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.

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.

Experimental analysis and modelling of in vitro proliferation of mesenchymal stem cells.

OBJECTIVES: Stem cell therapies based on differentiation of adult or embryonic stem cells into specialized ones appear to be effective for treating several human diseases. This work addresses the mathematical simulation of proliferation kinetics of stem cells. MATERIALS AND METHODS: Sheep bone marrow mesenchymal stem cells (phenotype characterized by flow cytometry analysis) seeded at different initial concentrations in Petri dishes were expanded to confluence. Sigmoid temporal profiles of total counts obtained through classic haemocytometry were quantitatively interpreted by both a phenomenological logistic equation and a novel model based on a one-dimensional, single-staged population balance approach capable of taking into account contact inhibition at confluence. The models' parameters were determined by comparison with experimental data on population expansion starting from single seeding concentration. Reliability of the models was tested by predicting cell proliferation carried out starting from different seeding concentrations. RESULTS AND DISCUSSION: It was found that the proposed population balance modelling approach was successful in predicting the experimental data over the whole range of initial cell numbers investigated, while prediction capability of phenomenological logistic equation was more limited.

Restricted diffusion of a freely diffusible second messenger: mechanisms underlying compartmentalized cAMP signalling.

It is becoming increasingly evident that the freely diffusible second messenger cAMP can transduce specific responses by localized signalling. The machinery that underpins compartmentalized cAMP signalling is only now becoming appreciated. Adenylate cyclases, the enzymes that synthesize cAMP, are localized at discrete parts of the plasma membrane, and phosphodiesterases, the enzymes that degrade cAMP, can be targeted to selected subcellular compartments. A-kinase-anchoring proteins then serve to anchor PKA (protein kinase A) close to specific targets, resulting in selective activation. The specific activation of such individual subsets of PKA requires that cAMP is made available in discrete compartments. In this presentation, the molecular and structural mechanisms responsible for compartmentalized PKA signalling and restricted diffusion of cAMP will be discussed.

Comparison of two brief neuropsychological batteries in people with multiple sclerosis.

BACKGROUND: We compared two brief neuropsychological batteries devised to assess people with multiple sclerosis (MS) and used them to assess the relationship between cognitive impairment and dinical characteristics. METHODS: We administered either the Brief Repeatable Battery of Neuropsychological Tests (BRBNT) or the Screening Examination for Cognitive Impairment (SEFCI) to 213 consecutive MS outpatients and 213 individually matched controls. RESULTS: Administration times were longer for BRBNT than SEFCI, for MS and controls (p=0.001). People with MS had lower scores in all individual tests than controls (p<0.001, BRBNT and SEFCI). By the criterion of poor performance on one or more tests, the sensitivity of BRBNT was 41.9% and that of SEFCI 31.5%. The corresponding figures by poor performance on two or more tests were 16.2% for BRBNT and 18.5% for SEFCI. The Buschke Selective Reminding and Paced Auditory Serial Addition were the tests best discriminating between people with MS and controls for BRBNT, and the Symbol Digit Modalities test for SEFCI. The only clinical variable independently associated with impaired performance on these batteries was EDSS. CONCLUSIONS: Both cognitive batteries were well accepted and easy to administer. Administration time for SEFCI was significantly shorter than for BRBNT; however, alternative forms for serial evaluation are available only for BRBNT. The BRBNT was slightly more sensitive in detecting impairment by the criterion of poor perfomance on one or more tests. EDSS score was the only clinical variable independently associated with cognitive impairment

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.

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.

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.

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.

Ascites, pleural, and pericardial effusions in acute pancreatitis. A prospective study of incidence, natural history, and prognostic role.

Ascites and pleural and pericardial effusions can be observed during acute pancreatitis. The aims of this study were to evaluate their incidence, natural history, and prognostic role in patients with acute pancreatitis. One hundred patients consecutively admitted with a diagnosis of acute pancreatitis were prospectively submitted to abdominal, pleural, and cardiac ultrasonography at admission and during follow-up. Ascites was found in 18 patients, pleural effusion in 20, and pericardial effusion in 17. Twenty-four patients of this series had severe pancreatitis; three of them died. All effusions disappeared spontaneously in patients who survived pancreatitis up to two months after dismissal. At multivariate analysis ascites and pleural effusion were demonstrated to be accurate independent predictors of severity. The respective odds ratios were 5.9 [95% confidence interval (CI), 1.5-23.0%) and 8.6 (95% CI, 2.3-32.5%). Furthermore the presence of pleural effusion, ascites, and pericardial effusion were associated with an increased incidence of pseudocyst during follow-up. Ascites and pleural and pericardial effusions are frequent during acute pancreatitis. Pleural effusion and ascites are accurate predictors of severity in these patients.

A quick procedure for the isolation of dimeric piratoxins-I and II, two myotoxins from Bothrops pirajai snake venom. N-terminal sequencing.

Two myotoxins, MP-I and MP-II, from Bothrops pirajai snake venom, have been purified by a quick high performance liquid chromatography (HPLC) procedure. Based on the HPLC coelution profile, amino acid composition, N-terminal sequence, polyacrylamide gel electrophoresis (PAGE) migration, as well as lack of phospholipase-A2 (PLA2) and proteolytic activities, MP-I and MP-II were identified as piratoxin-I (PrTX-I) and II (PrTX-II), respectively. This procedure affords, aside the reduced operation time, a high yield (35% of the applied sample in terms of A280nm) of MP-I, which is the major myotoxin of the venom. The N-terminal sequences of MP-I, MP-II, PrTX-I and PrTX-II, up to the 51st, 41st, 46th and 39th residues, respectively, have been determined, revealing MP-I (and hence PrTX-I) as a Lys-49 PLA2-like myotoxin. Both MP-I and MP-II have been shown, by SDS-PAGE, to occur in dimeric isoforms.

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).

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.