Biomedical subjects

N Cahir

Publications and source records attributed to N Cahir.

13 recordsLinked to original sources

PubMed2001

Development of an inhibin alpha subunit ELISA with broad specificity.

Inhibin immunoassays with a sufficiently broad specificity to detect all alpha subunit-containing forms are of value in detecting and monitoring various ovarian cancers. Assays to date with this specificity are not readily amenable to wide diagnostic application. The objective of this study was to develop a sensitive two-site ELISA using alpha subunit-directed monoclonal antibodies (Mabs) able to detect all forms of inhibin to replace a previously described alpha subunit-directed immunofluorometric assay (IFMA). In this study, the major inhibin epitopes in the two polyclonal antisera used in the alphaC IFMA were initially identified and Mabs were raised to these regions. These Mabs in conjunction with the inhibin alpha subunit R1 Mab (Groome) were used to develop alpha subunit ELISAs with high sensitivity. Application of these assays to human serum and human follicular fluid following fractionation by an immunoaffinity/preparative PAGE/electroelution procedure which separated inhibins according to their molecular weights, indicated that the specificity of the various ELISAs differed between Mab combinations with preferences noted for either the alpha subunit or dimeric forms. A combination of Mabs in an ELISA was identified which provided data which matched that obtained with the alphaC IFMA and which may be useful as a replacement inhibin assay in clinical studies.

Animals↗

PubMed2000

Relationship between serum inhibin A and B and ovarian follicle development after a daily fixed dose administration of recombinant follicle-stimulating hormone.

The aim of this study was to investigate the relationship of serum inhibin A and inhibin B to ovarian follicular development in women undergoing pituitary down-regulation and ovarian stimulation with a fixed daily dose of recombinant human FSH in an in vitro fertilization program. Thirty-eight patients were treated randomly with either 100 or 200 IU/day recombinant human FSH (Puregon) for a period of 9-14 days. Serum FSH, inhibin A, inhibin B, 17beta-estradiol, and follicular size and number were determined before FSH treatment and every second day from days 4-6 throughout FSH treatment. Serum FSH increased in a dose-related manner to reach a maximum by days 4-6 and remained unchanged over the duration of treatment. Serum inhibin A and 17beta-estradiol also increased with increasing FSH dose and continued to rise throughout the FSH treatment period. By contrast, serum inhibin B was increased by days 4-6 at both doses of FSH to reach a maximum by days 7-8, remaining unchanged thereafter. Serum inhibin B and, to a lesser extent, inhibin A correlated significantly with the number of oocytes retrieved even when assessed early (days 4-6) in the treatment period (inhibin B vs. number of oocytes: r = 0.89; P < 0.001; inhibin A vs. number of oocytes: r = 0.61; P < 0.05). Serum inhibin A, inhibin B, and 17beta-estradiol were weakly correlated with the number of follicles less than 11 mm when assessed on a daily basis; stronger correlations were observed with the greater than 11-mm follicles during the late stages of treatment. It is concluded that serum inhibin B levels determined during the early stages (e.g. days 4-6) of fixed dose FSH treatment provide an early indicator of the number of recruited follicles that are destined to form mature oocytes. In this context, serum inhibin B may be of predictive value in monitoring ovarian hyperstimulation treatment for in vitro fertilization.

Adult↗

PubMed1999

Inhibin forms in serum from postmenopausal women with ovarian cancers.

BACKGROUND AND OBJECTIVE: Previous studies have shown that serum inhibin as measured by alpha subunit-directed radioimmunoassay (RIA) and inhibin A ELISA was elevated in postmenopausal women with mucinous and granulosa cell cancers, with the RIA showing a more frequent elevation than the inhibin A ELISA. It was thus hypothesised that these cancers may also produce inhibin B or the free alpha subunit. The aim of the study was to identify the forms of inhibin found in a range of ovarian cancers using a range of inhibin assays with varying specificities. DESIGN: Serum samples obtained from women with ovarian cancer were assayed by inhibin B ELISA and Pro-alpha C subunit ELISA and compared with inhibin RIA and inhibin A ELISA. PATIENTS: Blood samples were obtained from 34 postmenopausal women (> 55 years) with no history of endocrine disease and from women with ovarian serous cystadenocarcinomas (n = 66), mucinous cystadenocarcinomas (n = 20), granulosa cell tumours (n = 9-11), miscellaneous ovarian cancers (n = 46) and non ovarian cancers (n = 23). MEASUREMENTS: Inhibin B and inhibin Pro-alpha C subunit levels were determined by ELISA and compared to values obtained by RIA and inhibin A ELISA. Cancers were discriminated from controls based on values obtained 2SD above the geometric mean of the control values. RESULTS: Granulosa cell tumours were detected by RIA and inhibin B ELISA (100%), Pro-alpha C ELISA (90%) and inhibin A ELISA (77%). Mucinous tumours were detected by RIA (70%), inhibin B ELISA (60%), Pro-alpha C ELISA (55%) and inhibin A (20%). Serous tumours were detected by RIA (35%) and the other assays (< 15%). Miscellaneous tumours were detected by RIA (41%) and other assays < 30%. CONCLUSIONS: Ovarian neoplasms may produce a variety of peptides related to the inhibins, including dimeric inhibin A and B. Inhibin B is detected in more ovarian cancers than inhibin A but does not discriminate as well as the alpha subunit directed assays. The higher discrimination index obtained with the RIA compared to the Pro-alpha C ELISA suggests that assays detecting all inhibin forms containing the alpha subunit and not just those detecting the Pro-alpha C subunit will provide the most useful detection method.

Aged↗

PubMed1999

Combined inhibin and CA125 assays in the detection of ovarian cancer.

BACKGROUND: The reproductive hormone inhibin has been used as a diagnostic marker of ovarian mucinous and granulosa cell cancers. The aims of this study were to develop a new inhibin immunofluorometric assay (alphaC IFMA) to replace an inhibin RIA as a diagnostic marker of these ovarian cancers and to assess whether the alphaC IFMA in combination with CA125, which detects serous cancers, leads to an improved biochemical diagnosis of all ovarian cancers. METHODS: Serum inhibin concentrations were determined in healthy postmenopausal women (n = 165) and women with ovarian cancers (n = 154), using an inhibin RIA and an alphaC IFMA, which detects inhibin forms containing the alphaC subunit as well as the free alphaC subunit. RESULTS: The alphaC IFMA gave a similar or better discrimination of mucinous (90% vs 71%) and granulosa cell (100% vs 100%) cancers compared with the inhibin RIA. Combination of CA125 and alphaC IFMA values by canonical variate analysis or by multiROC analysis showed that the percentage of all ovarian cancers detected was significantly increased compared with either CA125 or alphaC IFMA alone. CONCLUSIONS: The alphaC IFMA shows a similar or better specificity compared with the RIA, but with increased sensitivity. In combination with CA125, the alphaC IFMA provides an effective dual test for the detection of the majority (90%) of ovarian cancers.

Adenocarcinoma, Mucinous↗

PubMed1999

[Inhibin and ovarian cancer].

Previous observations from our laboratory have demonstrated that the levels of immunoreactive inhibin (ir-inh) are elevated in almost all patients with granulosa cell tumors and in the majority of postmenopausal women with mucinous ovarian cancers. The present report confirms these findings in a larger group of post-menopausal women. Immunohistochemistry for the inhibin alpha. beta A and beta B sununits shows predominantly epithelial staining in granulosa cell tumors and in the majority of mucinous cancers. Serous cystadenocarcinomas also frequently show positive staining. Studies seeking to identify G alpha i-2 or FSH receptor mutations have provided negative results in contrast to other reports. Further studies of the roles of the inhibin-related family of peptides in ovarian cancer diagnosis and monitoring are clearly indicated.

Adenocarcinoma, Mucinous↗

PubMed1998

Inhibin and ovarian cancer.

Previous observations from our laboratory have demonstrated that the levels of immunoreactive inhibin (ir-inh) are elevated in almost all patients with granulosa cell tumours and in the majority of postmenopausal women with mucinous ovarian cancers. The present manuscript confirms these findings in a larger group of postmenopausal women. Immunohistochemistry for the inhibin alpha, betaA and betaB subunits shows predominantly epithelial staining in granulosa cell tumours and in the majority of mucinous cancers. Serous cystadenocarcinomas also frequently show positive staining. Studies seeking to identify G alpha(i-2) or FSH receptor mutations have provided negative results in contrast to other reports. Further studies of the roles of the inhibin-related family of peptides in ovarian cancer diagnosis and monitoring are clearly indicated.

Aged↗

PubMed1998

Serum inhibins A and B fall differentially as FSH rises in perimenopausal women.

BACKGROUND: Serum FSH levels rise with increasing age in normal women, particularly as they enter the menopausal transition and progress to the postmenopausal state. The contributions of decreasing levels of inhibin-A (INH-A) and inhibin-B (INH-B) to this rise are presently unclear, as there are no reports of dimeric INH levels in relation to menopausal status. The present study was undertaken in order to provide preliminary data on relationships amongst the dimeric inhibins, oestradiol (E2) and FSH in normal subjects of defined menopausal status. METHODS: Single serum samples were obtained between cycle days 3 and 8 in regularly cycling women, or at random in those with irregular cycles or amenorrhoea, in 110 women, aged 48-59 years, in the third year of a prospective longitudinal study of the menopausal transition, 'The Melbourne Women's Mid-Life Health Project'. Samples were assayed for FSH, E2, INH-A, INH-B and immunoreactive inhibin (IR-INH) and results were analysed following logarithmic transformation. Undetectable values were assigned the limit of sensitivity of the respective assays. The relationships between hormones were evaluated as a function of menopausal stage. The latter was assigned as Stage 1, premenopausal (no reported change in menstrual cycle pattern), Stage 2, early peri-menopausal (reported change in menstrual cycle frequency in the preceding year with a bleed in the preceding 3 months), Stage 3, late peri-menopausal (no menses in the preceding 3-11 months) and Stage 4, postmenopausal (no menses in the preceding 12 months). RESULTS: The hormone concentrations in premenopausal subjects (geometric means, FSH 13.5 IU/l, E2 306 pmol/l, IR-INH 217 U/l, INH-A 96 ng/l, and INH-B 48 ng/l) were used as reference points for the other stages of menopausal status. Early peri-menopausal subjects had significantly lower levels of IR-INH (147 U/l) and INH-B (13.5 ng/l) in the presence of a small, statistically nonsignificant rise in FSH (to 21.4 U/l) and no significant change in E2 or INH-A. In late peri-menopausal subjects, IR-INH fell to 76 U/l, INH-A fell to 4.2 ng/l, whilst INH-B was not significantly different at 14 ng/l. FSH had risen significantly to 72.21 U/l. Oestradiol also fell significantly to 89 pmol/l. In the postmenopausal subjects there were no further significant changes in the peptide hormones or FSH, but E2 fell further to 41 pmol/l. There was a significant (P < 0.05) inverse correlation between FSH and E2 (R = -0.78), FSH and IR-INH (R = -0.66), FSH and INH-A (R = -0.53), FSH and INH-B (R = -0.29) while IR-INH and either INH-A or INH-B were positively correlated (R = +0.57 and +0.35, respectively). The data are consistent with negative feedback roles for both dimeric inhibins and E2 as contributors to the regulation of FSH secretion as menopausal status changes. CONCLUSIONS: The major significant endocrine event in women in the early peri-menopausal phase of the menopausal transition is a substantial fall in the circulating levels of inhibin-B with no significant change in inhibin-A or oestradiol. Progression to late peri-menopausal status is accompanied by a marked fall in inhibin-A and oestradiol and a rise in FSH without further change in inhibin-B. Inhibin-B, a marker of follicle number, is a significant factor in the endocrinology of the menopausal transition.

Cross-Sectional Studies↗

PubMed1997

The biological and immunological characterization of inhibin A and B forms in human follicular fluid and plasma.

In a previous study (see Ref.7), the molecular weight distribution of inhibin activity in fractionated human follicular fluid (hFF) and human male and female plasma/serum was determined by in vitro bioassay using ovine pituitary cells in culture and various specific inhibin A and inhibin alpha-subunit-directed immunoassays. It was shown, however, that the ovine in vitro bioassay detected inhibin B poorly. These findings are extended in the present study by the determination of the molecular weight profile of in vitro bioactivity using rat pituitary cells, which detects both inhibin A and B, a specific inhibin B enzyme-linked immunosorbent assay (ELISA), an RIA detecting the alpha N region of the alpha-subunit, an alpha-subunit ELISA (Pro-alpha C) directed to the inhibin forms containing the Pro sequence, and an alpha C subunit immunofluorometric assay that detects all inhibin forms. The profile in hFF of inhibin in vitro bioactivity, using rat pituitary cells in culture, significantly (P < 0.001) correlated with in vitro bioactivity using ovine pituitary cells (r = 0.85), inhibin A immunoactivity (r = 0.70), inhibin B immunoactivity (r = 0.89), and the combination of inhibin A + B immunoactivities (r = 0.93), with peaks of activity identified at 66K, 55K, 36K and 33K, consistent with presumed known mol wt forms of inhibin. Inhibin B profiles in fractionated serum from women stimulated with gonadotropins and male plasma consisted of two forms (66K and 36K), whereas inhibin A in female serum included, in addition, the 55K form. These findings indicated that higher molecular weight forms of inhibin B are present in biological samples, and their distribution differs from that of inhibin A, suggesting a differential processing of the precursor forms in the circulation. Pro-alpha C immunoactivity was identified in serum samples with prominent peaks at 36K and 29K (known Pro-alpha C subunit forms) and not with any high mol wt dimeric forms of inhibin. If this observation applies to a wider range of serum samples, the Pro-alpha C ELISA may provide an appropriate and specific assay for the measurement of free alpha-subunit. To compare immunoactivity levels between assays, the inhibins A, B, and Pro-alpha C standards were calibrated in terms of their alpha C subunit content, as determined by an alpha C subunit immunoassay, with the inhibin B standard containing 60% of the alpha C subunit content compared with either the inhibin A or Pro-alpha C standard. After adjustments of the various standards for this difference in alpha C subunit content, a comparison was undertaken of the combined levels of inhibins A, B, and Pro-alpha C immunoactivity across the hFF and serum chromatograms and compared with levels determined by the alpha-subunit-directed immunoassays. A high correlation (r = 0.59-0.96) was observed, indicating that the alpha-subunit immunoactivity in serum consists largely of a composite of presumed known molecular weight forms of inhibins A, B, and Pro-alpha C. It is concluded that: 1) inhibin in vitro bioactivity in hFF is largely attributed to the presence of 33-36K and 50-66K forms of inhibins A and B; and 2) inhibin alpha-subunit immunoactivity in hFF and serum is a composite of presumed known forms of inhibin A, inhibin B, and the alpha-subunit.

Animals↗

PubMed1996

Characterization of inhibin immunoreactivity in post-menopausal women with ovarian tumours.

BACKGROUND AND OBJECTIVE: We have previously reported elevated serum immunoreactive inhibin (INH) levels in patients with ovarian malignancies, particularly granulosa cell and mucinous tumours. The present study was designed to compare INH measurements using a heterologous radioimmunoassay with cross-reactivity for inhibin alpha-subunit derived peptides with measurements obtained using a new ELISA specific for dimeric inhibin-A. It was hypothesized that granulosa cell tumours may secrete significant quantities of inhibin-A whereas mucinous tumours were unlikely to do so because of the lack of a relation between INH and FSH measurements in the latter group. DESIGN: Serum samples obtained from women found to have ovarian cancer were assayed using the heterologous radioimmunoassay (the Monash assay) and using an ELISA specific for dimeric inhibin (the Groome assay) and the results were compared. PATIENTS: Samples for assay were available from 69 normal post-menopausal control women, 12 patients with mucinous tumours of the ovary, 26 with serous tumours, 7 with granulosa cell tumours and 8 with various other ovarian tumours. Patients were post-menopausal or had been subjected to bilateral oophorectomy at the time these samples were collected. MEASUREMENTS: The Monash and Groome assays were carried out as described previously. The upper limit of normal for post-menopausal women in the Monash assay was 122 U/l and for the Groome assay was calculated to be 32 ng/l. RESULTS: Among the 69 normal subjects, 4 were found to have elevated inhibin levels using the Monash RIA (133-190 U/l) and 4 were found to have elevated levels in the Groome ELISA (45.5-55.3 ng/l). Among 12 patients with mucinous tumours, 10 (83%) had elevated inhibin levels using the Monash assay but only 3 (25%) had elevated levels with the Groome assay (P < 0.005). Among 26 with serous tumours, 15 (58%) had elevated levels in the Monash assay but only 1 (4%) in the Groome assay (P < 0.001). Among 7 samples from patients with granulosa cell tumours, 100% were elevated in the Monash assay and 71% in the Groome assay (NS, non-significant). In a miscellaneous group of tumours all 8 had elevated levels in the Monash assay and 2 in the Groome assay (P < 0.001). CONCLUSIONS: It was concluded that in normal postmenopausal subjects, INH is generally undetectable or present at low levels, consistent with the loss of ovarian function. The majority of granulosa cell tumours appear to secrete significant amounts of dimeric inhibin-A, whereas mucinous tumours secrete predominantly other forms of INH, presumably related to the alpha-subunit. Serous tumours may also secrete inhibin-related peptides but not dimeric inhibin-A. The nature of the inhibin related peptides produced by epithelial ovarian cancers remains to be characterized.

Adenocarcinoma, Mucinous↗

PubMed1996

Biological and immunological characterization of inhibin forms in human plasma.

A number of immunoassay methods have been developed recently to detect specifically the bioactive alpha-beta A subunit inhibin dimer (inhibin A) in human plasma. However, the specificity of these assays in terms of their ability to detect the range of inhibin forms found in plasma and their relationship to bioactivity have not been investigated. Inhibin was fractionated from human follicular fluid (hFF) and serum/plasma from women stimulated with gonadotropins (IVF serum), and from postmenopausal and male plasma, using a combined immunoaffinity/preparative SDS-PAGE procedure. The molecular weight profile of inhibin was established by inhibin in vitro bioassay, three alpha-beta A subunit specific immunoassays, and three alpha subunit-directed immunoassays that detect the alpha subunit as well as inhibin A and B forms. In hFF inhibin forms of 33, 36, 55 and 66K were detected by in vitro bioassay and by most immunoassays except for 33 k inhibin, which was nondetectable by one alpha-beta A ELISA. The alpha subunit-directed assays also detected activity in the 29-31K region, in some assays in considerably high levels. In IVF serum in vitro bioactivity and immunoactivities were detected between 27 and 100K with the alpha-beta A assays failing to detect all bioactive forms. Alpha subunit-directed assays gave similar immunoactive profiles. Neither in vitro bioassay nor alpha-beta A assays detected activity in post-menopausal plasma or male plasma, while alpha subunit-directed assays showed peaks predominantly at 36 k, although at low levels. It is concluded that dimeric inhibin A specific assays detected bioactive inhibin forms in hFF and to a lesser extent in IVF serum. Alpha subunit-directed assays correlated poorly with in vitro bioassay in hFF because of the high alpha subunit levels in this sample. The higher correlation between these assays in IVF serum suggested that there was little free alpha subunit. The 36K form in male plasma may be free alpha subunit or inhibin B.

Animals↗

PubMed1995

Inhibin forms in human plasma.

In order to identify the molecular weight forms of bioactive and immunoactive inhibin in human plasma, plasma/serum was sequentially fractionated by immunoaffinity chromatography (using immobilised inhibin alpha subunit antiserum), reversed phase HPLC and preparative SDS-PAGE. The electroeluted gel fractions were assayed for inhibin in vitro bioactivity and immunoactivity, the latter by RIA. Initial experiments examined human follicular fluid as an inhibin-rich source. Bioactive and immunoactive fractions of 30, 35, 53, 65 and approximately 120 kDa were identified in addition to bio-inactive, immunoactive fractions of 26 kDa and 32 kDa. These molecular weights correspond to those of known inhibin forms and are attributed to differing degrees of glycosylation of the inhibin alpha subunit and variable processing of the alpha and beta inhibin subunits. Fractionation of male plasma pools revealed the presence of higher molecular weight immunoactive forms (55-120 kDa) as well as 28-31 kDa forms although the molecular weight distribution of activity between pools varied. To assess if the molecular weight pattern was modified by storage and/or subsequent fractionation, protease inhibitors were added initially to plasma and fractionated as above. The molecular weight distribution of immunoactivity was largely unaffected by the treatment, indicating that minimal processing had occurred. Postmenopausal serum itself showed low to undetectable activity. The addition of recombinant human 31 kDa inhibin to postmenopausal serum resulted in a molecular weight profile of inhibin immunoactivity consistent with the presence of 31 kDa inhibin. Fractionation of a serum pool from women undergoing gonadotrophin stimulation, in which inhibin levels were elevated, showed a range of bioactive and immunoactive inhibin forms over the 30-120 kDa range.(ABSTRACT TRUNCATED AT 250 WORDS)

Chromatography, Affinity↗

PubMed1994

Specific roles for the asparagine-linked carbohydrate residues of recombinant human follicle stimulating hormone in receptor binding and signal transduction.

FSH comprises two distinct subunits, both of which contain asparagine-linked carbohydrate residues, located at positions 52 and 78 on the alpha-subunit and positions 7 and 24 on the beta-subunit. These carbohydrate chains have been shown to regulate the biological activity of FSH, including signal transduction and receptor binding. However, the specific roles of the individual carbohydrate chains have been poorly defined. Using site-directed mutagenesis we disrupted the consensus sequences for glycosylation and expressed the mutated cDNAs in Chinese Hamster Ovary cells. Specifically deglycosylated FSH variants were secreted from all clonal cell lines expressing the mutated FSH cDNAs except for the cell line that lacked all four glycosylation sites. Analysis of the singly or doubly deglycosylated FSH mutants revealed that removal of the carbohydrate residue at position 78 on the alpha-subunit significantly increased the receptor binding affinity of human FSH by 72%. Removal of the other carbohydrate residues had no significant effect on receptor binding. The carbohydrate residue at position 52 on the alpha-subunit was found to play an essential role in signal transduction as its removal resulted in a significant decrease in potency to 26% of wild type levels. The other individual carbohydrate residues appear to play a minor role in signal transduction, although removal of each residue results in reduced maximal response. The removal of both alpha-subunit carbohydrates resulted in a significant decrease in biopotency, to 41% of wild type levels; whereas, the removal of both beta-subunit carbohydrate chains resulted in a significant increase in biopotency, to 216% of wild type levels. These studies have allowed the identification of site-specific roles for the carbohydrate residues of human FSH. Our data suggest that the carbohydrate residues play a greater role in determining the biological activity of FSH than has been suggested in similar studies of other glycoprotein hormones.

Animals↗

PubMed1991

The Psychology Student Stress Questionnaire.

Stressors of graduate psychology training remain relatively unexplored. The Psychology Student Stress Questionnaire (PSSQ) was developed to assess the impact of emotional, financial, and academic stressors of graduate psychology training on students. The PSSQ was administered, along with the Symptom Check List-90-R and the Health and Daily Living Form, to 133 graduate psychology students. Significant though limited correlations were obtained between the PSSQ and the two stress measures. Factor analysis of the PSSQ yielded seven underlying factors; time constraints accounted for the greatest variance in stress ratings. Female students had higher stress scores than males. These results suggest that the PSSQ could be useful in exploring student stress in graduate psychology training.

Achievement↗