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Biomedical subjects

P Sepulveda

Publications and source records attributed to P Sepulveda.

At least 19 recordsLinked to original sources
PubMed2002

In vivo evaluation of hydroxyapatite foams.

Hydroxyapatite (HA) is widely applied as bone graft material due to its osteoconductive potential and well-established biocompatibility. In this work, macroporous hydroxyapatite structures made through foaming of aqueous suspensions and gelcasting were tested for in vivo osteointegration. These foams are composed of a three-dimensional array of spherical pores with diameters of approximately 100-500 microm, interconnected by windows of smaller size in the range of 30-120 microm. The HA foams were implanted in the tibia of albino New Zealand rabbits and removed after a period of 8 weeks. Histological analysis revealed that the pores in the foams were partially or completely filled progressively with mature new bone tissue and osteoid after the implanted period. No immune or inflammatory reactions were detected. The high osteoconductive potential of the HA foams provides a potential structure for use as bone substitute in orthopedic, oral, and cranio-maxillofacial reconstructive surgery, and as dento-alveolar implants.

Animals↗
PubMed2002

In vitro dissolution of melt-derived 45S5 and sol-gel derived 58S bioactive glasses.

Effects of powder type, particle size (5-20 microm; 90-300 microm; 90-710 microm), and type of dissolution medium on the dissolution behavior of bioactive glasses were investigated in vitro using melt-derived 45S5 and sol-gel derived 58S bioactive glass powders. Dissolution studies were performed in simulated body fluid and in alpha-MEM based cell culture medium at 37 degrees C under dynamic conditions (1 Hz) for periods of 30 min, 1, 2, 4, 8, 17, and 22 h. The concentrations of elements dissolved from the glasses were evaluated using inductively coupled plasma analysis. The reacted powders were analyzed for bioactivity using Fourier transform infrared spectrometry to observe the formation of a calcium phosphate layer on the surface. The non-porous surfaces of melt-derived 45S5 glass powders exhibited lower dissolution rates and rate of surface layer formation than 58S gel-glass powders. The rates of dissolution for both types of powders were lower in culture medium, compared to simulated body fluid, and increased as the particle size decreased. Thus, particle size range, glass type, and powder volume fraction can be used as a means to control the release rate of active ions that stimulate the gene expression and cellular response for tissue proliferation and repair.

Biocompatible Materials↗
PubMed2001

Potential use of gelcasting hydroxyapatite porous ceramic as an implantable drug delivery system.

Hydroxyapatite (HA) ceramic in a porous configuration is suggested as a drug release system. A new technique for the production of this material, based on the foaming of suspensions and in situ polymerization (gelcasting method), resulted in a material whose characteristics are likely to make it useful as an implantable drug delivery system. Three batches of HA ceramic with different porosities were characterized by X-ray diffraction and scanning electron microscopy (SEM). Pore size and shape as well as density were determined. In vitro experiments were performed in order to evaluate the dissolution behavior of cisplatin in the system. X-ray diffraction analysis showed that the final product consisted of a single phase, indicating that the sintering process had not affected the structure of the HA. Energy dispersive X-ray analysis (EDX) showed absence of impurities. Pore diameters were in the range 15--34 microm. SEM showed that the material presented a highly interconnected spheroidal porous network with open micropores and closed macropores. In vitro experiments showed significant differences in the release rate of cisplatin between three different porosities.

Antineoplastic Agents↗
PubMed2001

A monoclonal antibody against the immunodominant epitope of the ribosomal P2beta protein of Trypanosoma cruzi interacts with the human beta 1-adrenergic receptor.

Monoclonal antibodies were raised against a recombinant ribosomal P2beta protein of Trypanosoma cruzi. One of these reacted with the C terminus of this protein (peptide R13, EEEDDDMGFGLFD) and epitope mapping confirmed that this epitope was the same as the one defined by the serum of immunized mice, and similar to the previously described chronic Chagas' heart disease (cChHD) anti-P epitope. Western blotting showed that the monoclonal antibody recognized the parasite ribosomal P proteins, as well as the human ribosomal P proteins. Electron microscopy showed that it stained different structures in parasite and human cells. Interestingly, surface plasmon resonance measurements indicated that the affinity for the parasite ribosomal P protein epitope (R13) was five times higher than for its human counterpart (peptide H13, EESDDDMGFGLFD). Since the human epitope contained an acidic region (EESDD) similar to the AESDE peptide recognized by cChHD patients in the second extra-cellular loop of the human beta1-adrenergic receptor, the biological activity of the antibody was assessed on neonatal rat cardiomyocytes in culture. The monoclonal antibody had an agonist-like effect. These results, together with the fact that the monoclonal reacted in Western blots with the different isoforms of the heart beta1-adrenergic receptor, confirm the possible pathogenic role of antibodies against the parasite ribosomal P protein based on their cross-reaction with the human beta1-adrenergic receptor.

Animals↗
PubMed2001

Characterization of melt-derived 45S5 and sol-gel-derived 58S bioactive glasses.

The ability of bioactive glasses to form a bond to living bone and also to stimulate bone-cell proliferation depends on the chemical composition and on the surface texture of the glasses. In this work, the differences in physical properties between the melt-derived 45S5 and sol-gel-derived 58S Bioglass powders of various particle-size ranges were studied. The powders were characterized for particle-size distribution by laser spectrometry, for specific surface area and porosity by nitrogen sorption analysis, and for morphological features by scanning electron microscopy. Melt-derived 45S5 powders exhibited a low-porosity texture with surface area in the range 0.15-2.7 m(2)/g. In contrast, the sol-gel-derived powders exhibited a highly mesoporous texture, with surface area in the range of 126.5-164.7 m(2)/g and a large fraction of 6-9 nm pore sizes. These differences in texture, as well as variations in chemical composition, account for significant changes in the resorption and in vivo responses.

Adsorption↗
PubMed2001

Dose-dependent behavior of bioactive glass dissolution.

The effect of glass dosage (0.001 g ml(-1) to 0.015 g ml(-1)) on the in vitro dynamic dissolution behavior of melt-derived 45S5 and sol-gel-derived 58S bioactive glasses, in simulated body fluid (SBF) at 37 degrees C, was evaluated. These glasses differ significantly in texture, especially the specific surface area and porosity, as a result of differences in manufacturing route. The concentrations of elements (Si, Ca, P, and Na) leached from the glasses into the dissolution medium, from 1 to 22 h, were evaluated with the use of induced coupled plasma analysis (ICP). The reacted powders were analyzed with the use of FTIR to observe the formation of a hydroxycarbonate apatite layer on the surface. The results show that the rate of HCA formation on both gel- and melt-derived bioactive glass powders in vitro depends on the concentration of the powders in solution. This result must be taken into account when carrying out in vitro cell-culture studies to simulate conditions in vivo and in experiments using extracts of the bioactive glass powders.

Apatites↗
PubMed2000

Production of porous hydroxyapatite by the gel-casting of foams and cytotoxic evaluation.

This study presents the manufacture of highly porous hydroxyapatite by a novel technique that employs the foaming of suspensions prior to the in situ polymerization of organic monomers contained in the compositions. This method produces strong gelled bodies with up to 90% porosity that can withstand machining in the green state. Complex-shaped components can be obtained if the process comprises casting in one of the processing steps. The organic additives are eliminated at temperatures above 300 degrees C, and sintering is carried out for consolidation of the ceramic matrix. Spherical interconnected cells with sizes ranging from 20 to 1000 micrometer characterize the porous structure, depending on the specimen density. Cytotoxicity tests were conducted on extracts from sintered HA foams based on a quantitative method of cell colony formation and the determination of cell death after indirect contact of the porous material with mammalian cells. This in vitro test of biological evaluation revealed that the original purity of the biomedical-grade hydroxyapatite powder was affected neither through processing nor by the employed reagents.

Animals↗
PubMed2000

DNA-Based immunization with Trypanosoma cruzi complement regulatory protein elicits complement lytic antibodies and confers protection against Trypanosoma cruzi infection.

A complement regulatory protein (CRP) of Trypanosoma cruzi was evaluated as a vaccine candidate in a murine model of experimental T. cruzi infection. Recombinant CRP derived from an Escherichia coli expression system and a plasmid encoding the full-length crp structural gene under the control of a eukaryotic promoter were used to immunize BALB/c mice. Immunization with both protein and DNA vaccines resulted in a Th1-type T-cell response, comparable antibody titers, and similar immunoglobulin G isotype profiles. Only mice immunized with the crp DNA plasmid produced antibodies capable of lysing the parasites in the presence of complement and were protected against a lethal challenge with T. cruzi trypomastigotes. These results demonstrate the superiority of DNA immunization over protein immunization with the recombinant CRP. The work also supports the further investigation of CRP as a component of a multigene, anti-T. cruzi DNA vaccine.

Animals↗
PubMed2000

Modulation of cardiocyte functional activity by antibodies against trypanosoma cruzi ribosomal P2 protein C terminus.

Antibodies against the Trypanosoma cruzi ribosomal P2beta protein (TcP2beta) have been associated with the chronic cardiac pathology of Chagas' disease in humans. Using synthetic peptides spanning the entire TcP2beta molecule, we investigated their epitope recognition by antibodies from mice chronically infected with T. cruzi and from mice immunized with two recombinant TcP2betas. We found clear differences in epitope recognition between antibodies from T. cruzi-infected mice and mice immunized with two different recombinant TcP2betas associated with different schedules of immunization. Major epitopes recognized by antibodies from mice immunized with recombinant glutathione S-transferase (GST) or histidine (Hist) fusion TcP2beta (GST-TcP2beta or Hist-TcP2beta) are located in the central and hinge regions of the molecule. Nevertheless, mice immunized with Hist-TcP2beta were also able to elicit antibodies against the TcP2beta C terminus, a region which is highly conserved in both T. cruzi and mammal ribosomal P proteins. Strikingly, antibodies from infected animals recognized only the TcP2beta C terminus. By using these antisera with distinct profiles of epitope recognition, it could be shown that only C terminus-specific antibodies were able to increase the beating frequency of cardiomyocytes from neonatal rats in vitro by selective stimulation of the beta1-adrenergic receptor. Thus, antibodies against the TcP2beta C terminus elicited in the absence of infection are able to modulate a functional activity of host cells through a molecular mimicry mechanism.

Amino Acid Sequence↗
PubMed1996

Ubiquitin-like epitopes associated with Candida albicans cell surface receptors.

We have recently reported the cloning of a Candida albicans polyubiquitin gene and the presence of ubiquitin in the cell wall of this fungus. The polyubiquitin cDNA clone was isolated because of its reactivity with antibodies generated against the candidal 37-kDa laminin-binding protein. In the present study, we have further investigated the relationship between ubiquitin and cell wall components displaying receptor-like activities, including the 37-kDa laminin receptor, the 58-kDa fibrinogen-binding mannoprotein, and the candidal C3d receptor. Two-dimensional electrophoretic analysis and immunoblot experiments with antibodies against ubiquitin and the individually purified receptor-like molecules confirmed that these cell surface components are ubiquitinated. In an enzyme-linked immunosorbent assay, polyclonal antisera to each receptor reacted with ubiquitin, thus demonstrating that the purified receptor preparations used as immunogens contained ubiquitin-like epitopes. It is proposed that ubiquitin may play a role in modulating the activity of these receptors and in the interaction of C. albicans cells with host structures.

Animals↗
PubMed1995

Efficiency of three different hemodialysis membranes for plasma porphyrin removal.

To assess the capability of three different membranes to remove porphyrins, plasma and dialysate porphyrin levels were fluorometrically measured in 10 patients with end-stage renal failure who were on hemodialysis. Three different hemodialysis membranes were used: cuprophan, polyacrylonitrile, and cellulose triacetate. Total plasma porphyrin concentrations decreased after dialysis, but to a lesser extent when using the cuprophan membrane (19%) than with the polyacrylonitrile (26%) or cellulose triacetate (30%) membranes (P < 0.01). However, since the free plasma porphyrin fraction remained unchanged, it can be assumed that the equilibrium between protein-bound and non-protein-bound (free) porphyrins is displaced toward the latter fraction. Dialysate porphyrin levels were lower (P < 0.01) when using the cuprophan membrane (10.1 micrograms/session) than when using polyacrylonitrile (17.8 micrograms/session) and cellulose triacetate (21.9 micrograms/session). Although most of the plasma porphyrins are protein bound, our results show that hemodialysis can remove significant amounts of non-protein-bound (free) porphyrins. The polyacrylonitrile and cellulose triacetate membranes had a greater capacity for porphyrin removal than cuprophan. Thus, two high-permeability membranes (polyacrylonitrile and cellulose triacetate) should be used whenever a reduction of plasma porphyrin levels is desired.

Acrylic Resins↗
PubMed1995

Absence of circadian variations in urinary porphyrin excretion in normal subjects.

The hypothesis that urinary porphyrins could be excreted in a circadian rhythm was examined. A group of 20 healthy subjects, 10 males and 10 females, aged 20-40 years, were used in this study. Urine was collected at 4-hr intervals over three 24-hr periods. Median porphyrin concentrations (microgram l-1 or microgram g-1 of creatinine) were similar in the six groups, but significant differences were observed in that excreted. Highest porphyrin excretion was detected in the afternoon (12.00-16.00 hr) and, in the evening (16.00-20.00 hr). pH values were also increased at evening but did not correlate with porphyrin excretion. Despite these temporal variations, no cycles indicating circadian periodicity were observed. Therefore, a 24-hr collection of urine is not necessary for the initial evaluation of porphyrin metabolism.

Adult↗
PubMed1993

Salivary porphyrin concentration in porphyria cutanea tarda.

To assess the clinical utility of saliva samples, serum, urine and saliva porphyrin concentration were fluorometrically measured in 31 patients with porphyria cutanea tarda. In comparison with normal values, porphyrin mean levels were 20-fold increased in serum and urine but only 3-fold in saliva. Saliva porphyrin concentration exhibited significant but weak correlations with porphyrin levels in serum (r = 0.69) and urine (r = 0.67). However, saliva porphyrins were not more closely correlated with protein-unbound serum porphyrins (r = 0.57). This finding suggests that saliva porphyrin concentration does not simply correspond to the filtered free fraction of serum porphyrins. Nevertheless, measurement of saliva porphyrins could represent a valuable non-invasive alternative to serum porphyrins in the monitoring of patients with porphyria cutanea tarda.

Fluorometry↗
PubMed1993

Clinical utility of fluorometric scanning of plasma porphyrins for the diagnosis and typing of porphyrias.

The fluorometric emission scanning (using excitation at 405 nm) of plasma samples, simply diluted five-fold in phosphate-buffered saline, allows the differentiation of three conditions according to their porphyrin content. The emission maximum at 626-628 nm is a specific finding in variegate porphyria, while in erythropoietic protoporphyria a characteristic peak is found at 636 nm. A fluorescence emission maximum at 618-622 nm corresponds to a third group that includes normal subjects, non-porphyria patients and patients suffering from acute intermittent porphyria, hereditary coproporphyria, congenital erythropoietic porphyria (Günther disease) and porphyria cutanea tarda. Therefore, this simple, quick and cheap screening test allows one to establish whether a patient with a photocutaneous syndrome has porphyria and whether this porphyria belongs to the types: variegate, protoporphyria or other cutaneous porphyrias.

Fluorometry↗
PubMed1975

Primary structure of porcine pepsin. II. Amino acid sequence of two cyanogen bromide fragments, CB3 and CB4.

The amino acid sequences of two cyanogen bromide fragments from porcine pepsin have been determined. Fragment CB3 which represents the NH2-terminal 80 residues of pepsin was assembled from the peptides purified from proteolytic digests of this fragment using alpha-chymotrypsin, thermolysin, and staphylococcal protease. Two chymotryptic peptides were isolated from the NH2-terminal region of this fragment. One of these contains 2 extra residues, Ala-Leu-, at the NH2 terminus. This peptide is apparently derived from a different cleavage site of pepsinogen in its conversion to pepsin. The second cyanogen bromide fragment, CB4, contains 47 residues. The sequence was established from the peptides resulting from proteolytic digests using alpha-chymotrypsin, alpha-lytic protease, and thermolysin. An isoleucyl residue at position 29 of fragment CB4 appears to be absent in some molecules. This represents a structural variant of pepsin.

Amino Acid Sequence↗
PubMed1975

Primary structure of porcine pepsin. III. Amino acid sequence of a cyanogen bromide fragment, CB2A, and the complete structure of porcine pepsin.

The complete amino acid sequence of porcine pepsin (EC 3.4.4.1) was constructed from the sequence of five cyanogen bromide fragments. The sequence of one of these fragments, CB2A, is reported here. The sequences of 4 other fragments are known from previous work. Porcine pepsin contains 327 residues with three structural variants. The active center aspartyl residue, which reacts with 1,2-epoxy-3-(p-nitrophenoxy)propane (Chen, K. C. S., and Tang, J. (1972) J. Biol. Chem. 247, 2566-2574), is located at residue 32. Another active site aspartyl residue, which reacts with diazo inactivators (Bayliss, R. S., Knowles, J. B., and Wybrandt, G. B. (1969) Biochem. J. 113, 377-386, IS LOCATED AT RESIDUE 215. The sequences around these 2 aspartyl residues are apparently homologous to each other. The sequences around the tryptophanyl residues at positions 39, 141, 181, and 300 are also homologous to one another. These homologous sequences could be genetic in origin. Fragment CB2A which contains 119 residues was constructed from the peptide sequences resulting from six proteolytic digestions and chemical cleavage at tryptophanyl bonds.

Amino Acid Sequence↗