Corticotrophin-releasing activity of desmopressin in Cushing's disease: lack of correlation between in vivo and in vitro responsiveness.

Desmopressin (DDAVP), an arginine vasopressin analogue, markedly stimulates ACTH secretion in patients with Cushing's disease, in contrast to its minimal effect in normal subjects. However, little is known about the mechanisms underlying this action and it appeared to be of interest to evaluate the effect of DDAVP on ACTH-secreting pituitary adenomas in vitro, in comparison with its effect in the same patients in vivo. Pituitary adenomas from 14 patients with Cushing's disease were incubated with DDAVP, corticotrophin-releasing hormone (CRH) and DDAVP together with vasopressin receptor antagonists or CRH. Incubation with DDAVP induced a modest dose-dependent increase in ACTH concentrations which appeared maximal at 10 nM. CRH stimulated ACTH to a greater extent compared with DDAVP and potentiated the effect of DDAVP alone. The DDAVP-induced ACTH increase appeared blunted by vasopressin V(2) and V(3) receptor antagonists. V(3) receptor gene expression was detected by RT-PCR in all adenoma samples except for two which were not responsive to DDAVP in vitro but responsive to the peptide in vivo. Surprisingly, no difference in the in vitro ACTH secretory response was observed between in vivo DDAVP-responsive (ACTH peak>150% baseline) and -unresponsive (ACTH peak<120% baseline) patients, suggesting that the pituitary adenoma is not the sole mediator of the ACTH-releasing effect of DDAVP. In conclusion, the marked stimulatory effect of DDAVP observed in patients with Cushing's disease appears to be mainly dependent on an extrapituitary action, possibly the inhibition of a corticotrophin release-inhibitory factor.

Paracrine delivery of IL-12 against intracranial 9L gliosarcoma in rats.

OBJECT: Interleukin-12 (IL- 12) has potential for the treatment of tumors because it can stimulate an antitumor immune response and possesses antiangiogenic properties. In the study reported here, the authors investigated the therapeutic role of locally delivered IL-12 in a malignant brain tumor model. METHODS: After genetically engineering 9L gliosarcoma cells to express IL-12 (9L-IL12 cells), the authors used these cells as a source of locally delivered cytokine. First, they investigated the behavior of these cells, which were implanted with the aid of stereotactic guidance into the rat brain, by using serial magnetic resonance imaging and histopathological examination. Second, they assessed the antitumor efficacy of proliferating, as well as nonproliferating (irradiated), 9L-IL12 cells by implanting these cells in animals challenged by wild-type 9L gliosarcoma (9Lwt) cells. The IL-12 expression in brain regions injected with 9L-IL12 was confirmed by reverse transcription-polymerase chain reaction. Last, the authors explored whether animals treated with 9L-IL12 cells developed an antitumor immunological memory by rechallenging the survivors with a second injection of 9Lwt cells. The authors demonstrated that local delivery of IL-12 into the rat brain by genetically engineered cells significantly prolongs survival time in animals challenged intracranially with a malignant glioma. CONCLUSIONS: These findings support continued efforts to refine local delivery systems of IL-12 in an attempt to bring this therapy to clinical trials.

Retroviral-mediated transfer of the galactocerebrosidase gene in neural progenitor cells.

Globoid cell leukodystrophy (GCL or Krabbe disease) is a recessive disease caused by mutations of the lysosomal enzyme galactocerebrosidase (GALC) and twitcher is the murine model of GCL. We have prepared retroviral packaging cell lines to transduce the GALC gene. Retroviral transduction restored GALC activity in GCL fibroblasts and increased such activity to very high levels in immortalized neural progenitor cells (ST14A cells). GALC activity was also normalized in twitcher fibroblasts co-cultured with ST14A cells over-expressing GALC, demonstrating that this enzyme is secreted and can be imported efficiently by GALC-deficient cells. These results give the necessary background to evaluate the therapeutic effect in twitcher of brain grafting of neural progenitor cells engineered to release high levels of GALC.

Concomitant deficiency of beta- and gamma-sarcoglycans in 20 alpha-sarcoglycan (adhalin)-deficient patients: immunohistochemical analysis and clinical aspects.

We have investigated the expression, using immunohistochemistry, of beta- and gamma-sarcoglycans in the muscles of 20 patients in whom previous screening had revealed a deficiency of alpha-sarcoglycan. alpha-, beta- and gamma-sarcoglycans were absent in 7 patients and variably reduced in 8 patients, in 2 of whom beta-sarcoglycan was more reduced than the alpha- and gamma-proteins. In 5 other patients with variably reduced alpha- and beta-sarcoglycans, gamma-sarcoglycan was completely absent. In all patients the distribution of hyposthenia at disease onset was similar, and predominantly involved pelvic girdle muscles; however, the age at onset and rate of disease progression were highly variable. In severely compromised patients, the onset of disease was before 10 years of age and gamma-sarcoglycan or all three sarcoglycans were absent from muscles. Immunohistochemical analysis of sarcoglycans should be part of routine screening for muscle dystrophies to identify patients with sarcoglycanopathy. Gene analysis is necessary to identify the primary defect; however, sarcoglycan immunohistochemistry may be useful for indicating which gene to investigate. Further biochemical characterization of the interactions between these proteins is required to fully elucidate their roles in causing severe, moderate or mild muscular dystrophy.

Acetylcholine receptor alpha-subunit isoforms are differentially expressed in thymuses from myasthenic patients.

A central role of the thymus in autosensitization to the acetylcholine receptor has been proposed to explain the immunopathogenetic processes in myasthenia gravis (MG). Two isoforms of the alpha-subunit of the acetylcholine receptor are known; they differ by a 25-amino-acid insertion coded by the P3A exon. We investigated the expression of the P3A exon by RNA polymerase chain reaction in fetal and adult human myoblasts and TE671 cells; both isoforms were expressed. Muscle biopsies from patients with MG, Duchenne muscular dystrophy, and polymyositis were also studied and it was again found that both isoforms were expressed, indicating that the P3A exon is not associated with autoimmune, degenerative, and inflammatory muscle diseases. When P3A expression was studied in thymus samples from normal subjects and from thymectomized MG patients, the P3A+ subunit was absent in 75% of patients with involuted thymus and in all patients with thymomas but was present in normal thymuses and in patients with hyperplasia. Differential expression of the alpha-subunit isoforms of the acetylcholine receptor within the thymus may play a role in the immune pathogenesis of MG.

Expression of transforming growth factor-beta 1 in dystrophic patient muscles correlates with fibrosis. Pathogenetic role of a fibrogenic cytokine.

Duchenne muscular dystrophy is a fatal disorder characterized by progressive muscular weakness, wasting, and severe muscle contractures in later disease stages. Muscle biopsy reveals conspicuous myofiber degeneration and fibrosis substituting muscle tissue. We quantitatively determined mRNA of the potent fibrogenic cytokine transforming growth factor-beta 1 by quantitative PCR in 15 Duchenne muscular dystrophy, 13 Becker muscular dystrophy, 11 spinal muscular atrophy patients, and 16 controls. Higher transforming growth factor-beta 1 expression was greater in Duchenne muscular dystrophy patients than controls (P = 0.012) and Becker patients (P = 0.03). Fibrosis was significantly more prominent in Duchenne muscular dystrophy than Becker muscular dystrophy, spinal muscular atrophy, and controls. The proportion of connective tissue in muscle biopsies increased progressively with age in Duchenne muscular dystrophy patients, while transforming growth factor-beta 1 levels peaked at 2 and 6 yr of age. Transforming growth factor-beta 1 protein was also detected by immunocytochemistry and immunoblotting. Our findings suggest that transforming growth factor-beta 1 stimulates fibrosis in Duchenne muscular dystrophy. Expression of transforming growth factor-beta 1 in the early stages of Duchenne muscular dystrophy may be critical in initiating muscle fibrosis and antifibrosis treatment could slow progression of the disease, increasing the utility of gene therapy.