Generation of anti-idiotypic reagents in the EGFRvIII tumor-associated antigen system

The use of anti-idiotype (anti-id) vaccines for immunotherapy of human cancers is attractive, as immunization with true anti-id reagents (Ab2 beta) has been shown to induce both cellular and humoral immunity, frequently when the original antigen does not, or when a state of anergy to the self-expressed tumor-associated antigen exists. The aim of this study was to investigate the potential of an anti-id vaccine approach to the glioma-associated antigen epidermal growth factor receptor variant III (EGFRvIII) for human clinical trials. By using conventional methodology, seven rat mAbs specific for the binding site of the murine anti-EGFRvIII-specific mAb Y10, as defined by the ability to inhibit the binding of mAb Y10 to EGFRvIII expressed on cells or as purified protein, were generated, and a subset (3/7) was found to be true Ab2 beta, as defined by the ability to induce the formation of antibody directed against EGFRvIII in two species (mouse and rabbit) when used as immunogen. The ability of these three Ab2 beta to elicit a protective anti-tumor response when used as a vaccine in the syngeneic, subcutaneous C57Bl/6-B16mseEGFRvIII tumor model was investigated. Following vaccination with one Ab2 beta mAb (2C7), 6/20 mice failed to develop tumor upon challenge, and 3/20 mice with outgrowing tumors exhibited dramatic regression of incipient tumors. Vaccination with a second mAb (5G8) resulted in one tumor-free survivor and one tumor regressor; vaccination with the third Ab2 beta mAb (7D3) did not confer protection, but did significantly increase the latency period until tumor outgrowth in all vaccinated recipients. The ability of Ab2 beta mAb 2C7 to induce an anti-EGFRvIII response in non-human primates was investigated by using the saponin adjuvant approved for human clinical trial, QS-21. Three of three macaques produced anti-EGFRvIII titers, as detected on EGFRvIII-expressing cells by both ELISA and fluorescence-activated cytometric analysis, following six immunizations with Ab2 beta mAb 2C7 and QS-21. The results obtained confirm that an anti-id response in the EGFRvIII antigen system can be induced in rodents, rabbits, and non-human primates, and it may prove a useful adjunct to immunotherapeutic approaches to EGFRvIII-positive gliomas, breast carcinomas, and non-small-cell lung tumors.     

Statistical Analysis of Multiplex Brain Gene Expression Images

Analysis of variance (ANOVA) was employed to investigate 9,000 gene expression patterns from brains of both normal mice and mice with a pharmacological model of Parkinson's disease (PD). The data set was obtained using voxelation, a method that allows high-throughput acquisition of 3D gene expression patterns through analysis of spatially registered voxels (cubes). This method produces multiple volumetric maps of gene expression analogous to the images reconstructed in biomedical imaging systems. The ANOVA model was compared to the results from singular value decomposition (SVD) by using the first 42 singular vectors of the data matrix, a number equal to the rank of the ANOVA model. The ANOVA was also compared to the results from non-parametric statistics. Lastly, images were obtained for a subset of genes that emerged from the ANOVA as significant. The results suggest that ANOVA will be a valuable framework for insights into the large number of gene expression patterns obtained from voxelation.     

Interactions of inositol 1,4,5-trisphosphate (IP(3)) receptors with synthetic poly(ethylene glycol)-linked dimers of IP(3) suggest close spacing of the IP(3)-binding sites

The distances between the inositol 1,4,5-trisphosphate (IP(3))-binding sites of tetrameric IP(3) receptors were probed using dimers of IP(3) linked by poly(ethylene glycol) (PEG) molecules of differing lengths (1-8 nm). Each of the dimers potently stimulated (45)Ca(2+) release from permeabilized cells expressing predominantly type 1 (SH-SY5Y cells) or type 2 (hepatocytes) IP(3) receptors. The shortest dimers, with PEG linkers of an effective length of 1.5 nm or less, were the most potent, being 3-4-fold more potent than IP(3). In radioligand binding experiments using cerebellar membranes, the shortest dimers bound with highest affinity, although the longest dimer (8 nm) also bound with almost 4-fold greater affinity than IP(3). The affinity of monomeric IP(3) with only the PEG attached was 2-fold weaker than IP(3), confirming that the increased affinity of the dimers requires the presence of both IP(3) motifs. The increased affinity of the long dimer probably results from the linked IP(3) molecules binding to sites on different receptors, because the dimer bound with greater affinity than IP(3) to cerebellar membranes, where receptors are densely packed, but with the same affinity as IP(3) to purified receptors. IP(3) and the IP(3) dimers, irrespective of their length, bound with similar affinity to a monomeric IP(3)-binding domain of the type 1 IP(3) receptor expressed in bacteria. Short dimers therefore bind with increased affinity only when the receptor is tetrameric. We conclude that the four IP(3)-binding sites of an IP(3) receptor may be separated by as little as 1.5 nm and are therefore likely to be placed centrally in this large (25 x 25 nm) structure, consistent with previous work indicating a close association between the central pore and the IP(3)-binding sites of the IP(3) receptor.     

Processing of an apicoplast leader sequence in Plasmodium falciparum and the identification of a putative leader cleavage enzyme

The plastid (apicoplast) of the malaria-causing parasite Plasmodium falciparum was derived via a secondary endosymbiotic process. As in other secondary endosymbionts, numerous genes for apicoplast proteins are located in the nucleus, and the encoded proteins are targeted to the organelle courtesy of a bipartite N-terminal extension. The first part of this leader sequence is a signal peptide that targets proteins to the secretory pathway. The second, so-called transit peptide region is required to direct proteins from the secretory pathway across the multiple membranes surrounding the apicoplast. In this paper we perform a pulse-chase experiment and N-terminal sequencing to show that the transit peptide of an apicoplast-targeted protein is cleaved, presumably upon import of the protein into the apicoplast. We identify a gene whose product likely performs this cleavage reaction, namely a stromal-processing peptidase (SPP) homologue. In plants SPP cleaves the transit peptides of plastid-targeted proteins. The P. falciparum SPP homologue contains a bipartite N-terminal apicoplast-targeting leader. Interestingly, it shares this leader sequence with a Delta-aminolevulinic acid dehydratase homologue via an alternative splicing event.     

Programmed cell death in the developing limb

The sculpturing of shape in the developing limb together with the regression of the tail in anuran tadpoles constitute, perhaps, the most paradigmatic processes of programmed cell death. The study of these model systems has been of fundamental importance to support the idea that cell death is a physiological behavior of cells in multicellular organisms. Furthermore, different experimental approaches, including comparative analyses of the pattern of cell death in different avian species (i.e. chick interdigits versus duck interdigital webs) and in chick mutants with different limb phenotypes, provided the first evidence for the occurrence of a genetic program underlying the control of cell death. Two well known research groups in the field of limb development, the USA group headed first by John Saunders and next by John Fallon and the group of Donald Ede and Richard Hinchliffe in the U.K. provided a remarkable contribution to this topic. In spite of the historical importance of the developing limb in establishing the concept of programmed cell death, this model system of tissue regression has been largely neglected in recent studies devoted to the analysis of the molecular control of self-induced cell death (apoptosis). However, a considerable amount of information concerning this topic has been obtained in the last few years. Here we will review current information on the control of limb programmed cell death in an attempt to stimulate further molecular studies of this process of tissue regression.     

Stage of the estrous cycle at the time of pregnant mare's serum gonadotropin injection affects pre-implantation embryo development in vitro in the mouse

The present study aims to analyze in the mouse the effect of the stage of the estrous cycle at the time of pregnant mare's serum gonadotropin (PMSG) injection on fertilization of ovulated cumulus-enclosed oocytes and later embryo development in vitro to the blastocyst stage. Quality of blastocysts was evaluated by staining and counting of total number of nuclei, mitotic index, percentage of apoptotic nuclei, and cell allocation to the inner cell mass (ICM) and trophectoderm (TE) lineage. Superovulation of hybrid (C57Bl/6JIco female x CBA/JIco male) female mice of 4-6 weeks of age was induced by a priming injection of PMSG at different stages of the estrous cycle followed after a 48-hr interval by human chrorionic gonadotropin. Our data indicate that injection of PMSG at the estrus phase gives the best outcome whereas injection of PMSG at the diestrus-1 or diestrus-2 phase provides the worst results. In fact, (1) total number of oocytes ovulated, number of ovulated oocytes enclosed by cumulus cells, and number of TE cells in day-5 blastocysts were significantly lower in diestrus-1 females than in estrus, diestrus-2 and proestrus mice; (2) percentage of day-5 blastocysts and total number of cells in day-5 blastocysts were lower in diestrus-1 and diestrus-2 females than in estrus and proestrus mice; and (3) percentage of apoptotic nuclei in day-5 blastocysts was lower in estrus mice than in diestrus-1, diestrus-2, or proestrus females. These data endorse previous studies suggesting that administration of gonadotropins in mice should be synchronized with the innate estrous cycle of females.     

Predictive variables of in vitro fertilization and pre-implantation embryo development in the mouse

The present study aims to analyze the cause-effect relationships among several in-vitro fertilization and pre-implantation embryo development variables in the mouse. Superovulation of hybrid (C57Bl/6JIco female X CBA/JIco male) female mice of 4-6 weeks of age was induced by a priming injection of pregnant mare's serum gonadotropin at the estrus stage of the estrous cycle followed after a 48-hr interval by human chrorionic gonadotropin. Ovulated cumulus-enclosed oocytes were inseminated with sperm from hybrid males of 12-16 weeks of age. The multiple linear regression analyses performed indicated that (a) total number of ovulated oocytes is a good predictor of both fertilization frequency and total number of cells in day-5 blastocysts; (b) fertilization frequency predicts percentage of day-5 blastocysts; (c) total number of cells in day-5 blastocysts is predicted by percentage of day-5 blastocysts; and (d) total number of cells in day-5 blastocysts predicts percentage of apoptotic cells, number of inner cell mass (ICM) and trophectoderm (TE) cells, and ICM/TE ratio in day-5 blastocysts. Mitotic index in day-5 blastocysts was positively correlated with total number of ovulated oocytes, percentage of ovulated cumulus-enclosed oocytes, fertilization frequency, percentage of day-5 blastocysts and total number of cells in day-5 blastocysts. On the contrary, it was negatively correlated with percentage of apoptotic cells in day-5 blastocysts.     

DNA damage in female workers exposed to pesticides in banana plantations at Limón,Costa Rica

Pesticide use in Costa Rica is very high and all year round. A high percentage of what is sprayed remains in the environment and in the living organisms around. This situation brings contamination and health problems to people in contact with them. The onset of adverse effects may be in the short or the long term, and symptoms vary widely, from headaches to cancer. Much research in this area has been devoted to acute or chronic effects, and not until recently to the genotoxic effect of pesticides. This study evaluated the genotoxic effect of pesticides used in banana packing activities, using the comet assay (single cell electrophoresis) as the biological marker in lymphocytes. This was a case-control double blind study of 30 exposed women from 15 banana farms and 28 women not occupationally exposed to pesticides from the same geographic area. Results show damage to single stranded DNA after working from 5 to 15 years (R2 = 0.12). In Costa Rica we do not have an historical record of the kind of pesticides used in banana farms, the period of time and for how long were they used. This prevented further analysis concerning dose, frequency of exposure and use of new or old kind of pesticides in the farms in relation to DNA damage. The comet assay is of value in the genetic monitoring of pesticide exposed populations.     

Marine science in Revista de Biologia Tropical in its 50th anniversary

The first paper published in the Revista de Biología Tropical (RBT) on anything related to marine science was in 1963. Since then the number of marine-related papers has increased to 637, which represents 27% of the total production of RBT (excluding the Supplements), and 33% since 1979. Most publications are Full Articles on Ecology (135 papers). The marine ecosystem of which there is more publications is the coral reefs (28); and fish is the most studied taxonomic group (165). Almost half of the Supplements are marine related (12). The RBT must continue its efforts to maintain itself as a leading marine science publication in Latin America.