruzi antibodies in serum samples from both groups of patients were compared by enzyme-linked immunosorbent assay (ELISA) employing either one of four antigenic fractions: mouse laminin (LAM), which reacts through Gal alpha 1-3Gal epitopes expressed on trypomastigote surface: whole intact trypomastigotes (TCT); trypomastigotes excreted/secreted antigens (TESA); and epimastigote alkaline extract (EAE). The selection of T. cruzi antigen preparations was based on their relative content of surface and internal antigens found in trypomastigote forms. The proportion of IgG reactive to carbohydrate epitopes was assessed through the decay of IgG reactivity from acute and chronic sera after m-periodate oxidation of solid-phase bound antigens. Trypomastigote and TESA antigens recognized by IgG from acute and chronic sera were also compared by immunoblotting. ELISA and immunoblotting data showed that: (1) the proportion of IgG directed to trypomastigote surface antigens was higher in acute than in chronic sera, whereas the opposite was found for internal antigens, (2) acute sera contained a higher percentage of IgG reactive to trypomastigote carbohydrate epitopes than chronic sera, and (3) anti-T. cruzi IgA was found exclusively in acute sera and led to 100% positivity when LAM, TCT, and TESA were employed as antigens. IgA ELISA with these antigens and IgG immunoblotting pattern with TESA could be useful as serological markers for the acute phase of human Chagas disease.     

Synopsis of recent developments in venomous snake systematics, No. 2

OBJECTIVE: The aim of this study was to investigate various outcome measures of stimulation with highly purified subcutaneous follicle-stimulating hormone (Fertinex, a urofollitropin) compared with first- and second-generation urinary human menopausal gonadotropin standards (Pergonal, Metrodin). STUDY DESIGN: Retrospective analysis was restricted to our most efficient in vitro fertilization age group (23-34 years). Data from Institute for Assisted Reproduction in vitro fertilization cycles 1 through 11 with Pergonal, Metrodin, or both were tabulated for hormonal values, oocyte quality, and embryo outcome as baseline data. Patients in cycles 12 through 13 were treated with Fertinex and Pergonal or Fertinex alone and then reviewed for the same parameters. RESULTS: Two hundred thirty-eight in vitro fertilization records with embryo transfer were analyzed. Clinical pregnancy rates per embryo transfer in an optimal age group were similar despite use of first- through third-generation urinary gonadotropin preparations: Pergonal and Metrodin, 67%; Metrodin, 64%; Fertinex and Pergonal, 62%; and Fertinex, 54%. There were no discernible differences in hormonal response, oocyte recovery, or embryonic growth. CONCLUSION: Administered subcutaneously, the third-generation urinary gonadotropin preparation Fertinex is effective in in vitro fertilization treatment in young women.     

Glycoform composition of serum gonadotrophins through the normal menstrual cycle and in the post-menopausal state

The heterogeneity of follicle stimulating hormone (FSH) and luteinizing hormone (LH) was investigated in five women aged 29.4 +/- 3.2 years (mean +/- SD) throughout their menstrual cycles and in five post-menopausal women aged 53.8 +/- 5.6 years. Chromatofocusing (pH range 7-4) revealed menstrual cycle stage- and postmenopausal-related differences in the serum gonadotrophin charge. There were differences in the proportion of FSH with an isoelectric point (pl) > 4.3 across phases of the menstrual cycle (P = 0.019): midcycle (MC) 50%; early to mid-follicular (EMF) 36%; late follicular (LF) 37%, luteal (L) 29% and following the menopause (PM) 17%. There was no significant difference in the proportion of LH with pl > 6.55 between midcycle (53%) and EMF, LF or L phases (36, 43 and 32% respectively); although all were greater than that found in the menopause (13%). Concanavalin A chromatography revealed less (P < 0.005) complex FSH and LH glycoforms at midcycle (63 and 13%) than in the EMF, LF and L phases (90 and 18; 90 and 20 and 93 and 24% respectively). Menopausal gonadotrophins were least complex (FSH 34%, LH 4%). There was a direct relationship between serum FSH and FSH pl/complexity, and less acidic FSH was associated with reduced FSH complexity. Increased oestradiol was associated with basic FSH isoforms during the menstrual cycle and reduced follicular phase FSH complexity. We conclude that changes in gonadotrophin glycoforms occur through the menstrual cycle which are related to changes in the prevailing steroid environment. Following the menopause oestrogenic loss resulted in acidic, relatively simple glycoforms.     

Immunological characterisation of the acrosomal filament in the marine shrimp Sicyonia ingentis

Human alpha-galactosidase A (alpha-Gal A) is the lysosomal glycohydrolase that cleaves the terminal alpha-galactosyl moieties of various glycoconjugates. Overexpression of the enzyme in Chinese hamster ovary (CHO) cells results in high intracellular enzyme accumulation and the selective secretion of active enzyme. Structural analysis of the N -linked oligosaccharides of the intracellular and secreted glycoforms revealed that the secreted enzyme's oligosaccharides were remarkably heterogeneous, having high mannose (63%), complex (30%), and hybrid (5%) structures. The major high mannose oligosaccharides were Man5-7GlcNAc2 species. Approximately 40% of the high mannose and 30% of the hybrid oligosaccharides had phosphate monoester groups. The complex oligosaccharides were mono-, bi-, 2,4-tri-, 2,6-tri- and tetraantennary with or without core-region fucose, many of which had incomplete outer chains. Approximately 30% of the complex oligosaccharides were mono- or disialylated. Sialic acids were mostly N -acetylneuraminic acid and occurred exclusively in alpha2, 3-linkage. In contrast, the intracellular enzyme had only small amounts of complex chains (7.7%) and had predominantly high mannose oligosaccharides (92%), mostly Man5GlcNAc2 and smaller species, of which only 3% were phosphorylated. The complex oligosaccharides were fucosylated and had the same antennary structures as the secreted enzyme. Although most had mature outer chains, none were sialylated. Thus, the overexpression of human alpha-Gal A in CHO cells resulted in different oligosaccharide structures on the secreted and intracellular glycoforms, the highly heterogeneous secreted forms presumably due to the high level expression and impaired glycosylation in the trans- Golgi network, and the predominately Man5-7GlcNAc2 cellular glycoforms resulting from carbohydrate trimming in the lysosome.     

The dental management of patients with natural rubber latex allergy

PURPOSE: The use of highly purified follicle-stimulating hormone (Metrodin-HP) was compared with that of a preparation containing follicle-stimulating hormone and luteinizing hormone (Pergonal) for production of superovulation in an IVF program. METHODS: We used the Oxford Fertility Unit database to identify patients undergoing their first cycle of IVF, using either Metrodin-HP or Pergonal. Patients were treated with a standardized drug protocol and were stratified by age and cause of infertility. Ovarian stimulation with either Metrodin-HP (Serono Laboratories) or human menopausal gonadotropin (hMG; Pergonal; Serono Laboratories) after pituitary desensitization commenced in the midluteal phase of the preceding cycle. Monitoring was performed by ultrasound and serum estradiol measurement prior to transvaginal oocyte recovery, followed by IVF and transfer of no more than three embryos. RESULTS: For Metrodin-HP versus Pergonal, the rates of egg retrieval (98 vs 94%), fertilization (89 vs 92%), clinical pregnancy (32.9 vs 23.4%), miscarriage (4.1 vs 4.5%), live birth (26 vs 18.5%), and ovarian hyperstimulation syndrome (5.5% vs 5.9%) were similar in both groups. The apparent increase in clinical pregnancy and live birth with Metrodin-HP did not reach statistical significance. The dosages of gonadotropins used were comparable. Estradiol levels measured on day 8 of stimulation were significantly lower in the Metrodin-HP group than in the Pergonal group, but the difference did not reach statistical significance on the day of hCG administration. Significantly more follicles (greater than 12 mm) were obtained in the Metrodin-HP group, but the numbers of eggs recovered and fertilized were similar in the two groups. CONCLUSIONS: These findings demonstrate that highly purified FSH (Metrodin-HP) is as effective and successful as hMG (Pergonal) for ovarian stimulation in a standard IVF regimen. Exogenous luteinizing hormone (LH) is not required for satisfactory ovarian stimulation in IVF. Measurement of estradiol may be less helpful in the monitoring of Metrodin-HP cycles, but the level reached on the day of hCG administration can still be used to predict, and hence avoid, ovarian hyperstimulation syndrome.     

Characterisation, calibration and comparison by international collaborative study of international standards for the calibration of therapeutic preparations of FSH

Therapeutic preparations of FSH, used primarily for treatment of infertility, are calibrated by in vivo bioassay against international standards (IS) derived from different sources deemed appropriate to their use according to pharmacopoeial monographs. Menotrophins, which have been used for several decades to treat infertility, have been calibrated against the IS for urinary FSH and LH (ISU) but are now being replaced by highly purified urinary FSH or rDNA-derived FSH (rFSH). The aim of this study was to evaluate two preparations of human rFSH and one preparation of highly purified urinary FSH as candidate WHO IS for bioassay in an international collaborative study by 27 laboratories in 12 countries, and to characterise them in a range of in vitro bioassays and immunoassays. The biological activity of the three candidate standards was confirmed by all laboratories using all assays contributed to the study. Dose-response relationships by in vivo bioassay for any of the candidate standards did not differ significantly from that for the ISU. Dose-response relationships obtained in in vitro bioassays and immunoassays were also broadly similar among these preparations although dose-response lines for some preparations appeared to be non-parallel in some immunoassays. For each of the three candidate IS, estimates of the relative potency in terms of ISU by in vivo bioassay did not differ significantly between laboratories. In contrast estimates by immunoassays and in vitro bioassays showed significant differences between laboratories. Estimates of relative potency of the highly purified candidate IS materials in terms of one another exhibited less inter-laboratory variability than estimates in term of ISU. Each of the candidate standards showed adequate stability to serve as an IS. On the basis of the results of this study rFSH (code 92/642) was established as the first IS for FSH, human, recombinant for bioassay with an assigned unitage of 138 IU per ampoule and urinary FSH (code 92/512) was established as the first IS for FSH, human, urinary (urofollitropin) for bioassay with an assigned unitage of 121 IU per ampoule, based on their respective calibration by in vivo bioassay in terms of ISU. These assignments of unitage maintain continuity of unitage for preparations in therapeutic use and also appear to be consistent with one another.     

An analysis of a class of DNA sequence reading molecules

The bacterial insertion sequence IS21 contains two genes, istA and istB, which are organized as an operon. IS21 spontaneously forms tandem repeats designated (IS21)2. Plasmids carrying (IS21)2 react efficiently with other replicons, producing cointegrates via a cut-and-paste mechanism. Here we show that transposition of a single IS21 element (simple insertion) and cointegrate formation involving (IS21)2 result from two distinct non-replicative pathways, which are essentially due to two differentiated IstA proteins, transposase and cointegrase. In Escherichia coli, transposase was characterized as the full-length, 46 kDa product of the istA gene, whereas the 45 kDa cointegrase was expressed, in-frame, from a natural internal translation start of istA. The istB gene, which could be experimentally disconnected from istA, provided a helper protein that strongly stimulated the transposase and cointegrase-driven reactions. Site-directed mutagenesis was used to express either cointegrase or transposase from the istA gene. Cointegrase promoted replicon fusion at high frequencies by acting on IS21 ends which were linked by 2, 3, or 4 bp junction sequences in (IS21)2. By contrast, cointegrase poorly catalyzed simple insertion of IS21 elements. Transposase had intermediate, uniform activity in both pathways. The ability of transposase to synapse two widely spaced IS21 ends may reside in the eight N-terminal amino acid residues which are absent from cointegrase. Given the 2 or 3 bp spacing in naturally occurring IS21 tandems and the specialization of cointegrase, the fulminant spread of IS21 via cointegration can now be understood.     

Characterization of sugar chain structures of human alpha-fetoprotein by lectin affinity electrophoresis. ibarahp@oka.urban.ne.jp

Alpha-Fetoprotein (AFP) glycoforms, defined as AFP with different chemical structures of carbohydrate, were analyzed by affinity electrophoresis with several lectins of known specificities against complex-type oligosaccharides. Serum AFP samples from cord blood on full-term delivery and from patients with hepatocellular carcinoma and extrahepatic malignancies including gastrointestinal tumors and yolk sac tumors were used. Two-dimensional lectin affinity electrophoresis and also lectin affinity chromatography coupled with lectin affinity electrophoresis were employed. More than ten AFP glycoforms were identified or characterized using the above-mentioned AFP samples. Known specificities of the lectins against complex-type oligosaccharides were refined or their additional specificities were found in this study. Lectin appeared to have specificity against carbohydrates by recognizing not only specific residues but also the whole carbohydrate molecule containing the residues, resulting in differential affinities for the lectin.     

Identification and characterization of glycosylation sites in human serum clusterin

Clusterin is a ubiquitous, heterodimeric glycoprotein with multiple possible functions that are likely influenced by glycosylation. Identification of oligosaccharide attachment sites and structural characterization of oligosaccharides in human serum clusterin has been performed by mass spectrometry and Edman degradation. Matrix-assisted laser desorption ionization mass spectrometry revealed two molecular weight species of holoclusterin (58,505 +/- 250 and 63,507 +/- 200). Mass spectrometry also revealed molecular heterogeneity associated with both the alpha and beta subunits of clusterin, consistent with the presence of multiple glycoforms. The data indicate that clusterin contains 17-27% carbohydrate by weight, the alpha subunit contains 0-30% carbohydrate and the beta subunit contains 27-30% carbohydrate. Liquid chromatography electrospray mass spectrometry with stepped collision energy scanning was used to selectively identify and preparatively fractionate tryptic glycopeptides. Edman sequence analysis was then used to confirm the identities of the glycopeptides and to define the attachment sites within each peptide. A total of six N-linked glycosylation sites were identified, three in the alpha subunit (alpha 64N, alpha 81N, alpha 123N) and three in the beta subunit (beta 64N, beta 127N, and beta 147N). Seven different possible types of oligosaccharide structures were identified by mass including: a monosialobiantennary structure, bisialobiantennary structures without or with one fucose, trisialotriantennary structures without or with one fucose, and possibly a trisialotriantennary structure with two fucose and/or a tetrasialotriantennary structure. Site beta 64N exhibited the least glycosylation diversity, with two detected types of oligosaccharides, and site beta 147N exhibited the greatest diversity, with five or six detected types of oligosaccharides. Overall, the most abundant glycoforms detected were bisialobiantennary without fucose and the least abundant were monosialobiantennary, trisialotriantennary with two fucose and/or tetrasialotriantennary. Clusterin peptides accounting for 99% of the primary structure were identified from analysis of the isolated alpha and beta subunits, including all Ser- and Thr-containing peptides. No evidence was found for the presence of O-linked or sulfated oligosaccharides. The results provide a molecular basis for developing a better understanding of clusterin structure-function relationships and the role clusterin glycosylation plays in physiological function.     

Treatment of urinary incontinence in women in general practice: observational study

OBJECTIVE: To examine what is attainable when treating urinary incontinence in women in general practice. DESIGN: Observational study with 12 months' follow up. Interview and clinical examination before, during, and after treatment of women seeking help for urinary incontinence in general practice. SETTING: General practice in the rural district of Rissa, Norway. SUBJECTS: 105 women aged 20 or more with urinary incontinence. INTERVENTIONS: Treatment with pelvic floor exercises, electrostimulation, oestrogen, anticholinergic drugs, bladder training, and protective pads. MAIN OUTCOME MEASURES: Subjective and objective measures of urinary incontinence; number of patients referred to a specialist. RESULTS: After 12 months' follow up 70% (69/99) of the women were cured or much better; the mean score on a 100 mm visual analogue scale decreased from 37 to 20 mm; and the proportion of women who were greatly bothered by their incontinence decreased by 62%. 20% (20/98) of women became continent, and the percentage of women with severe incontinence decreased from 64% (63/99) to 28% (27/98). Mean leakage per 24 hours measured by a pad test decreased from 28 g at the start of treatment to 13 g after 12 months. The number of light weight pads or sanitary towels decreased from 1.6 to 0.6 a day. In all, 17/105 (16%) patients were referred to a specialist. CONCLUSIONS: Urinary incontinence in women can be effectively managed in general practice with fairly simple treatment. Most women will be satisfied with the results.     

Gonadotrophin heterogeneity and biopotency: implications for assisted reproduction

The extensive heterogeneity of the gonadotrophin hormones, follicle stimulating hormone (FSH) and luteinizing hormone (LH), is due primarily to the heterogeneous nature of their carbohydrate side-chains, in particular sialic acid residues. In this review, we discuss the role of carbohydrate chains in receptor binding and activation, biological activity, and metabolic half-life. The synthesis and secretion of the various glycoforms of both FSH and LH appear to be under endocrine control with gonadotrophin-releasing hormone (GnRH), oestradiol and testosterone playing important roles. Evidence for different glycoforms having variable biopotency or different encoded functions is increasing, and the production and secretion of more or less acidic gonadotrophin species in different physiological states may represent an important mechanism whereby the pituitary regulates gonadal cell and organ function. This has potential importance for the development of new pharmaceutical reagents and new therapeutic regimens in assisted reproduction. It is envisaged that the use of existing and new forms of FSH/LH will allow patients to be treated in a more controlled and physiological manner, with treatment regimens individualized to the needs of the patient.     

Results from in vitro fertilization and intracytoplasmic sperm injection with purified gonadotrophins (Metrodin HP)

Purified urinary follicle-stimulating hormone (uFSH-HP; Metrodin HP, Serono Ltd.) was compared with a combination of pure FSH and human menopausal gonadotrophin (hMG; Pergonal, Serono Ltd.) in patients undergoing standard in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). In standard IVF, pure FSH gave a significantly higher pregnancy rate per started cycle than did the combination with hMG (35 vs. 18%, p < 0.05). No differences between standard IVF and ICSI were seen which could be associated with hormonal stimulation in an open non-randomized series of patients. In 11 ICSI cycles, the use of recombinant FSH (Gonal F, Serono Ltd.) resulted in 4 ongoing pregnancies.     

Biological and immunochemical characterization of recombinant human thyrotrophin

Recombinant human thyroid-stimulating hormone (recTSH) has recently been engineered to detect metastatic lesions in patients operated on for thyroid cancer. In this report, we have compared the microheterogeneity, carbohydrate (CHO) content, mitogenic potency and immunoreactivity of the biotechnology product to those of human TSH of pituitary origin (pitTSH). Compositional analysis revealed that recombinant (rec) TSH produced in Chinese hamster ovary cells was overglycosylated compared with the native hormone (21 and 14%, respectively) with a higher amount of sialic acid and lack of N-acetylgalactosamine. Electrofocusing followed by immunoblotting resolved recTSH into six glycoforms with pIs ranging from 6.0 to 8.6, which were converted to a major species of pI 8.9 by sialidase treatment. pitTSH contained five main isoforms of pI 6.5-8.2 distinct from those of recTSH and partially resistant to sialidase. Binding activity of both human TSHs to porcine thyroid membrane receptors was found to be similar, but recTSH appeared to be 20% active compared to pitTSH in eliciting cAMP production and cell growth in rat FRTL-5 cells. Immunoreactivity of the recombinant hormone was investigated using polyclonal and monoclonal antibodies raised against the native hormone or synthetic peptide sequences of its subunits. While rec- and pitTSH were recognized to a similar extent by anti-protein antibodies, they exhibited a different binding pattern to antipeptide antibodies. Serial dilution of anti-alpha 1-25, anti-alpha 26-51, anti-beta 96-112 antisera bound recTSH to a greater extent than pitTSH, while anti-beta 31-51 and anti-beta 53-76 displayed similar recognition toward both preparations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Selective alterations in binding kinetic parameters and allosteric regulation of N-methyl-D-aspartate receptors after prolonged seizures in the developing rat brain

To investigate the effect of chronic smoke exposure on pulmonary macrophages (PM), the expression of seven different surface and intracellular molecules of PM was studied in induced sputum (IS) samples from healthy volunteers--nine smokers and seven non-smokers. Sputum was induced by inhalation of nebulized saline (3.5% NaCl). Cell viability and total cell counts (TCC) were performed immediately. Cell differentials were determined on May-Grunwald Giemsa-stained cytospin preparations. The PM were immunologically characterized by use of the following monoclonal antibodies: RFD1, RFD7, CD11b, CD54, CD68, CD71 and HLA-DR. The stainings were performed with a three-step, indirect immuno-alkaline phosphate method. Viability and TCC did not differ between the groups. Smokers had a higher percentage of macrophages (P < 0.05) and a lower proportion of neutrophils (P < 0.05). The percentage of macrophages expressing RFD1, HLA-DR, CD71 (P < 0.01 for all) and CD54 (P < 0.05) was significantly lower in smokers, whereas the remaining markers were expressed equally in the two groups. The results indicate that smoking induces a decrease in the expression by PM of surface molecules known to be associated with the antigen-presenting function.     

The V4-34 encoded anti-i autoantibodies recognize a large subset of human and mouse B-cells

Autoantibodies to the i, I and Pr2 carbohydrate determinants bind red blood cells, preferentially at low temperature in vitro. Using multiparameter flow cytometric analyses, we demonstrate that each of these autoantibodies also react with human and mouse lymphocytes at physiologic temperatures. The anti-Pr2 autoantibody recognizes a glycoprotein determinant(s) expressed by a subset of both T and B lymphocytes. In contrast, the binding of anti-i and anti-I antibodies each is restricted to B-lymphocytes. The anti-i autoantibody binds to over 50% of all B cells, whereas the anti-I antibody reacts with less than 10% of either tonsillar or blood B cells. Prior studies identified that the B cell isoform of CD45 (B220) has the linear poly-N-acetyllactosamine that forms the "i" determinant. Because anti-B220 antibodies recently have been reported to influence T-dependent B-cell isotype switching, we tested each antibody for its ability to influence the production of secondary Ig isotypes by murine splenocytes co-cultured with a stimulator helper T cell clone. We find that addition of anti-i antibody increases the proportion of B cells secreting secondary Ig isotypes. In contrast, the anti-I antibody had no such effect. These findings imply that stimulation of B cells through the highly conserved carbohydrate determinant that forms the "i" antigen may be of physiologic importance in T-dependent B-cell differentiation.