Presencia del marcador lmp-1 del virus epstein-barr en linfomas de caninos / Presence of lmp-1 protein of epstein-barr virus in lymphomas of canine origin

RESUMEN El virus Epstein-Barr (EBV) es un virus de alta prevalencia en humanos que se asocia con tumores de la línea linfoide B. En caninos se dispone de pocos reportes sobre la presencia del EBV y su rol en esta especie. El objetivo del presente estudio fue determinar la presencia de la proteína latente de membrana del EBV (LMP-1) en tejidos obtenidos de 20 linfomas de caninos cuyo diagnóstico se había realizado durante un periodo de 10 años, entre 2004 y 2014. Los linfomas se reclasificaron mediante las nuevas clasificaciones histopatológicas para linfomas y se sometieron a inmunohistoquímica (IHQ) con los anticuerpos anti-CD79a, anti-CD3, anticuerpos específicos para linfocitos B y T, además de un anti-LMP-1 como marcador de la presencia del EBV. Se encontró que el linfoma más común fue el linfoma nodal de zona T con un 75% de los casos. Al realizar la inmunomarcación se encontraron 18 casos positivos a CD3, 2 casos positivos a CD79a y 6 casos positivos a LMP-1, lo que representa el 30% de positividad del EBV en linfomas. El análisis Ji cuadrado demostró significancia estadística entre la presencia del virus y la presencia del linfoma lo que sugiere, no solamente que el virus está circulando en la población canina, sino que además puede tener relación con la ocurrencia de esta neoplasia. Con relación a las variables demográficas, sólo en la raza Golden Retriever se demostró relación con la presencia del linfoma, pero no con la presencia del virus.

ABSTRACT Epstein Barr virus (EBV) is a human high prevalent virus associated with lymphoid B cells tumors development. In canines, few reports have been published regarding the presence of the virus in dogs but its role in this species remains unclear. The aim of this study was to determine the presence of LMP-1 protein of EBV in 20 canine lymphomas tissues which were previously diagnosed in a period of time between 2004 -2014. Lymphomas were reclassified in accordance with the new histopathological classifications for lymphomas and were stained by IHQ with anti-CD79a, anti-CD3 and anti-LMP-1; in addition, specific antibodies for B lymphocytes, T lymphocytes and EBV biomarker, respectively. It was found that the most common lymphoma was T-zone lymphoma in 75% of the cases of the study. The distribution of the cases regarding the immunostaining was: 18 positive cases with anti-CD3, 2 positive cases with anti-CD79a and 6 positive cases with anti- LMP-1. Positive cases of LMP-1 as a biomarker of the presence of EBV corresponded to the 30% of the cases of the study. Chi-square test showed statistical significance between the presence of the virus and the presence of lymphomas, which suggests not only that the virus is circulating in the canine population but also that could have implications in the development of the disease. Regarding demographic parameters, only the Golden Retriever breed showed a relationship with the presence of lymphoma, but not with the presence of the virus.

Immunological and structural characterization of an epitope from the Trypanosoma cruzi KMP-11 protein.

The K1 peptide is an HLA-A*0201-restricted cytotoxic epitope derived from the Trypanosoma cruzi KMP-11 protein, this being the etiological agent of Chagas' disease. This work describes the K1 peptide's secondary structure and its recognition by sera from chagasic patients. Circular dichroism and NMR spectroscopy analysis revealed that the K1 peptide adopts an alpha-helical conformation. Fifty-six percent of individuals had anti-K1 and 86% anti-KMP-11 antibodies by ELISA in the chronic Chagas' group and 28 and 68% in the indeterminate Chagas' group, respectively. By contrast, no reactivity was observed in sera from healthy individuals and tuberculosis patients. Antibody response subclass specificity to the K1 peptide was IgG1 and IgG3. Taken together these results support the idea that the K1 peptide acts as a B-cell-inducer epitope during Chagas' disease.

Evaluation of IFN-gamma production by CD8 T lymphocytes in response to the K1 peptide from KMP-11 protein in patients infected with Trypanosoma cruzi.

The cellular response mediated by MHC class I restricted CD8+ T cells has been shown to be crucial in the control of Chagas disease. The K1 peptide derived from T. cruzi KMP-11 protein has a high binding affinity to the HLA-A*0201 molecule. Nevertheless, it is not known whether this peptide is processed and displayed as an MHC class I epitope during natural infection by T. cruzi. The aim of this study was to evaluate, by ELISPOT assay, the ability of K1 peptide to activate CD8+ T lymphocytes to produce IFN-gamma. Therefore, CD8+ T lymphocytes from 22 HLA-A*0201+ individuals, 12 chronic chagasic patients and 10 uninfected controls, were analysed. The results revealed that two of the chagasic patients had IFN-gamma-secreting CD8+ T cells that were able to respond to K1 peptide with a relative frequency of 110 and 230 per million CD8+ T cells. In contrast, none of HLA-A*0201+ uninfected controls responded to K1 peptide. Responses to HLA-A*0201 restricted peptide from the influenza matrix protein were found in six chagasic patients and four uninfected controls with an average frequency of 175 and 111 cells per million CD8+ T cells, respectively. Moreover, a flow cytometric assay for degranulation showed that chagasic responders had K1-specific cytotoxic CD8+ T cells. It is shown here for the first time that the K1 peptide is efficiently processed, presented and recognized by CD8+ T lymphocytes during the natural course of Chagas disease.

Molecular characterization of the kinetoplastid membrane protein-11 genes from the parasite Trypanosoma rangeli.

Trypanosomatids are early divergent parasites which include several species of medical interest. Trypanosoma rangeli is not pathogenic for humans but shows a high immunological cross-reactivity with Trypanosoma cruzi, the causative agent of Chagas' disease that affects more than 17 million people throughout the world. Recent studies have suggested that T. cruzi KMP-11 antigen could be a good candidate for the induction of immunoprotective cytotoxic responses against T. cruzi natural infection. In the present paper the genes coding for the T. rangeli kinetoplastid membrane protein-11 have been characterized. The results show that the locus encoding this protein is formed by 4 gene units measuring 550 nucleotides in length, organized in tandem, and located in different chromosomes in KP1(+) and KP1(-) strains. The gene units are transcribed as a single mRNA of 530 nucleotides in length. Alignment of the T. rangeli KMP-11 deduced amino acid sequence with the homologous KMP-11 protein from T. cruzi revealed an identity of 97%. Interestingly, the T and B cell epitopes of the T. cruzi KMP-11 protein are conserved in the T. rangeli KMP-11 amino acid sequence.