Spontaneous and tetracosactide-induced anti-ACTH antibodies in man.

CONTEXT: During a clinical trial of regular tetracosactide depot injections, four of 13 patients with autoimmune Addison's disease (AAD) developed adverse reactions immediately following tetracosactide injections. We wished to investigate whether these adverse effects could be due to the production of circulating antitetracosactide (ACTH1-24 ) antibodies. DESIGN: Anti-ACTH binding activity was investigated using immunoblotting and ELISA on sera from participants in the trial (n = 13; baseline and after tetracosactide exposure), 131 unrelated patients with AAD, 92 patients with Graves' disease (GD), 15 patients with isolated ACTH deficiency and 102 controls. Immunohistochemistry of human pituitary tissue sections was also performed using pooled sera. RESULTS: Bands at approximately 4 and 6 kDa, corresponding to ACTH1-24 and full-length ACTH1-39, respectively, were found in 10 of 13 (77%) of sera from trial patients exposed to tetracosactide, including all those who had an adverse reaction. This is in contrast with healthy control sera, which showed no binding. The same 10 subjects also showed high levels of binding to tetracosactide by ELISA, along with 21% of patients with AAD, 14% of patients with GD (both P < 0·001 compared to controls) and 1 isolated ACTH deficiency patient (7% of 15). These sera also recognized native ACTH in human pituitary sections. CONCLUSION: Our study demonstrates that repeated administration of depot tetracosactide can lead to anti-ACTH1-24 autoreactivity. In addition, a significant number of patients with AAD and GD also had similar, spontaneous, anti-ACTH reactivity. The presence of these antibodies could mediate some of the adverse effects or explain the well-described phenomenon of resistance to chronic ACTH therapy.

Comparative localization of corticotropin and corticotropin releasing factor-like peptides in the brain and hypophysis of a primitive vertebrate, the sturgeon Acipenser ruthenus L.

The sturgeon is a primitive actinopterigian fish that, unlike modern teleosts, possess a portal vascular system that connects a true median eminence with the anterior pituitary as in mammals. The occurrence and localization of corticotropin and corticotropin releasing factor-like immunoreactivies were examined in the brain of the sturgeon (Acipenser ruthenus L.) by immunocytochemistry with antisera raised against synthetic non-conjugated human corticotropin, and rat/human corticotropin releasing factor. In the hypothalamus, corticotropin-immunoreactive parvicellular perikarya were found in the infundibular nucleus and in dendritic projections to the infundibular recess. In addition, ependymofugal corticotropin-immunoreactive fibres were found to terminate in the ventral hypothalamus. Corticotropin releasing factor-immunoreactive neurons were found in the rostral portion of the ventral hypothalamus (tuberal nucleus), and in the vicinity of the rostral aspect of the lateral recess. These cells projected to the dorsal hypothalamus, the ventral hypothalamus, the median eminence, the anterior and posterior telencephalon, the tegmentum mesencephali, and the pars nervosa of the pituitary. An affinity-purified UI antiserum failed to stain the sturgeon hypothalamus. Corticotrophs in the rostral pars distalis of the pituitary were also corticotropin-immunoreactive. In the neurointermediate lobe, only about 50% of cells of the pars intermedia appeared to be corticotropin-positive, the rest appeared unstained. These results suggest that the presence of corticotropin-like and corticotropin releasing factor-like peptides in the brain is a relatively early event in vertebrate evolution, already occurring in Chondrostean/Actinopterigian fishes, as exemplified by A. ruthenus. The close spatial relationship between corticotropin releasing factor immunoreactivity and corticotropin immunoreactivity in the ventral hypothalamus of A. ruthenus supports a possible interaction between the two systems in that area of the sturgeon brain. The pars intermedia might be an important site for corticotropin synthesis, even though the possibility cannot be excluded that the antiserum was recognizing the proopiomelanocortin molecule. The occurrence of corticotropin releasing factor immunoreactivity in the region of median eminence/pars intermedia of the sturgeon suggests that the sturgeon corticotropin releasing factor might regulate the adenohypophyseal release of proopiomelanocortin products in the same manner as in other vertebrates. The presence of extrahypothalamic corticotropin releasing factor-immunoreactive projections suggests further neuromodulatory functions for this peptide in A. ruthenus.

Receptor-specific antibodies by immunization with "antisense" peptides?

Synthetic peptides whose sequences are specified by RNA complementary to the mRNA coding for peptide hormones have been reported to be useful antigens for the generation of receptor-specific antibodies. We have synthesized an eikositetrapeptide whose sequence corresponds to the complementary strand of the mRNA coding for the sequence of human ACTH(1-24). This "antisense" ACTH(1-24) peptide, "HTCAh," was coupled to bovine serum albumin or thyroglobulin prior to injection into rabbits. The complex proved to be very antigenic, inducing antisera of high titer and specificity. The antisera were tested in ACTH and MSH binding and bioassays, with or without prior purification of IgG molecules. None of the antisera displayed any effect in these assays, nor did they bind to blotted MSH/ACTH receptor protein from Cloudman S91 melanoma cells or to ACTH antibodies. The HTCAh peptide itself did not display measurable association to tritiated or iodinated ACTH(1-24), nor did it displace ACTH(1-24) in a receptor binding assay. However, the peptide bound to a low affinity site of mouse B16 melanoma cells which was independent of the MSH/ACTH binding site and induced melanin formation in these cells, but only at relatively high peptide concentration. Thus, in our hands, the antisense peptide approach using HTCAh as antigen did not lead to receptor-specific antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)

Multiple immunolabeling in histology: a new method using thermo-inactivation of immunoglobulins.

We describe a new method for successive immunofluorescence detection of multiple peptides in single paraffin tissue sections. Immunoglobulins were inactivated at 130 degrees C after each labeling step while tissues were protected with a mixture of phosphate-buffered saline and glycerin. The feasibility of the method is demonstrated by double-staining of rat pituitary corticotrophs with anti-ACTH[1-24] and anti-ACTH[17-39] antiserum. The method is applicable to brain tissue, since single neurons of ovine paraventricular hypothalamus could be triple stained with anti-vasopressin, anti-neurophysins I and II, and anti-CRF antiserum. The usual absorption controls and crossreactivity tests, as well as peptide staining, can be performed on the same tissue section. This new labeling procedure should prove useful in endocrinology, clinical pathology, and the neurosciences.

[1H-NMR study of the peptide corresponding to the ACTH-like sequence of the variable region of human G1 Eu heavy chain immunoglobin]. / 1H-IaMR-issledovanie peptida, sootvetstvuiushchego AKTG-podobnoi posledovatel'nosti variabel'noi chasti tiazheloi tsepi immunoglobulina G1 Eu cheloveka.

Synthetic decapeptide corresponding to ACTH-like sequence of the variable part of the heavy chain of immunoglobulin G1 Eu was studied by two-dimensional 1H-NMR spectroscopy (400 MHz). A complete assignment of signals in the peptide spectrum was made. The decapeptide was shown not to have any ordered spatial structure, and was characterized by a high extent of flexibility of the oligopeptide chain, except for the peptide bond with an N-terminal residue.

Immunochemical identification of an alpha 1-acid glycoprotein-antigenic determinant on carcinoembryonic antigen (CEA) and non-specific cross-reacting antigen (NCA).

An immunochemical characterization of carcinoembryonic antigen (CEA) and NCA (non-specific cross-reacting antigen) was performed. Positive reactions of CEA and NCA (Mr 60 000) with some antibodies to alpha 1-acid glycoprotein (AG) were observed. Thus, both antigens may contain immune determinants in common with alpha 1-acid glycoprotein. CEA showed positive reactivity with anti-NCA. NCA showed positive reactivity with either polyclonal or monoclonal antibodies to CEA, but negative reactivity with auto-antibodies to CEA. 125I-Tetracosapeptide (synthetic peptide-24 corresponding to the amino terminal sequence of CEA) failed to react with any antisera against CEA, NCA and AG. 125I-AG also showed no immuno-reaction with any antibody against CEA, NCA and tetracosapeptide. These results suggest that some monoclonal antibodies to CEA are directed against a common antigenic determinant of both CEA and NCA in addition to AG and tetracosapeptide, and that the auto-antibody to CEA is directed against a unique immune determinant which is not common to NCA. Thus, CEA appears to contain a unique determinant not found in NCA. Similarities in the composition of both amino acids and carbohydrates of CEA and NCA suggest that CEA is 'big-big' AG and NCA is 'big' AG.

Adrenocorticotropin radioimmunoassay: properties of antisera against synthetic ACTH(1-24) and its clinical application.

Two antisera against synthetic ACTH(1-24) developed in rabbit showed strikingly different affinities toward the ACTH molecule. Both antisera (A-6 and A-7) were highly specific for the COOH-terminal region of ACTH(1-24). Antisera A-6 recognized ACTH(1-39) poorly. Radioimmunoassays (RIAs) using these antisera permitted the rapid (less than or equal to 18 h) quantitation of ACTH(1-24) (A-6) or ACTH(1-39) (A-7) at picogram levels. ACTH levels were determined on silicic acid extracts of rat and human plasma samples by the RIA specific for mid-region of ACTH(1-39) (A-7) and compared with that obtained by an ACTH(34-39) (C-terminal) RIA. In nearly all cases the C-terminal/mid-region ACTH ratios were less than 1.0, indicating that C-terminus of ACTH is more readily degraded by tissue or blood peptidases than are internal sequences. A solid-phase immunoadsorbent RIA specific for the extreme COOH-terminus of ACTH(1-24) was developed by coupling antiserum (A-6) to Sepharose 4B. This assay exhibited the same specificity as the soluble antiserum, yet tolerated relatively high concentrations of protein. Although the assay was suitable for rapid quantitation of ACTH(1-24), a decrease in sensitivity was observed in comparison to a conventional assay.

[Antibody to 1-24 ACTH and hypofunction of the adrenal cortex in a patient who was unilaterally adrenalectomized for Cushing's syndrome (author's transl)].

We report here on a patient who was unilaterally adrenalectomized for Cushing's syndrome, and who developed antibodies to 1-24 ACTH. A 49-year-old nurse had undergone right adrenalectomy for adrenal adenoma. After surgery, she was treated with 0.5 mg of 1-24 ACTH-Z together with glucocorticoid replacement therapy for 40 days. Thereafter she was given 0.25-0.5 mg of ACTH-Z every other day for 4 months. ACTH-Z was then stopped for a year but glucocorticoid therapy was continued. About one year prior to this admission, 1 mg of ACTH-Z was again initiated 1 to 2 times a week. Glucocorticoid therapy was not withdrawn during the four years after adrenalectomy. She was admitted for the purpose of withdrawal of glucocorticoids. Her serum was found to bind labeled ACTH. This labeled ACTH was competitively displaced from binding by unlabeled hormones. Finally, reaction with specific antihuman Ig demonstrated an antibody of the IgG class. The titer of the antibodies gradually decreased after the discontinuation of ACTH-Z, but it is still present in measurable quantity in her serum. The clinical significance of the circulating anti-ACTH antibody in her serum is discussed.

Human corticotropin (ACTH) radioimmunoassay with synthetic 1--24 ACTH.

A corticotropin antiserum was obtained from rabbits immunized with synthetic 1--24 corticotropin conjugated with bovine serum albumin. The antiserum did not cross react with synthetic alpha-melanotropin or with synthetic beta-endorphin and had a cross reactivity of 0.23% with human beta-lipotropin. We developed a radioimmunoassay with the antiserum obtained, in which we used polyethylene glycol in conjunction with a second precipitating antibody for fast (15-min) separation of antibody-bound and free corticotropin. The assay had a sensitivity of 16 ng/L and was validated on patients with various pituitary and adrenal diseases. From 103 normal subjects, the median value for corticotropin in specimens collected during the morning was 34 ng/L of plasma; the upper 95% confidence limit of the normal range was 98 ng/L.

[Immunohistochemical study of the adenohypophysis of the monkey Macaca irus with the use of antibodies anti 1-24-ACTH, anti 17-39-ACTH, anti alpha-and beta-endorphins, anti beta-LPM]. / Etude immunohistochimique de l'adénohypophyse du singe Macacus irus à l'aide d'anticorps anti1-24ACTH, anti17-39ACTHanti alpha et beta-endorphines, anti beta-LPH.

Indirect immunofluorescence technique with anti1-24- and anti17-39 ACTH, anti alpha- and anti beta-endorphins, anti beta-LPH sera has allowed us to detect a cellular type in the anterior lobe of the hypophysis of Macacus irus which react simultaneously with these five antisera. These cells are especially localized in the ventro-medial zone, but there are also present in the pars distalis, under the glandular capsule, and in the lateral lobes, amid the other cellular types. The cells of the intermediate lobe react on the whole with anti1-24-, these antisera are also immunoreactive with the anti alpha- and anti17-39ACTH and anti beta-LPH ; SOME CELLS, WHich react with anti beta-endorphin antisera. The adenohypophysis of Macacus irus contains therefore two categories of cells reacting with the above mentioned antisera : one of this type, localized in the anterior lobe and in the intermediate lobe, react simultaneously with the five antisera, the other type, localized only in the intermediate lobe does not react with the antiendorphins antisera.

[Studies on the antigenicity of synthetic ACTH (beta1-24-corticotropin) in normal subjects (author's transl)]. / Untersuchungen zur Antigenität von synthetischem ACTH (beta1-24-Corticotropin) bei freiwilligen Versuchspersonen

9 healthy volunteers were subjected to a 3-week treatment with synthetic ACTH. Antibody response against beta1-24-corticotropin (Synacthen) was tested by passive transfer in the Prausnitz-Küstner reaction, complement fixation, passive hemagglutination and agar-gel diffusion. Failure of the healthy persons to produce reaginic or non-reaginic antibodies is compared titerature.