Preventing endotoxin-stimulated alveolar macrophages from decreasing epithelium Na+ channel (ENaC) mRNA levels and activity.

The acute respiratory distress syndrome is characterized by impairment of the alveolar-capillary barrier. Our laboratory has shown that distal lung epithelial cell (DLEC) amiloride-sensitive Na+ transport is impaired by in vitro coculture with endotoxin (lipopolysaccharide)-stimulated alveolar macrophages (AM) through an L-arginine-dependent mechanism. To investigate the effect of this model on mRNA levels of the rat epithelial Na+ channel, mature fetal rat DLEC monolayers were incubated for 16 h with rat AM (1 x 10(7)) and lipopolysaccharide (10 microg/mL), or the cell-free supernatant of lipopolysaccharide-stimulated rat AM. Such exposure resulted in a profound decrease in mRNA expression for all subunits (alpha, beta, and gamma) of the rat epithelial Na+ channel, without affecting 18S RNA levels. This effect was prevented by the antioxidant N-acetylcysteine. In separate experiments, confluent DLEC monolayers were exposed to lipopolysaccharide-stimulated AM supernatant for 16 h with or without N-acetylcysteine and DTT and studied in Ussing chambers. As previously demonstrated in our laboratory, AM supernatant resulted in a significant (p < 0.05) impairment of DLEC Na+ transport, as reflected by a decrease in the amiloride-sensitive component of short-circuit current (control, 3.96 +/- 0.18 microA/cm2 versus supernatant, 2.34 +/- 0.56 microA/cm2; p < 0.05). This effect was significantly reversed by N-acetylcysteine (3.55 +/- 0.48 microA/cm2), but not by DTT (1.87 +/- 0.21 microA/cm2). N-acetylcysteine, but not DTT, increased DLEC thiol levels. These studies elucidate mechanisms by which activated AM impair alveolar epithelial barrier function in an in vitro model of acute lung injury.

Lower limb ischemia: phase 1 results of salvage perfusion.

Revascularization of an ischemic lower extremity is associated with high morbidity (20-30%) and perioperative mortality (10-20%) regardless of the mode of intervention, surgical or thrombolytic, considered to be due to polymorphonuclear (PMN) activation and mediator release. In this study, the safety and feasibility of cell-free extracorporeal perfusion of the limb with a solution designed to minimize both local and systemic injury was tested. Methods. Patients with severe limb ischemia (sensory/motor loss, rest pain/gangrene) were studied prospectively by random assignment into the treatment arm (n = 14) or control arm (n = 21). Surgical management consisted of restorative procedure, thrombectomy or embolectomy (n = 21), or reconstruction (n = 14). Reperfusion of the ischemic limb was achieved with hypertonic, hyperoncotic perfusate containing anti-oxidants delivered via the arterial tree (mean volume 1835 +/- 824 ml) with initial venous drainage (mean volume 775 +/- 263 ml) in the restorative group. Means were compared by paired t test. Results. No adverse systemic effects were detected after limb perfusion (electrolytes, coagulation, platelet function, CBC). Rapid lactate wash-out was observed within 30 min of perfusion (preperfusion 3.2 +/- 4.1 mM, 30 min postperfusion 0.7 +/- 0.71 mM, P < 0.01). Blunting of PMN activation was shown by chemiluminescence (CL) analysis (preischemic CL: 0.68 +/- 0.2; 30 min CL: 0.47 +/- 0. 2; P < 0.013). F2-isoprostanes, a marker of free radical-mediated systemic lipid peroxidation, were significantly reduced in patients treated with study perfusion method (70.55 +/- 39.54 versus control 194.38 +/- 25.24, P < 0.005). Mortality with treatment was 0/14 versus 5/21 in the control. Complication frequency: MI 0/14 vs 3/21; renal 0/14 vs 1/21; leg edema 1/14 vs 5/21; amputations 2/14 vs 1/21. Conclusion. Modification of limb perfusion in patients with severe limb ischemia, using our simple and rapid (15-20 min) method provides beneficial systemic effects.

HIV infection in vitro enhances the activity of src-family protein tyrosine kinases.

OBJECTIVE: We examined the effect of HIV infection on src-family protein tyrosine kinase (PTK) activity to determine if alterations in src-family PTK activity could contribute to the HIV-related chronic immune system activation observed in patients infected with HIV. METHODS: Jurkat, a CD4+ human T lymphocyte cell line was infected with HIV IIIB. Kinase activity was determined by in vitro immune complex kinase assays using antibodies specific for the src-family PTKs, p56lck, p59fyn and p60c-src expressed in T lymphocytes. PTK protein and total phosphotyrosine levels were assessed by Western blotting. The role of the gp120-CD4-Lck interaction in HIV-related PTK activation was determined using gp 120-treated Jurkat cells and HIV-infection of JCaM 1.6 cells, a Jurkat-derived cell line that lacks p56lck. RESULTS: Cells infected with HIV for 24 h exhibited increased levels of total tyrosine phosphorylation and enhanced src-family PTK activity without altered levels of expression of src-family kinases. The activity of Lck and Fyn was enhanced within 30 min of infection. HIV-related src-family PTK activation was not a function of the gp120-CD4-Lck interaction and occurred in the presence of 10 mmol/l zidovudine indicating that reverse transcriptase and activation of the HIV genome is not required. CONCLUSIONS: HIV-related activation of src-family PTK is a response of the cell to early stages of the virus life cycle, possibly either membrane fusion or viral uncoating. These results indicate that endogenous src-family PTKs may play a role in HIV-related immune activation and dysfunction. Moreover, activation of src-family PTK may be a mechanism used by the virus to facilitate some aspect of its own life cycle.

An octapeptide analogue of HIV gp120 modulates protein tyrosine kinase activity in activated peripheral blood T lymphocytes.

Following infection with HIV, patients exhibit lymphocyte dysfunction before the loss of CD4+ T cells. The major HIV surface glycoprotein, gp120, can modulate lymphocyte function in vitro; however, the mechanism by which gp120 affects T lymphocyte signal transduction is controversial. We have used Peptide T, a synthetic octapeptide derived from a conserved, CD4 binding region of gp120, to examine gp120-related modulation of lymphocyte signal transduction. Activation of lymphocytes through the T cell receptor (TCR) in collaboration with cell surface accessory molecules results in rapid increases in tyrosine phosphorylation, probably through the recruitment and activation of src-family protein tyrosine kinases (PTK) such as lck and fyn which have been implicated in mediating the proximal signalling events mediated through the TCR. To identify potential mechanisms by which gp120 could modulate the function of T lymphocytes, we determined the effect of Peptide T on normal, activated peripheral blood lymphoblasts. Treatment of normal, activated peripheral blood lymphoblasts with Peptide T (10(-9) M) for 60 min transiently reduced levels of protein tyrosine phosphorylation (ptyr). Reduction in levels of cellular ptyr was associated with transient inhibition of the activity of total cellular and CD4-associated p56lck kinase activity (80%). Peptide T also induced a small delayed reduction in the p59fyn activity (up to 42%). Despite the decrease in total cellular ptyr levels, pp60c-src kinase activity was increased 11-fold following treatment with Peptide T. Peptide T pretreatment also induced tyrosine phosphorylation of a 48-kD CD4-associated protein, indicating that Peptide T may have multiple effects. Peptide T did not alter the levels of total cellular p56lck enzyme, nor did it directly inhibit the activity of purified p56lck. These results are consistent with a Peptide T-dependent modulation of PTK regulation, and support the potential of gp120 to interfere with T lymphocyte signal transduction in activated T lymphocytes.

The effect of heat shock on immediate post-preservation lung function.

Exposure of animals to transient hyperthermia causes the induction of highly conserved proteins, heat shock proteins (HSPs), which are thought to by cytoprotective against a variety of injuries, including ischemia. We tested the hypothesis that heating donor animals prior to harvest would improve pulmonary preservation. Anaesthetized New Zealand White rabbits underwent radiant heating to 42.5-43.5 degrees C (rectal) 8 h prior to harvest of the lungs. The lungs were harvested without flush and stored for 18 h at 4 degrees C. The left lung were perfused ex vivo with fresh blood for 10 min. Blood gases, pulmonary artery (Ppa) and airway (P(aw) pressures, and wet/dry ratios (W/D) were measured. Control animals were treated identically except without heating. All heated animals had HSP72 at lung harvest and 18 h later, whereas no control had detectable levels of HSP72 at either time. In Experiment 1 (n = 12, VT 20 ml, F1O2 0.21, 30 bpm, PEEP 0.5 cm H2O), PO2 in the heated group was 57.6 +/- 7.3 mmHg (mean +/- SEM) vs. 51.6 +/- 5.7 in the controls (NS). In Experiment 2 (n = 8, VT 15 ml, F1O2 0.21, 35 bpm, PEEP 2 cm H2O), PO2 of the heated group was 63.5 +/- 6.5 vs. 83.1 +/- 9.5 in the controls (NS). Ppa after 10 min was not significantly different in the heated group in Experiment 1 (16.7 +/- 0.9 mmHg vs. 24.2 +/- 3.7 in controls) or in Experiment 2 (19.5 +/- 1.8 vs. 11.3 +/- 2.9 in controls). Wet/dry ratios were not different in either Experiment 1 (6.4 +/- 0.4 vs. 5.8 +/- 0.2 in controls) or Experiment 2 (5.0 +/- 0.2 vs. 5.0 +/- 0.5).(ABSTRACT TRUNCATED AT 250 WORDS)

Tratamento cirurgico dos adenomas hiperfuncionais das paratiroides. / Surgical treatment of hyperfunctional adenomas of the parathyroid

Os autores apresentam dados clinicos, bioquimicos, radiologicos, cirurgicos, posoperatorios precoces e tardios de 12 casos de hiperparatiroidismo (HPT) (11 primarios e 1 secundario), operados na Secao de Cirurgia Endocrina do HSPE (11 casos) e no Hospital de Itaquera (1 caso). No diagnostico clinico, chamam a atencao para as lesoes osseas presentes em cerca de 83% dos doentes e as renais, em cerca de 66%. Dos exames laboratoriais, a calcemia total encontrava-se elevada em todos os doentes portadores de adenoma (11/11). Em todos os 11 casos de HPT primario, o adenoma localizava-se no pescoco, sendo, em 2, intratiroidianos. Em 7 casos, para facilitar a identificacao do adenoma, foi utilizado o azul de toluidina intravenoso; em 5 casos, o resultado foi excelente; num caso, nao se encontrou o adenoma na regiao cervical, nem no torax; o doente faleceu, sendo encontrado o adenoma dentro da tiroide. Em 10 dos casos de HPT primario, houve regressao do quadro clinico, laboratorial e radiologico apos a cirurgia. O tempo medio de seguimento pos-operatorio foi de 6,8 anos (1 a 13)