Nuclear estradiol receptor in the adult rat uterus: a new exchange assay

A protamine exchange assay has been developed to measure uterine nuclear estrogen receptor in mature rats exposed to estradiol (E). After ovariectomized-adrenalectomized mature rats are injected with E, estrogen receptor (RnE) is extracted from uterine nuclei with 0.6 M potassium chloride, diluted, and quantitatively precipitated with protamine sulfate. The precipitate is subjected to a ligand exchange with radiolabeled estradiol (E), with or without unlabeled diethylstilbesterol, to determine nonspecific binding. At 37 degrees C complete exchange of E for E in RnE is observed at 2.5 h; virtually no receptor degradation occurs up to at least 5 h. Exchange does not occur at 4 degrees C. Using the protamine assay, the depletion of cytoplasmic estrogen receptor (Rc) and the accumulation of RnE were studied at various doses of E at specific time points. Increasing doses of E result in a decrease of Rc with an equal increase of RnE. At the highest dose of E (10 mug) Rc is completely depleted within 10 min, by 6 h it is 25% replenished, and by 24 h returns to slightly above control levels. Within 10 min after the injection, RnE increases to 80-90% of the original cytoplasmic level of receptor (approximately 2-3 pmol/mg of DNA or approximately 1.5 pmol/100 mg of uterus). At 6 h RnE is 75% depleted and it is completely absent at 24 h. The protamine assay permits precise quantitative studies of nuclear estrogen receptor and avoids the problems of receptor degradation and excessive nonspecific binding often found in exchange reactions at elevated temperatures.     

EGF-induced redistribution of erbB2 on breast tumor cells: flow and image cytometric energy transfer measurements

The effects of the CCK(B) antagonists, CAM1028 and CI988 and a CCK(A) antagonist, CAM1481, were studied on the anxiety-related behaviour produced by withdrawal from chronic ethanol treatment, using the elevated plus maze. Cessation of chronic ethanol administration produced a profile, in both mice and rats, consistent with increase in anxiety-related behaviour. In mice, SC administration of CAM1028 or CI988 reduced the decrease in the time spent on the open arms, the number of entries into these arms and the increases in the latencies to first open arm entry, after withdrawal from the ethanol treatment. The increases in stretched attend postures and head dips from the closed arms and the central square seen during the withdrawal phase, were also decreased by the CCK(B) antagonists, but the decreases in the number of rears and in general activity were unaffected. The doses of CAM1028 and CI988 tested were 0.1 and 1 mg/kg; for some of the withdrawal-induced changes in behaviour only the 1 mg/kg dose was effective. In contrast, the CCK(A) antagonist, CAM1481, at the same doses, had little effect on the anxiety-related behaviour produced by withdrawal from chronic ethanol treatment, although it did decrease the changes in the number of rears and the head dipping behaviour. In rats, the majority of the changes produced by withdrawal from chronic ethanol treatment were decreased by CAM1028 at 1 mg/kg, although the decreases in open arm entries, rearing behaviour and in overall activity were unaffected. CAM1028, CI988 and CAM1481 had no effects on the behaviour of control mice or rats in the plus-maze. The results show that CCK(B) antagonists were effective in decreasing the majority of the anxiogenic effects of withdrawal from chronic ethanol treatment.     

Lack of local suppression in orally tolerant CD8-deficient mice reveals a critical regulatory role of CD8+ T cells in the normal gut mucosa

We found that feeding keyhole limpet hemocyanin (KLH) to CD8-deficient (CD8-/-) mice induced oral tolerance that was comparable in both magnitude and quality to that induced in wild-type (wt) mice. The tolerance was dose dependent, and only higher doses of KLH caused significant reduction in specific Ab and T cell responses. Both Th1 and Th2 CD4+ T cell functions were affected. Feeding KLH together with cholera toxin (CT) adjuvant, however, abrogated the induction of oral tolerance equally well in CD8-/- and wt mice. On the contrary, CT adjuvant was unable to abrogate already established oral tolerance in both CD8-/- and wt mice. Most importantly, whereas Ag feeding induced hyporesponsiveness in systemic as well as in local gut IgA responses in wt mice, a lack of local suppression was evident in orally tolerant CD8-/- mice following oral immunizations. Thus, contrary to the situation in wt mice, Ag feeding induces systemic, but not local, gut IgA hyporesponsiveness in CD8-/- mice, suggesting that CD8+ T cells in the normal gut mucosa exert an important down-regulatory function. In wt mice the local suppression extended to an unrelated Ag, OVA, given together with KLH and CT adjuvant, i.e., bystander suppression. Based on these results we propose that tolerance induced by feeding Ag is highly compartmentalized, requiring CD8+ T cells for local suppression of IgA responses, whereas systemic tolerance may affect CD4+ T cells of both Th1 and Th2 types independently of CD8+ T cells. Finally, the adjuvant effect of CT abrogates induction, but not established, oral tolerance through a mechanism that does not require CD8+ T cells.     

Operative videothoracoscopy in the surgical treatment of penetrating firearms wounds of the chest

We prospectively analyzed our experience with operative videothoracoscopy (OVT) performed in a field military hospital in cases of penetrating firearms wounds of the thorax (PFAWT) sustained in Chechnya. From February to April 1996, we treated 206 wounded patients, of whom 37 (18.0%) had sustained chest injuries. PFAWT were present in 23 soldiers, accounting for 62.2% of all chest injuries. Twelve injuries were confined to the thorax, eight patients had associated injuries, and three soldiers had thoracoabdominal injuries. Nineteen patients had pleural drainage performed during medical evacuation. The thoracic injuries were right-sided (17), involved bullets or shell splinters (23); were through and through (16), represented solitary wounds (19), and were associated with internal organ injuries (21). Fifteen patients had indications for OVT when they were delivered from the battle-field 1.5 to 22 hours after injury. All patients manifested signs of hemorrhagic shock and hemodynamic instability. Indications for OVT were ongoing intrapleural bleeding (6), clotted hemothorax (6), or marked air leakage (3) preventing lung inflation with the OP-02 apparatus (field modification). OVT revealed 12 lung wounds, nine of which were multiple wounds, pleural bleeding in 6 patients, clotted hemothorax in 11 patients, and foreign bodies in 5 patients. Two patients underwent thoracotomy, one for suspicion of heart injury and the second because we could not adequately visualize and control bleeding revealed at OVT to be from the intercostal artery in the left costovertebral angle. Eight of 23 patients had no indication for operative videothoracoscopy and were managed with continued pleural aspiration and drug therapy. Wedge resection of the lung using an Endo-GIA-30 stapler was necessary in two patients because of parenchymal destruction and bleeding. Evacuation of clotted blood by fragmentation and aspiration was satisfactory in all cases. Satisfactory manual suturing of selected lung injuries was obtained largely with intracorporeal knot tying. The duration of the procedures ranged from 40 to 90 minutes. No morbidity nor mortality was encountered in patients undergoing OVT. Postoperative pain was minimized by using OVT placement of catheters in the chest wall soft tissue with local administration of 2% Trimecain. Patients were able to stand in 10 to 12 hours and to walk by the end of the first postoperative day. All patients who underwent OVT were evacuated without drains by the third or fourth postoperative day, all tolerating sitting and standing positions. We conclude that early OVT in the military field hospital for continued bleeding, clotted hemothorax, and continued major air leakage has several advantages in military patients with PFAWT: early definition and management of organ injury; identification and control of bleeding in most instances; earlier and more accurate assessment for thoracotomy; vigorous lavage and removal of projectiles such as bone fragments and evacuation of clotted hemothorax; early debridement with suture closure of the thoracic wall canal; and minimal postoperative pain with dramatically reduced suppurative sequelae and bronchopleural fistulae.     

Prediction of major depression and dysthymia from CES-D scores among ethnic minority adolescents

Two tumour cell clones, 6D1 and 4C2 cells, which are defective both in the major histocompatibility gene complex (MHC) class I expression and in the endogenous antigen presentation, are recovered with interferon (IFN)-gamma treatment. The present study describes the ultrastructure of these cells by using scanning and transmission electron microscopy in relation to the effect of IFN-gamma treatment. The general morphology of these cells was found to be similar to each other and comparable to that of a tumour cell clone, 4A1 cells, of the same origin, normal in MHC class I expression; they exhibited a fibroblast-like appearance and had many blebs on all the cell surfaces, with desmosome-like junctions between cells. On IFN-gamma treatment, surface fine blebs appeared less, and mitochondria became more densely stained. Expression of MHC class I molecules on the cell surface was much higher in the IFN-gamma treated 6D1 and 4C2 cells than in untreated cells, when estimated by immunoelectron microscopy. The addition of an epitope peptide to these cells did not enhance the class I expression, which differed from other antigen presentation-defective cells such as RMA-S cells, nor change the cell surface morphology.