Mutation detection of the human glucocorticoid receptor alpha gene area coding for the hormone-binding domain by denaturing gradient gel electrophoresis.

Mutations in the hormone-binding domain of the human glucocorticoid receptor alpha (hGRalpha) gene have been detected in a variety of glucocorticoid resistance syndromes. Using the denaturing gradient gel electrophoresis technique, we developed a sensitive method for the detection of alterations in the gene area coding for the whole hormone-binding domain and part of the DNA-binding domain of the hGRalpha. This method can be applied for screening of glucocorticoid receptor gene alterations in glucocorticoid-dependent diseases.

Evaluation of inflammatory cytokines in malignant and benign pleural effusions.

We measured the levels of inflammatory cytokines interleukin-1alpha (IL-1alpha), interleukin-1beta (IL-1beta), interleukin-2 (IL-2), interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor-alpha (TNF-alpha) in pleural effusions and serum in 65 consecutive patients: 32 with malignant pleural effusion (MPE) (group A), and 33 with inflammatory benign pleural effusion (BPE) (group B). Serum levels of 15 healthy individuals served as control. Concentrations of IL-1alpha were higher in serum compared to pleural fluid in both groups (47.1+/-33.9 vs. 25.9+/-1.7 fmol/ml, p<0.001, in group A; and 39.9+/-30.9 vs. 25.4+/-16.3 fmol/ml, p<0.02, in group B). Similarly, concentrations of IL-1beta and IL-2 were significantly higher in serum compared to pleural fluid in both groups. In contrast, IL-6, IL-8 and TNF-alpha were found at high concentration in MPE in comparison to serum IL-6: 171.8+/-60.4 vs. 7. 2+/-7 fmol/ml (p<0.001), IL-8: 1175.15+/-2385.6 vs. 285.2+/-187.2 pg/ml (p<0.05), TNF-alpha: 204.9+/-82.9 vs. 79.4+/-31.9 fmol/ml (p<0. 001). Similarly, pleural concentrations of IL-6, IL-8 and TNF-alpha were higher in BPE patients in comparison to serum IL-6: 124.3+/-56. 2 vs. 8.6+/-6.4 fmol/ml (p<0.001) IL-8: 2109.2+/-4121.5 vs. 291. 6+/-197.9 pg/ml (p<0.02), TNF-alpha: 183.8+/-28.2 vs. 86.2+/-23.9 fmol/ml (p<0.001). These data suggest that IL-6, IL-8 and TNF-alpha might be secreted locally at the site of active disease both in benign and malignant pleural effusions.

Significance of alpha-2-macroglobulin, alpha-1-acid glycoprotein, and C-reactive protein in pleural effusion differentiation.

BACKGROUND: The differentiation between exudates and transudates is fundamental when investigating the cause of pleural effusions. Acute-phase proteins could be potentially useful markers in this discrimination. OBJECTIVE: The present study was designed to evaluate whether the acute-phase proteins: alpha(2)-macroglobulin (AMG), alpha(1)-acid glycoprotein (AAG) and C-reactive protein (CRP) are useful in investigating the pleural effusions. METHODS: We prospectively measured the concentrations of the above proteins in the serum and pleural fluid of 84 consecutive patients with various diseases using a nephelometric assay. RESULTS: Pleural effusion AMG, AAG and CRP were all significantly elevated in the group of patients with exudates compared to patients with transudates (p < 0.001, p < 0.001 and p < 0.01, respectively). An AAG value >63 mg/dl in a pleural effusion is predictive of an exudate with a sensitivity of 90% and a specificity 85%. Similarly, an AMG value >44 mg/dl in a pleural effusion is predictive of an exudate with a sensitivity and a specificity of 90% and 60%, respectively. Moreover, pleural AAG was significantly higher in cancerous exudates than in exudates and transudates of all other cause taken together (p < 0.001). Finally, to differentiate the same pleural effusion, the cut-off value of 1.0 mg/dl of pleural CRP has a sensitivity and a specificity of 74% and 74%, respectively. CONCLUSIONS: We conclude that both AAG and AMG concentrations in pleural effusions have a high sensitivity and are therefore useful parameters in distinguishing exudates from transudates, but the latter is inferior due to its unacceptably low specificity.

Evaluation of ferritin, interleukin-6, interleukin-8 and tumor necrosis factor alpha in the differentiation of exudates and transudates in pleural effusions.

In an attempt to define diagnostic criteria for the differentiation of pleural exudates from transudates, we measured ferritin (FER), interleukin-6 (IL-6), interleukin-8 (IL-8) and tumor necrosis factor-alpha (TNF-alpha) in pleural effusions and blood serum in 84 consecutive patients with pleural effusions of various etiologies. Concentrations of FER, IL-8 and TNF-alpha were significantly higher in serum and pleural effusion in patients with exudates than in patients with transudates. Serum concentrations of IL-6 were not significantly increased in pleural exudate patients (9.78 +/- 17.12 fmol/L) compared to transudate patients (4.05 +/- 2.33 fmol/L), while significant differences were found between pleural exudates and transudates (p < 0.001). Increased levels of FER were found in serum and pleural effusion of cancer patients in comparison to non cancer patients (p < 0.001 and p < 0.001, respectively). Serum concentrations of IL-6, IL-8, and TNF-alpha were not significantly increased in cancer compared to non-cancer patients, while increased concentrations of IL-6 and IL-8 were found in pleural fluid of patients with cancer in comparison to non-cancer patients. Finally/ no statistically significant differences were found in serum and pleural TNF-alpha concentrations among patients with cancer and patients with non-cancer effusion. We conclude that FER, IL-6, IL-8 and TNF-alpha concentrations in pleural effusions are useful markers in differentiating exudates from transudates.

Mechanisms accounting for the impaired natural-killer cell activity in refractory anaemia with excess of blasts.

Natural killer (NK) cells were analyzed in 38 untreated patients with refractory anaemia with excess of blasts (RAEB), using cytotoxicity assays and immunofluorescence with monoclonal antibodies. We found that patients with RAEB have normal numbers of peripheral blood and bone marrow NK cells. NK cells from RAEB patients express very low natural-killer cell activity (NKa) which may be increased significantly with recombinant alpha-interferon and recombinant interleukin-2, although it remains below the lower limit of the control range. The cells exhibit normal tumour cell binding capacity, but fail to release sufficient amounts of natural-killer cytotoxic factors (NKCFs) upon their interaction with NK-sensitive K562 cell targets or their stimulation with phytohaemagglutinin. Our results suggest that defective NKa in RAEB patients may be due, at least in part, to impaired release of functionally active NKCFs. This disturbance is probably the result of some intrinsic defect of RAEB NK cells in NKCF production, storage, and/or release. The possibility of an impairment in the activation signal provided by the stimulatory K562 cells cannot be excluded, although it seems unlikely. We postulate that this abnormality might represent a manifestation of dysplastic haemopoiesis. Further studies are certainly needed to investigate whether other defective mechanisms are also implicated in the determination of the low NKa in patients with RAEB.