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Biomedical subjects

J Amaral

Publications and source records attributed to J Amaral.

At least 19 recordsLinked to original sources
PubMed2004

Surface studies on acrylic bone cement.

Poly(methyl methacrylate) (PMMA) is used to fill the gap between the prosthesis and the surrounding bone in cemented arthroplasties. Biocompatibility problems related to bone cement application limit the clinical success of these cemented arthroplasties. Being the cement surface in close connection with the living bone, it is reasonable to assume that surface properties such as, surface composition and surface energy, will play a role in the biomaterial performance. X-ray photoelectron spectroscopy (XPS) analysis and surface energy studies were carried out during 4 months, in order to assess a possible correlation between aging time and surface changes. The aging of PMMA, in a biological model fluid, strongly influences the composition and wettability of the cement surface. These changes may be explained through the hydrolysis of PMMA ester groups and the subsequent hydrogen bonding. Although our study does not exactly reproduce the in vivo environment surrounding a prosthesis, it suggests that the changes in the composition and wettability of the surface may modulate the host response towards the implant, thus contributing to its loosening.

Bone Cements↗
PubMed2002

The effect of ethanol on acrylic bone cement.

Prosthesis loosening is a major problem associated with the use of poly(methyl methacrylate) (PMMA) bone cement that may be related to a peri-implant vacuolisation commonly observed at bone-cement interface. Methyl methacrylate (MMA) monomer may be one of the cement components partly responsible for the mentioned vacuolisation due to a cytotoxic effect associated to this compound. Alcoholism has been related to bone necrosis in predisposed individuals. Furthermore, ethanol has been shown to clean material with adherent cement debris during cleaning procedure in laboratory. Consequently, we have decided to study whether ethanol will also be related to an increased liberation of MMA from the polymer matrix. 'In vitro' release studies using PMMA plates were conducted to access the role of ethanol on the liberation of the monomer. Contact angle measurements and surface tension estimation were also carried out in order to find a possible effect of ethanol on surface cement properties. Results suggest that ethanol, even in small quantities, enhances the leaching of the monomer from the polymer matrix, but does not considerably change the wettability properties of the cement surface.

Acrylic Resins↗
PubMed2001

The influence of vacuum mixing on methylmethacrylate liberation from acrylic cement powder.

Polymethylmethacrylate (PMMA) bone cement is a biomaterial used to anchor prostheses during joint replacement surgery. Residual methylmethacrylate monomer (MMA) may be related with the cytotoxic effect of PMMA. The aim of the present paper was to investigate the effect of two different cement mixing methods: hand stirring at atmospheric pressure and under partial vacuum (0.330 and 0.154 bar) on residual monomer liberation in phosphate buffer saline solution from acrylic cement powder. Residual MMA content was determined by high-performance liquid chromatography. Mathematical models were applied to experimental dissolution data revealing that monomer release was significantly reduced in bone cement powder obtained at 0.154 bar vacuum pressure compared to the other mixing conditions. The kinetic models applied are consistent with a simple diffusion mechanism of the monomer from the polymer matrix.

Acrylates↗
PubMed2001

Study on the scale-up of human IgG3 purification using protein A affinity chromatography.

The purification of human IgG3 subclass out of IgG (Immunoglobulin-G) was studied using protein A-Sepharose affinity chromatography. The effect of operational parameters such as flow rate, ionic strength, pH and size of sample was investigated, and the process was scaled-up 10-fold. The use of 0.5 m NaCl in the loading buffer had a dramatic effect in the purity of IgG3 recovered in the flowthrough fraction (values in the order of 97% were consistently obtained). This was attributed to a more effective binding of IgG subclasses 1, 2 and 4 to protein A (well known classical mechanism based in Fc fragment) and in some extent to a decrease in the binding of subclass 3 to protein A by the alternative mechanism based in the Fab fragment. The increase in residence time also increased in a relevant way the purity of IgG3. This is attributed to an increased effectiveness of the mechanisms mentioned above. The recovery yields in the IgG3 rich fraction were in the range 21-32% and are possibly a consequence of binding to protein A by the alternative mechanism and also due to deactivation during processing.

Chromatography, Affinity↗
PubMed2001

Importance of ICOS-B7RP-1 costimulation in acute and chronic allograft rejection.

Primary T cell activation requires B7-CD28 and CD40-CD154 costimulation, but effector T cell functions are considered to be largely independent of these costimulatory pathways. Although blockade of costimulation with cytolytic T lymphocyte-associated antigen 4-immunoglobulin (CTLA-4-Ig) or monoclonal antibody (mAb) to CD154 prolongs allograft survival, chronic rejection follows, which suggests that additional key costimulatory pathways are active in vivo. We found that both antibody to inducible costimulator (anti-ICOS) and an ICOS-Ig fusion protein suppressed intragraft T cell activation and cytokine expression and prolonged allograft survival in a manner similar to that in ICOS-/- allograft recipients. The combination of anti-ICOS therapy and cyclosporin A led to permanent engraftment. In addition, ICOS-B7RP-1 costimulation was required for the development of chronic rejection after CD40-CD154 blockade. These data demonstrate a key role for the ICOS-B7RP-1 pathway in acute and chronic rejection and highlight the benefits of targeting this pathway in combination with the use of conventional immunosuppressive agent.

Animals↗
PubMed2001

Gallbladder muscle dysfunction in patients with chronic acalculous disease.

BACKGROUND & AIMS: The mechanisms responsible for the abnormalities of gallbladder emptying in patients with chronic acalculous gallbladder disease (AGD) have not been elucidated. This study was designed to determine whether a muscle defect could explain this gallbladder dysfunction. METHODS: Gallbladder contraction induced by a continuous intravenous cholecystokinin octapeptide (CCK-8) infusion was determined by ultrasonography in control subjects, patients with AGD, pigment stones, and cholesterol stones. Muscle cells were obtained by enzymatic digestion. (125)I-CCK-8 binding and [(35)S]guanosine triphosphate gamma S (GTP gamma S) binding studies were performed. RESULTS: In vivo gallbladder contraction induced by CCK-8 was significantly lower in AGD (29.4%) and cholesterol stones (28.8%) than in pigment stones (59.8%) and normal controls (57.8%; P < 0.01). In vitro muscle cell contraction induced by CCK-8 was also lower in AGD than in pigment stones. It remained impaired in AGD after stimulation with the G-protein activators GTP gamma S and AlF(4) and with the second messenger 1,2-dioctanoyl-sn-glycerol. However, GTP gamma S binding induced by CCK-8 and vasoactive intestinal polypeptide and the binding capacity of CCK receptors were not different between AGD and pigment stones. CONCLUSIONS: These findings suggest that there is a good correlation between in vivo and in vitro gallbladder response to CCK-8 in patients with AGD. Unlike those found in cholesterol stones, the muscle defects in AGD appear to reside in the contractile apparatus.

Cell Membrane↗
PubMed2001

Chronic oesophagitis in the cat.

BACKGROUND: Our understanding of the pathophysiology of gastro-oesophageal reflux disease (GERD) in man is limited. The aim of the present study was to establish a long-term (>1 year) animal model for reflux oesophagitis which would allow us to study various aspects of the development of chronic reflux oesophagitis. METHODS: Myotomy was carried out in the gastro-oesophageal junction in eight cats; seven other cats were sham-operated. Before the operation, and every 2 months thereafter, oesophagoscopy was carried out, biopsies were taken for histology, and manometry was performed to determine the lower oesophageal sphincter pressure (LESP). The cats were killed 1 year after the operation. RESULTS: The myotomy operation resulted in a significantly decreased LESP. In oesophageal biopsies from these cats, there was a varying degree of oesophagitis starting already 2 months after surgery. In six of the eight myotomized cats there was hyperplasia of the stratum basale, and cardiac type metaplasia was observed in two cats. The control cats showed no significant changes in LESP or in the histology of the oesophagus. CONCLUSIONS: In cats followed for more than a year, myotomy in the gastro-oesophageal junction results in reflux oesophagitis similar to that seen in patients with chronic gastro-oesophageal reflux.

Animals↗
PubMed2000

In vitro release studies of methylmethacrylate liberation from acrylic cement powder.

Bone cement or polymethylmethacrylate (PMMA) is commonly used for anchoring cemented prosthesis to the bone. Cytotoxic effect of culture media exposed to PMMA powder may be related with long term problems associated with acrylic cement application, being the monomer (methylmethacrylate) one of the cement's component partly responsible for the cytotoxic effect. The present work reports the studies of monomer release from acrylic bone cement powder under different experimental conditions: setting time of PMMA (in solution and air) and different culture media composition. High-performance liquid chromatography was used for the determination of residual monomer. Mathematical models were applied to experimental dissolution data revealing that monomer release is lightly affected by the studied variables. The monomer release seems to be a surface phenomena, suggesting that the possible actions of monomer will mainly be due to the initial loss of non polymerized monomer rather than to further depolymerization of the already polymerized cement.

Bone Cements↗
PubMed2000

Defect of receptor-G protein coupling in human gallbladder with cholesterol stones.

Human gallbladders with cholesterol stones (ChS) exhibit an impaired muscle contraction and relaxation and a lower CCK receptor-binding capacity compared with those with pigment stones (PS). This study was designed to determine whether there is an abnormal receptor-G protein coupling in human gallbladders with ChS using (35)S-labeled guanosine 5'-O-(3-thiotriphosphate) ([(35)S]GTPgammaS) binding, (125)I-labeled CCK-8 autoradiography, immunoblotting, and G protein quantitation. CCK and vasoactive intestinal peptide caused significant increases in [(35)S]GTPgammaS binding to Galpha(i-3) and G(s)alpha, respectively. The binding was lower in ChS than in PS (P < 0.01). The reduced [(35)S]GTPgammaS binding in ChS was normalized after the muscles were treated with cholesterol-free liposomes (P < 0.01). Autoradiography and immunoblots showed a decreased optical density (OD) for CCK receptors, an even lower OD value for receptor-G protein coupling, and a higher OD for uncoupled receptors or Galpha(i-3) protein in ChS compared with PS (P < 0.001). G protein quantitation also showed that there were no significant differences in the Galpha(i-3) and G(s)alpha content in ChS and PS. We conclude that, in addition to an impaired CCK receptor-binding capacity, there is a defect in receptor-G protein coupling in muscle cells from gallbladder with ChS. These changes may be normalized after removal of excess cholesterol from the plasma membrane.

Autoradiography↗
PubMed1999

Excess membrane cholesterol alters human gallbladder muscle contractility and membrane fluidity.

BACKGROUND & AIMS: The relationship between muscle contractility, plasma membrane cholesterol, and fluidity was investigated in human gallbladders with gallstones. METHODS: Isolated gallbladder muscle cells were used to measure contraction. Plasma membranes of gallbladder muscle were purified in a sucrose gradient and measured for cholesterol content and cholesterol/phospholipid mole ratio. Membrane fluidity was determined by using fluorescence polarization and was expressed as the reciprocal of anisotropy. RESULTS: The maximal contraction induced by cholecystokinin octapeptide was significantly less in gallbladders with cholesterol stones than in those with pigment stones. The membrane cholesterol content and cholesterol/phospholipid mole ratio were significantly higher in gallbladders with cholesterol stones than in those with pigment stones. Membrane anisotropy was also higher than in gallbladders with pigment stones, reflecting lower membrane fluidity in gallbladders with cholesterol stones. After muscle cells from cholesterol stone gallbladders were incubated with cholesterol-free liposomes for 4 hours, cholecystokinin octapeptide-induced contraction, membrane cholesterol content and cholesterol/phospholipid ratio, and membrane fluidity returned to normal levels. CONCLUSIONS: Gallbladder muscle from patients with cholesterol stones has increased membrane cholesterol/phospholipid mole ratio and decreased membrane fluidity resulting in impaired muscle contractility. These abnormalities are corrected by removing the excess cholesterol from the plasma membranes.

Cell Membrane↗
PubMed1999

CCK receptor dysfunction in muscle membranes from human gallbladders with cholesterol stones.

Human gallbladders with cholesterol stones exhibit impaired muscle contraction induced by agonists that act on transmembrane receptors, increased membrane cholesterol content, and abnormal cholesterol-to-phospholipid ratio compared with those with pigment stones. The present study was designed to investigate the functions of the CCK receptor of gallbladder muscle membranes by radioreceptor assay and cross-linking. 125I-labeled CCK-8 binding was time-dependent, competitive, and specific. Scatchard analysis showed that the maximum specific binding (Bmax) was significantly decreased in cholesterol compared with pigment stone gallbladders (0.18 +/- 0. 07 vs. 0.38 +/- 0.05 pmol/mg protein, P < 0.05). In contrast, the affinity for CCK was higher in cholesterol than pigment stone gallbladders (0.18 +/- 0.06 vs. 1.2 +/- 0.23 nM). Similar results were observed in binding studies with the CCK-A receptor antagonist [3H]L-364,718. Cross-linking and saturation binding studies also showed significantly less CCK binding in gallbladders with cholesterol stones. These abnormalities were reversible after incubation with cholesterol-free liposomes. The Bmax increased (P < 0.01) and the dissociation constant decreased (P < 0.001) after incubation with cholesterol-free liposomes. In conclusion, human gallbladders with cholesterol stones have impaired CCK receptor binding compared with those with pigment stones. These changes are reversed by removal of the excess membrane cholesterol. These receptor alterations may contribute to the defective contractility of the gallbladder muscle in patients with cholesterol stones.

Bile Pigments↗
PubMed1998

Intracellular RNA cleavage by the hairpin ribozyme.

Studies involving ribozyme-directed inactivation of targeted RNA molecules have met with mixed success, making clear the importance of methods to measure and optimize ribozyme activity within cells. The interpretation of biochemical assays for determining ribozyme activity in the cellular environment have been complicated by recent results indicating that hammerhead and hairpin ribozymes can cleave RNA following cellular lysis. Here, we report the results of experiments in which the catalytic activity of hairpin ribozymes is monitored following expression in mammalian cells, and in which post-lysis cleavage is rigorously excluded through a series of biochemical and genetic controls. Following transient transfection, self-processing transcripts containing active and inactive hairpin ribozymes together with cleavable and non-cleavable substrates were generated within the cytoplasm of mouse OST7-1 cells using T7 RNA polymerase. Unprocessed RNA and products ofintracellular cleavage were detected and analyzed using a primer-extension assay. Ribozyme-containing transcripts accumulated to a level of 4 x 10(4) copies per cell, and self-processing proceeded to an extent of >75% within cells. Cellular RNA processing was blocked by mutations within the ribozyme (G8A, G21U) or substrate (DeltaA-1) that, in vitro , eliminate cleavage without affecting substrate binding. In addition to self-processing activity, trans -cleavage reactions were supported by the ribozyme-containing product of the self-processing reaction, and by the ribozyme linked to the non-cleavable substrate analog. Ribozyme activity was present in extracts of cells expressing constructs with active ribozyme domains. These results provide direct biochemical evidence for the catalytic activity of the hairpin ribozyme in a cellular environment, and indicate that self-processing ribozyme transcripts may be well suited for cellular RNA-inactivation experiments.

Animals↗
PubMed1997

Gallbladder relaxation in patients with pigment and cholesterol stones.

BACKGROUND & AIMS: Gallbladders with cholesterol stones show a defective contraction in response to agonists. The aim of this study was to investigate the muscle relaxation of human gallbladders with cholesterol or black pigment gallstones. METHODS: Gallbladder relaxation was measured in vitro using muscle strips and single muscle cells. Relaxation was expressed as percent inhibition of either basal active tension in strips or maximal cell contraction induced by diacylglycerol. The production of cyclic nucleotides was determined using a 125I-labeled radioimmunoassay kit. RESULTS: Frequency-dependent relaxation evoked by electrical field stimulation was significantly lower in gallbladders with cholesterol stones than in gallbladders with pigment stones. Relaxation and adenosine 3',5'-cyclic monophosphate (cAMP) production induced by isoproterenol, vasoactive intestinal peptide, and forskolin were also significantly decreased in gallbladders with cholesterol stones. However, the relaxation in response to 8-bromo-cAMP, nitric oxide (NO), and the NO donor S-nitroso-N-acetylpenicillamine (SNAP), which circumvent plasma membrane receptors and directly activate intracellular mechanisms, was similar in gallbladders with cholesterol and pigment stones. Guanosine 3',5'-cyclic monophosphate production induced by NO and SNAP was also similar. CONCLUSIONS: Human gallbladder muscle from specimens with cholesterol stones show an impaired relaxation and lower cAMP production compared with specimens with pigment stones. The muscle defect(s) responsible for this impairment seem to be in the plasma membranes.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1997

Different pathways mediate cholecystokinin actions in cholelithiasis.

Smooth muscle from gallbladders with cholesterol stones exhibits impaired response to cholecystokinin (CCK). This study investigated whether the impaired response is mediated by different signal-transduction pathways responsible for CCK-induced contraction in prairie dog and human gallbladders with cholesterol stones. Gallbladder muscle cells were isolated enzymatically to study contraction. Protein kinase C (PKC) activity was measured by examining the phosphorylation of a specific substrate peptide from myelin basic protein Ac-MBP-(4-14). Gallbladder muscle cells from high-cholesterol-fed prairie dogs contracted less in response to CCK octapeptide (CCK-8) than those from the control group. However, inositol-1,4,5-trisphosphate (IP3), diacylglycerol, and guanosine 5'-O-(3-thiotriphosphate) induced the same magnitudes of contraction in these two groups. In control prairie dog and human gallbladders, the maximal contraction caused by 10(-8) M CCK-8 was blocked by the calmodulin antagonist CGS9343B but not by the PKC inhibitor H-7. Conversely, in gallbladders with cholesterol stones from prairie dogs or human patients, the maximal contraction induced by 10(-8) M CCK-8 was blocked by H-7 and chelerythrine but not by CGS9343B. In these gallbladders CCK-8 caused a significant PKC translocation from the cytosol to the membrane. High CCK concentrations may activate the calmodulin-dependent pathway in functionally normal gallbladder muscle and the PKC-dependent pathway in muscle from gallbladders with cholesterol stones. The defect of gallbladder muscle after cholesterol feeding and stones might reside in the steps before G protein activation.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1995

Percutaneous anterior discectomy under ultrasound guidance.

The object of this experiment was to demonstrate that, by using a peritoneal fluid infusion coupled with ultrasound guidance, a safe anterior entry into the L5-S1 disc of the pig could be made. The goal was to develop a procedure with the advantages of a laparoscopic approach to the disc which has been published to allow removal of extruded herniations, that a neurosurgeon could perform without a general surgeon in attendance. The procedure was carried out under general anesthesia and fluoroscopic control. One liter of normal saline was then infused into the abdomen of a pig. Under ultrasound guidance and laparoscopic confirmation, a Nucleotome was placed into the L5-S1 disc and the disc was aspirated. The results showed that the disc was safely entered in all 5 pigs. The disc was successfully removed in 4 of 5 pigs. In all animals the bowel floated out of the pelvis, obviating the need for bowel manipulation. Laparoscopic ultrasound was needed for visualization in 3 pigs, while external ultrasound was used in two. Our conclusion is that peritoneal fluid infusion and ultrasound guidance, when applied to laparoscopic anterior discectomy, eliminates the need for retroperitoneal dissection and bowel manipulation, significantly simplifying the laparoscopic discectomy procedure.

Animals↗
PubMed1995

Direct G protein activation reverses impaired CCK signaling in human gallbladders with cholesterol stones.

Human gallbladders were used to investigate the mechanisms of the impaired contraction induced by cholecystokinin (CCK) associated with cholesterol stones. Single muscle cells were isolated enzymatically with collagenase. Inositol 1,4,5-trisphosphate was measured by high-performance liquid chromatography. Diacylglycerol was assayed by thin-layer chromatography. CCK stimulation showed decreased muscle contraction and production of inositol 1,4,5-trisphosphate and diacylglycerol in gallbladders with cholesterol stones compared with those with pigment stones. Exogenous calmodulin induced maximal contraction of 22.4 +/- 0.5 and 21.0 +/- 0.6% in gallbladders with cholesterol and pigment stones, respectively. Similar findings were observed with a synthetic diacylglycerol analogue. Two G protein activators, aluminum fluoride and guanosine 5'-O-(3-thiotriphosphate), evoked similar responses in these two types of gallbladders, with maximal contractions of 21.3 +/- 0.4 and 23.3 +/- 0.5%, respectively, in those with cholesterol stones and 20.9 +/- 0.8 and 22.6 +/- 0.4%, respectively, in those with pigment stones. These results suggest that receptor-dependent ligands like CCK cannot fully activate the intracellular pathways, which, however, can be fully stimulated by circumventing receptors with G protein activators or second messengers. After G protein activation, the pathways appear to be functionally intact. The defect might then reside in the receptor or in the interaction between receptors and G proteins.

Aluminum Compounds↗
PubMed1994

Cholecystokinin-coupled intracellular signaling in human gallbladder muscle.

BACKGROUND/AIMS: It has been shown that cholecystokinin (CCK) contracts the gallbladder muscle by utilizing intracellular calcium, but the intracellular pathways have not been elucidated. The present study was designed to characterize the signal transduction pathways that mediate CCK-induced contraction of human gallbladder muscle. METHODS: Single muscle cells were isolated from human gallbladders by enzymatic digestion with collagenase. Permeable cells were obtained by incubation with saponin. Protein kinase C (PKC) activity was determined by measuring the phosphorylation of a specific substrate peptide from myelin basic protein, Ac-MBP(4-14). RESULTS: The inositol-1,4,5-trisphosphate (IP3) antagonist heparin blocked the contractions induced by CCK. The PKC inhibitor H-7 blocked the contractions caused by low, but not high, concentrations of CCK and IP3. In contrast, the calmodulin inhibitor CGS9343B blocked the contractions induced by high, but not low, doses of CCK and IP3. Furthermore, exogenously activated calmodulin blocked the PKC-mediated contraction induced by diacylglycerol. Direct measurements of PKC activity showed that low, but not high, CCK concentrations caused PKC translocation. CONCLUSIONS: CCK contracts the gallbladder muscle via IP3-mediated calcium release. CCK activates the PKC pathway at low concentrations, whereas it activates the calmodulin pathway at high concentrations, which in turn inhibits the activation of PKC.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗