Pedro Ostoa-Saloma

The Role of Cytokines in Breast Cancer Development and Progression

Cytokines are highly inducible, secretory proteins that mediate intercellular communication in the immune system. They are grouped into several protein families that are referred to as tumor necrosis factors, interleukins, interferons, and colony-stimulating factors. In recent years, it has become clear that some of these proteins as well as their receptors are produced in the organisms under physiological and pathological conditions. The exact initiation process of breast cancer is unknown, although several hypotheses have emerged. Inflammation has been proposed as an important player in tumor initiation, promotion, angiogenesis, and metastasis, all phenomena in which cytokines are prominent players. The data here suggest that cytokines play an important role in the regulation of both induction and protection in breast cancer. This knowledge could be fundamental for the proposal of new therapeutic approaches to particularly breast cancer and other cancer-related disorders.

Catalytic classes of proteinases of Entamoeba histolytica.

Endopeptidase inhibitors were used to determine the catalytic classes of proteinases present in extracts of Entamoeba histolytica (strain HM 1:IMSS) axenically grown in vitro. Cysteine proteinases account for most of the proteolytic activity; one or more proteinases with different catalytic mechanisms are also present but could not be unambiguously assigned to a particular catalytic class. Proteinases in amebic lysates were resolved by polyacrylamide gel electrophoresis with sodium dodecyl sulfate. The detergent was exchanged with Triton X-100 and the proteolytic activity in the gels was demonstrated by overlaying it on another gel containing the substrate. Four lysis zones were observed corresponding to molecular weights of 66,000, 56,000, 40,000 and 27,000. The first cannot be classified yet, but the last three showed properties consistent with those of cysteine proteinases. Finally, a novel technique is described which uses purified human alpha-2-macroglobulin to trap, purify and characterize proteases from amebic lysates. The results obtained with this technique confirm those of the overlay technique, since both methods reveal four distinct proteinases in the two different amebic preparations examined.

Genetic Vaccination against Murine Cysticercosis by Using a Plasmid Vector Carrying Taenia solium Paramyosin

ABSTRACT A plasmid vector carrying the immunoprotective amino-terminal fragment of Taenia solium paramyosin (VW2-1) was designed for genetic vaccination studies. Mice that were genetically immunized with VW2-1 and challenged by intraperitoneal inoculation of Taenia crassiceps cysticerci showed 43 to 48% reductions in the parasite burden, values which were similar to values obtained previously when the recombinant protein was used.

Progesterone induces scolex evagination of the human parasite Taenia solium: evolutionary implications to the host-parasite relationship.

Taenia solium cysticercosis is a health problem in underdeveloped and developed countries. Sex hormones are involved in cysticercosis prevalence in female and male pigs. Here, we evaluated the effects of progesterone and its antagonist RU486 on scolex evagination, which is the initial step in the development of the adult worm. Interestingly, progesterone increased T. solium scolex evagination and worm growth, in a concentration-independent pattern. Progesterone effects could be mediated by a novel T. solium progesterone receptor (TsPR), since RU486 inhibits both scolex evagination and worm development induced by progesterone. Using RT-PCR and western blot, sequences related to progesterone receptor were detected in the parasite. A phylogenetic analysis reveals that TsPR is highly related to fish and amphibian progesterone receptors, whereas it has a distant relation with birds and mammals. Conclusively, progesterone directly acts upon T. solium cysticerci, possibly through its binding to a progesterone receptor synthesized by the parasite.

Cloning, Characterization, and Functional Expression of Taenia Solium Calreticulin

Calreticulin is an endoplasmic reticulum protein involved in the homeostasis of intracellular Ca++ and other physiological processes. A complementary DNA clone containing the complete coding sequence of Taenia solium calreticulin (TsCRT) was isolated and characterized. Recombinant TsCRT was expressed in bacteria as a 50-kDa protein that specifically bound calcium when tested in a radioassay. The deduced amino acid sequence has 47–50% identity with other reported calreticulins. Poor recognition of TsCRT by human and pig sera with confirmed cysticercosis discourages its use for diagnosis of the disease. However, further characterization and localization studies could provide insights into the role of TsCRT in T. solium physiology and host–parasite interactions.

Proteinases of Entamoeba histolytica associated with different subcellular fractions

Crude lysates of Entamoeba histolytica (strain HM 1:IMSS) analyzed by substrate gel electrophoresis in 12.5% acrylamide separating gels with reducing agents showed six hydrolysis zones with apparent molecular weights of 73,000 (high), 45,000, 36,000 (intermediate), 30,000, 26,000 and 23,000 (low molecular weight proteinases). Amebic lysates fractionated using the procedure of Aley et al. or the procedure of Rosenberg and Gitler and analyzed by the same method show all enzymes in the fractions with the soluble components and only the intermediate and low molecular weight proteinases in the fraction containing internal vesicles or membranes and plasma membrane. Some of these proteinases seem to be integral membrane proteins since they resist treatment with high salt, high urea buffer. All fractions are capable of digesting azocasein. Fractionation of amebic lysates by hydrophobic chromatography using phenyl-Sepharose or phase separation of amebic extracts with Triton X-114 show that proteinases with high, intermediate and low molecular weight behave as hydrophilic proteins while only proteinases of intermediate and low molecular weight behave as hydrophobic proteins. These results suggest that some proteinases are segregated in different compartments of the cell.

Trypanosoma cruzi SHSP16: Characterization of an α-crystallin small heat shock protein

This report describes the characterization of a member of the alpha-crystallin small heat shock protein family in a trypanosomatid, which was isolated from the human pathogen Trypanosoma cruzi. One alpha-crystallin small heat shock protein gene was identified in a database search. The coding region is located in an open reading frame of 429bp encoding a protein of 142 amino acids. The amino acid sequence was deduced from the isolated gene. The protein has an alpha-crystallin domain characteristic of the alpha-crystallin small heat shock proteins and a molecular weight of 15.9kDa, so the protein was designated SHSP16. Analysis of the nucleotide sequences of four different T. cruzi strains showed two different sequences, which correspond to the two main T. cruzi genetic groups. Gene expression analysis by RT-PCR showed increased transcription of the gene after the parasite was exposed to heat stress. Recombinant SHSP16 showed molecular chaperone activity in vitro, because it inhibited the thermal aggregation of the mitochondrial malate dehydrogenase enzyme.

Cholera Toxin B-Subunit Gene Enhances Mucosal Immunoglobulin A, Th1-Type, and CD8+ Cytotoxic Responses When Coadministered Intradermally with a DNA Vaccine

ABSTRACT A plasmid vector encoding the cholera toxin B subunit (pCtB) was evaluated as an intradermal genetic adjuvant for a model DNA vaccine expressing the human papillomavirus type 16 L1 capsid gene (p16L1) in mice. p16L1 was coadministered with plasmid pCtB or commercial polypeptide CtB as a positive control. Coadministration of pCtB induced a significant increment of specific anti-L1 immunoglobulin A (IgA) antibodies in cervical secretions (P < 0.05) and fecal extracts (P < 0.005). Additionally, coadministration of pCtB enhanced the production of interleukin-2 and gamma interferon by spleen cells but did not affect the production of interleukin-4, suggesting a Th1-type helper response. Furthermore, improved CD8+ T-cell-mediated cytotoxic activity was observed in mice vaccinated with the DNA vaccine with pCtB as an adjuvant. This adjuvant effect was comparable to that induced by the CtB polypeptide. These results indicate that intradermal coadministration of pCtB is an adequate means to enhance the mucosa-, Th1-, and CD8+-mediated cytotoxic responses induced by a DNA vaccine.

Immunodiagnosis of Neurocysticercosis: Ways to Focus on the Challenge

Neurocysticercosis (NCC) is a disease of the central nervous system that is considered a public health problem in endemic areas. The definitive diagnosis of this disease is made using a combination of tools that include imaging of the brain and immunodiagnostic tests, but the facilities for performing them are usually not available in endemic areas. The immunodiagnosis of NCC is a useful tool that can provide important information on whether a patient is infected or not, but it presents many drawbacks as not all infected patients can be detected. These tests rely on purified or semipurified antigens that are sometimes difficult to prepare. Recent efforts have focused on the production of recombinant or synthetic antigens for the immunodiagnosis of NCC and interesting studies propose the use of new elements as nanobodies for diagnostic purposes. However, an immunodiagnostic test that can be considered as “gold standard” has not been developed so far. The complex nature of cysticercotic disease and the simplicity of common immunological assumptions involved explain the low scores and reproducibility of immunotests in the diagnosis of NCC. Here, the most important efforts for developing an immunodiagnostic test of NCC are listed and discussed. A more punctilious strategy based on the design of panels of confirmed positive and negative samples, the use of blind tests, and a worldwide effort is proposed in order to develop an immunodiagnostic test that can provide comparable results. The identification of a set of specific and representative antigens of T. solium and a thorough compilation of the many forms of antibody response of humans to the many forms of T. solium disease are also stressed as necessary.

Cloning, characterization and functional expression of a cyclophilin of Entamoeba histolytica.

Full-length Entamoeba histolytica cyclophilin gene (EhCyp) was isolated, characterized and recombinantly expressed in bacterial cells. The deduced amino acid sequence of EhCyp shows 60-70% identity with cyclophilins from other organisms and has conserved the cyclophilin signature motifs and residues involved in cyclosporin A binding. Upstream of the 501 bp open reading frame of EhCyp, sequences resembling the putative consensus E. histolytica CE1, CE2 and CE3 regulatory elements were found. Northern blot assays revealed a single transcript of 0.63 kb. The transcription start was determined by primer extension at position -13 relative to the initial ATG codon. Cyclosporin A binding and peptidyl-proplyl cis-trans isomerase activities characteristic of cyclophilin were detected in soluble extracts of E. histolytica trophozoites and in the recombinant protein. In both cases, the isomerase activity was inhibited by nanomolar concentrations of cyclosporin A. Treatment of cultured trophozoites with cyclosporin A decreased their proliferation with a 50% inhibition value of 1 microg/ml and was lethal in doses over 50 microg/ml.