The molecular basis of alkaptonuria.

Alkaptonuria (AKU) occupies a unique place in the history of human genetics because it was the first disease to be interpreted as a mendelian recessive trait by Garrod in 1902. Alkaptonuria is a rare metabolic disorder resulting from loss of homogentisate 1,2 dioxygenase (HGO) activity. Affected individuals accumulate large quantities of homogentisic acid, an intermediary product of the catabolism of tyrosine and phenylalanine, which darkens the urine and deposits in connective tissues causing a debilitating arthritis. Here we report the cloning of the human HGO gene and establish that it is the AKU gene. We show that HGO maps to the same location described for AKU, illustrate that HGO harbours missense mutations that cosegregate with the disease, and provide biochemical evidence that at least one of these missense mutations is a loss-of-function mutation.

[Evaluation of the contralateral breast with magnetic resonance in patients with newly diagnosed unilateral breast cancer]. / Evaluación de la mama contralateral mediante resonancia magnética en pacientes con diagnóstico reciente de cáncer mamario unilateral.

Breast Magnetic Resonance (BMR) imaging is a useful tool in the evaluation of breast cancer before surgical treatment. BMR imaging plays an important role in the evaluation of the extension of the malignant lesions, and the study of multifocality and multicentricity. BMR may have a role in the detection of synchronous contralateral breast cancer that is occult to conventional imaging methods (mammography and ultrasonography). In this study we review 13 series of different authors in which they have used BMR in the evaluation of the contralateral breast in patients with newly diagnosed breast cancer. Two thousand five hundred and eleven patients were evaluated with BMR and 123 contralateral cancers, that were occult to conventional methods, were detected with this technique (4,9 %). Therefore, BMR imaging of the breast is useful as a complementary tool because of its high sensitivity in local staging of a breast cancer and its ability in the detection of synchronous contralateral breast cancer in patients with newly diagnosed breast cancer.

Evaluation of CD7 and terminal deoxynucleotidyl transferase (TdT) expression in CD34+ myeloblasts from patients with myelodysplastic syndrome.

There is an emerging use of flow cytometry to evaluate patients with myelodysplastic syndrome (MDS). We have studied CD7 and TdT expression in the CD34+ myeloid blast cell population in 55 bone marrow samples of patients with MDS. CD7 and/or TdT were detected in 38 out of 55 patients (69%). CD7 expression was not related to other bad prognosis data but conversely, we found an association between TdT+ CD34 myeloblasts and high-risk MDS patients according to the International Prognostic Scoring System. Therefore, CD7 and TdT may help to establish the diagnosis of MDS and, TdT expression also seems to be a useful marker in distinguishing risk groups.

Chromosomal changes pattern and gene amplification in T cell non-Hodgkin's lymphomas.

T cell non-Hodgkin's lymphomas are a heterogeneous group of lymphomas with poor prognosis, and whose genetic alterations are not well understood. Comparative genomic hybridization (CGH) is a technique that allows the identification of DNA imbalances without cytogenetic studies. We have studied 37 samples from 29 T cell non-Hodgkin's lymphomas (25 peripheral and four lymphoblastic lymphomas) by CGH in order to detect DNA sequence copy number changes of putative importance in the biology and prognosis of these neoplasms. We detected abnormal CGH profiles in 16/27 (59%) of samples at diagnosis, a ratio that increased to 66% (23/37) when we included the relapsed samples. The most common recurrent changes were gains related to the X chromosome, either the whole chromosome or partially the Xq26-27 bands (19%). Other recurrent changes included gains of bands 9q34, gains of chromosomes 17, 19, and 20, and complete or partial deletions of chromosome 13 (10%). Cancer-related genes located at Xq26-28 region were analyzed by Southern blot and fluorescence in situ hybridization (FISH). Low level amplification of some of these genes was detected by this technique confirming the results obtained by CGH in this region. The detection of abnormal CGH profiles in these T cell lymphomas could have clinical implications. Patients with abnormal CGH profiles showed significant associations with advanced stage of disease, overexpression of P53, and higher proliferative index.

Molecular and clinical study of 61 Angelman syndrome patients.

We analyzed 61 Angelman syndrome (AS) patients by cytogenetic and molecular techniques. On the basis of molecular findings, the patients were classified into the following 4 groups: familial cases without deletion, familial cases with submicroscopic deletion, sporadic cases with deletion, and sporadic cases without deletion. Among 53 sporadic cases, 37 (70%) had molecular deletion, which commonly extended from D15S9 to D15S12, although not all deletions were identical. Of 8 familial cases, 3 sibs from one family had a molecular deletion involving only 2 loci, D15S10 and GABRB3, which define the critical region for AS phenotypes. The parental origin of deletion, both in sporadic and familial cases, was exclusively maternal and consistent with a genomic imprinting hypothesis. Among sporadic and familial cases without deletion, no uniparental disomy was found and most of them were shown to inherit chromosomes 15 from both parents (biparental inheritance). A discrepancy between cytogenetic and molecular deletion was observed in 14 (26%) of 53 patients in whom cytogenetic analysis could be performed. Ten (43%) of 23 patients with a normal karyotype showed a molecular deletion, and 4 (13%) of 30 patients with cytogenetic deletion, del(15) (q11q13), showed no molecular deletion. Most clinical manifestations, including neurological signs and facial characteristics, were not distinct in each group except for hypopigmentation of skin or hair. Familial cases with submicroscopic deletion were not associated with hypopigmentation. These findings suggested that a gene for hypopigmentation is located outside the critical region of AS and is not imprinted.

Cytogenetic and molecular studies of siblings with ataxia telangiectasia followed for 7 years.

We present cytogenetic, immunologic, and molecular data obtained over 7 years from a family with ataxia telangiectasia (AT) including 2 affected children and their unaffected sibling, and their obligate heterozygous parents. In a period of 3 years, the T lymphocytes from both AT patients showed clonal rearrangements of chromosomes 7 and 14 at specific bands (7p13, 7q35, 14q12, and 14q32), where loci for the Ig and TCH genes are located. A molecular study was carried out on peripheral blood and bone marrow samples from both patients using Southern blot and PCR for Ig and TCR genes. A monoclonal population for TCR gamma was observed in one of the two affected children, but only in peripheral blood.

In vivo sustained release of adenoviral vectors from poly(D,L-lactic-co-glycolic) acid microparticles prepared by TROMS.

We have prepared and characterised injectable adenovirus-loaded polymeric microparticles to be used for in vitro and in vivo gene transfer studies. Microparticles were prepared by the water-in-oil-in-water solvent evaporation method using a novel system where the emulsification process is carried out by the turbulent injection of the phases in the total recirculation one-machine system (TROMS) apparatus. In vitro studies were performed to assess the amount of infectious adenovirus released from the microparticles, showing that these microparticles release higher amounts of infectious adenovirus than microparticles prepared by standard emulsification techniques. We also tested whether sustained release in vivo could overcome the short-lived gene expression profile which is typical of adenovirus delivery into muscle. Intramuscular injection of adenovirus-loaded microparticles in immunocompetent mice showed transgene (beta-galactosidase) expression for at least 7 weeks in two out of four muscles injected with adenovirus-loaded microparticles prepared by TROMS, but not in control muscles injected with purified adenovirus stocks.

Influence of the surface characteristics of PVM/MA nanoparticles on their bioadhesive properties.

The aim of this work was to investigate the influence of the cross-linkage of poly(methylvinylether-co-maleic anhydride) (PVM/MA) nanoparticles with increasing amounts of 1,3-diaminopropane (DP) and, eventually, bovine serum albumin (BSA) on their gastrointestinal transit and bioadhesive properties. The fluorescently-labelled formulations were orally administered to rats and, at different times, the amount of nanoparticles in both the lumen content and adhered to the gut mucosa were quantified. The gut transit was evaluated by calculating the gastric (k(ge)) and intestinal (k(ie)) emptying rates. The adhered fraction of nanoparticles in the whole gut was plotted versus time and, from these curves, the intensity, capacity and extent of the adhesive interactions were estimated. The bioadhesive potential of PVM/MA was much higher when formulated as nanoparticles (NP) than in the solubilised form in water. However, k(ge) and k(ie) increased by increasing the extent of cross-linkage of nanoparticles with DP, while the capacity to develop adhesive interactions and the intensity of the adhesive phenomenon were significantly higher for non-hardened than for DP-cross-linked carriers. In contrast, the BSA-coating of cross-linked nanoparticles significantly decreased k(ge) and k(gi), whereas the intensity of the bioadhesive phenomenon was significantly higher than for NP. In summary, the adhesivity of the nanoparticles appears to modulate their gastrointestinal transit profile.

High-performance liquid chromatographic determination of amphotericin B in plasma and tissue. Application to pharmacokinetic and tissue distribution studies in rats.

A sensitive HPLC method with piroxicam as internal standard was developed for assaying amphotericin B in plasma and tissue. An Ultrabase-C18 column and a simple mobile phase consisting of an acetonitrile-acetic acid (10%)-water (41:43:16) mixture were used. The flow-rate was 1 ml/min and the effluent was monitored at 405 nm. The linearity of the assay method was up to 1000 ng/ml and 100 micrograms/g for plasma and tissue, respectively. Intra-day and inter-day RSDs were below 10% for all the sample types. This HPLC assay has been applied to determine amphotericin B in plasma and tissue samples taken during pharmacokinetic and tissue distribution studies in rats.

Determination of oligonucleotide ISIS 2922 in nanoparticulate delivery systems by capillary zone electrophoresis.

ISIS 2922 is an antisense oligonucleotide with antiviral activity against cytomegalovirus. However, its rapid degradation in biological fluids and its low capacity for diffusion across cell membranes limit its therapeutical use. One possibility to overcome these drawbacks consists of using nanoparticles as drug carriers. The aim of this study was to develop an analytical method for determining the amount of ISIS 2922 loaded into albumin nanoparticles. For this purpose, capillary zone electrophoresis (CZE) was performed on a fused-silica capillary filled with borate buffer (12.5 mM, pH 9.5). Paracetamol was used as an internal standard and a diode-array detection system was set at 270 nm. Under these conditions, the limit of quantitation of ISIS 2922 was 1.27 microg and the precision and accuracy of the method did not exceed 7%. Moreover, the use of paracetamol as internal standard and the quantification by means of a 'corrected area' procedure enabled us to reduce the peak variability and accurately determine the amount of oligonucleotide loaded in the albumin nanoparticles. In summary, this assay is a selective and sensitive CZE method for the accurate quantitation of ISIS 2922 oligonucleotide in albumin nanoparticles.

Serum leptin concentration in patients infected with human immunodeficiency virus.

OBJECTIVE: To assess the potential effect of serum leptin levels in human immunodeficiency virus (HIV)-related wasting. METHODS: Morning serum leptin levels of 94 randomly chosen HIV-infected patients were measured and correlated with age, sex, weight, height, body mass index (BMI), routine blood chemistries (SMA 18), complete blood cell count, HIV viral load, and CD4/CD8 ratio. RESULTS: The mean serum leptin level was 7.0 +/- 6.9 ng/mL. Leptin levels were significantly higher in the 38 female patients than in the 56 male patients (10.0 +/- 8.4 ng/mL versus 5.0 +/- 4.9 ng/mL; P<0.001). Leptin levels were positively correlated with BMI (r = 0.71; P<0.05). The correlation of leptin levels with BMI was improved when the results were analyzed stratified by the sex of the patients (r = 0.74 for female patients; r = 0.81 for male patients). CONCLUSION: This study showed that the serum leptin levels in HIV-infected patients with BMI between 18 and 25 kg/m 2 were comparable to leptin levels in lean, healthy subjects. Leptin distribution was positively correlated with BMI, as expected. These data do not support the hypothesis for a major role of serum leptin in HIV-related wasting.

Ganciclovir-loaded albumin nanoparticles: characterization and in vitro release properties.

Ganciclovir is one of the most widely used antiviral drug for the treatment of cytomegalovirus retinitis. Due to its short half-life in the vitreous, frequent administrations are necessary to maintain the therapeutic levels. In this context, the aim of this study was to characterise and in vitro evaluate the drug release properties of three different formulations of ganciclovir-loaded albumin nanoparticles. These carriers were prepared by a coacervation method and chemical cross-linking with glutaraldehyde. Depending on the step where the drug and/or cross-linking agent were added three different formulations were obtained, named models A, B and C. For model A nanoparticles, ganciclovir was incubated with the just-formed albumin nanoparticles. For the other two types of nanoparticulate formulations, the drug was added to a solution of albumin (model B) and glutaraldehyde (model C) prior to the formation of the carriers by coacervation. In all cases, the size of the different nanoparticulate formulations was comprised between 200 and 400 nm and the yield ranged from 50%, in model A, to 65% in model B. Concerning the ganciclovir loading, model B nanoparticles offered the higher capacity to carry this antiviral drug (around 30 microg ganciclovir/mg nanoparticle). On the contrary, the drug loading calculated for model A nanoparticles was only 14.6 microg/mg. The in vitro release profiles of the nanoparticles showed a biphasic pattern, with an initial and rapid release, followed by a slower step for up 5 days. This burst effect was especially relevant in model A (around 60% in 1 h), followed by model B (40%) and less important in model C (20%). The addition of trypsin to the release medium did not have a significant influence on the release characteristics. However, the release of the drug was increased in acidic or basic mediums, due to the disruption of the covalent binding between ganciclovir and the protein matrix via glutaraldehyde. This strong linkage was also confirmed by TLC experiences. In summary, a first step of incubation between the drug and the protein, prior to the preparation of nanoparticles, enabled us to obtain albumin carriers able to release ganciclovir in a sustained way.

Bioadhesive potential of gliadin nanoparticulate systems.

The objective of this work was to prepare, characterise and evaluate the adhesive potential of gliadin nanoparticulate carriers. Firstly, lectin-nanoparticle conjugates were obtained by the carbodiimide (CDI) covalent binding of Dolichos biflorus lectin (DBA) to the surface of gliadin nanoparticles (NP) containing carbazole (as a model lipophilic drug). The DBA binding efficiency was favoured in mild acidic conditions. Similarly, a CDI concentration of about 0.63 mg/mg nanoparticles, acting during at least 1 h, provided binding efficiencies of about 50% bulk lectin. Under optimised experimental conditions, the DBA conjugates showed a size of around 500 nm and the amount of loaded carbazole and the DBA content were calculated to be around 15 and 23.5 microg/mg, respectively. The bioadhesive activity of NP and DBA conjugates was determined in samples of small and large rat intestinal mucosa. The amount of adsorbed NP was calculated to be around 8 and 4 g/m(2) in the small and large intestine, respectively. This high capacity to interact with the mucosa may be explained by gliadin composition. In fact, gliadin is rich in neutral and lipophilic residues. Neutral amino acids can promote hydrogen bonding interactions with the mucosa, while the lipophilic components can interact with the biological tissue by hydrophobic interactions. The bioadhesive activity of DBA conjugates was calculated to be about 2 g/m(2) in the small intestine and greater than 4 g/m(2) in the caecum and distal colon. These degrees of interaction were always significantly higher than those obtained with controls. Finally, DBA did not provide the specificity for interaction with Peyer's patches. In summary, gliadin nanoparticles show a high capacity of non-specific interaction with the intestine, whereas DBA binding to the surface of these carriers provided a greater specificity for colonic mucosa.

Optimization of topical cidofovir penetration using microparticles.

Cidofovir is a new class of antiviral agent with potent in vitro and in vivo activity against a broad spectrum of herpes viruses. The aim of this work was to obtain a prolonged therapeutic effect of cidofovir in the basal epidermis after its topical application. For this purpose, poly(lactide-co-glycolide) (PLGA) microparticles were prepared by solvent evaporation and spray-drying methods. Microparticles prepared by spray-drying showed a encapsulation efficiency of 80%. Conversely, for all the microspheres prepared by the W/O/W solvent evaporation method the encapsulation efficiency was low. Also, microparticles prepared by spray-drying showed a higher burst release. Skin penetration and distribution experiments were carried out with cidofovir-loaded microparticles prepared by spray-drying, since these carriers presented the best characteristics in terms of size and encapsulation efficiency. A cidofovir solution in 0.2% PVA served for comparison. Penetration experiments were carried out in Franz type diffusion cells with an available diffusion area of 1.76 cm(2), using porcine skin. The results obtained showed that the amount of cidofovir penetrated, over a 24 h time period, was higher with the drug solution than with microparticles. Cidofovir distribution in porcine skin, after topical application of microparticles and drug solution for 24 h, was determined by horizontal slicing of the skin. The profiles obtained for the two formulations showed that the quantity of cidofovir retained in the skin decreased with the depth. Besides the amount of cidofovir found in the basal epidermis (120-150 microm) was much higher with microparticles than with the control solution. These data showed that cidofovir-loaded microparticles could improve cidofovir topical therapy since these vehicles increased drug retention in the basal epidermis and decreased its penetration through the skin.

In vitro evaluation of gentamicin released from microparticles.

In this study, the preparation, characterization and drug release behaviour of gentamicin (GM)-loaded poly(D,L-lactide-co-glycolide) microspheres are described. The microspheres were produced using a double emulsion solvent evaporation technique. All the microspheres preparation resulted in spherical shape and the mean diameter was 3 microm (for empty microspheres) and between 5 and 9 microm for microparticles loaded with GM. The encapsulation efficiency (EE) ranged from 3.4 to 90% depending on the formulation. Increasing the volume of the external aqueous phase, increased the EE. Encapsulation also depended on the pH value of the internal aqueous phase, the highest value was achieved when maintained the internal aqueous phase at pH 6, where GM was more soluble. Moreover, increasing nominal GM loading yielded lower encapsulation efficiencies. The release profiles of GM from microparticles resulted in biphasic patterns. After an initial burst, a continuous drug release was observed for up to 4 weeks. Finally, the formulations with higher loading released the drug faster.

Probability tree algorithm for general diffusion processes.

Motivated by path-integral numerical solutions of diffusion processes, PATHINT, we present a tree algorithm, PATHTREE, which permits extremely fast accurate computation of probability distributions of a large class of general nonlinear diffusion processes.

[A clinical, cytogenetic and molecular study of 10 patients with the Prader-Willi syndrome]. / Estudio clínico, citogenético y molecular en 10 pacientes con síndrome de Prader-Willi.

BACKGROUND: The Prader-Willi syndrome (PWS) is a neurogenetic disorder associated with abnormalities in the chromosomal region 15q11-13 of paternal origin. Most cases (65-85%) have a deletion involving the paternally derived chromosome and the remainder (20-25%) have a maternal uniparental disomy. Some patients have a defect in the imprinting process. We report the results of molecular, cytogenetic and clinical studies on 10 PWS patients. PATIENTS AND METHODS: 18 suspected patients were classified as PWS typical or not typical as they fulfilled or not the clinical criteria for PWS. Cytogenic studies-high resolution chromosome banding analyses (HRGTG) and fluorescence in situ hybridization (FISH) -and molecular chromosome genetic analyses--microsatellite markers and Southern blotting--were carried out from peripheral blood lymphocytes. RESULTS: PWS was confirmed in 10 probands. 8 fulfilled the clinical criteria for PWS and showed cytogenetic and/or molecular abnormalities. In 2 patients without clinical or cytogenetic data, diagnosis was confirmed by molecular methods only. Cytogenetic and molecular findings describe a characteristic clinical picture of PWS. CONCLUSIONS: Cytogenetic techniques (FISH and HRGTG) confirmed PWS diagnosis in 40% of cases, microsatellite studies in 70% of them and Southern blotting (the metilation test) in 100% cases. Southern blotting is the method of choice for rapid diagnostic testing of patients suspected of having PWS.

Pharmacokinetic profiles of prazepam and 14C-prazepam in rat.

Pharmacokinetics of Prazepam and 14C-Prazepam were studied in rat. Prazepam was measured in blood and plasma by a gas-liquid chromatography assay with an electron capture detector. Its major metabolite, Desmethyldiazepam, was also determined in blood in the same way. Total radioactivity was measured in plasma by scintillation spectrometry. Pharmacokinetic analysis were carried out by two ways; according to compartmental pharmacokinetic models and by statistic moments.

[Controlled liberation of active principles by means of new galenic formulations]. / Liberación controlada de principios activos mediante el empleo de formulaciones galénicas.

Drugs inside a conventional galenic form are distributed between specific biological targets and other anatomical tissues. With the aim to obtain a more rational and a better therapeutic, one of the most promising possibilities by using the concept of vectorization: association of an active principle to an appropriate vector with the object to increase its action efficiency and efficacy. By this means, they do not just increase the affinity of the drug to the target but also active principle gets protected from a potentially hostile environment (hydrolytic enzymes, acid pH, etc.). The success in the extension of the applications of the vectorización depends more and more of an appropriate design, for what the fundamental objective of this revision will be the one of presenting the general characteristics and some of the current applications in these new galenic forms.

Development and validation of a HPLC method for the determination of cyclosporine a in new bioadhesive nanoparticles for oral administration.

A simple and reliable high performance liquid chromatography method was developed and validated for the rapid determination of cyclosporine A in new pharmaceutical dosage forms based on the use of poly (methylvinylether-co-maleic anhydride) nanoparticles. The chromatographic separation was achieved using Ultrabase C18 column (250×4.6 mm, 5 µm), which was kept at 75°. The gradient mobile phase consisted of acetonitrile and water with a flow rate of 1 ml/min. The effluent was monitored at 205 nm using diode array detector. The method exhibited linearity over the assayed concentration range (22-250 µg/ml) and demonstrated good intraday and interday precision and accuracy (relative standard deviations were less than 6.5% and the deviation from theoretical values is below 5.5%). The detection limit was 1.36 µg/ml. This method was also applied for quantitative analysis of cyclosporine A released from poly (methylvinylether-co-maleic anhydride) nanoparticles.