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Biomedical subjects

G Guillén

Publications and source records attributed to G Guillén.

At least 19 recordsLinked to original sources
PubMed2006

[Hirschsprung-neurointestinal dyspasia: diferential diagnosis and reliability of diagnostic procedures].

The suspicion of Hirschsprung's disease (HD) or Neurointestinal Dysplasia (NID) arises with the appearance of a common symtomatology : delay in meconium evacuation, abdominal distention, vomiting, intestinal occlusion and ultimately, chronic constipation. The need to perform a correct differential diagnosis between both pathologies is essential, given that the treatment of HD is surgical while NID is expectant. The objective of this paper is to define the necessity of using or not all the diagnostic tests simultaneously to obtain a precise diagnosis. The authors performed a complete clinical review of a series of 42 HD and 18 NID analysing the results of diagnostic procedures (Radiology / transition zone, anorrectal manometry / absence of anal inhibitory reflex, and suction biopsy / AcHE study). The authors concluded that is necessary to perform all the three diagnostic procedures simultaneously in all patients with symtomatology given that not one test has the capacity to provide a diagnosis alone. Anorectal manometry has proven to be the most reliable test to diagnose HD. The authors agree with the guidelines published by Meier Ruge in 2004 to diagnose NID.

Constipation↗
PubMed2005

Memory immune response generated in Cercopithecus aethiops against meningococcal polysaccharide serogroup C conjugate vaccine.

The chemical conjugation of bacterial polysaccharide to carrier proteins has proved to be an efficient tool to improve the immunological response against these bacterial antigens. In this study, we characterized the antibody response generated in a non-human primate model against the meningococcal capsular polysaccharide serogroup C (CCPS) conjugated to the P64k protein. Similar to licensed vaccines the CCPS conjugate is able to generate a good memory immune response with antibody titers threefold higher than the free CCPS. Three different ELISA protocols were used to measure the antibody response and the importance of the coating antigen was demonstrated. The ELISA using the derivatized CCPS showed the best results and had a high correlation with the bactericidal activity. The antibodies elicited showed a high protective capacity when assayed in the infant rat protection model.

Animals↗
PubMed2005

Immunological characterization of two hepatitis B core antigen variants and their immunoenhancing effect on co-delivered hepatitis B surface antigen.

The hepatitis B virus (HBV) core and surface antigens are potent immunogens in animal models and humans. They have been used in vaccine studies for prevention or therapy of HBV diseases and also as carrier molecules in new developments. In this study we explored the nasal immunogenicity of two different variants of the recombinant complete nucleocapsid (HBcAg) as well as their adjuvant effect on hepatitis B surface antigen (HBsAg). To characterize the immune response, the serum IgG antibody response was tested during one year against both antigens, and the serum and vaginal secretions were tested for recognition of linear epitopes of HBcAg for both HBcAg variants. The results obtained evidenced that the intranasal immunogenicity of both HBcAg variants was similar and high, developing early and long lasting IgG responses. A similar recognition pattern to all sera and vaginal washes samples was generated by the two variants of HBcAg, also similar to a pool of human anti-HBcAg positive sera. A synergistic effect in the enhancement of the immunogenicity for both antigens was evidenced in the combined formulation after nasal administration. Taken together, these results would be of interest in the design of more potent therapeutic and preventive vaccines complementing systemic and mucosal responses.

Amino Acid Sequence↗
PubMed2004

Discriminative capacity of calcaneal quantitative ultrasound and of osteoporosis and fracture risk factors in postmenopausal women with osteoporotic fractures.

Bone fragility fractures constitute the principal complication of osteoporosis. The identification of individuals at high risk of sustaining osteoporotic fractures is important for implementing preventive measures. The purpose of this study is to analyze the discriminative capacity of a series of osteoporosis and fracture risk factors, and of calcaneal quantitative ultrasound (QUS), in a population of postmenopausal women with a history of osteoporotic fracture. A cross-sectional analysis was made of a cohort of 5195 women aged 65 or older (mean +/- SD: 72.3 +/- 5.4 years) seen in 58 primary care centers in Spain. A total of 1042 women (20.1%) presented with a history of osteoporotic fracture. Most fractures (93%) were non-vertebral. Age-adjusted odds ratios corresponding to each decrease in one standard deviation of the different QUS parameters ranged from 1.47 to 1.55 (P < 0.001) for fractures. The age-adjusted multivariate analysis yielded the following risk factors independently associated with a history of osteoporotic fracture: number of fertile years, a family history of fracture, falls in the previous year, a history of chronic obstructive airway disease, the use of antiarrhythmic drugs, and a low value for any of the QUS parameters. The area under the receiver operating characteristic curve of the best model was 0.656. In summary, a series of easily assessable osteoporotic fracture risk factors has been identified. QUS was shown to discriminate between women with and without a history of fracture, and constitutes a useful tool for assessing fracture risk. Various of the vertebral and hip fracture risk factors frequently cited in North American and British populations showed no discriminative capacity in our series--thus suggesting that such factors may not be fully applicable to our population and/or to the predominant type of fractures included in the present study.

Aged↗
PubMed2004

Development of a nasal vaccine for chronic hepatitis B infection that uses the ability of hepatitis B core antigen to stimulate a strong Th1 response against hepatitis B surface antigen.

There are estimated to be 350 million chronic carriers of hepatitis B infection worldwide. Patients with chronic hepatitis B are at risk of liver cirrhosis with associated mortality because of hepatocellular carcinoma and other complications. An important goal, therefore, is the development of an effective therapeutic vaccine against chronic hepatitis B virus (HBV). A major barrier to the development of such a vaccine is the impaired immune response to HBV antigens observed in the T cells of affected patients. One strategy to overcome these barriers is to activate mucosal T cells through the use of nasal vaccination because this may overcome the systemic immune downregulation that results from HBV infection. In addition, it may be beneficial to present additional HBV epitopes beyond those contained in the traditional hepatitis B surface antigen (HbsAg) vaccine, for example, by using the hepatitis B core antigen (HBcAg). This is advantageous because HBcAg has a unique ability to act as a potent Th1 adjuvant to HbsAg, while also serving as an immunogenic target. In this study we describe the effect of coadministration of HBsAg and HBcAg as part of a strategy to develop a more potent and effective HBV therapeutic vaccine.

Administration, Intranasal↗
PubMed2003

Carboxy-terminally truncated Dengue 4 virus envelope glycoprotein expressed in Pichia pastoris induced neutralizing antibodies and resistance to Dengue 4 virus challenge in mice.

We have expressed a recombinant Dengue 4 virus envelope glycoprotein (E4rec), truncated at its C-terminus by 53 amino acids, in Pichia pastoris. The presence of E4rec was confirmed by Western-blot using anti-DEN 4 hyper immune mouse ascitic fluid. E4rec migrated during SDS-PAGE as a 64 kDa protein. Treatment with endoglycosidases showed that the E protein was modified by the addition of short mannose chains and the absence of hyperglycosylation. When administered to BALB-C mice, E4rec elicited a DEN 4 neutralizing antibody response haemagglutination inhibition antibodies and specific memory T cell response. Mice immunized were also significantly protected against lethal DEN 4 virus challenge (86.6%, p < 0.001).

Animals↗
PubMed2002

A recombinant envelope protein from Dengue virus purified by IMAC is bioequivalent with its immune-affinity chromatography purified counterpart.

Semi-purified DEN-4 envelope protein, obtained in Pichia pastoris, was capable of generating neutralising and protecting antibodies after immunisation in mice. Here we compared two purification processes of this recombinant protein using two chromatographic steps: immune-affinity chromatography and immobilised metal ion adsorption chromatography (IMAC). The protein purified by both methods produced functional antibodies reflected by titres of haemagglutination inhibition and neutralisation. IMAC could be used as an alternative for high scale purification.

Animals↗
PubMed2001

Immunogenicity of recombinant class 1 protein from Neisseria meningitidis refolded into phospholipid vesicles and detergent.

The possibility of eliciting bactericidal antibodies against a recombinant class 1 protein (P1) from Neisseria meningitidis, joined to the first 45 amino acids of the neisserial LpdA protein (PM82), was examined. P1 was produced in Escherichia coli as intracellular inclusion bodies, from which it was purified and reconstituted by (a) inclusion into phospholipid vesicles and detergent and (b) refolding in 0.1% SDS. When Balb/c mice were immunised, high titres of subtype-specific bactericidal antibodies against P1 were obtained in both cases. These results suggest that in spite of being a denaturing agent, it is possible to use SDS to reconstitute the P1 protein in a conformation that exposes the immunodominat regions.

Animals↗
PubMed2001

Safety and preliminary immunogenicity of the recombinant outer membrane protein P64k of Neisseria meningitidis in human volunteers.

P64k is a meningococcal protein from Neisseria meningitidis that has been obtained by recombinant DNA technology. Recombinant P64k has been extensively characterized by physicochemical and immunological methods. Lately this protein has been found to act as a versatile immunological carrier for weak antigens in mice. In the present work, a Phase I clinical trial was carried out in healthy volunteers who received three inoculations of either placebo or recombinant P64k (20 or 50 microg). No severe adverse events occurred during the trial. Only mild adverse events in ten volunteers were observed. At 1 month after the third dose, 15 out of 18 volunteers (83.3%) who received the recombinant antigen had a P64k-specific antibody titre > or =1:100, as detected by ELISA. A fourth dose, given 9 months after the third one, elicited a potent booster immune response in P64k vaccinees. Accordingly, these P64k formulations were considered safe and immunogenic in healthy human volunteers.

Adult↗
PubMed2001

Biochemical characterization of profilin from seeds of Phaseolus vulgaris L.

The isoform composition of the 14.4 kDa profilin polypeptide was analyzed in seeds, leaves, flowers, roots and root-nodules from Phaseolus vulgaris L. Isoforms of pIs approximately 4.4-5 were present in all the tissues analyzed. The biochemical features of the protein present in seed tissue were determined. Seed profilin bound to Phenyl-Sepharose under low salt conditions which suggested a hydrophobic interaction; however, it was not associated with microsomal membranes nor it partitioned as a hydrophobic protein in Triton X-114. Fractions eluting from poly-L-proline or Phenyl-Sepharose columns contained well detectable amounts of profilin but no actin, suggesting that most of the protein was not present as profilactin in the seed. However, seed profilin appeared to be in some kind of complex since several molecular weight species were observed on native gels. In addition, profilin was found preferentially in the embryo axis and light microscopic immunolocalization showed a cytoplasmic distribution in this tissue.

Blotting, Western↗
PubMed2000

P64k meningococcal protein as immunological carrier for weak immunogens.

Previously, the P64k meningococcal protein, an antigen of 64 kDa expressed in Escherichia coli, has been extensively characterized. We have successfully conjugated several synthetic peptides and meningococcal group C polysaccharide to P64k. In three out of four model peptides, the murine humoral immune response against the homologous peptide, evaluated after three doses of conjugate, was higher in the animals immunized with the coupled peptide than in those that received free peptide. The fourth and largest was immunogenic by itself. Similarly, the antigroup C polysaccharide levels reached by conjugated polysaccharide were significantly higher than those produced against unconjugated polysaccharide. As a carrier for one of the peptides, P64k was compared with bovine serum albumin (BSA) and tetanus toxoid (TT), being able to induce slightly higher or similar antipeptide antibody levels than these well-establish protein carriers. Our results suggest that recombinant P64k protein could be a readily available immunological carrier, as efficient as other commonly used large carrier molecules.

Amino Acid Sequence↗
PubMed1999

Characterisation of the lpdA gene from Neisseria meningitidis by polymerase chain reaction, restriction fragment length polymorphism and sequencing.

P64k protein from Neisseria meningitidis is well recognised in sera from individuals convalescent from meningococcal disease or vaccinated with the Cuban antimeningococcal vaccine VA-MENGOC-BC. The presence of the protein in more than 80 meningococcal strains has also been verified. It is immunogenic in animal models and the antibodies elicited show bactericidal activity against meningococci. To further investigate at the molecular level whether lpdA, the gene coding for P64k protein, is conserved among different N. meningitidis strains, a total of 20 strains isolated from different geographic areas were differentiated on the basis of restriction fragment length polymorphism (RFLP) patterns after polymerase chain reaction (PCR) amplification of the lpdA gene and restriction endonuclease digestion with HpaII. Although a total of five different PCR-RFLP patterns were present, nucleotide sequence determination showed that identity levels were as high as 93-99% among the N. meningitidis strains analysed.

Amino Acid Sequence↗
PubMed1999

Profilin in Phaseolus vulgaris is encoded by two genes (only one expressed in root nodules) but multiple isoforms are generated in vivo by phosphorylation on tyrosine residues.

Actin-binding proteins such as profilins participate in the restructuration of the actin cytoskeleton in plant cells. Profilins are ubiquitous actin-, polyproline-, and inositol phospholipid-binding proteins, which in plants are encoded by multigene families. By 2D-PAGE and immunoblotting, we detected as much as five profilin isoforms in crude extracts from nodules of Phaseolus vulgaris. However, by immunoprecipitation and gel electrophoresis of in vitro translation products from nodule RNA, only the most basic isoform of those found in nodule extracts, was detected. Furthermore, a bean profilin cDNA probe hybridised to genomic DNA digested with different restriction enzymes, showed either a single or two bands. These data indicate that profilin in P. vulgaris is encoded by only two genes. In root nodules only one gene is expressed, and a single profilin transcript gives rise to multiple profilin isoforms by post-translational modifications of the protein. By in vivo 32P-labelling and immunoprecipitation with both, antiprofilin and antiphosphotyrosine-specific antibodies, we found that profilin is phosphorylated on tyrosine residues. Since chemical (TLC) and immunological analyses, as well as plant tyrosine phosphatase (AtPTP1) treatments of profilin indicated that tyrosine residues were phosphorylated, we concluded that tyrosine kinases must exist in plants. This finding will focus research on tyrosine kinases/tyrosine phosphatases that could participate in novel regulatory functions/pathways, involving not only this multifunctional cytoskeletal protein, but other plant proteins.

Blotting, Western↗
PubMed1998

A novel cancer vaccine composed of human-recombinant epidermal growth factor linked to a carrier protein: report of a pilot clinical trial.

BACKGROUND: There is evidence of a relationship between epidermal growth factor (EGF) and tumor cell proliferation, such as the overexpression of EGF receptor (EGF-R) in different human tumors, which makes this system an interesting target for cancer treatment. Up to now, passive immunotherapy with monoclonal antibodies against the EGF-R has been assayed in clinics. Our approach consists of active immunotherapy with human EGF (hu-EGF). We conducted a pilot clinical trial to define the safety, toxicity and immunogenicity of vaccination with hu-EGF coupled to a carrier protein. PATIENTS AND METHODS: Ten patients with histologically-proven malignant carcinomas (colon, lung, stomach and prostate) in advanced clinical stages were enrolled. Patients were immunized twice (on days 0 and 15) with hu-EGF linked to either tetanic toxoid (TT, five patients) or P64K Neisseria Meningitidis recombinant protein (P64k, five patients), intradermically, using aluminium hydroxyde as adjuvant. RESULTS: In both groups 60% of patients developed anti-EGF antibody titers without evidence of toxicity. Secondary reactions were very mild, limited to erythema and itching at the site of injection, which disappeared without medication. CONCLUSIONS: We conclude that the proposed vaccination with hu-EGF was well tolerated and that antibody titers against self EGF were developed. The results of this trial may be useful in the design of new clinical trials with higher dose immunization protocols and using more effective adjuvants.

Aged↗
PubMed1998

Expression in Escherichia coli of the lpdA gene, protein sequence analysis and immunological characterization of the P64k protein from Neisseria meningitidis.

By making use of recombinant DNA technology it is possible to characterize meningococcal outer membrane proteins (OMPs) capable of stimulating a host immune response. The lpdA gene, which codes for an OMP (P64k) from Neisseria meningitidis, was cloned in Escherichia coli. The recombinant protein was recognized by sera from patients convalescing from meningococcal disease. The monoclonal antibodies obtained against the recombinant protein recognized the natural protein on a Western blot, and monoclonal antibody 114 was assayed in ELISA with a panel of 85 N. meningitidis strains. The protein was recognized in 81 strains (95.3%); the strains that were not recognized were neither epidemic nor isolated from systemic disease. The complete amino acid sequence of P64k was obtained by automatic sequencing and MS.

Amino Acid Sequence↗
PubMed1997

Sequence analysis of the structural tbpA gene: protein topology and variable regions within neisserial receptors for transferrin iron acquisition.

The gene coding for the 98-kDa meningococcal outer membrane transferrin binding protein 1 (TbpA) from strain B385 was cloned and sequenced. Sequence comparison among its deduced aminoacid sequence and those from TbpA and the closely related LbpA (lactoferrin binding protein) gene from three different meningococcal strains, and four isolates from two other bacterial pathogens, showed that TbpA variability is confined to five specific segments, designated VR1 (199-287), VR2 (306-381), VR3 (480-546), VR4 (618-651) and VR5 (681-708). The third VR was the most variable among strains both at the nucleotide and amino acid levels. Six additional tbpA genes from different meningococcal strains were cloned and its VR3 sequence determined. On the basis of this data we were able to cluster tbpA genes in two groups: D (bearing a deletion in VR3) and N (nondeleted); all N and D strains belonging to the groups of high or low molecular weight transferrin receptor isotype, respectively. However, by phenogram analysis, the prototypical strain M982 (Group II) was clustered with M990 (B16B6 isotype, Group I). These results point to the existence of important exposed regions as well as to the possibility of horizontal gene exchange involving this locus. A topology model with 14 exposed loops and 28 membrane spanning segments was postulated. According to this tentative analysis, TbpA as well as LbpA proteins should form a gated channel in the neisserial outer membrane. The variable regions were located in the fifth, sixth, eighth, 10th and 11th loops respectively. Among TbpAs VR1, VR2, and VR3 resulted the most relevant regions.

Amino Acid Sequence↗