To define domains of the human MxA GTPase involved in GTP hydrolysis and antiviral activity, we used two monoclonal antibodies (mAb) directed against different regions of the molecule. mAb 2C12 recognizes an epitope in the central interactive region of MxA, whereas mAb M143 is directed against the N-terminal G domain. mAb 2C12 greatly stimulated MxA GTPase activity, suggesting that antibody-mediated crosslinking enhances GTP hydrolysis. In contrast, monovalent Fab fragments of 2C12 abolished GTPase activity, most likely by blocking intramolecular interactions required for GTPase activation. Interestingly, intact IgG molecules and Fab fragments of 2C12 both prevented association of MxA with viral nucleocapsids and neutralized MxA antiviral activity in vivo. mAb M143 had no effect on MxA function, indicating that this antibody binds outside functional regions. These data demonstrate that the central region recognized by 2C12 is critical for regulation of GTPase activity and viral target recognition.z 1999 Federation of European Biochemical Societies.

Objective: With increasing life span osteoporosis becomes a more recognized problem in patients with cystic fibrosis (CF). The aim of this cross-sectional study in 75 adult patients with CF (mean age 25.3 years) was to assess the prevalence of low bone mineral density (BMD) by dual-energy x-ray absorptiometry (DEXA) and, for the first time, by quantitative ultrasound (QUS), and to identify predicting factors. Design and Methods: Bone status was assessed at the lumbar spine (L2-L4) and the femoral neck by DEXA, and at the calcaneus by QUS (stiffness index). These data were correlated with a variety of clinical and anthropomorphic variables. Biochemical markers of bone turnover such as osteocalcin, bone-specific alkaline phosphatase, crosslinks in urine, 25-hydroxy vitamin D (25-OH vitamin D), parathyroid hormone, calcium and free testosterone were determined by standard assays. Results: The mean BMD T score (^S.E.M.) was 21:4^0:17 at the lumbar spine, and 20:54^0:16 at the femoral neck. The mean T score of the calcaneal stiffness index was 20:83^0:19: Based on a lumbar spine T score ,22.5 by DEXA, 27% of the patients had osteoporosis. Multiple regression analysis showed that the forced expiratory volume in one second (FEV 1 ) and the use of oral glucocorticoids were independent predictors of low lumbar spine BMD, whereas body mass index (BMI) and the use of oral glucocorticoids were independent predictors of low femoral neck BMD. The stiffness index correlated moderately with BMD (0.49-0.62, P , 0:0001). QUS had a sensitivity and specificity of only 57% and 89% respectively for diagnosing 'osteoporosis' (based on a femoral neck T score ,22.5 by DEXA). Positive and negative predictive values were 36% and 95% respectively. Conclusions: Low BMD is frequent in adults with CF and is most strongly correlated with disease severity (BMI, FEV 1 ) and the use of glucocorticoids. Calcaneal QUS might help to screen out patients with a normal BMD, but sensitivity and specificity were not sufficiently high to replace DEXA in these patients.

Objective: The insulin tolerance test (ITT) is regarded as the gold standard for the evaluation of pituitary ACTH and growth hormone reserve. However, the intended critical hypoglycemia results in considerable discomfort and requires close surveillance during the test. Design and methods: In a pilot study, we evaluated whether the ITT could be markedly simplified, made less hazardous and more convenient by routine i.v. low-dose glucose administration after hypoglycemia has been achieved. Sixteen healthy subjects (three females, 13 males) were tested twice in a randomized, single-blinded fashion, receiving 0.15 IU insulin/kg body weight as an i.v. bolus. After hypoglycemia (serum glucose less than 2.2 mmol/l) had been achieved, 500 ml isotonic saline (protocol A (A)), or 500 ml 5% glucose solution (protocol B (B)) were infused over 30 min. Results: Compared with saline, glucose infusion shortened the period of hypoglycemia from 31 þ 14 to 17 þ 6 min (P , 0.01). In addition, prolonged duration of hypoglycemia (.45 min) was reduced (6 subjects in protocol A vs none in protocol B). Despite shorter duration of hypoglycemia, all subjects had adequate stimulated cortisol (.500 nmol/l) and hGH (. 5 mg/l) levels. Mean peak concentrations of plasma ACTH (24^12 pmol/l (A) vs 21^8 pmol/l (B)), serum cortisol (690^83 nmol/l vs 634^83 nmol/l) and serum hGH (26^16 mg/l vs 22^13 mg/l) were slightly, but not significantly lower. In contrast, glucose infusion significantly reduced peak plasma epinephrine levels at 45 min (4.96^4.91 pmol/l (A) vs 1.53^1.1 pmol/l (B), P , 0.05) and ameliorated discomfort, as evaluated by a visual analog scale (P , 0.05). Conclusions: Taken together, our pilot study suggests that, while the duration of hypoglycemia is shortened and acute epinephrine response is reduced, low-dose infusion of glucose does not significantly alter peak cortisol and growth hormone responses during ITT. Studies with a larger number of subjects and patients with suspected hypopituitarism are needed to further evaluate this modified protocol.

images in clinical medicineT h e n e w e ng l a n d j o u r na l o f m e dic i n e n engl j med 365;25 nejm.org december 22, 2011 e46 A 23-year-old man presented with a 1-year history of diabetes mellitus, hypertension, and hypogonadism and a weight gain of 22 kg. Abdominal striae were present on physical examination (Panel A). An endocrine evaluation showed a corticotropin-dependent hypercortisolism: his morning cortisol level after an overnight dexamethasone suppression test was 38 µg per deciliter (1048 nmol per liter) (reference range, <1.8 µg per deciliter [<49.7 nmol per liter]) and his corticotropin level was 287 pg per milliliter (63.2 pmol per liter) (reference range, <46 pg per milliliter [<10.1 pmol per liter]). Endogenous cortisol production could not be suppressed with the administration of high-dose dexamethasone and could not be stimulated with the administration of corticotropin-releasing hormone, a finding that suggested a diagnosis of Cushing's syndrome resulting from the ectopic secretion of corticotropin. Axial computed tomographic images of the chest revealed an anterior mediastinal tumor (Panel B, arrow). The mass was assumed to be a thymic carcinoid tumor and was completely resected. Histologic analysis suggested a tumor with a nested, or zellballen, pattern of growth (Panel C, left inset, hematoxylin and eosin), strong and diffuse positivity for synaptophysin (Panel C, upper-right inset), and S-100 positivity for sustentacular cells (Panel C, bottom-right inset, arrows); there was no histologic evidence of cancer. These findings confirmed a diagnosis of corticotropin-producing mediastinal paraganglioma. Postoperative levels of serum cortisol and corticotropin were low. On follow-up 3 months later, the patient showed nearly complete clinical recovery.

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