Thymosin beta-4 prenatal administration improves fetal development and halts side effects due to preterm delivery.

OBJECTIVE: Thymosin beta 4 (TB4) is the most abundant member of the beta-thymosin family in humans. The main physiological role of TB4 is the regulation of actin polymerization. TB4 is also involved in angiogenesis, cell survival, cell migration and fetal development. The aim of this study was to evaluate the activity of TB4 as a fetal growth promoter when administered during pregnancy. MATERIALS AND METHODS: Our protocols have been carried out in full conformity with the rules and guidelines expected for this kind of trial. 10 pregnant mice received the same injection regimen. Only 6 of these 10 are part of this experiment because they were pregnant. At 10:00 a.m. on day E14 and E17 of gestation mice were weighed and treated with an intraperitoneal injection of TB4 (Regene RX, Rockville, MD, USA; 6 mg/kg in PBS). RESULTS: The mothers treated with TB4 for two days precisely E14 and E17, showed a higher cranio-caudal length when compared to control newborns. At histology, maternal TB4 treatment was associated with more advanced development of lungs, heart, kidney, cerebral cortex and notochord. CONCLUSIONS: Our study shows that TB4 administration during gestation may act as a powerful fetal growth promoter, by accelerating the development of newborn organs and tissues.

Changes in GABA(A) receptor gene expression induced by withdrawal of, but not by long-term exposure to, zaleplon or zolpidem.

The effects of long-term treatment with and subsequent withdrawal of the two hypnotic drugs zaleplon and zolpidem on the abundance of gamma-aminobutyric acid type A (GABA(A)) receptor subunit mRNAs in cultured rat cerebellar granule cells were investigated. Incubation of neurons with either drug at a concentration of 10 microM for 5 days did not significantly affect the amounts of mRNAs encoding the alpha(1), alpha(4), beta(1), beta(2), beta(3), gamma(2)L, or gamma(2)S subunits. As expected, similar treatment with the nonselective benzodiazepine diazepam resulted in a decrease in the abundance of alpha(1), beta(2), gamma(2)L, and gamma(2)S subunit mRNAs as well as an increase in that of the beta(1) subunit mRNA. Withdrawal of zaleplon or zolpidem, like that of diazepam, induced a marked increase in the amount of the alpha(4) subunit mRNA. In addition, discontinuation of treatment with either hypnotic drug resulted in a decrease in the amounts of alpha(1), beta(2), gamma(2)L, and gamma(2)S subunit mRNAs as well as an increase in that of the beta(1) subunit mRNA. These effects of zaleplon and zolpidem on GABA(A) receptor gene expression are consistent with the reduced tolerance liability of these drugs, compared with that of diazepam, as well as with their ability to induce both physical dependence and withdrawal syndrome.

Changes in GABAA receptor gamma 2 subunit gene expression induced by long-term administration of oral contraceptives in rats.

The effects of oral contraceptives (OCs) on neurosteroid concentrations were evaluated in female rats and women. In rats, ethynylestradiol and levonorgestrel (0.030 and 0.125 mg, respectively, subcutaneously) administered daily for 6 weeks reduced the concentrations of pregnenolone (-41%) progesterone (-74%) and allopregnanolone (-79%) in the cerebral cortex; the plasma concentrations of these steroids were also reduced but by smaller extents. OC administration for 3 months also reduced the serum concentrations of pregnenolone, progesterone and allopregnanolone in women. Chronic administration of OCs in rats increased the abundance of gamma-aminobutyric acid type A (GABA(A)) receptor gamma 2L and gamma 2S subunit mRNAs and the relative protein in the cerebral cortex, while the amounts of various alpha and beta subunit mRNAs were unaffected. Ovariectomy did not modify the effect of OCs administration on the concentrations of neurosteroids in the rat cerebral cortex (but not in the plasma) as well as on the GABA(A) receptor gene expression, suggesting a direct effect of OCs in brain. Finally, rats treated with OCs exhibited an anxiety-like behavior in the elevated plus-maze test. These results indicate that long-term treatment with OCs induced a persistent reduction in the concentrations of pregnenolone, progesterone and its GABA(A) receptor-active metabolite, allopregnanolone, both in rats and women. In rats this effect was associated with a plastic adaptation of GABA(A) receptor gene expression in the rat cerebral cortex.

Echocardiographic detection of right-sided cardiac thrombi in pulmonary embolism.

Echocardiography has proved useful in detecting right-sided heart thrombi in cases of pulmonary embolization. We found echocardiographic evidence of right-sided cardiac thrombi in six of seven patients affected by pulmonary embolism referred to our hospital within the past five months. In one patient with clinical evidence of pulmonary and paradoxic embolization, echocardiography revealed, besides thrombi within the inferior cava and right atrium, a transient, wide, systolic movement of the valvula foraminis ovalis toward the left atrium, suggesting an interatrial communication that was confirmed by contrast echocardiography. Five patients had a good outcome, with disappearance of the thrombi following IV heparin therapy, and one patient died.

Echocardiographic recognition of mediastinal masses.

The aim of our study was to assess the ability of echocardiography to recognize mediastinal masses. We studied 50 patients: 25 with acute lymphoblastic leukemia, nine with lymphomas, four with pleural effusion and previously known neoplastic diseases, ten with nonlymphomatous mediastinal masses, and two with compression of the superior vena cava. In 33 of 50 patients, computed tomography (CT) showed mediastinal masses, and in one, an aneurysm of the thoracic descending aorta. All patients had previously had chest x-ray and two-dimensional echocardiographic (2-DE) examinations in order to recognize the presence of masses. Two-DE was found to have similar specificity (94.1 percent) but higher sensitivity (90.9 percent vs 60.6 percent) if related to radiographic examination. The positive predictive value was 96.7 vs 95.2 percent and the negative predictive value was 84.2 percent vs 55.1 percent. In particular, 2-DE was more useful than chest x-ray examination when a concomitant pleural or pericardial effusion was present. Moreover, abnormalities of cardiac function due to compression or infiltration and the follow-up of patients were better evaluated.

Increase in expression of the GABA(A) receptor alpha(4) subunit gene induced by withdrawal of, but not by long-term treatment with, benzodiazepine full or partial agonists.

The effects of long-term exposure to, and subsequent withdrawal of, diazepam or imidazenil (full and partial agonists of the benzodiazepine receptor, respectively) on the abundance of GABA(A) receptor subunit mRNAs and peptides were investigated in rat cerebellar granule cells in culture. Exposure of cells to 10 microM diazepam for 5 days significantly reduced the amounts of alpha(1) and gamma(2) subunit mRNAs, and had no effect on the amount of alpha(4) mRNA. These effects were accompanied by a decrease in the levels of alpha(1) and gamma(2) protein and by a reduction in the efficacy of diazepam with regard to potentiation of GABA-evoked Cl- current. Similar long-term treatment with 10 microM imidazenil significantly reduced the abundance of only the gamma(2)S subunit mRNA and had no effect on GABA(A) receptor function. Withdrawal of diazepam or imidazenil induced a marked increase in the amount of alpha(4) mRNA; withdrawal of imidazenil also reduced the amounts of alpha(1) and gamma(2) mRNAs. In addition, withdrawal of diazepam or imidazenil was associated with a reduced ability of diazepam to potentiate GABA action. These data give new insights into the different molecular events related to GABA(A) receptor gene expression and function produced by chronic treatment and withdrawal of benzodiazepines with full or partial agonist properties.

Fractionation of Bothrops pirajai snake venom: isolation and characterization of piratoxin-I, a new myotoxic protein.

Whole desiccated venom of Bothrops pirajai was fractionated on a gel filtration (Sephadex G-75) column. Phospholipase A2, arginine esterase and clotting activity profiles of the six fractions (SI to SVI) obtained were determined. Fraction SIV from the gel filtration column was subjected to chromatography on SP-Sephadex C-25. It was resolved into five subfractions (SIV-SP1, to SIV-SP5). Fractions SIV-SP1, SIV-SP2 and SIV-SP3 showed phospholipase A2 activity but, among these fractions, only SIV-SP3 was homogeneous. Induction of myonecrosis by SIV-SP3, SIV-SP4 and SIV-SP5 was demonstrated by their ability to release serum creatine kinase, and for SIV-SP5, to induce histological alterations in the injected mouse muscle. Chemical characterization by determination of mol. wts, isoelectric focusing and direct manual sequencing of the N-terminal region was performed for SIV-SP3, SIV-SP4 and SIV-SP5. When compared with bothropstoxin-I, the myotoxin SIV-SP5 showed the same total number of amino acid residues (121) and constant molar ratio for all but three amino acids. We have named this toxin piratoxin-I (PrTX-I).

Crystallization of piratoxin I, a myotoxic Lys49-phospholipase A2 homologue isolated from the venom of Bothrops pirajai.

Large single crystals of piratoxin I. a Lys49-PLA2 homologue with low enzymatic activity, have been obtained. The crystals belong to the orthorhombic system space group P2(1)2(1)2(1), and diffract X-rays to a resolution of 2.8 A. Preliminary analysis reveals the presence of two molecules in the crystallographic asymmetric unit.

Biochemical and histopathological alterations induced in rats by Tityus serrulatus scorpion venom and its major neurotoxin tityustoxin-I.

Intravenous injection into the rat of sublethal doses of Tityus serrulatus scorpion venom (100 micrograms protein/kg) or its major neurotoxin tityustoxin-I (TsTX-I, 20 micrograms/kg) caused, 30-180 min after injection, statistically significant increases in the serum levels of aspartate aminotransferase, amylase, creatine kinase and lactate dehydrogenase, as well as hyperglycemia, a high level of plasma free fatty acids and a low level of liver glycogen. The in vitro serum levels of the above enzymes did not change. For alanine aminotransferase, gamma-glutamyl transferase and alkaline phosphatase, neither in vitro nor in vivo alterations were observed. The whole venom and TsTX-I caused hepatic congestion with hemolysis and hydropic degeneration. Other histological lesions included edema and congestion with subpleural hemorrhage in the lungs, hypertrophy of fibers with degeneration areas in the heart, and congestion and hemorrhage in the kidneys. In the salivary glands, alterations to the acini and ductules were visible. In the adrenal glands no morphological alterations could be detected at the studied doses. The results suggest that the in vivo enzymatic and histopathological alterations are due to tissue lesions evoked by the whole venom and TsTX-I. An indirect effect, however, induced by stimulation of acetylcholine and catecholamine release in the postganglionic nerve terminals, cannot be excluded.

Isolation of a polypeptide from Phoneutria nigriventer spider venom responsible for the increased vascular permeability in rabbit skin.

Fractionation of Phoneutria nigriventer venom by Sephadex G-10 followed by ion-exchange chromatography yields a fraction (fraction XIII) which increases microvascular permeability in rabbit skin in vivo by activating the tissue kallikrein-kinin system. One polypeptide (PNV3) with the ability to increase microvascular permeability in the rabbit skin in vivo was isolated from fraction XIII and biochemically characterized. PNV3 has 132 amino acid residues with a calculated mol. wt of 14,475. This polypeptide showed the following N-terminal sequence: AVFAIQDQPC. Amino acid analysis indicated the presence of six disulfide bridges and a high content of Glx (20%). Pairwise comparison of PNV3 amino acid sequence with 27 other spider venom polypeptides and proteins indicated that PNV3 presents high similarity (60-70%) with other toxins (Tx2.1, Tx2.5 and Tx2.6) isolated from P. nigriventer venom.

Constrictive pericarditis versus restrictive cardiomyopathy: the role of Doppler echocardiography in differential diagnosis.

Doppler ultrasound recordings of velocities of flow across the mitral and tricuspid valves and in the hepatic veins, and their variation with respiration, were recorded in seven patients with constrictive pericarditis and in six patients with restrictive cardiomyopathy. Deceleration of mitral and tricuspid flow was also evaluated during apnea. Color flow Doppler was performed in order to evaluate mitral and tricuspid regurgitation. Eight healthy adults served as controls. The patients with constrictive pericarditis showed higher peak diastolic velocities of mitral flow, as well as marked increase of velocity of flow at the onset of expiration and decrease at the onset of inspiration. Reciprocal respiratory variation of the velocities were also observed across the tricuspid valve. The patients with restrictive cardiomyopathy showed moderate or severe mitral and tricuspid regurgitation. They also showed shorter deceleration of flow across the mitral and tricuspid valves during apnea. The pattern of flow in the hepatic veins showed reversal during systole with accentuated reversion during inspiration. These results suggest that patient with constrictive pericarditis and restrictive cardiomyopathy can be differentiated by comparing Doppler echocardiographic data, along with changes induced by respiration.

Echocardiographic evidence of intrathoracic masses due to localized blast crisis in a patient affected by chronic granulocytic leukemia.

We report a case of intrathoracic blast crisis in a patient affected by chronic granulocytic leukemia. Two-dimensional echocardiography performed to exclude pericardial effusion showed tumor masses in area of the atrioventricular groove and by the side of the left ventricular wall, which were confirmed by computed tomography.

Cardiac toxicity of 5-fluorouracil. Report of a case of spontaneous angina.

We report a case of a patient with colon carcinoma and liver metastasis who presented chest pain after 5-fluorouracil (5-FU) administration. Clinical electrocardiographic evolution was similar to that observed in Prinzmetal's angina, and chest pain promptly resolved with nifedipine. These data suggest that coronary spasm may be the cause of cardiotoxicity due to 5-FU, and that calcium antagonists may probably be used in the prevention or treatment of 5-FU cardiotoxicity.

Decellularized ovine arteries as small-diameter vascular grafts.

Atherosclerosis and its complications still represent the leading cause of death in the developed countries. While autologous blood vessels may be regarded as the best solution for peripheral and coronary bypass, they are unavailable in most patients. Even though tissue engineering techniques are often applied to the development of small-diameter vascular grafts, limiting factors of this approach are represented by the lack of essential extracellular matrix proteins and/or poor biomechanical properties of the scaffolds used. Along these lines, the aim of this study was to develop a decellularization protocol for ovine carotids to be used as suitable small-diameter vascular grafts. Samples were treated either with sodium dodecyl sulphate (SDS) or with Trypsin and Triton X-100; a final nuclease digestion was performed for both protocols. Morphological analyses demonstrate complete removal of nuclei and cellular components in treated vessels, also confirmed by significant reduction in wall thickness and DNA content. Essential extracellular matrix proteins such as collagen, elastin, and fibronectin are well preserved after decellularization. From a mechanical point of view, Trypsin and Triton X-100 treated arteries show elastic modules and compliance comparable to native carotids, whereas the use of SDS makes samples stiffer, with a significant decrease in the compliance mean value and an increase in longitudinal and circumferential Young's modules. It is demonstrated that the treatment where Trypsin and Triton X-100 are combined guarantees complete decellularization of carotids, with no significant alteration of biomechanical and structural properties, thus preserving a suitable environment for adhesion, proliferation, and migration of cells.

Evaluation of the use of human Mesenchymal Stem Cells for acute toxicity tests.

In vitro cytotoxicity tests are typically carried out with transformed, immortalized cell lines or primary cells. Immortalized cells are readily available and easily maintained, although they usually show anomalous behavior and phenotypes, which do not reflect the mechanisms observed in their normal homologous cells. Primary cells are indeed considered a better option as model systems for predicting toxicological behavior, although they are limited in quantity and suffer from batch-to-batch variation due to the need to isolate them freshly for each study. In particular, human Mesenchymal Stem Cells (hMSCs) have never been adopted in order to develop in vitro model systems for acute toxicity tests of chemicals. Therefore, the aim of this study was to verify the possibility of using hMSCs as an alternative method to estimate in vivo starting dose for acute toxicity. As suggested by ICCVAM, 12 reference chemicals were assessed in the present study and a Neutral Red Uptake assay was performed. It is shown for the first time that MSCs isolated from human bone marrow can be confidently used in this area of toxicology. MSCs represent a good promise for the development of in vitro human assays and could ultimately replace, improve or overtake current predictive models in toxicology.

Experimental analysis and modelling of in vitro HUVECs proliferation in the presence of various types of drugs.

OBJECTIVES: This study focuses on experimental analysis and corresponding mathematical simulation of in vitro HUVECs (human umbilical vein endothelial cells) proliferation in the presence of various types of drugs. MATERIALS AND METHODS: HUVECs, once seeded in Petri dishes, were expanded to confluence. Temporal profiles of total count obtained by classic haemocytometry and cell size distribution measured using an electronic Coulter counter, are quantitatively simulated by a suitable model based on the population balance approach. Influence of drugs on cell proliferation is also properly simulated by accounting for suitable kinetic equations. RESULTS AND DISCUSSION: The models' parameters have been determined by comparison with experimental data related to cell population expansion and cell size distribution in the absence of drugs. Inhibition constant for each type of drug has been estimated by comparing the experimental data with model results concerning temporal profiles of total cell count. The reliability of the model and its predictive capability have been tested by simulating cell size distribution for experiments performed in the presence of drugs. The proposed model will be useful in interpreting effects of selected drugs on expansion of readily available human cells.

In vitro ovine articular chondrocyte proliferation: experiments and modelling.

This study focuses on analysis of in vitro cultures of chondrocytes from ovine articular cartilage. Isolated cells were seeded in Petri dishes, then expanded to confluence and phenotypically characterized by flow cytometry. The sigmoidal temporal profile of total counts was obtained by classic haemocytometry and corresponding cell size distributions were measured electronically using a Coulter Counter. A mathematical model recently proposed (1) was adopted for quantitative interpretation of these experimental data. The model is based on a 1-D (that is, mass-structured), single-staged population balance approach capable of taking into account contact inhibition at confluence. The model's parameters were determined by fitting measured total cell counts and size distributions. Model reliability was verified by predicting cell proliferation counts and corresponding size distributions at culture times longer than those used when tuning the model's parameters. It was found that adoption of cell mass as the intrinsic characteristic of a growing chondrocyte population enables sigmoidal temporal profiles of total counts in the Petri dish, as well as cell size distributions at 'balanced growth', to be adequately predicted.