Chondrocyte differentiation for auricular cartilage reconstruction using a chitosan based hydrogel.

Tissue engineering with the use of biodegradable and biocompatible scaffolds is an interesting option for ear repair. Chitosan-Polyvinyl alcohol-Epichlorohydrine hydrogel (CS-PVA-ECH) is biocompatible and displays appropriate mechanical properties to be used as a scaffold. The present work, studies the potential of CS-PVA-ECH scaffolds seeded with chondrocytes to develop elastic cartilage engineered-neotissues. Chondrocytes isolated from rabbit and swine elastic cartilage were independently cultured onto CS-PVA-ECH scaffolds for 20 days to form the appropriate constructs. Then, in vitro cell viability and morphology were evaluated by calcein AM and EthD-1 assays and Scanning Electron Microscopy (SEM) respectively, and the constructs were implanted in nu/nu mice for four months, in order to evaluate the neotissue formation. Histological analysis of the formed neotissues was performed by Safranin O, Toluidine blue (GAG's), Verhoeff-Van Gieson (elastic fibers), Masson's trichrome (collagen) and Von Kossa (Calcium salts) stains and SEM. Results indicate appropriate cell viability, seeded with rabbit or swine chondrocyte constructs; nevertheless, upon implantation the constructs developed neotissues with different characteristics depending on the animal species from which the seeded chondrocytes came from. Neotissues developed from swine chondrocytes were similar to auricular cartilage, while neotissues from rabbit chondrocytes were similar to hyaline cartilage and eventually they differentiate to bone. This result suggests that neotissue characteristics may be influenced by the animal species source of the chondrocytes isolated.

Mechanical and structural response of a hybrid hydrogel based on chitosan and poly(vinyl alcohol) cross-linked with epichlorohydrin for potential use in tissue engineering.

The development and characterization of a hybrid hydrogel based on chitosan (CS) and poly(vinyl alcohol) (PVA) chemically cross-linked with epichlorohydrin (ECH) is presented. The mechanical response of these hydrogels was evaluated by uniaxial tensile tests; in addition, their structural properties such as average molecular weight between cross-link points (Mcrl), mesh size (DN), and volume fraction (v(s)) were determined. This was done using the equivalent polymer network theory in combination with the obtained results from tensile and swelling tests. The films showed Young's modulus values of 11 ± 2 MPa and 9 ± 1 MPa for none irradiated and ultraviolet (UV) irradiated hydrogels, respectively. The cell viability was assessed using Calcein AM and Ethidium homodimer-1 assay and environmental scanning electron microscopy. The 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan thiazolyl blue formazan (MTT Formazan assay) results did not show cytotoxic effects; this was in good agreement with nuclear magnetic resonance and fourier transform infrared spectroscopies; their results did not show traces of ECH. This indicated that after the crosslinking process, there was no free ECH; furthermore, any possibility of ECH release in the construct during cell culture was discarded. The CS-PVA-ECH hybrid hydrogel allowed cell growth and extracellular matrix formation and showed adequate mechanical, structural, and biological properties for potential use in tissue engineering applications.

A mouse model to study the alterations in haemostatic and inflammatory parameters induced by Lonomia achelous caterpillar haemolymph.

A mouse model was established to reproduce the haemorrhagic syndrome which occurs in humans after accidental contact with the hairs of the caterpillar Lonomia achelous (LA) and measures the haemostatic and inflammatory alterations that occur as a result of this contact. Mice were injected intradermally with different doses (0.4, 0.8 and 1.6 mg/animal) of L. achelous haemolymph (LAH). Haematological (haemoglobin, haematocrit, platelet count, differential leukocyte count), haemostatic (fibrinogen, plasminogen, factor XIII [FXIII], fibrinolytic activity) and inflammatory parameters (tumour necrosis factor alpha [TNF-α], nitric oxide [NO]) were measured at different times up to 48 h. C57BL/6 mice responded to LAH injection, in terms of these parameters, in a manner similar to that seen in humans, whereas the BALB/c mice were unresponsive. In C57BL/6 mice injected with LAH, time course measurements showed: a) a reduction in the haemoglobin, haematocrit, fibrinogen, FXIII and plasminogen levels, b) no effect on the platelet count and c) immediate leukocytosis and an increase in the fibrinolytic activity in plasma. An inflammatory response (TNF-α) was observed within 1 h post-injection, followed by a more persistent increase in serum NO. These findings suggest that C57BL/6 mice represent a useful model of the haemorrhagic syndrome observed in humans who have suffered contact with the caterpillar, permitting a deeper understanding of the role of the inflammatory response in the haematological and haemostatic manifestations of this syndrome.

Resistencia a los antimicrobianos de staphylococcus aureus aislados de lesiones de piel y tejido blandos / Resistence at the antimicrobial of staphylococcus aureus isolator of lesions of skin and tissure soft

Entre 1994 y 1998 se identificaron 135 cepas de S. aureus en lesiones de piel, de pacientes del Servicio de Dermatología del Instituto de Biomedicina. Se determinó el patrón de resistencia antimicrobiana siguiendo las normas de la NCCLS, encontrándose los siguientes resultados: penicilina, 94 por ciento de resistencia; oxacilina, 22 por ciento; clindamicina, 19 por ciento; eritromicina, 30 por ciento; ciprofloxacina, 8 por ciento; trimetoprim-sulfametoxazol, 16 por ciento; rifampicina, 39 por ciento; vancomicina, 0 por ciento; amoxacilina-ácido clavulánico, 29 por ciento; y gentamicina, 10 por ciento. Sólo amoxacilina significativa entre ambulatorios y hospitalizados, 20 vs 57 por ciento, respectivamente (P>0,05). La resistencia a trimetoprim-sulfametoxazol fue mayor en los pacientes con úlceras, al compararlas con furunculosis-foliculitis (P<0,05). Las cepas estudiadas muestran alta resistencia a la mayoría de los antibióticos probados, tanto en pacientes ambulatorios como hospitalizados. Es importante conocer la susceptibilidad de S. aureus a los antimicrobianos, para garantizar el éxito del tratamiento y evitar el uso indiscriminado de los mismos, a fin de minimizar el problema de resistencia bacteriana

Genetic variation among four Mexican populations (Huichol, Purepecha, Tarahumara, and Mestizo) revealed by two VNTRs and four STRs.

Allele distributions of two polymorphisms with variable number of tandem repeats (VNTR), D1S80 and APOB, and four polymorphisms with short tandem repeats (STR), VWA, TH01, CSF1PO, and HPRTB, were analyzed in three Mexican ethnic groups: Huichol, Purepecha, and Tarahumara. Genotype distribution was in agreement with Hardy-Weinberg expectations for each locus and ethnic group. Heterozygosity (H), power of discrimination, and probability of exclusion were estimated. The three groups presented some distinctive genetic features: (1) a diminished genetic diversity (H = 66.8% to 73.4%) and mean number of alleles by locus (5.8 to 6.3) in comparison with Mexican mestizos (H = 78.3%, 10.5 alleles/locus), and (2) uneven allele distributions as evidenced by "distinctive alleles" with high frequencies, especially in the Tarahumara and the Huichol. Genetic relatedness analysis included data from a previously typed mestizo population, the largest and most widely distributed population in Mexico. Allele distribution differentiation was observed among all four groups, except between mestizo and Purepecha (p > 0.05), which was interpreted as indicating a larger Spanish component in the Purepecha as a result of gene flow effects. Although intrapopulation inbreeding (FIS) was not significant, heterozygote deficiency in the total population (FIT) and divergence among populations (FST) were significant (p < 0.05). Genetic distances displayed a closer relationship among mestizos, Purepechas, and Huichols in relation to Tarahumaras. Correlation between the observed genetic features and the geographic isolation level points to genetic drift as the main cause of differentiation among these Mexican populations.

Invertebrate compounds acting on the hemostatic mechanism.

Physiological secretions from some invertebrates have toxic effects on mammalian blood coagulation and fibrinolytic systems. Some of these effects occur because the substances contained in the secretions resemble the components of the hemostatic system. Some of the substances have been characterized, and have been found to have similar molecular weights or sequences, which may indicate a common ancestry. The components can be divided into five groups: antithrombic agents (group I); inhibitors and activators of the prothrombinase complex (group II); substances that affect platelet function (group III); substances that affect the fibrinolytic mechanism (group IV); and a group of miscellaneous agents whose activities are difficult to group together (group V). In group I special mention of the antithrombin agents in Hirudo medicinalis should be made. In group II, the agents affecting the prothrombinase complex are antistasin from Haementeria officinalis, ghilanten from Haementeria Ghiliani and the tick anticoagulant protein from Ornithodoros moubata, a factor V activator/inhibitor from Lonomia achelous and factor II and factor X activators from L. achelous and Lonomia obliqua. Examples of factors which affect platelet function (group III) are glossina from the black fly Glossina morsitans, calin from H. medicinalis, decorsin (a desintegrin) from Macrobdella decorsa, and FAGA from Stichopus japonicus selenka. The first three of these are inhibitors of platelet aggregation, and the last is an inducer. The plasminogen activators (group IV) from the L. achelous caterpillar and Eutriatoma maculata trigger the fibrinolytic system, whereas hementin from H. officinalis and hementerin from Haementeria depressa are directly fibrinolytic. The last group of substances (group V) include those with factor-XIIa-like activity from D. farinae, kallikrein-like activity and a factor XIII degrading enzyme from L. achelous, destabilase from H. medicinalis and prolixin S (nitroforin 2, or anti-factor-IXa) from Rhodnius prolixus. Some of these components have been well characterized, cloned and prepared in recombinant form, and seem to be very promising from the therapeutic point of view.

[Evaluation of methods for the diagnosis of Helicobacter pylori infection]. / Evaluacion de los metodos para diagnostico de infeccion por Helicobacter pylori.

UNLABELLED: Helicobacter pylori is a gram negative curved bacillus recognized as etiologic agent of chronic gastritis and an important factor in the development of duodenal ulcer and gastric cancer. The purpose of this work is to evaluate three microbiologic methods for diagnosing H. pylori infection. We studied 375 samples from 218 patients, who consulted at the Department of Gastroenterology of the Hospital Vargas and were candidates for endoscopy. Samples of gastric mucous tissue were taken at level of the antrum and each sample was stained with Gram, tested for urease and cultured. RESULTS: whit H. pylori positive 123 patients with duodenal ulcer at the endoscopy (91.1%), 17 had gastric ulcer (76.5%), 40 gastritis and/or duodenitis (60.0%), 1 duodenal ulcer and gastric (100.0%) and 37 normal endoscopies (56.8%). Evaluating the three methods used we found that of the 286 H. pylori positive samples, the Gram stain detected 282 (sensibility 98.6% and specificity 96.6%); the urease test 276 (sensibility 96.5% and specificity 98.6%) and culture was positive in 255 samples (sensibility 89.2% and specificity 100.0%). These results show that both the Gram stain and Urease test are useful for diagnosing H. pylori infection due to their high sensitivity and specificity, rapidness, low cost for the Gram stain, and easy interpretation for Urease test. Culture, even though less sensitivity++, represents the most unquestionable diagnosis and permits carrying out susceptibility to antimicrobials tests.

Six new cases of a caterpillar-induced bleeding syndrome.

We describe six new cases of a hemorrhagic diathesis induced by contact with Lonomia achelous caterpillars. Onset of clinical bleeding varied between a few hours and 10 days post-exposure. Laboratory coagulation tests showed prolonged PT, PTT and ThT; normal platelets and a marked decrease of fibrinogen, factor V, plasminogen and factor XIII (including its subunits A and S). Factors VII, II and alfa 2 anti-plasmin were variably affected. In addition, activation of the fibrinolytic system and the generation of a procoagulant effect could also be demonstrated. Two cases developed severe hemorrhagic diathesis and one of them died of a cerebral hemorrhage. Different aspects of this rare syndrome are discussed in relation to its complex physiopathology and the variability observed in all clinical and laboratory manifestations. Therapeutic recommendations and some possible hazards following replacement transfusions are also considered.

An A alpha Ser-434 to N-glycosylated Asn substitution in a dysfibrinogen, fibrinogen Caracas II, characterized by impaired fibrin gel formation.

We have identified a unique N-glycosylated Asn substitution for a Ser at position 434 of the A alpha chain of an abnormal fibrinogen designated fibrinogen Caracas II. This dysfibrinogen was characterized by impaired fibrin monomer aggregation. Since there were 4 Thr residues immediately following the mutation, a new Asn-X-Thr/Ser-type consensus sequence, Asn-Thr-Thr arose for N-glycosylation of the Asn. The extra oligosaccharide was found to consist mainly of a disialylated biantennary structure comprising 81.9%, while a neutral and a monosialylated biantennary oligosaccharide represented only 3.6% and 14.5%, respectively. The mutation resides in the carboxyl-terminal region of the A alpha chain, which could fold back to form an extra small globular region located near the central region of the molecule (Erickson, H.P., and Fowler, W.E. (1983) Ann. N. Y. Acad. Sci. 408, 146-163; Weisel, H.P., Stauffacher, C.V., Bullitt, E., and Cohen, C. (1985) Science 230, 3124-3133). Therefore, the participation of this region, referred to as an additional central domain or an alpha domain, in fibrin gel formation is strongly implicated.

Unreliability in the estimation of the molecular weight of fibrinogen degradation products (FDPs) by polyacrylamide/sodium dodecyl sulphate electrophoresis (SDS/PAGE).

Evidence is presented indicating that using different SDS/PAGE techniques the molecular weight estimation and the appearance of fibrinogen degradation products on the gel may vary according to the type used. It is suggested that changes in the structure and shape of the fragments, due to asymmetry in the degradation, may explain the differences.

Studies on the degradation of fibrinogen by proteolytic enzymes from the larvae of Lanomia achelous (Cramer).

Fibrinogen degradation products formed by the action of purified haemolymph and saliva of a Saturnidae caterpillar of the Lonomia genus were studies by immunoelectrophoresis and polyacrylamide/SDS gel electrophoresis. The pattern of degradation differ form the one described for plasmin, trypsin, brinase, and ochrase. The most striking difference being the rapid loss of the alpha chain in spite of the presence of the protease inhibitor aprotinin, and/or denaturalizing agents such as 8 M Urea and 2% SDS.