Bioaccumulation of resveratrol metabolites in myocardial tissue is dose-time dependent and related to cardiac hemodynamics in diabetic rats.

BACKGROUND AND AIMS: Trans-resveratrol (RSV) is a natural compound occurring in different foods and plants, which in vivo is rapidly conjugated with glucuronic acid and sulfate. Despite its demonstrated cardioprotective activity, the bioaccumulation of RSV or its metabolites in cardiac tissue is still unknown. METHODS AND RESULTS: Diabetic rats were randomized to 1, 3 or 6 weeks of RSV treatment at two different doses (1 or 5 mg/kg/day). A dose and time-dependent accumulation was observed, with no detectable levels of RSV metabolites found in heart tissues after 1 week and significant concentrations of RSV-3-sulfate and RSV-3-glucuronide after 6 weeks of treatment (0.05 nmol/g of tissue and 0.01 nmol/g of tissue, respectively). Tissue accumulation of RSV metabolites was accompanied by an improvement of cardiac function in long-term diabetes, when myocardial morpho-functional damage is more evident, with an almost complete recovery of all hemodynamic parameters, at the highest RSV dose. CONCLUSION: Even if a higher concentration of RSV in tissues cannot be ruled out after constant oral administration, an accumulation coherent with what is usually evaluated in cell based mechanistic studies is largely unattainable and the RSV unconjugated form would not be present in this paradigm. The current investigation provides data on myocardial tissue concentrations of RSV metabolites, after short/medium term RSV treatment. This knowledge constitutes a basic requirement for future studies aimed at reliably defining the molecular pathways underlying RSV-mediated cardioprotective effects and opens up new perspectives for research focused on testing phenolic compounds as adjuvants in degenerative heart diseases.

Development and validation of an high performance liquid chromatography-tandem mass spectrometry method for the determination of imatinib in rat tissues.

An high performance liquid chromatography-tandem mass-spectrometry (HPLC-MS/MS) method was developed and validated for the determination in rat heart and liver of the tyrosine kinase inhibitor imatinib (IM), an anticancer drug approved for the treatment of chronic myeloid leukemia and gastrointestinal stromal tumors. Extraction of the drug from tissues was performed by solvent extraction and the obtained extracts were analyzed by HPLC-MS/MS in selected reaction monitoring mode. The developed method was validated according to the criteria for bioanalytical method, showing good performances in terms of lower limit of quantification (LLOQ=0.02µgml(-1)), linearity (R(2)=0.998), repeatability (RSD<3%), reproducibility (RSD<13%) and recovery (RR>89%). The developed method was then applied to the analysis of heart and liver of rats treated with different doses of IM, with and without the simultaneous administration of carvedilol, a beta-blocking agent with cardioprotective effect, in order to evaluate tissue levels of the tyrosine kinase inhibitor. The obtained results revealed that the amount of IM in the rat heart was significantly affected by the administered dose, whereas carvedilol had no effect on IM concentrations. Thus, we have developed a method that allows the detection of IM traces in complex tissues such as the heart and liver and that may be proposed for the determination of the drug in other clinically relevant biological samples.

Resident cardiac stem cells.

The introduction of stem cells in cardiology provides new tools in understanding the regenerative processes of the normal and pathologic heart and opens new options for the treatment of cardiovascular diseases. The feasibility of adult bone marrow autologous and allogenic cell therapy of ischemic cardiomyopathies has been demonstrated in humans. However, many unresolved questions remain to link experimental with clinical observations. The demonstration that the heart is a self-renewing organ and that its cell turnover is regulated by myocardial progenitor cells offers novel pathogenetic mechanisms underlying cardiac diseases and raises the possibility to regenerate the damaged heart. Indeed, cardiac stem progenitor cells (CSPCs) have recently been isolated from the human heart by several laboratories although differences in methodology and phenotypic profile have been described. The present review points to the potential role of CSPCs in the onset and development of congestive heart failure and its reversal by regenerative approaches aimed at the preservation and expansion of the resident pool of progenitors.

Spectrum of F8 gene mutations in haemophilia A patients from a region of Italy: identification of 23 new mutations.

SUMMARY: Haemophilia A (HA) is an X-linked recessive bleeding disorder caused by a lack or decrease of coagulation factor VIII activity. The molecular diagnosis of HA is challenging and a variety of different mutations have been identified throughout the F8 gene. Our aim was to detect the causative mutation in 266 HA patients from Emilia-Romagna region (Italy) and in all suspected carriers. Molecular analysis of F8 in 201 HA patients (152 index cases) was performed with a combination of several indirect and direct molecular approaches, such as long distance polymerase chain reaction, multiplex ligation-dependent probe amplification, denaturing high performance liquid chromatography and direct sequencing. The analysis revealed 78 different mutations, 23 of which were novel, not having been reported in national or international databases. The detection rate was 100%, 86% and 89% in patients with severe, moderate and mild HA, respectively. The information provided by this registry will be helpful for monitoring the treatment of HA patients in Emilia-Romagna and also for reliable genetic counselling of affected families in the future.

Novel association of HLA-haplotypes with primary sclerosing cholangitis (PSC) in a southern European population.

AIMS: In patients with with primary sclerosing cholangitis we investigated the major histocompatibility complex (MHC) genes and mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. METHODS: In 64 PSC patients and 183 normal controls of the same population (Northern Italy), allelic polymorphisms at the DNA level were investigated in MHC region genes: HLA-DRB1, HLA-DQB1 and HLA-B, tumour necrosis factor A (TNFA), and in CFTR gene, with polymerase chain reaction-based methodologies. RESULTS: Frequencies of DRB1*01, DQA1*0101, DQB1*0102 (14 vs. 8%, p<0.05), DRB1*16, DQA1*0102, DQB1*0502 (8 vs. 3%, p<0.025) and DRB1*04, DQA1*03, DQB1*0301 (10 vs. 4%, p<0.005) haplotypes were more elevated in PSC patients. The frequency of patients positive for HLA DRB1*01, *1601 or *04 related haplotypes was significantly increased (32 vs. 14%, p<0.00025). DRB1*07, DQA1*0201, DQB1*02 haplotype frequency was significantly decreased (4 vs. 15%, p<0.001). After removing HLA-DRB1*01, *1601, *04 related haplotype sharing patients, HLA-DRB1*03, DQA1*0501, DQB1*02 haplotype frequency was significantly increased (32 vs. 14%, p<0.01). TNFA2 allele frequency was significantly increased in PSC patients (23 vs. 14%, p<0.025), as well as the TNFA2 homozygous genotype (9 vs. 0.5%, p=0.0013). No mutations were found on the CFTR gene and the allelic frequency of the 5T polymorphism in intron 8 was not increased. CONCLUSION: These data suggest that the role of genes in the HLA region is relevant, but not necessarily disease-specific and it might be different in populations with divergent ancestries.

Medical applications of shape memory alloys.

Shape memory alloys (SMA) are materials that have the ability to return to a former shape when subjected to an appropriate thermomechanical procedure. Pseudoelastic and shape memory effects are some of the behaviors presented by these alloys. The unique properties concerning these alloys have encouraged many investigators to look for applications of SMA in different fields of human knowledge. The purpose of this review article is to present a brief discussion of the thermomechanical behavior of SMA and to describe their most promising applications in the biomedical area. These include cardiovascular and orthopedic uses, and surgical instruments.

Medical applications of shape memory alloys

Shape memory alloys (SMA) are materials that have the ability to return to a former shape when subjected to an appropriate thermomechanical procedure. Pseudoelastic and shape memory effects are some of the behaviors presented by these alloys. The unique properties concerning these alloys have encouraged many investigators to look for applications of SMA in different fields of human knowledge. The purpose of this review article is to present a brief discussion of the thermomechanical behavior of SMA and to describe their most promising applications in the biomedical area. These include cardiovascular and orthopedic uses, and surgical instruments

Inhibitory effect of somatostatin on neutral amino acid transport in isolated brain microvessels.

In the presence of somatostatin-14 or some of its receptorial agonists, the uptake of large neutral amino acids by isolated brain microvessels was found to be inhibited up to 50%, no other transport system being affected. Although the luminal and abluminal sides of brain endothelial cells are both capable of taking up large neutral amino acids, only uptake from the abluminal side appears to be inhibited by somatostatin. The involvement of a type-2 somatostatin receptor was suggested by assays with a series of receptor-specific somatostatin agonists, and was confirmed by the release of inhibition caused by a specific type-2 receptor antagonist. A type-2-specific mRNA was indeed shown to be present in both bovine brain microvessels ex vivo and primary cultures of endothelial cells from rat brain microvessels.

Fluorescent in situ hybridisation on tissue sections: a quantitative approach with confocal laser scanning microscopy.

The use of fluorescent detection methods in association with digital microscopy technologies is an innovative approach for tissue localisation of messenger RNA. The success of such methods relies on the tissue preservation, local availability of the probe and on the existence of high resolution tridimensional analysis systems. Cryostatic sections, mild denaturation, short oligonucleotide probes (20mer) and confocal laser scanning microscopy allow the fulfillment of all these conditions avoiding photobleaching and tissue autofluorescence. In this paper, we describe in detail a method for in situ hybridisation set up with digoxigenin-coupled oligonucleotide complementary to beta-actin mRNA as a probe and an anti-hapten fluorescent antibody as second step for detecting specific hybridisation. Fluorescence was analysed by means of a confocal laser scanning microscope (CLSM) that provides images with low out-of-focus blurring also with relatively low numerical aperture (NA) objectives. We propose also an easy method to perform semi-quantitative thresholding analysis which allows to discriminate between background and specific signal.

Deltorphin transport across the blood-brain barrier.

In vivo antinociception studies demonstrate that deltorphins are opioid peptides with an unusually high blood-brain barrier penetration rate. In vitro, isolated bovine brain microvessels can take up deltorphins through a saturable nonconcentrative permeation system, which is apparently distinct from previously described systems involved in the transport of neutral amino acids or of enkephalins. Removing Na+ ions from the incubation medium decreases the carrier affinity for deltorphins (-25%), but does not affect the Vmax value of the transport. The nonselective opiate antagonist naloxone inhibits deltorphin uptake by brain microvessels, but neither the selective delta-opioid antagonist naltrindole nor a number of opioid peptides with different affinities for delta- or mu-opioid receptors compete with deltorphins for the transport. Binding studies demonstrate that mu-, delta-, and kappa-opioid receptors are undetectable in the microvessel preparation. Preloading of the microvessels with L-glutamine results in a transient stimulation of deltorphin uptake. Glutamine-accelerated deltorphin uptake correlates to the rate of glutamine efflux from the microvessels and is abolished by naloxone.

The presence of posttransplant HLA-specific IgG antibodies detected by enzyme-linked immunosorbent assay correlates with specific rejection pathologies.

Posttransplant monitoring of anti-HLA antibodies with routine techniques gives unsatisfactory results due to a variety of technical limitations. We investigated how a new alternative technique correlates with posttransplant clinical events. A total of 313 nonselected serum samples from 136 patients were screened by an ELISA utilizing captured soluble HLA class I antigens. We observed the absence of anti-HLA antibody production in acute rejection cases responding to standard antirejection therapy. On the other hand, we showed a clear presence of these antibodies in acute rejection episodes not responding to standard therapy (P<0.0001) and in chronic rejection (P<0.001). We conclude that routine posttransplant monitoring by ELISA offers early risk assessment that is crucial for proper immunosuppression and for antirejection therapy choice.

X-linked Alport syndrome: an SSCP-based mutation survey over all 51 exons of the COL4A5 gene.

The COL4A5 gene encodes the alpha5 (type IV) collagen chain and is defective in X-linked Alport syndrome (AS). Here, we report the first systematic analysis of all 51 exons of COL4A5 gene in a series of 201 Italian AS patients. We have previously reported nine major rearrangements, as well as 18 small mutations identified in the same patient series by SSCP analysis of several exons. After systematic analysis of all 51 exons of COL4A5, we have now identified 30 different mutations: 10 glycine substitutions in the triple helical domain of the protein, 9 frameshift mutations, 4 in-frame deletions, 1 start codon, 1 nonsense, and 5 splice-site mutations. These mutations were either unique or found in two unrelated families, thus excluding the presence of a common mutation in the coding part of the gene. Overall, mutations were detected in only 45% of individuals with a certain or likely diagnosis of X-linked AS. This finding suggests that mutations in noncoding segments of COL4A5 account for a high number of X-linked AS cases. An alternative hypothesis is the presence of locus heterogeneity, even within the X-linked form of the disease. A genotype/phenotype comparison enabled us to better substantiate a significant correlation between the degree of predicted disruption of the alpha5 chain and the severity of phenotype in affected male individuals. Our study has significant implications in the diagnosis and follow-up of AS patients.

Pharmacokinetic profile of the induction dose of propofol in chronic renal failure patients undergoing renal transplantation.

STUDY OBJECTIVE: To define the pharmacokinetic profile of the induction dose of propofol in chronic renal failure patients. DESIGN AND SETTING: Determination of propofol blood concentrations after the bolus dose of 2 mg.kg-1 bw injected in 30 seconds in a peripheral vein in a group of chronic renal failure (CRF) patients and in a group of normal patients (controls). PATIENTS: 10 CRF patients (7 males, 3 females, mean age 47 +/- 8 years old, mean body weight 66 +/- 8 kg) candidates to cadaveric renal transplantation and free from major hepatic diseases (study group); 8 ASA I patients (5 males, 3 females), without major cardiorespiratory, hepatic, renal, hematologic or metabolic diseases undergoing minor elective surgical procedures lasting from 50 to 90 minutes (control group). MEASUREMENTS: a) propofol blood concentrations by means of HPLC; b) derived pharmacokinetic parameters (calculated by means of Siphar, version 4.0, Societé de informatique médicale, Simed, Paris, 1991); c) cardiovascular parameters (heart rate, central venous pressure, invasive arterial pressure). MAIN RESULTS: The decay of propofol whole blood concentrations, distribution, redistribution and elimination half lives were similar in CRF and in control patients. On the contrary, significantly different in CRF patients were propofol blood concentrations from two to ten minutes following the induction dose (lower), the area under concentration- time curve (AUC) (smaller), the mean resident time (longer), the total body clearance (greater), the volumes of distribution at steady state and during the elimination phase (larger). The larger volumes of distribution are closely correlated with the significantly lower albumin concentrations in the uremic patients. An accelerated hepatic biotransformation is one of the possible explanations for the greater total body clearance of propofol in the uraemic patients: in fact an increased glucuronyltrasferase activity and glucuronoconjugation induced by phenols has been demonstrated in uraemia. On the other hand, large volumes of distribution are often associated with elevated total body clearance. The only significant change in the cardiovascular profile was a reduction of 17 +/- 8% of the systolic blood pressure one minute after the administration of the induction dose of propofol, whereas heart rate, arterial and central venous pressures were rather stable after intubation and at skin incision: proper vascular filling before the induction of anaesthesia has probably played a crucial role in maintaining hemodynamic stability. CONCLUSIONS: From the data gathered in this study, propofol can be considered a suitable anaesthetic agent for the induction of general anaesthesia in uraemic patients. In our opinion these data could constitute a basis for future protocols of total intravenous anaesthesia with propofol in uremic patients.

Major COL4A5 gene rearrangements in patients with juvenile type Alport syndrome.

Mutations in the COL4A5 gene, which encodes the a5 chain of type IV collagen, are found in a large fraction of patients with X-linked Alport syndrome. The recently discovered COL4A6, tightly linked and highly homologous to COL4A5, represents a second candidate gene for Alport syndrome. We analyzed 177 Italian Alport syndrome families by Southern blotting using cDNA probes from both COL4A5 and COL4A6. Nine unrelated families, accounting for 5% of the cases, were found to have a rearrangement in COL4A5. No rearrangements were found in COL4A6, with the exception of a deletion encompassing the 5' ends of both COL4A5 and COL4A6 genes in a patient with Alport syndrome and leiomyomatosis. COL4A5 rearrangements were all intragenic and included 1 duplication and 7 deletions. Polymerase chain reaction (PCR) analysis was carried out to characterize deletion and duplication boundaries and to predict the resulting protein abnormality. The two smallest deletions involved a single exon (exons 17 and 40, respectively), while the largest ones spanned exons 1 to 36. The clinical phenotype of patients in whom a rearrangement in COL4A5 was detected was severe, with progression to end-stage renal failure in juvenile age and hypoacusis occurring in most cases. These data have some important implications in the diagnosis of patients with Alport syndrome.

TCR-beta chain gene rearrangement and expression in human T-cell development and in leukemia.

T-cell receptor TCR-beta gene expression is an early event during human ontogenesis since the majority of thymocytes express cytoplasmic beta chain as early as the 15th week of gestation, when a complete VDJ rearrangement and functional 1.3-kb beta gene transcript are detectable. We report here our contribution with those of others on the analysis of TCR-beta gene ontogenesis. By sequencing beta gene transcripts we have demonstrated that beta gene N-regions increase dramatically in the thymus after the 20th week and that the period between 20-30 weeks is of critical importance for the acquisition of N-diversity. A correlation between TdT and N-region expression also exists. An ordered expression of TdT and cytoplasmic beta chain occurs in humans starting around the 20th week, similar to the sequence of coordinated expression of TdT and cytoplasmic mu chains detectable in B-cell precursors. TCR-beta gene behavior in T-cell neoplasms, in 'biphenotypic' leukemias and in B-ALL is also discussed. An interesting study of seven cases of B-ALL with complete V(D)J beta gene rearrangement is analyzed, as is its implication for further analysis in B-cell leukemia.

Changes in circulating levels of atrial natriuretic factor (ANF) during orthotopic liver transplantation in humans.

Atrial natriuretic factor (ANF) is a 28 amino acid peptide secreted by the atrial cardiocytes. Clearance is via the lung (50%) and the liver (25%). The main stimulus to ANF secretion is atrial distension but vasoconstrictors, sympathetic stimulation, catecolamines and tachycardia are able to enhance its circulating blood levels. ANF blood concentrations were measured during orthotopic liver transplantation in six postnecrotic cirrhotic patients. Significant increases in ANF blood levels occurred at the end of the anhepatic phase (P < or = 0.02 vs baseline) associated with low cardiac filling pressures (P < or = 0.02 vs baseline) and increased systemic vascular resistances (P < or = 0.02 vs preanhepatic phase). Aldosterone blood levels showed a similar behaviour, increasing significantly (P > or = 0.001 vs baseline) at the end of the anhepatic phase. ANF fell after reperfusion of the graft and returned towards baseline values at the end of the procedure. Since most of the total body clearance of ANF is performed by the lungs, its sharp increase at the end of the anhepatic phase could be considered a counterregulatory response to vasoconstricting stimulation and to fluid-sparing mechanisms in the presence of relative hypovolaemia. Its decrease after reperfusion could be related to volume normalization and partly to the enhanced clearance performed by the newly grafted liver.

Effect of inorganic phosphate on hypoxanthine transport in isolated brain microvessels.

In isolated brain microvessels, used as an in vitro model of the blood-brain barrier, the rate of hypoxanthine uptake was modulated by the presence of inorganic phosphate. A single high-capacity, low-affinity transport system was apparently active in a phosphate-free medium (Vmax = 840 pmol/mg protein/min, Km = 750/uM); in the presence of 10 mM phosphate, there was also a low-capacity, high-affinity system (Vmax = 47 pmol/mg protein/min, Km = 27/uM). The phosphate-dependent component was inactive in the absence of glucose or of Na+ ions, or upon addition of phloretine (but was scarcely affected by 2,4-dinitrophenol). This activity was apparently coupled to the intracellular phosphoribosyltransferase-catalyzed conversion of purines into the corresponding nucleotides: when inorganic phosphate was present in the suspending medium, labeled hypoxanthine was transported with higher efficiency and was readily converted to inosine monophosphate and to other related nucleotides. In the absence of phosphate ions, hypoxanthine was instead metabolized to xanthine and uric acid.

T-cell receptor beta-chain gene rearrangement and expression during human thymic ontogenesis.

T-cell receptor (TCR) beta-chain proteins appear early (approximately 15th week of gestation) during human thymic ontogenesis. These beta-chain proteins, which appear before terminal deoxynucleotidyl transferase (TdT), could be an expression of a fully rearranged (V-D-J), incompletely rearranged (D-J), or germline TCR beta-chain gene. The aims of this study, performed from the 15th week onward, were the following: (1) to investigate whether or not TCR beta gene rearranges at an early stage during human thymic ontogenesis; (2) to investigate whether complete presumptive functional (1.3 kb) TCR beta gene transcript is present at these early stages of development, or if incomplete (1 kb) or germ-line (1.1 kb) transcripts are expressed; (3) to examine the phenotype of TCR beta-chain+ cells with two-color fluorescence using monoclonal antibody (MoAb) beta F1 and MoAbs that recognize CD1, CD2, CD3, CD4, CD8, CD5, and CD7 antigens (rabbit anti-calf TdT antiserum was used to detect TdT); and (4) to demonstrate whether or not beta gene N-diversity regions are detectable as early as the 15th week and whether or not N-nucleotide insertions correlate to TdT expression. Fifteen- to 22-week fetal thymuses and pediatric thymuses were investigated. We demonstrated that TCR beta-chain gene rearranged as early as the 15th week in human thymus and that a complete functional TCR beta gene transcript was expressed at these early stages of human development. No other analyses to date have investigated TCR beta gene expression in early human thymus using molecular biology techniques. No significant differences were detectable between phenotypic analysis of fetal and pediatric samples, except for TdT expression, which appeared after the 20th week. Essentially all mCD3+ (OKT3+) cells were beta-chain+ at the different weeks investigated. A significant percentage of CD1+ cells were beta-chain+, and the percentage increased along with the age of development. After the 20th week, we identified three main populations: TdT+, cCD3+, beta F-(early thymic precursors); TdT+, CD1+, beta F1+ (intermediate maturity cortical thymocytes); and TdT-, mCD3+, beta F1++ (mature medullary thymocytes). Given these values, we may consider beta-chain expression an ordered process. beta gene N-nucleotide insertions were correlated to TdT expression, since N-regions increased considerably after the 20th week. A further increase of N-nucleotide insertions was detected from the 22nd week to the 32nd week.

Effect of the cationic polypeptide polylysine on neutral amino acid transport in isolated brain microvessels.

The functionality of isolated brain microvessels - used as anin vitro model of the blood-brain barrier - can be influenced by interaction with cationic proteins. The various polylysines (Mr ranging from 0.9 to 180 kDa) tested affected the activity of both the Na(+)-dependent ("A") and the Na(+)-independent ("L") systems for neutral amino acid transport. Exposure to the 180 kDa polylysine caused a conspicuous inhibition of both transport systems, associated to an increased passive permeability. There was a constant, Mr-dependent, inhibition of the the L-system-mediated uptake of hydrophobic neutral amino acids. The activity of the A-system was enhanced, upon exposure to polymers larger than 22 kDa reaching its peak at 68 kDa and and declining at higher Mr values. The effect which was Na(+)-ions dependent and abolished by phloretine, could be essentially ascribed to an increased affinity of the MeAIB for its carrier (Km value decreasing from 265 to 169µM in presence of 68 kDa polylysine).