Catedra de Inmunologia

Cabral, Argentina

Catedra de Inmunologia

Cabral, Argentina

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Giglio J.,Catedra de Radioquimica | Fernandez S.,Catedra de Radioquimica | Pietzsch H.-J.,Helmholtz Center Dresden | Dematteis S.,Catedra de Inmunologia | And 3 more authors.
Nuclear Medicine and Biology | Year: 2012

The evaluation of oxygenation status of solid tumors is an important field of radiopharmaceutical research. With the aim to develop new potential 99mTc-radiopharmaceuticals for imaging hypoxia, we have synthesized two novel isocyanide derivatives of metronidazole, which has demonstrated high affinity for hypoxic tumors in vitro and in vivo. Methods: Metronidazole derivatives 4-isocyano-N-[2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl]butanamide (M1) and 1-(4-isocyanobutanoyl)-4-[2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl]piperazine (M2) were synthesized, and labeling was performed through preparation of their corresponding 99mTc-(4+1) complexes, 99mTc-NS3M1 and 99mTc-NS3M2. The structure of the technetium complexes was corroborated by preparation and characterization of the corresponding rhenium complexes. We have studied the main physicochemical properties (stability, lipophilicity and plasma protein binding). Biological behavior in HCT-15 cells both in oxia and in hypoxia was assessed. Biodistribution in normal mice and in animals bearing induced 3LL Lewis murine lung carcinoma was also studied. Results: Metronidazole derivatives were successfully synthesized. Labeling with high radiochemical purity was achieved for both ligands. 99mTc complexes were stable in labeling milieu and human plasma. However, presence of the piperazine linker in M2 resulted in higher lipophilicity and protein binding. Although cell uptake in hypoxic conditions was observed for both radiotracers, 99mTc-NS3M2 biodistribution was considered unsuitable for a potential radiopharmaceutical due to high liver uptake and poor blood clearance. However, 99mTc-NS3M1 demonstrated a very favorable in vivo profile both in normal mice and in mice bearing induced tumors. Conclusion: Selective uptake and retention in tumor together with favorable tumor/muscle ratio make 99mTc-NS3M1 a promising candidate for further evaluation as potential hypoxia imaging agent in tumors. © 2012 Elsevier Inc..


Kim H.-J.,University of California at Davis | McCoy M.R.,University of California at Davis | Majkova Z.,University of California at Davis | Dechant J.E.,University of California at Davis | And 4 more authors.
Analytical Chemistry | Year: 2012

Some unique subclasses of Camelidae antibodies are devoid of the light chain, and the antigen binding site is comprised exclusively of the variable domain of the heavy chain (VHH). Although conventional antibodies dominate current assay development, recombinant VHHs have a high potential as alternative reagents for the next generation of immunoassay. We expressed VHHs from an immunized alpaca and developed a VHH-based immunoassay using 3-phenoxybenzoic acid (3-PBA), a major metabolite of pyrethroid insecticides as a model system. A phage VHH library was constructed, and seven VHH clones were selected by competitive binding with 3-PBA. The best immunoassay developed with one of these VHHs showed an IC 50 of 1.4 ng/mL (limit of detection (LOD) = 0.1 ng/mL). These parameters were further improved by using the phage borne VHH, IC 50 = 0.1 ng/mL and LOD = 0.01 ng/mL. Both assays showed a similar tolerance to methanol and dimethylsulfoxide up to 50% in assay buffer. The assay was highly specific to 3-PBA and its 4-hydroxylated derivative, 4-hydroxy 3-PBA, (150% cross reactivity) with negligible cross reactivity with other tested structural analogues, and the recovery from spiked urine sample ranged from 80 to 112%. In conclusion, a highly specific and sensitive VHH for 3-PBA was developed using sequences from immunized alpaca and phage display technology for antibody selection. © 2011 American Chemical Society.


The aim of this work consisted to study the clinical and hematological effects that included blood coagulation and antibody production of a group of three horses immunized for the production of monovalent serum against Bothrops alternatus venom, in order to correlate the results with the humoral immune response of the animals. The results showed that immunization of horses with the essayed protocol induce slight clinic alterations and discrete changes in some parameters of the hemogram. Results also showed variations in the neutralizing capacity of the serum on different venom components, particularly toxins that affect blood coagulation. Also, it was verified a direct relationship between the rate of white blood cells and serum immunoglobulin production. Furthermore, they also indicate that the three-month rest period may be considered as too long as it causes a marked decrease in antibody titles, losing the serum its neutralizing capacity to venom compounds. Nevertheless, after re-administration of venom horses provided a rapid reposition of anti-toxins against B. alternatus venom, tolerating high venom doses during this second period of immunization.


Rossotti M.A.,Catedra de Inmunologia | Carlomagno M.,Catedra de Inmunologia | Gonzalez-Techera A.,Catedra de Inmunologia | Hammock B.D.,Cancer Center | And 2 more authors.
Analytical Chemistry | Year: 2010

The impact of the use of herbicides in agriculture can be minimized by compliance with good management practices that reduce the amount used and their release into the environment. Simple tests that provide real time on-site information about these chemicals are a major aid for these programs. In this work, we show that phage anti-immunocomplex assay (PHAIA), a method that uses phage-borne peptides to detect the formation of antibody-analyte immunocomplexes, is an advantageous technology to produce such field tests. A monoclonal antibody to the herbicide clomazone was raised and used in the development of conventional competitive and noncompetitive PHAIA immunoassays. The sensitivity attained with the PHAIA format was over 10 times higher than that of the competitive format. The cross-reactivity of the two methods was also compared using structurally related compounds, and we observed that the two-site binding of PHAIA "double-checks" the recognition of the analyte, thereby increasing the assay specificity. The positive readout of the noncompetitive PHAIA method allowed adaptation of the assay into a rapid and simple format where as little as 0.4 ng/mL clomazone (more than 10-fold lower than the proposed standard) in water samples from a rice field could be easily detected by simple visual inspection. © 2010 American Chemical Society.


Iriarte A.,Laboratorio Of Organizacion Y Evolucion Del Genoma | Iriarte A.,Laboratorio Of Evolucion | Arbildi P.,Catedra de Inmunologia | La-Rocca S.,Catedra de Inmunologia | And 2 more authors.
Acta Tropica | Year: 2012

Glutathione transferase enzymes (GSTs) constitute a major detoxification system in helminth parasites and have been related to the modulation of host immune response mechanisms. At least three different GSTs classes have been described in Platyhelminthes: Mu, Sigma and Omega. Mining the genome of . Echinococcus multilocularis and the ESTs databases of . Taenia solium and . E. . granulosus identified two new GSTs from the cestode . E. granulosus, named EgGST2 and EgGST3. It also revealed that the Omega class of GSTs was absent from the Taenidae family. EgGST2 and EgGST3 are actively expressed in the parasite. In order to know the origin of these new GSTs, . in silico analyses were performed. While EgGST2 is classified as belonging to the Sigma class, the data obtained for EgGST3 allowed a less clear interpretation. The study of the evolutionary relatedness based on the C-terminal domain sequence, gene structure conservation and three-dimensional structure predictions, suggests that EgGST3 is derived from the Platyhelminthes' Sigma-class cluster. Interestingly, the N-terminal domain displays some characteristic Omega-class residues, including a Cys residue that is likely to be involved in the catalytic mechanism. We discuss different evolutionary scenarios that could explain the observed patterns. © 2012 Elsevier B.V..


Harispe L.,Seccion Bioquimica | Garcia G.,Catedra de Inmunologia | Arbildi P.,Catedra de Inmunologia | Pascovich L.,Catedra de Inmunologia | And 4 more authors.
Acta Tropica | Year: 2010

Glutathione transferases (GSTs) are believed to be a major detoxification system in helminths. We describe the expression and functional analysis of EgGST, a cytosolic GST from Echinococcus granulosus, related to the Mu-class of mammalian enzymes. EgGST was produced as an enzymatically active dimeric protein (rEgGST), with highest specific activity towards the standard substrate 1-chloro-2,4-dinitrobenzene (CDNB; 2.5 μmol min -1 mg -1), followed by ethacrynic acid. Interestingly, rEgGST displayed glutathione peroxidase activity (towards cumene hydroperoxide), and conjugated reactive carbonyls (trans-2-nonenal and trans,trans-2,4-decadienal), indicating that it may intercept damaging products of lipid peroxidation. In addition, classical GST inhibitors (cybacron blue, triphenylthin chloride and ellagic acid) and a number of anthelmintic drugs (mainly, hexachlorophene and rafoxanide) were found to interfere with glutathione-conjugation to CDNB; suggesting that they may bind to EgGST. Considered globally, the functional properties of rEgGST are similar to those of putative orthologs from Echinococcus multilcularis and Taenia solium, the other medically important cestodes. Interestingly, our results also indicate that differences exist between these closely related cestode GSTs, which probably reflect specific biological functions of the molecules in each parasitic organism. © 2009 Elsevier B.V. All rights reserved.


Ferreira A.M.,Catedra de Inmunologia | Minarrieta L.,Catedra de Inmunologia | Lamas Bervejillo M.,Catedra de Inmunologia | Rubbo H.,University of the Republic of Uruguay
Free Radical Biology and Medicine | Year: 2012

Lipid nitration has been observed during oxidative/nitrative stress conditions generating a variety of biomolecules capable of modulating cellular responses. This concept has grown as a result of studies with nitro-derivatives of long-chain unsaturated fatty acids containing a nitroalkene group (nitro-fatty acids). This review focuses on the interactions of nitro-fatty acids with members of the peroxisome proliferator-activated receptors (PPARs) family. These nuclear receptors belong to a superfamily of ligand-activated transcription factors, which serve as sensors of lipophilic molecules and regulate the expression of a set of genes involved in glucose and lipid metabolism. Here we discuss how nitro-fatty acids bind and activate PPARs, including the current knowledge of the molecular interactions and cell signaling events involved as well as their therapeutic potential associated with chronic inflammation and metabolic disorders. © 2012 Elsevier Inc. All rights reserved.


Saiz C.,University of the Republic of Uruguay | Castillo V.,University of the Republic of Uruguay | Fontan P.,University of the Republic of Uruguay | Bonilla M.,Catedra de Inmunologia | And 3 more authors.
Molecular Diversity | Year: 2014

In this study, we report a strategy using dynamic combinatorial chemistry for targeting the thioredoxin (Trx)-reductase catalytic site on Trx glutathione reductase (TGR), a pyridine nucleotide thiol-disulfide oxido-reductase. We chose Echinococcus granulosus TGR since it is a bottleneck enzyme of platyhelminth parasites and a validated pharmacological target. A dynamic combinatorial library (DCL) was constructed based on thiol-disulfide reversible exchange. We demonstrate the use of 5-thio-2-nitrobenzoic acid (TNB) as a non-covalent anchor fragment in a DCL templated by E. granulosus TGR. The heterodimer of TNB and bisthiazolidine (2af) was identified, upon library analysis by HPLC (IC -{50} 50 = 24 \upmu μ M). Furthermore, 14 analogs were synthetically prepared and evaluated against TGR. This allowed the study of a structure-activity relationship and the identification of a disulfide TNB-tricyclic bisthiazolidine (2aj) as the best enzyme inhibitor in these series, with an IC -{50} 50 = 14 \upmu μ M. Thus, our results validate the use of DCL for targeting thiol-disulfide oxido-reductases. © 2013 Springer Science+Business Media Dordrecht.


Carlomagno M.,Catedra de Inmunologia | Lassabe G.,Catedra de Inmunologia | Rossotti M.,Catedra de Inmunologia | Gonzalez-Techera A.,Catedra de Inmunologia | And 2 more authors.
Analytical Chemistry | Year: 2014

Short peptide loops selected from phage libraries can specifically recognize the formation of hapten-antibody immunocomplexes and can thus be used to develop phage anti-immunocomplex assays (PHAIA) for noncompetitive detection of small molecules. In this study, we generated recombinant chimeras by fusing anti-immunocomplex peptides selected from phage libraries to the N- or C-termini of core streptavidin and used them to setup phage-free noncompetitive assays for the herbicide clomazone (MW 240 Da). The best conditions for refolding were optimized by a high throughput screening allowing to obtain tens of mg of purified protein per liter of culture. The noncompetitive assay developed with these chimeras performed with a 50% saturating concentration (SC50) of 2.2 ± 0.3 ng/mL and limit of detection (LOD) of 0.48 ng/mL. Values that are 13- and 8-fold better that those obtained for the SC50 and LOD of the competitive assay setup with the same antibody. Apart from the first demonstration that recombinant peptide-streptavidin chimeras can be used for sensitive immunodetection of small molecules with a positive readout, this new assay component is a highly standardized reagent with a defined stoichiometry, which can be used in combination with the broad option of existing biotinylated reagents offering a great versatility for the development of conventional immunoassay and biosensors. The utility of the test was demonstrated analyzing the clomazone runoff during the rice growing season in northern Uruguay. © 2014 American Chemical Society.


PubMed | Catedra de Inmunologia
Type: | Journal: Molecular and biochemical parasitology | Year: 2016

In the cestode parasite Echinococcus granulosus, three phylogenetically distant cytosolic glutathione transferases (GSTs) (EgGST1, 2 and 3) were identified. Interestingly, the C-terminal domains of EgGST3 and EgGST2 but not EgGST1, exhibit all amino acids involved in Sigma-class GST dimerization. Here, we provide evidence indicating that EgGST2 and EgGST3 naturally form a heterodimeric structure (EgGST2-3), and also we report the enzymatic activity of the recombinant heterodimer. EgGST2-3 might display novel properties able to influence the infection establishment. This is the first report of a stable heterodimeric GST built up by phylogenetically distant subunits.

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