Engineered variants of human glutamic acid decarboxylase (GAD) and autoantibody epitope recognition.

Of the two homologous forms of glutamic acid decarboxylase, GAD65 and GAD67, only GAD65 is a common target of autoimmunity. Epitope profiles of autoantibodies to GAD65 (GADA) in 140 type 1 diabetes, adult-onset diabetes mellitus (AODM), and thyroid diseases (TD) were studied. Probes were GAD65, GAD65/67 hybrids (displaying separately GAD65 residues 1-95, 96-444, and 445-585), delta GAD65 (a truncated GAD65 spanning residues 69-585), and GAD67. delta GAD65 and GAD65 detected 137 and 125 positive patients, respectively. The hybrids reacted with 113 sera and in 3 cases disclosed cryptic epitopes. Eighteen patients reacted with GAD67, indicating GAD65-GAD67 cross-reactivity. Most patients recognized both middle and C-terminal epitopes, had low reactivity against N-terminal epitopes, and seldom displayed reactivity limited to the N or C terminus. Compared with type 1 and AODM, TD patients showed a greater prevalence of multiple reactivity and higher incidence of GAD67 positivity.

Replacement of methionine-161 with threonine eliminates a major by-product of human glutamate decarboxylase 65-kDa variant expression in Escherichia coli.

Most insulin-dependent diabetes mellitus patients gen-erate conformational autoantibodies to the islet-cell 65-kDa variant of human glutamate decarboxylase (GAD65), and several immunochemical tests for the early detection of type-1 diabetes rely on GAD65 antibody (GADA) assessment using properly folded recombinant GAD65 as the antigen. In addition, preventive therapies based on tolerization by GAD65 administration may be available in the near future. Therefore, there exists a strong interest in a facile and economically sound expression procedure for this antigen. Several attempts to produce, in native form, wild-type GAD65 in Escherichia coli have failed. However, this difficulty was recently surmounted in our laboratory by expressing GAD65 as a fusion protein with thioredoxin [Papouchado, Valdez, Ghiringhelli, Poskus and Ermácora (1997) Eur. J. Biochem. 246, 350-359]. In this work, a new GAD65 hybrid gene was prepared by joining engineered cDNA obtained from human and rat tissues. The new gene was modified additionally to finally code for human GAD65 with a single amino-acid substitution: Met-161-->Thr. This change impeded the co-expression of a 48-kDa by-product from an internal translation site. Also, a second 58-kDa by-product was identified as a GAD65 C-terminal proteolytic fragment that co-purifies with thioredoxin-M161T GAD65. The new GAD65 variant was expressed and easily purified, yielding an antigen that performed equally or better than wild-type GAD65 in the reference radiobinding assay for GADA. The procedure provides an inexpensive source of large amounts of fully active and immunochemically competent GAD65.

Increased adrenocortical responsiveness to exogenous ACTH in oral contraceptive users.

To evaluate the effects of changing steroid milieu on adrenocortical function, basal levels and responses of cortisol, 17-hydroxyprogesterone (17PO), androstenedione (A), dehydroepiandrosterone (DHEA), and testosterone to exogenous synthetic ACTH were investigated in six normal women during the early follicular (EF) and midluteal (ML) phases of the menstrual cycle and in five women on an oral contraceptive (OC) agent (35 micrograms ethinyl estradiol and 1 mg ethynodiol diacetate, Demulen). Baseline serum steroid and cortisol binding globulin (CBG) levels were measured on days 3-7 and 21-23 of the menstrual cycle in the normal subjects and on days 3-7 of OC treatment cycles. ACTH stimulation (10 micrograms m-2 i.v. bolus) was performed following dexamethasone suppression (0.5 mg p.o. q 6 h X 4). Basal levels of cortisol and CBG as well as cortisol responses to ACTH were increased in OC users relative to normal women tested during both the EF and ML phases of the cycle. In addition, 17PO levels were increased during the ML phase both before and following dexamethasone suppression compared to levels present in the EF phase and in OC users, no doubt because of increased ovarian steroidogenesis.

Increased cortisol production in women runners.

Because we previously found increased basal serum cortisol levels in women runners, we examined adrenocortical function in amenorrheic running women (AR), eumenorrheic running women (R), and normal nonexercising women (NC) in further detail. Mean 24-h urinary cortisol levels were significantly elevated (P less than 0.001) in six AR [45.1 +/- 7.2 (+/- SEM) micrograms/24 h] and eight R (38.5 +/- 6.9 micrograms/24 h) compared to four NC (13.9 +/- 2.8 micrograms/24 h). After adrenal suppression with 2 mg dexamethasone, integrated responses and absolute maximal elevations in serum cortisol levels in response to 10 micrograms/m2 exogenous ACTH (1-24) administered as an iv bolus dose, were not significantly different among six AR, six R, and six NC. This dose of ACTH results in maximal steroid release. The disappearance rates of cortisol (5 mg, iv) after dexamethasone suppression were similar in four AR, five R, and four NC and corresponded to a two-compartment model with mean half-lives of 4.9 and 93.8 min, respectively. Cortisol-binding globulin levels were also similar among the groups. These data document higher cortisol secretion and suggest increased ACTH secretion in running women.

Conservative treatment of ectopic pregnancy with methotrexate.

Six subjects with distal ampullary ectopic pregnancies were treated with four doses of intravenous methotrexate (1.0 mg/kg) followed by four doses of leucovorin (0.1 mg/kg, intramuscularly). The diagnosis was established in all cases by laparoscopy following sonography and radioimmunoassay for serum beta subunit of human chorionic gonadotropin. Subjects were followed with daily quantitative serum beta-human chorionic gonadotropin radioimmunoassay and sonography. Five of the six subjects experienced resolution of their ectopic pregnancy without additional surgical treatment. One subject underwent salpingectomy following treatment. Morbidity also included three patients with mild stomatitis or gastritis, and two patients had transient elevations of serum transaminase levels. Two patients had protracted courses and received blood transfusions. The most abrupt response and most uncomplicated courses were experienced in the three subjects with initial human chorionic gonadotropin levels below 1000 mIU/ml. This preliminary experience suggests that methotrexate may be an effective alternative for the treatment of early ectopic pregnancy.

Congenital adrenal hyperplasia and luteal dysfunction.

A case of 21-hydroxylase deficiency diagnosed and first treated at the age of 3 years is presented. Although pubertal development was delayed, full reproductive function was attained, with menses occurring at 14-33-day intervals. Infertility evaluation at age 20 revealed elevated 17-hydroxyprogesterone (17Po) and androgen concentrations and reduced luteal phase progesterone levels consistent with inadequate luteal function. This was corrected with additional corticosteroid replacement. Normal pregnancy and delivery followed.

Complete development of secondary sex characteristics in a case of 46,XY pure gonadal dysgenesis.

Although pubertal development is unusual in 46,XY gonadal dysgenesis, it may occur in association with gonadal tumors. The authors report a case of 46,XY gonadal dysgenesis in a 17-year-old girl remarkable for H-Y+ phenotype and bilateral gonadoblastomas accompanied by dysgerminomatous change and nearly complete female secondary sex development. Endocrinologic activity of the gonads was indicated by marked decrease in sex steroid production after gonadectomy. Occurrence of gonadal neoplasia in this case is consistent with the observation that neoplastic transformation is likely in H-Y+ cases of 46,XY gonadal dysgenesis.

Triple-X syndrome and premature ovarian failure.

Two patients with 47,XXX karyotypes and premature ovarian failure are described. Both presented with episodes of amenorrhea, elevated levels of circulating follicle-stimulating hormone and luteinizing hormone, and decreased estrogen concentrations. However, evidence of follicular activity existed in both cases. Why some women with 47,XXX karyotypes suffer from premature ovarian failure and why others have normal fertility and normal lengths of reproductive function is not known. Reports of immunologic disorders in patients with the triple-X syndrome and data showing that control of the lymphocyte may be related to the X chromosome point to a possible link between premature ovarian failure in 47,XXX women and immunologic abnormalities.