Biomedical subjects

S Casares

Publications and source records attributed to S Casares.

At least 19 recordsLinked to original sources

PubMed2003

A binding event converted into a folding event.

We have designed a chimeric protein by connecting a circular permutant of the alpha-spectrin SH3 domain to the proline-rich decapeptide APSYSPPPPP with a three-residue link. Our aim was to obtain a single-chain protein with a tertiary fold that would mimic the binding between SH3 domains and proline-rich peptides. A comparison of the circular-dichroism and fluorescence spectra of the purified chimera and the SH3 circular permutant showed that the proline-rich sequence occupies the putative SH3 binding site in a similar conformation and with comparable interactions to those found in complexes between SH3 and proline-rich peptides. Differential scanning calorimetry indicated that the interactions in the binding motif interface are highly cooperative with the rest of the structure and thus the protein unfolds in a two-state process. The chimera is more stable than the circular permutant SH3 by 6-8 kJ mol(-1) at 25 degrees C and the difference in their unfolding enthalpy is approximately 32 kJ mol(-1), which coincides with the values found for the binding of proline-rich peptides to SH3 domains. This type of chimeric protein may be useful in designing SH3 peptide ligands with improved affinity and specificity.

Amino Acid Sequence↗

PubMed2003

Sensitive detection of GM1 lipid rafts and TCR partitioning in the T cell membrane.

The cholesterol-rich lipid rafts on T cell membrane play important role in the formation of T cell receptor (TCR) signalosome upon receptor ligation. Analytical studies on the kinetics of lipid rafts formation and recruitment of protein receptors to lipid rafts are still limited by the use of a large number of cells. Herein, we describe a strategy for detecting fine alterations in the amount and distribution of glycosphingolipid (GM1) lipid rafts, and in the formation of GM1-TCR complexes in detergent-insoluble and -soluble compartments of the T cell membrane from a relative low number of cells. Using this strategy, we found that the GM1 moiety was physically associated with TCR in both detergent-insoluble and -soluble fractions. Shortly after ligation of CD3/TCR complex with a soluble CD3- epsilon mAb, the TCR was found mainly in the detergent-soluble fraction of the T cell membrane.

Animals↗

PubMed2003

Structural cooperativity in the SH3 domain studied by site-directed mutagenesis and amide hydrogen exchange.

We have studied the effects produced by site-directed mutagenesis upon energetic and structural cooperativity in the Src homology region 3 domain of alpha-spectrin. The mutation of Asn47 to Gly or Ala in the distal loop brings about significant changes to the global stability of the domain in spite of not affecting its structure to any great extent. The binding affinity for a proline-rich peptide is also largely diminished in both mutant domains. We have compared the apparent Gibbs energies of the amide hydrogen-deuterium exchange (HX) between the wild-type and the Gly47 mutant. The observed changes in the Gibbs energy of HX indicate a remarkable energetic cooperativity in this small domain. Regions of the domain's core have a high cooperativity with the position of the mutation, indicating that their HX occurs mainly in states in which the distal loop is unstructured. More flexible regions, which undergo HX mainly by local motions, show a lower but still considerable cooperativity with the distal loop. We conclude that there is an important correlation between regional stability and cooperativity in this small domain.

Deuterium↗

PubMed2002

The temperature dependence of the hydrogen exchange in the SH3 domain of alpha-spectrin.

The amide hydrogen-deuterium exchange (HX) in the Src homology region 3 (SH3) domain of alpha-spectrin has been measured by nuclear magnetic resonance as a function of temperature between 8 and 46 degrees C. The analysis of the temperature dependence of HX from a statistical thermodynamic point of view has allowed us to estimate the enthalpies and entropies of the conformational processes leading to HX. The results indicate that under native conditions the domain undergoes a wide variety of conformational fluctuations, ranging from local motions, mainly located in loops, turns and chain ends and involving only low enthalpy and entropy, to extensive structural disruptions affecting its core and involving enthalpies and entropies that come fairly close to those observed during global unfolding.

Deuterium↗

PubMed2002

pH dependence of the hydrogen exchange in the SH3 domain of alpha-spectrin.

Using nuclear magnetic resonance we have measured the hydrogen exchange (HX) in the Src homology region 3 (SH3) domain of alpha-spectrin as a function of pH*. At very acidic pH* values the exchange of most residues appears to occur via global unfolding, although several residues show abnormally large Gibbs energies of exchange, suggesting the presence of some residual structure in the unfolded state. At higher pH* HX occurs mainly via local or partial unfoldings. We have been able to characterize the coupling between the electrostatic interactions in this domain and the conformational fluctuations occurring under native conditions by analyzing the dependence upon pH* of the Gibbs energy of exchange. The SH3 domain seems to be composed of a central core, which requires large structural disruptions to become exposed to the solvent, surrounded by smaller subdomains, which fluctuate independently.

Deuterium↗

PubMed2001

CpG motifs of DNA vaccines induce the expression of chemokines and MHC class II molecules on myocytes.

Determining how an immune response is initiated after in vivo transfection of myocytes with plasmids encoding foreign antigens is essential to understand the mechanisms of intramuscular (i. m.) genetic immunization. Since myocytes are facultative antigen-presenting cells lacking MHC class II and co-stimulatory molecules, it was assumed that their unique role upon DNA vaccination is to synthesize and secrete the protein encoded by the plasmid. Here we describe that i. m. injection of unmethylated CpG motifs induced the expression of chemokines (monocyte chemotactic protein-1) and MHC class II molecules on myocytes. Our results indicate that immunostimulatory DNA sequences (CpG motifs) of DNA vaccines augment synthesis of chemokine by myocytes with subsequent recruitment of inflammatory cells secreting IFN-gamma, a potent cytokine that up-regulates the expression of MHC class II molecules on myocytes. A myoblast cell line triple transfected with plasmids encoding MHC class II molecules and an immunodominant CD4 T cell epitope of influenza virus presented the endogenously synthesized peptide and activated specific T cells. These findings suggest that one mechanism for the immunogenicity of DNA vaccines consists in the presentation of peptides to CD4 T cells by in vivo plasmid-transfected myocytes.

Animals↗

PubMed2001

Antigen-specific downregulation of T cells by doxorubicin delivered through a recombinant MHC II--peptide chimera.

As the number of drugs with potential therapeutic use for T-cell-mediated diseases increases, there is a need to find methods of delivering such drugs to T cells. The major histocompatibility complex (MHC)--peptide complexes are the only antigen-specific ligands for the T-cell receptor (TCR) expressed on T cells, and they may be an appropriate drug delivery system. We engineered a soluble bivalent MHC class II-peptide chimera on the immunoglobulin scaffold (I-E(d)alpha beta/Fc gamma 2a/HA110-120, DEF) that binds stably and specifically to CD4 T cells recognizing the HA110-120 peptide. Doxorubicin, a powerful antimitogenic anthracycline, was enzymatically assembled on the galactose residues of a DEF chimera. The DEF-gal-Dox construct preserved both the binding capacity to hemagglutinin (HA)-specific T cells, and the drug toxicity. Brief exposure of HA-specific T cells to DEF-gal-Dox construct in vitro was followed by drug internalization in the lysosomes, translocation to the nucleus, and apoptosis. Administration of DEF-gal-Dox to mice expressing the TCR-HA transgene reduced the frequency of TCR-HA T cells in the spleen and thymus by 27% and 42%, and inhibited HA proliferative capacity by 40% and 60%, respectively. It has not been demonstrated previously that pharmacologically active drugs able to modulate T-cell functions can be delivered to T cells in an antigen-specific manner by soluble, bivalent MHC II-peptide chimeras.

Animals↗

PubMed2001

Enzymatically mediated engineering of multivalent MHC class II-peptide chimeras.

We previously reported the genetic engineering of the first soluble, bivalent major histocompatibility complex (MHC) class II-peptide ligand for T-cell receptor (TCR). This ligand binds stably and specifically to cognate T-cells and exhibits immunomodulatory effects in vitro and in vivo. The increase in valence of MHC class II-peptide ligands was shown to parallel their avidity for cognate TCRs and potency in stimulating cognate T-cells. We describe a new enzymatic method to increase the valence of MHC-peptide ligands by cross-linking the N-glycan moieties of dimeric MHC II-peptide units through a flexible, bifunctional polyethylene glycol linker. Using this method, we generated covalently stabilized tetravalent and octavalent MHC II-peptide ligands which bound stably and specifically to cognate TCR and preserved their structural integrity in blood and lymphoid organs for 72 h. Depending on the TCR/CD4 occupancy and degree of TCR/CD4 co-clustering, the multivalent MHC II-peptide ligands polarized efficiently the antigen-specific CD4(+) T-cells toward type 2 cell differentiation or induced T-cell anergy and apoptosis. The enzymatically mediated engineering of multivalent MHC-peptide ligands for cognate TCRs may provide rational grounds for the development of new therapeutic agents endowed with strong modulatory effects on antigen-specific T-cells.

Animals↗

PubMed2001

Insights into the pathogenesis of type 1 diabetes: a hint for novel immunospecific therapies.

Type 1 diabetes is an organ-specific autoimmune disease whose incidence is increasing worldwide. At present, there is no effective therapy to prevent or cure this disease. The genetic background (MHC and non-MHC genes) and environmental factors (pathogens, drugs, and diet) are critical for the initiation of the autoimmune response against the pancreatic beta-cells. Recognition of the pancreatic autoantigens by T cells in a predetermined environment of antigen-presenting cells, costimulation, and cytokines is crucial for the selective activation of diabetogenic or protective/regulatory T cells. Once the autoimmune process is triggered, epitope spreading and sustaining the autoimmune responses by continuous antigen stimulation leads to expansion of effector cells, which launch the attack on the beta-cells. Despite of some controversy, most of the studies in humans and animal models suggest that CD4 (Th1) T cells are directly involved in the autoimmune attack by secretion of pro-inflammatory cytokines and recruitment of cytotoxic CD8 T cells. Secretion of anti-inflammatory cytokines by Th2 cells is protective against the disease. Therapy with peptides derived from major target antigens, such as glutamic acid decarboxylase 65 or proinsulin, can prevent the disease in animal models by rising protective Th2 cells. Herein, we review the recent progress in the immunopathogenesis of Type 1 diabetes and insights into the development of new diagnostic tools and antigen-specific immunomodulators, such as MHC-peptide chimeras.

Animals↗

PubMed2001

T-cell tolerance and autoimmune diabetes.

Herein we describe the major signaling events that occur in T-cells upon T-cell receptor (TCR) engagement, and the mechanisms responsible for the induction of T-cell anergy that may ultimately lead to the development of immunospecific therapies in T-cell mediated autoimmune diseases. A new type of antigen presenting molecule (dimeric MHC class-II/peptide, DEF) endowed with antigen-specific immunomodulatory effects such as induction of Th2 polarization and T-cell anergy is also described as a potential antidiabetogenic agent. According to our preliminary results, the MHC II/peptide-based approach may provide rational grounds for further development of antigen-specific immunotherapeutic agents such as human-like MHC lI/peptide chimeras endowed with efficient down-regulatory effects in CD4 T-cell-mediated autoimmune diseases such as Type 1 diabetes, multiple sclerosis, primary biliary cirrhosis, and rheumatoid arthritis.

Animals↗

PubMed2001

Modulation of CD4 T cell function by soluble MHC II-peptide chimeras.

Peptides antigens of 8 to 24 amino acid residues in length that are derived from processing of foreign proteins by antigen presenting cells (APC), and then presented to T cells in the context of major histocompatibility complex molecules (MHC) expressed by APC, are the only physiological ligands for T cell receptor (TCR). Co-ligation of TCR and CD4 co-receptor on T cells by MHC II-peptide complexes (signal 1) leads to various T cell functions depending on the nature of TCR and CD4 co-ligation, and whether costimulatory receptors (signal 2) such as CD28, CTLA-4, CD40L are involved in this interaction. Recently, the advance of genetic engineering led to the generation of a new class of antigen-specific ligands for TCR, i.e., soluble MHC class I-, and MHC class II-peptide chimeras. In principle, these chimeric molecules consist of an antigenic peptide which is covalently linked to the amino terminus of alpha-chain in the case of MHC I, or beta-chains in the case of MHC II molecules. Conceptually, such TCR/CD4 ligands shall provide the signal 1 to T cells. Since soluble MHC-peptide chimeras showed remarkable regulatory effects on peptide-specific T cells in vitro and in vivo, they may represent a new generation of immunospecific T cell modulators with potential therapeutic applicability in autoimmune and infectious diseases. This review is focused on the immunomodulatory effects of soluble, MHC class II-peptide chimeras, and discuss these effects in the context of the most accepted theories on T cell regulation.

Adjuvants, Immunologic↗

PubMed1999

Antigen-specific signaling by a soluble, dimeric peptide/major histocompatibility complex class II/Fc chimera leading to T helper cell type 2 differentiation.

Interaction between a T cell receptor (TCR) and various ligands, i.e. , anti-TCR antibodies, superantigens, peptides, or altered peptide ligands in the context of major histocompatibility complex (MHC) molecules can trigger different T helper cell (Th) effector functions. Herein, we studied the T cell response induced by a soluble, dimeric peptide/MHC class II chimera, namely hemagglutinin (HA)110-120/I-E(d)alphabeta/Fcgamma2a (DEF). We have previously demonstrated that the soluble DEF molecule binds stably and specifically to HA110-120-specific TCRs expressed by a T cell hybridoma. Administration of DEF in vivo induced differentiation of resting and activated peptide-specific T cells toward a Th2 response, as indicated by the increase of interleukin (IL)-4, IL-10, and specific immunoglobulin (Ig)G1 antibodies and decrease of IL-2, specific IgG2a antibodies, and cytotoxic T lymphocyte activity. In contrast to HA110-120 peptide presented by the DEF molecule to T cells, the nominal synthetic peptide induced a predominant Th1 response, and the PR8 virus-derived HA110-120 peptides induced a mixed Th1/Th2 response. Independent of antigen processing, soluble DEF was almost 2 logs more potent in stimulating cognate T cells than the nominal peptide. Polarization of cognate T cells toward the Th2 response occurred upon interaction of soluble DEF with TCR and CD4 molecules followed by early activation of p56(lck) and ZAP-70 tyrosine kinases, and negative signaling of the signal transducer and activator of transcription (STAT)4 pathway of Th1 differentiation. DEF-like molecules may provide a new tool to study the mechanisms of signaling toward Th2 differentiation and may also provide a potential immunotherapeutic approach to modulate autoreactive T cells toward protective Th2 immune responses.

Animals↗

PubMed1999

The native state conformational ensemble of the SH3 domain from alpha-spectrin.

The folding/unfolding equilibrium of the alpha-spectrin SH3 domain has been measured by NMR-detected hydrogen/deuterium exchange and by differential scanning calorimetry. Protection factors against exchange have been obtained under native conditions for more than half of the residues in the domain. Most protected residues are located at the beta-strands, the short 3(10) helix, and part of the long RT loop, whereas the loops connecting secondary structure elements show no measurable protection. Apparent stability constants per residue and their corresponding Gibbs energies have been calculated from the exchange experiments. The most stable region of the SH3 domain is defined by the central portions of the beta-strands. The peptide binding region, on the other hand, is composed of a highly stable region (residues 53-57) and a highly unstable region, the loop between residues 34-41 (n-Src loop). All residues in the domain have apparent Gibbs energies lower than the global unfolding Gibbs energy measured by differential scanning calorimetry, indicating that under our experimental conditions the amide exchange of all residues in the SH3 domain occurs primarily via local unfolding reactions. A structure-based thermodynamic analysis has allowed us to predict correctly the thermodynamics of the global unfolding of the domain and to define the ensemble of conformational states that quantitatively accounts for the observed pattern of hydrogen exchange protection. These results demonstrate that under native conditions the SH3 domain needs to be considered as an ensemble of conformations and that the hydrogen exchange data obtained under those conditions cannot be interpreted by a two-state equilibrium. The observation that specific regions of a protein are able to undergo independent local folding/unfolding reactions indicates that under native conditions the scale of cooperative interactions is regional rather than global.

Calorimetry, Differential Scanning↗

PubMed1999

Antineoplastic efficacy of doxorubicin enzymatically assembled on galactose residues of a monoclonal antibody specific for the carcinoembryonic antigen.

We have developed a novel procedure to couple enzymatically the antineoplastic agent doxorubicin (Dox) on the galactose residues of a monoclonal antibody specific for the tumor-associated carcinoembryonic antigen. The synthesis of the immunoconjugate consists of covalent attachment of the NH2 terminus of Dox to oxidized galactose residues of desialylated monoclonal antibody, followed by concurrent stabilization of Schiff bases by mild reduction with pyridine borane. The immunoconjugate preserved both antibody specificity and drug cytotoxicity. At equimolar concentrations, the immunoconjugate was 8 times more cytotoxic against two carcinoembryonic antigen-expressing carcinoma cell lines, LoVo and SW-480, than Dox alone. The intracellular drug accumulation was 8-8.5 times higher than that obtained with free Dox, and >50% of the drug delivered by the conjugate was retained for 24 h in the tumor cells. Only 4 days after treatment with a single dose of immunoconjugate carrying 2.5 ng of Dox, LoVo and SW-480 tumor transplants on the chorioallantoic membrane of embryonated hen eggs showed reduced tumor-induced angiogenesis and tumor progression by half, with no detectable damage to surrounding tissues. In contrast, the same amount of free drug induced insignificant changes in tumor progression and tumor-induced angiogenesis. Enzymatically mediated, glycosidic coupling of antineoplastic agents to antibodies specific for tumor-associated antigens may represent a novel platform for the development of more efficient anticancer agents with reduced side effects.

Animals↗

PubMed1999

Characterization of mutated protein encoded by partially duplicated fibrillin-1 gene in tight skin (TSK) mice.

Fibrillin-1 (Fbn-1) is a ubiquitous protein present in the extracellular matrix of various organs and it is a major component of microfibrils embedded in the core of elastic fibers. In humans, mutations or deletions of the Fbn-1 gene are associated with several genetic diseases. In addition, several microsatellite alleles near Fbn-1 gene were found associated with diffuse scleroderma. In TSK/+ mice, which develop a scleroderma-like syndrome, the Fbn-1 gene exhibits an inframe duplication of exons 17-40. In this study, we report that the synthesis and secretion of wild-type Fbn-1 in TSK/+ is higher than that of the mutated Fbn-1 protein excluding the possibility that TSK genetic defect is due to a loss of the wild allele. We also demonstrate for the first time that TGF-beta, which plays a crucial role in skin fibrosis, binds to both wild-type and mutated Fbn-1. The amount of bound TGF-beta was higher in mutated than wild-type Fbn-1 and appears related to the number of TGF-beta binding motifs.

Animals↗

PubMed1999

Induction of antibody response by DNA immunization of newborn baboons against influenza virus.

Previous studies showed that DNA immunization of newborn mice with plasmids expressing influenza virus antigens induced protective immunity. We have now extended the study of neonatal responsiveness to DNA vaccines to nonhuman primates. Baboons immunized as neonates with plasmids expressing type A influenza virus hemagglutinin (HA) and nucleoprotein (NP) in doses ranging from 40 microg to 1 mg per plasmid per dose developed virus-specific humoral responses. The titer and kinetics of appearance of virus-specific IgG antibodies were dose dependent. Specific antibodies were detected by enzyme-linked immunosorbent assay (ELISA) as early as 1 month after birth in baboons immunized with the highest and intermediate doses of vaccine. Virus-neutralizing antibodies were detected in the group of baboons immunized with the highest dose. The specificity of virus-neutralizing antibodies was found to be directed against homologous determinants of HA; however, the IgG antibodies also cross-reacted with HA of a drift variant. Thus, DNA vaccination of newborn baboons with a prototype vaccine against influenza virus resulted in induction of specific humoral immunity.

Animals↗

PubMed1999

Escape from self-tolerance leads to neonatal insulin-dependent diabetes mellitus.

Double transgenic (dTg) mice expressing the hemagglutinin (HA) of influenza virus under the insulin promoter and the TCR specific for the immunodominant CD4 T cell epitope of HA (HA110-120) develop insulin-dependent diabetes mellitus (IDDM). In order to gain information on the breaking down of neonatal self-tolerance we studied the occurrence of IDDM after birth. Our results showed that newborn mice develop fulminant IDDM characterized by occurrence of insulitis as early as 3 days after birth, followed by hyperglycemia by 7 days, and significant hypoinsulinemia by 28 days. The neonatal breakdown of self-tolerance of T cells positively selected in the thymus is supported by the facts that: (i) peripheral HA110-120 specific T cells from neonates are fully functional and proliferated upon stimulation with the nominal peptide, and (ii) peptide-specific T cells were accumulated in the pancreas of dTg mice as early as 3 days after birth. Our results demonstrate that diabetes occurring in young dTg mice is due to early activation of self-reactive T cells immediately after birth. Accumulation of specific T cells in the target organ leads to destruction of pancreatic beta-cells and IDDM. These mice may provide a useful model to evaluate new strategies for the prevention of diabetes.

Animals↗

PubMed1998

Cellular mechanisms involved in protection against influenza virus infection in transgenic mice expressing a TCR receptor specific for class II hemagglutinin peptide in CD4+ and CD8+ T cells.

Mice transgenic for a TCR that recognizes peptide110-120 of hemagglutinin of PR8 influenza virus in the context of MHC class II I-Ed molecules express the transgenes in both CD4+ and CD8+ T cells. We have found that these TCR-hemagglutinin (TCR-HA) transgenic mice display a significantly increased resistance to the primary infection with PR8 virus compared with the wild-type mice. The TCR-HA transgenic mice mounted significant MHC type II and enhanced MHC type I-restricted cytotoxicity as well as increased cytokine responses in both spleen and lungs after infection with PR8 virus. In contrast, the primary humoral response against PR8 virus was not significantly different from that of the wild-type mice. In vivo depletion and adoptive cell transfer experiments demonstrated that both CD4+ and CD8+ TCR-HA+ T cell subsets were required for the complete clearance of pulmonary virus following infection with a dose that is 100% lethal in wild-type mice. Whereas CD4+ TCR-HA+ T cells were necessary for effective activation and local recruitment of CD8+ T cells, CD8+ TCR-HA+ T cells showed a Th1-biased pattern and MHC type II-restricted cytotoxicity. However, in the absence of in vivo expression of MHC type I molecules on the infected cells, the protection conferred by the TCR-HA+ T cells was impaired, indicating that the enhanced MHC class I-restricted cytotoxicity due to TCR-HA+ CD4+ Th cells was a critical element for clearance of the pulmonary virus by the transgenic mice.

Animals↗