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Biomedical subjects

A Alcina

Publications and source records attributed to A Alcina.

At least 19 recordsLinked to original sources
PubMed2006

The 1858T PTPN22 gene variant contributes to a genetic risk of type 1 diabetes in a Ukrainian population.

The 1858T variant of the protein tyrosine phosphatase gene, PTPN22, is associated with an increased risk of several autoimmune diseases. The aim of this study has been to investigate the possible association of 1858C-->T PTPN22 polymorphism and type 1 diabetes (T1D) in Caucasians from Ukraine. Overall, the distribution of 1858 PTPN22 genotypes differed significantly between the T1D patient group (n = 296) and the control group (n = 242) (P = 0.0036). When both groups were classified according to sex, the TT genotype and T allele showed a statistically significant higher frequency in T1D female patients (5.9 and 22.8%, respectively) in comparison with the female controls (0 and 11.9%) (P = 0.008 for both analyses). The patients with the TT genotype were significantly younger at the onset of T1D compared with those with genotypes TC and CC (P = 0.035 and 0.019, respectively). In our Ukrainian Caucasian cohort, we confirmed the association between T1D and the PTPN22,1858T allele.

Adolescent↗
PubMed2006

OAS1 gene haplotype confers susceptibility to multiple sclerosis.

Multiple sclerosis (MS) is associated with genetic susceptibility and unknown environmental triggers, possible viral infections, but the specific etiological mechanism that subsequently develops into an inflammatory/autoimmune cascade of events is poorly understood. Recently, genetic variants of 2',5'- oligoadenylate synthetase 1 (OAS1) gene, a critical enzyme involved in innate antivirus response, have been associated with differential enzyme activity and type 1 diabetes in both case-control and family studies. We hypothesized that polymorphisms in the OAS1 gene could influence the susceptibility to MS. To test this hypothesis, we conducted a case-control study of 333 patients with MS and 424 healthy controls and genotyped two OAS1 single nucleotide polymorphisms (SNPs) by restriction fragment length polymorphism method: rs 10774671, A/G SNP altering the splicing site at the seventh exon, and rs 3741981, a nonsynonymous (Ser162Gly) A/G SNP in the third exon. Haplotype but not single-marker analysis revealed an association of the haplotype created by the G allele at rs 10774671 and the A allele at rs 3741981 with the susceptibility to MS (P value = 8.8 x 10(-5)). Subjects carrying this haplotype had an increased risk of MS comparing with those not carrying it (odds ratio = 4.7, 95% confidence interval 2.1-10.9). Our findings indicate that the OAS1 gene polymorphisms may confer susceptibility to MS or serve as markers of functional variants and suggest that OAS1 activity is involved in the etiology of the disease. Future studies in a larger sample and association analysis with functional variants will clarify the role of the OAS1 gene in the susceptibility to MS.

2',5'-Oligoadenylate Synthetase↗
PubMed2002

Analysis of -631 and -475 interleukin-2 promoter single nucleotide polymorphisms in multiple sclerosis.

We have analysed the interleukin-2 (IL-2) promoter single nucleotide polymorphisms -475 A/T and -631 G/A, relative to the initiation codon, in patients with multiple sclerosis (MS) and in healthy controls. Both groups showed a very low frequency of T at -475 and A at -631. Our results suggest that these polymorphisms do not contribute to MS susceptibility.

Genetic Predisposition to Disease↗
PubMed2001

The -174/-597 promoter polymorphisms in the interleukin-6 gene are not associated with susceptibility to multiple sclerosis.

Interleukin-6 (IL-6) has been implicated in the etiology of experimental autoimmune encephalomyelitis (EAE) in transgenic animals and contributes to neuropathology in humans. A single nucleotide polymorphism (SNP) at position -174 in the IL-6 gene promoter (IL-6pr) appears to influence IL-6 expression. Complete linkage disequilibrium was observed between the -174 and the -597 alleles. The aim of this study was to investigate the possible influence of -174/-597 IL-6pr polymorphisms on susceptibility to multiple sclerosis (MS). Genotyping of the -597 variant was performed by an RFLP method in 131 MS patients [88 relapsing-remitting (RR-MS), 43 secondary progressive (SP-MS)] and 157 healthy subjects. No differences were found between MS patients and controls with respect to the distribution of -597 IL-6pr genotypes. Neither was found when genotypes were analyzed according to the clinical course of the disease (RR-MS or SP-MS). Future studies focusing on complex transcriptional interactions between the IL-6pr and 3' flanking region polymorphic sites will be necessary to determine the IL-6 haplotype influence on susceptibility to MS.

Adult↗
PubMed2001

Allelic expression and interleukin-2 polymorphisms in multiple sclerosis.

We have investigated the association of two single nucleotide polymorphisms (SNPs) at positions -384 and 114 in the human interleukin-2 (hIL-2) with multiple sclerosis (MS). For two of the -384 genotypes (G/T, T/T), we observed an association with the susceptibility to secondary progressive (SP) course of MS (P=0.005 and P=0.013, respectively). Expression level differences of the IL-2 alleles (between one- and three-fold) were not attributable to the -384 promoter polymorphism. These data indicate for the first time the relevance of the il-2 gene locus in human MS and its possible involvement in other autoimmune diseases.

Age of Onset↗
PubMed2000

Allelic selection of human IL-2 gene.

The allelic expression of mouse IL-2 cannot be definitely extrapolated to what might happen in humans. Therefore, we investigated the regulation of allelic expression of the IL-2 gene in non-genetically manipulated human T lymphocytes by following natural allelic polymorphisms. We found a phenotypically silent punctual change in the human IL-2 at position 114 after the first nucleotide of the initiation codon, which represents a dimorphic polymorphism at the first exon of the IL-2 gene. This allowed the study by single-cell PCR of the regulation of the human IL-2 allelic expression in heterozygous CD4(+) T cells, which was found to be tightly controlled monoallelically. These findings may be used as a suitable marker for monitoring the IL-2 allelic contribution to effector activities and in immune responses against different infections or in pathological situations.

Alleles↗
PubMed1999

The cloning and expression of Pfacs1, a Plasmodium falciparum fatty acyl coenzyme A synthetase-1 targeted to the host erythrocyte cytoplasm.

Plasmodium is unable to carry out de novo fatty acid synthesis and has to obtain these compounds from their host for subsequent activation by thioesterification with coenzyme A. This activity is catalyzed by a fatty acyl-CoA synthetase enzyme (EC 6.2.1.3). Here, we describe a novel gene from P. falciparum whose recombinant purified product from baculovirus-transfected insect cell line had the enzymatic activity of a long-chain fatty acyl-CoA synthetase. It was named pf acs1, since it belongs to a multi-member gene family as revealed by the sequence of several clones and a multi-band pattern in Southern blots. The sequence specifies a product of 820 amino acid residues. It was transcribed and expressed in infected erythrocytes having an apparent molecular mass of 100 kDa. Immuno-labeling of infected erythrocytes with a specific antibody against the carboxy-terminal part of the PfACS1 localized the product early after the erythrocyte invasion in vesicle-like structures budding off the parasitoforous membrane toward the red cell cytoplasm. Its unique carboxy- terminal structure of 70 extra amino acid residues, longer than any other reported acyl-CoA synthetase, is probably related to its localization in the cytoplasm of the host erythrocyte. The phylogenetic relationship among other AMP-forming enzymes, placed PfACS1 closer to Saccharomyces cerevisiae, sharing significant amino acid identities, especially in the conserved signature motif that modulates fatty acid substrate specificity and ATP/AMP-binding domains. Taking into account the importance of this enzymatic activity for the parasite, its extra-cellular location inside the infected erythrocyte, and the divergence with respect to the homologous human enzymes, it may be an important protein as a potential target candidate for chemotherapeutic antimalaria drugs.

Amino Acid Sequence↗
PubMed1999

Induction of autoantibodies to different interleukin-2 allotypes.

We report the development of an in vivo system to induce the generation, and study the potential role, of autoantibodies to the lymphokine interleukin-2 (IL-2). To elicit IL-2 autoantibodies, mice were immunized with purified fusion proteins containing the N-terminal region of different IL-2 allotypes, where major changes have been observed. This part of the IL-2 molecule includes a conserved sequence with an essential residue for interacting with the beta-chain of the heterotrimeric IL-2 receptor. Mice bearing an RF IL-2 allotype, immunized with several N-terminal IL-2 fusion proteins, produced IgG antibodies against Mus musculus, C57BL/6, Mus spretus and the self molecule RF IL-2, but there were large differences among then in reactivity. These N-terminal IL-2 immunogens break the maintenance of self tolerance possibly by the introduction of new T cell epitopes on self IL-2. The immunized mice developed a complex set of immunopathologies such as splenomegaly, haemolytic anaemia and lymphoadenopathy with a long latency period after the last immunization. These pathologies resembled those described for IL-2-deficient mice (IL-2(-/-)) and mice injected with anti-IL-2 receptor alpha-antibody. Human IL-2 autoantibodies have been detected in several immune-affected situations and therefore this model would be of interest to study the potential evolution of these autoantibodies in relation to immunopathology. The production of these autoantibodies against conserved epitopes of mouse IL-2 may facilitate studies on the structural homologies between different IL-2 allotypes and from various species, and could be applied to other cytokines.

Amino Acid Sequence↗
PubMed1998

High expression in bacteria and purification of polymorphic mouse interleukin 2 molecules.

Recently, besides the known mouse interleukin 2 (IL-2) molecule, six other IL-2 alleles have been found in different mouse strains. In order to study their in vivo and in vitro biological activities large quantities are required. We cloned the corresponding IL-2 cDNAs into a pET7-7/BL21(DE3) bacterial system, a T7-RNA-polymerase-dependent expression vector, producing between 30 to 100 mg of IL-2 per litre of culture. The purification step is based on the resolution properties of the sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) technique and the capability of the IL-2 to regain its activity after SDS denaturation. These purified IL-2 molecules supported the growth of an IL-2-dependent cell line, CTL-L2, in a similar way to a commercial mouse IL-2 control. However, RF IL-2 allele, which is also expressed in NOD mice had a relatively lower growth activity in the CTL-L2 assay. These IL-2 molecules can be obtained in a purified form and totally recovered their activity after elimination of the SDS and 2-mercaptoethanol used in the extraction procedure.

Alleles↗
PubMed1998

Antiparasitic effects of the intra-Golgi transport inhibitor megalomicin.

The macrolide antibiotic megalomicin (MGM) has been shown to inhibit vesicular transport between the medial- and trans-Golgi, resulting in the undersialylation of cellular proteins (P. Bonay, S. Munro, M. Fresno, and B. Alarcón, J. Biol. Chem. 271:3719-3726, 1996). Due to the effects of MGM on the Golgi and on the replication of enveloped viruses, we decided to test whether it has any antiparasitic activity. The results showed that MGM has potent activity against the epimastigote stage of Trypanosoma cruzi, producing a 50% inhibitory concentration (IC50) of 0.2 microg/ml. Furthermore, MGM was also active against the intracellular replicative, amastigote form of T. cruzi, completely preventing its replication in infected murine LLC/MK2 macrophages at a dose of 5 microg/ml. Although less potent, MGM was also active against Trypanosoma brucei epimastigotes (IC50, 2 microg/ml) and Leishmania donovani and Leishmania major promastigotes (IC50, 3 and 8 microg/ml, respectively). MGM also blocked intracellular replication of the asexual stage of Plasmodium falciparum-infected erythrocytes at 1 microg/ml. Finally, MGM was active in an in vivo model, resulting in the complete protection of BALB/c mice from death caused by acute T. brucei infection and significantly reducing the parasitemia. These results suggest that MGM is a potential drug for the treatment of veterinary and human parasitic diseases.

Animals↗
PubMed1996

Glutamine and tetrapeptide repeat variations affect the biological activity of different mouse interleukin-2 alleles.

Mouse interleukin-2 (IL-2) was thought to be encoded by a single allele. We have recently described N-terminal differences in five IL-2 molecules from nine mouse strains analyzed (Matesanz, F., Alcina, A. and Pellicer, A., Immunogenetics 1993. 38: 300). In this study, we isolated and sequenced the cDNA of three polymorphic IL-2 molecules and constructed two recombinant IL-2 molecules to cover representative structural changes and to address the functional significance of these changes using human and mouse cellular assays in vitro. Apart from punctual codon changes, major differences include an expanding CAG codon (translated into glutamine) and the presence of the tetrapeptide Pro-Thr-Ser-Ser repeated 1, 2, or 3.5 times which is also present once in human IL-2. This tetrapeptide repeat includes an O-glycosylation site. These recombinant IL-2 proteins were expressed at high levels in bacteria and purified by preparative SDS-PAGE with a complete activity recovery. Differences in growth-inducing activity on mouse primary splenocytes were observed in some of them, although no differences were observed in proliferative stimulation of CTLL cells. In human peripheral blood lymphocytes and the T cell line Kit-225, the growth stimulation capacity was inversely dependent on the size of the glutamine stretch and the number of tetrapeptide repeats. These results suggest an evolutionary adaptation of the mouse IL-2/IL-2 receptor system that maintains polyglutamine extensions in the IL-2 molecule. In summary, mouse IL-2 polymorphism results in different bioactivities which may determine susceptibility or resistance to disease.

Alleles↗
PubMed1992

A new cDNA sequence for the murine interleukin-2 gene.

We have amplified by PCR and sequenced the first exon of the interleukin 2 gene from the RF/J mouse strain DNA. When we compared the RF/J first exon sequence with the one reported previously, we found several differences. These differences are also reflected in the deduced amino acid sequence and they have been localized in the first 23 amino acids of the mature polypeptide. The finding of this new IL-2 sequence shows that there is more than one allele for the mouse IL-2 molecule and raises the possibility of functional differences between alleles.

Amino Acid Sequence↗
PubMed1992

A Trypanosoma cruzi membrane protein shares an epitope with a lymphocyte activation antigen and induces crossreactive antibodies.

Chagas' disease results from the infection of the protozoan parasite Trypanosoma cruzi and affects several million people in South America. Several alterations of the immune response have been described in this disease, such as severe immunosuppression of both cellular and humoral responses and massive polyclonal stimulation with the generation of autoantibodies crossreacting with host cells and tissues. We have obtained monoclonal antibodies (mAbs) from T. cruzi-infected mice that recognized a 50/55-kD antigen (GP50/55) on the T. cruzi membrane, but not in other parasites of the family Trypanosomatidae. One of these GP50/55-specific mAbs (C10) crossreacts with a 28-kD antigen (p28) expressed on the membrane of greater than 85% of activated mouse T and B lymphocytes, after in vitro activation with concanavalin A, Salmonella typhosa lipopolysaccharide, phorbol dibutyrate ester, or antigen, and on several murine T and B lymphocyte cell lines. Human T and B lymphocytes also express upon activation with phytohemagglutinin or Staphylococcus aureus Cowan I (SAC) a similar antigen recognized by mAb C10, although in a lower proportion of cells (30-40%). Furthermore, this mAb was able to suppress mouse and human T and B cell proliferation to any of those stimuli. In addition, sera from chagasic patients and T. cruzi-infected mice, but not from control patients or littermates, contain antibodies that recognize a similar p28 antigen on B lymphocytes. Furthermore, the immunoglobulin fractions of some chagasic sera also suppress the proliferation of human T lymphocytes. These results suggest a possible pathological role of autoantibodies as an alternative mechanism for T. cruzi-associated immunosuppression.

Animals↗
PubMed1991

Characterization of a glycosyl-phosphatidylinositol-anchored membrane protein from Trypanosoma cruzi.

Four monoclonal antibodies (MAbs) specific for Trypanosoma cruzi were obtained. Flow cytometry analysis showed that these four MAbs stained the membranes of the three main morphological forms of T. cruzi: amastigotes, trypomastigotes, and epimastigotes. The four MAbs seemed to recognize the same 50- to 55-kDa antigen that was revealed by immunoblotting. Competition experiments revealed that they defined at least two different epitopes on the molecule. The antigen was detected on the external surface of the membrane by immunoelectron microscopy. Several experiments indicated that the 50- to 55-kDa antigen recognized by these four MAbs was a glycosyl-phosphatidylinositol-anchored membrane protein. (i) The antigen could be removed from the cell surface by treatment with proteases, NaOH, HNO2, and phosphatidylinositol-specific phospholipase C (PI-PLC). (ii) The phase distribution of the antigen in Triton X-114 solutions changed drastically upon treatment with PI-PLC. The antigen was found mainly in the detergent phase in nontreated samples and in the aqueous phase in PI-PLC-digested samples. (iii) A cross-reacting determinant that was found in other glycosyl-phosphatidylinositol-anchored membrane proteins appeared after PI-PLC treatment.

Animals↗
PubMed1988

Early and late heat-induced proteins during Leishmania mexicana transformation.

During in vitro transformation of L. mexicana from promastigotes to amastigotes at 37 degrees C, a higher growing temperature, transforming parasites showed two different sets of polypeptides. One set of proteins was synthesized at the beginning of temperature-shift and corresponded to the so called "heat shock proteins" (hsp) being expressed by promastigotes mostly. The second set was expressed later on was specifically associated with the amastigotes stages and was strongly similar to the pattern of polypeptides synthesized by amastigotes from infected peritoneal macrophages. These results suggest hsp may play a function at a defined time in the transformation of the parasite.

Animals↗
PubMed1988

The heat-shock response in Trypanosoma cruzi.

When Trypanosoma cruzi epimastigotes are exposed to temperatures of 37-41 degrees C there is a drastic decline in total protein synthesis. Analysis of the proteins synthesized at 41 degrees C by one-dimensional gel electrophoresis showed three major bands of Mr 83,000, 70,000 and 60,000. A similar pattern of heat-shock proteins was found in two different strains of T. cruzi (Tulahuen and GM strains) and in exponentially growing or in stationary epimastigotes. Actinomycin D prevented the appearance of these polypeptide bands, suggesting that the heat-shock proteins in T. cruzi epimastigotes are induced at the level of transcription. Analysis of the proteins synthesized by metacyclic forms at different temperatures suggests that heat-shock proteins in these cells are already synthesized at 27 degrees C. Elevation of temperature above 37 degrees C blocks the synthesis of most proteins in metacyclic forms except for major bands of Mr 83,000, 70,000, 60,000 and 55,000. More detailed analyses by high-resolution two-dimensional gel electrophoresis of the proteins synthesized at 27 degrees C or 37 degrees C by epimastigotes indicates that the heat-shock protein pattern is more complex than that demonstrated by one dimension, and at least ten new polypeptides are identified in two-dimensional gels. A similar analysis of metacyclic forms shows that most if not all the proteins present at 39 degrees C are also present at 27 degrees C. This result led us to the suggestion that the differentiation of T. cruzi to metacyclic forms involves the induction of heat-shock proteins, which prepares the parasite to infect the mammalian host.

Animals↗
PubMed1988

A tubulin-related 55 kilodalton surface antigen recognized by different Trypanosoma cruzi stage-specific monoclonal antibodies from infected mice.

Thirteen monoclonal antibodies (MAbs) specific for the membrane of live Trypanosoma cruzi have been obtained from BALB/c infected mice. Most of them had greater avidity for intact than for disrupted parasites. According to the staining by indirect immunofluorescence of the different live developmental stages of the parasite the MAbs could be divided into several groups. Three of them were trypomastigote specific, one amastigote-specific and two epimastigote-specific. The rest reacted with either all stage forms or with various combinations of the different stages. However, despite the fact that they seemed to correspond to stage-specific antibodies, ten of them reacted with the same 55/50 kDa antigen by immunoblotting. Similarly, a 55 kDa protein was immunoprecipitated from these MAbs. By contrast, a single band or a dimer of about 25 kDa was the predominant antigen(s) immunoprecipitated by the same MAbs in absence of protease inhibitors. This smaller protein may arise from proteolysis of the 55 kDa band. This protein is related to tubulin since tubulin (a 55 kDa protein) but not other cytoskeleton proteins blocked the binding of these MAbs to T. cruzi, and some MAbs react with pig alpha-tubulin by immunoblotting.

Animals↗