Pharmacological characterization of mediators and vagal influence in the acute allergic bronchoconstriction in guinea pigs

Summary The allergic bronchoconstriction in guinea pigs has been attributed mainly to the release of mast cell mediators. Histamine has been involved in the first minutes of the anaphylactic reaction and new-formed compounds in the subsequent response. In this asthma model the vagal influence has been sparsely investigated. In the present work we evaluated the pharmacological modification of the acute allergic bronchoconstrictor response in guinea pigs sensitized to ovalbumin through aerosol exposure. Pyrilamine (20 g/kg), diethylcarbamazine (a lipoxygenase inhibitor, 10 mg/kg) and dexamethasone (4 mg/kg) each reduced the antigen-induced bronchoconstriction throughout the 30 min studied. Indomethacin (3.1 mg/kg) did not modify the response to the antigen. Atropine (2 mg/kg) plus bilateral vagotomy also diminished this response from 5 min onward. On the other hand, from 5 min ahead pyrilamine-resistant bronchoconstriction was partially inhibited by dexamethasone, and it was almost completely blocked during all of the response when atropine plus bilateral vagotomy were added to dexamethasone. Dipyridamole (an inhibitor of the adenosine uptake, 0.4 mg/kg) enhanced the bronchoconstriction, though this was significant only in the 2–5 min time-interval of the response. These results suggest that histamine and vagal influence play an important role in the whole response to antigen, that other mediators, probably leukotrienes, participate in this response from 5 min onward, and that adenosine could exert a potentiation effect on this response. Send offprint requests to L. M. Montaño at the Instituto Nacional de Enfermedades Respiratorias     

A fast and simple in situ PCR method to detect human papilloma virus infection in archival paraffin-embedded tissues

We describe a simple and fast method for the detection and localization of low copy numbers of HPV DNA in formalin-fixed and paraffin-embedded archival tissues. We have developed a protocol for direct bl situ-PCR in order to demonstrate its convenience in rapid and reproducible assessment of HPV infection in unknown biopsies. The morphological aspect of the tissues has been maintained, despite the multiple steps of fixation, permeabilization and thermal cycling, and positivity has been detected only in virus target cells.     

Effects of the Calcium Antagonist Felodipine on Resting and Stress Testing Blood Pressure in Essential Hypertension

The current study analyzed the effects of different doses of the calcium channel blocker felodipine on cardiovascular response to a set of standardized laboratory tasks. We randomly allocated 21 essential hypertensive patients to receive extended release felodipine 5 mg, felodipine 10 mg and placebo, each given once daily for 2 weeks, according to a double-blind 3-period design. At the end of each treatment period, patients were examined at resting baseline and while performing a mental arithmetic test, a handgrip test and a cycle ergometry test. Compared to placebo, the average fall in resting blood pressure (BP) was of 7.9 ± 5.6/6.1 ± 4.5 mm Hg with felodipine 5 mg (p < 0.01) and of 15.1 ± 5.8/13.9 ± 4.5 mm Hg with felodipine 10 mg (p < 0.001). During mental arithmetic, BP decrease was 11.6 ± 8.1/9 ± 5 mm Hg with felodipine 5 mg (p < 0.01) and 20.4 ± 8.1/15.3 ± 5 mm Hg with felodipine 10 mg (p < 0.001). During handgrip test, BP was significantly reduced after both felodipine doses by 11.7 ± 9.3/9.5 ± 6.5 mm Hg (p < 0.05) and 22.1 ± 9.3/22.4 ± 6.5 mm Hg (p < 0.001), respectively. During cycle ergometry, systolic BP was significantly reduced after felodipine 10 mg by 20.1 ± 9.4 mm Hg (p < 0.001), whereas the fall induced by felodipine 5 mg (7.7 ± 9.4 mm Hg) was not statistically significant (p > 0.05); diastolic BP was significantly reduced by both felodipine doses [average fall of 6.6 ± 5.8 mm Hg (p < 0.05) after felodipine 5 mg and of 12.7 ± 5.8 mm Hg (p < 0.001) after felodipine 10 mg]. There was no treatment effect on the magnitude of systolic BP reactivity from baseline during either mental arithmetic, handgrip test or cycle ergometry (all, p > 0.05). Heart rate values were significantly higher after both felodipine doses than after placebo, either at rest or during stress testing (all, p < 0.05). These data suggest that felodipine, especially at higher doses, may be effective in lowering BP not only at rest but also during exposure to commonly recurring stressful situations.     

Response of multicellular tumor spheroids to liposomes containing TNF-α

Summary This publication describes a new model to investigate the influence of tumor necrosis factor- (TNF-) on a three-dimensional glial cell aggregate under defined, standardized, reproducible conditions using the glioma cell line A 172.The cells are initially grown as normal monolayer culture until they reach a cell density of up to 1×10⁶. Subsequently they are grown as spheroids by the liquid overlay technique. Spheroids grown in this way were divided into ten groups of more than 50 cell aggregates. Three groups were coincubated with free TNF- in increasing dosages (100 ng/ml, 200 ng/ml and 1000 ng/ml); three groups were incubated with empty liposomes (0.2 mg/ml, 0.4 mg/ml and 2 mg/ml); three groups received liposomes which had been loaded with TNF-, and one group, which received no treatment, served as control.The diameter of the spheroids ranged from 80 m to 350 m. There was no significant difference in growth between the 3 groups treated with free TNF-. Comparing spheroids treated with TNF- with those which had been coincubated with empty liposomes, there was a significant difference (p<0.001) in growth, which correlated with the amount of liposomes. Similarly, free TNF- had a significantly (P<0.001) stronger growth-inhibiting effect as compared to liposomes loaded with TNF-. Comparing the groups treated with liposomes only to those treated with liposomes loaded with TNF-, the latter exhibited a more marked (although not significantly) growth-inhibiting effect.The preliminary conclusion is that the major growth-inhibiting effect seems to be mediated by the liposomes. This phenomenon is in agreement with results obtained in monolayer cultures.     

Cryopreserved,cultured, allogenic,human epidermal grafts for the treatment of chronic ischemic ulcers: A report on two cases

Cultured, allogenic, human epidermal grafts have been successfully used to treat extensive burns, donor sites, leg ulcers, and eroded areas in recessive dystrophic epidermolysis bullosa. The following report describes the use of cryopreserved cultured human epidermal allografts in the treatment of long-standing ischemic ulcers unresponsive to conventional medical therapy in two patients who had previously undergone unsuccessful vascular reconstructive surgery. A marked reduction in ulcer size as well as rapid pain relief was achieved in both patients. In selected cases the use of epidermal grafts as a biologic topical therapy could represent an alternative to other medical treatments. However, thus far, only a few cases have been reported and the uncertain short- and long-term results as well as the excessive costs may limit the widespread application of this treatment modality.     

Isolation perfusion in extracorporeal circulation with interleukin-2 and lymphokine-activated killer cells in the treatment of in-transit metastases from limb cutaneous melanoma

Background: Therapies of advanced melanoma patients with interleukin-2 (IL-2) and cytotoxic lymphocytes have produced interesting results, but a larger diffusion of these treatments is limited by the severe side effects due to IL-2 systemic infusion. A strictly regional administration of IL-2 and cells by an isolation perfusion (IP) in extracorporeal circulation (ECC) for the treatment of regional melanoma metastases could improve tolerability and efficacy of this specific modality of immunotherapy. Methods: Ten patients were submitted to adoptive immunotherapy with IL-2 and lymphokine-activated killer (LAK) cells by IP in ECC. The schedule of treatment included the first course of a 5-day systemic administration of IL-2 (Proleukin, EuroCetus 9–12 × 10⁶ IU/M²/day continuous infusion); autologous LAK cells were obtained via leukapheresis and after in vitro activation were given (range 8–28 × 10⁹) along with IL-2 (120-2,400 IU/ml of perfusion priming) to the affected limb by IP; IL-2 (9–12×10⁶ IU/m²/day) was also administered by systemic continuous infusion for 5 days starting on the day after IP. Results: All patients concluded the treatment without any major local or systemic toxicities. Clinical responses included one complete and six partial remissions; three patients had stable disease. All patients are alive. Follow-up after IP ranged from 12 to 35 months (median: 22). The analysis of circulating lymphocytes revealed the rapid disappearance of LAK cells, suggesting their extravasation and/or endothelial adhesion in perfused tissues. Conclusions: IP with IL-2 and LAK cells is a new approach for the treatment of in-transit metastases due to cutaneous melanoma. The treatment appears to be feasible and reliable. Further biological and immunological studies should permit amelioration of the present modality of treatment.     

Pregnancies in an 18-Year Follow-up after Biliopancreatic Diversion

Background: 239 pregnancies occurred in 1136 women who had undergone biliopancreatic diversion (BPD). Methods: There were 73 abortions, and 14 pregnancies are presently in their course. The 152 term pregnancies (six twins) occurred in 129 women 2-173 months (mean 42.7) after BPD. Mean age and current excess weight were 31.4 years (20-42) and 29.1% (-6.9-78.2), and mean excess weight loss was 72.9% (30.4-110.5). Results: Mean weight gain during pregnancy was 6.2 kg (-21-25). In 32 patients (21%), parenteral nutritional support was needed. In all the other patients (79%), the usual supplementations were given. Of the newborns, 122 were delivered at term (84.7%) with a mean weight of 2842.4 g (1760-4600 g) and a mean length of 48.5 cm (43-59 cm), while the 22 preterm babies (15.3%) weighed 2151.1 g (1400-3850 g) and had a length of 44.6 cm (33-56 cm). Forty infants (27.8%) were small for gestational age but 17 of them weighed more than 2500 g. Eleven twins (one abortion at 26th week) were also delivered, with a mean weight of 2088.6 g (1200-3100 g) and a mean length of 45.6 cm (35-50 cm). Delivery was spontaneous in 85 instances (56%), while vacuum extractor was used in one, and 66 cesarean sections were performed. There were two birth malformations, one infant died after surgery for meconium obstruction and two died from unknown causes. Of the 129 women, 35 had been infertile before BPD. Conclusions: Disappearance of infertility and decrease of pregnancy risk are to be considered among the beneficial effects of weight reduction following BPD.