Impact of MICA 3'UTR allelic variability on miRNA binding prediction, a bioinformatic approach.

MicroRNAs (miRNAs) are small non-coding RNAs that participate as powerful genetic regulators. MiRNAs can interfere with cellular processes by interacting with a broad spectrum of target genes under physiological and pathological states, including cancer development and progression. Major histocompatibility complex major histocompatibility complex class I-related chain A (MICA) belongs to a family of proteins that bind the natural-killer group 2, member D (NKG2D) receptor on Natural Killer cells and other cytotoxic lymphocytes. MICA plays a crucial role in the host's innate immune response to several disease settings, including cancer. MICA harbors various single nucleotide polymorphisms (SNPs) located in its 3'-untranslated region (3'UTR), a characteristic that increases the complexity of MICA regulation, favoring its post-transcriptional modulation by miRNAs under physiological and pathological conditions. Here, we conducted an in-depth analysis of MICA 3'UTR sequences according to each MICA allele described to date using NCBI database. We also systematically evaluated interactions between miRNAs and their putative targets on MICA 3'UTR containing SNPs using in silico analysis. Our in silico results showed that MICA SNPs rs9266829, rs 1880, and rs9266825, located in the target sequence of miRNAs hsa-miR-106a-5p, hsa-miR-17-5p, hsa-miR-20a-5p, hsa-miR-20b-5p, hsa-miR-93, hsa-miR-1207.5p, and hsa-miR-711 could modify the binding free energy between -8.62 and -18.14 kcal/mol, which may affect the regulation of MICA expression. We believe that our results may provide a starting point for further exploration of miRNA regulatory effects depending on MICA allelic variability; they may also be a guide to conduct miRNA in silico analysis for other highly polymorphic genes.

Heat-killed Helicobacter pylori upregulates NKG2D ligands expression on gastric adenocarcinoma cells via Toll-like receptor 4.

BACKGROUND: Natural killer (NK) cells are paramount for immunity against infectious agents and tumors. Their cytokine and cytolytic responses can be mediated by natural killer group 2, member D (NKG2D), an activating receptor whose ligands (NKG2DL) expression is induced in conditions of cell stress and malignant transformation. Since sustained expression of NKG2DL MICA is related to lower survival rates in gastric adenocarcinoma patients, and Helicobacter pylori infection contributes to tumorigenesis; we asked whether H. pylori stimulus could promote NKG2DL expression on human gastric adenocarcinoma cells. METHODS: Heat-killed H. pylori (HKHP) was used to stimulate MKN45 cells before analysis of NKG2DL and Toll-like receptor 4 (TLR4) protein levels by flow cytometry and transcripts by real-time PCR. LPS from Rhodobacter sphaeroides and inhibitory peptide Pepinh MYD were used to inhibit TLR4/MyD88 signaling pathway to assess its participation on NKG2DL expression. NK cell-mediated cytotoxicity was measured by lactate dehydrogenase (LDH) and CD107a mobilization assays. RESULTS: Stimulation of MKN45 cells with HKHP increased MICA, ULBP4 (another NKG2DL), and TLR4 at the protein and transcriptional levels. MICA, but not ULBP4 expression, was upregulated in a TLR4/MyD88-dependent manner. Furthermore, the presence of NKG2DL on the surface of HKHP-stimulated MKN45 cells enabled NK cell cytotoxic activation. CONCLUSIONS: Our data indicate that induction of NKG2DL expression on gastric adenocarcinoma cells by H. pylori promotes an immune response that may ultimately contribute to either gastric tissue damage, as a consequence of persistent activation of immunity, or tumor immune evasion due to chronic NKG2DL expression.

Major Histocompatibility Complex Class I-Related Chain A (MICA) Allelic Variants Associate With Susceptibility and Prognosis of Gastric Cancer.

Gastric cancer (GC) is the fifth most prevalent type of cancer worldwide. Gastric tumor cells express MICA protein, a ligand to NKG2D receptor that triggers natural killer (NK) cells effector functions for early tumor elimination. MICA gene is highly polymorphic, thus originating alleles that encode protein variants with a controversial role in cancer. The main goal of this work was to study MICA gene polymorphisms and their relationship with the susceptibility and prognosis of GC. Fifty patients with GC and 50 healthy volunteers were included in this study. MICA alleles were identified using Sanger sequencing methods. The analysis of MICA gene sequence revealed 13 MICA sequences and 5 MICA-short tandem repeats (STR) alleles in the studied cohorts We identified MICA*002 (*A9) as the most frequent allele in both, patients and controls, followed by MICA*008 allele (*A5.1). MICA*009/049 allele was significantly associated with increased risk of GC (OR: 5.11 [95% CI: 1.39-18.74], p = 0.014). The analysis of MICA-STR alleles revealed a higher frequency of MICA*A5 in healthy individuals than GC patients (OR = 0.34 [95% CI: 0.12-0.98], p = 0.046). Survival analysis after gastrectomy showed that patients with MICA*002/002 or MICA*002/004 alleles had significantly higher survival rates than those patients bearing MICA*002/008 (p = 0.014) or MICA*002/009 (MICA*002/049) alleles (p = 0.040). The presence of threonine in the position MICA-181 (MICA*009/049 allele) was more frequent in GC patients than controls (p = 0.023). Molecular analysis of MICA-181 showed that the presence of threonine provides greater mobility to the protein than arginine in the same position (MICA*004), which could explain, at least in part, some immune evasion mechanisms developed by the tumor. In conclusion, our findings suggest that the study of MICA alleles is crucial to search for new therapeutic approaches and may be useful for the evaluation of risk and prognosis of GC and personalized therapy.

Optimization of culture conditions for the expression of three different insoluble proteins in Escherichia coli.

Recombinant protein expression for structural and therapeutic applications requires the use of systems with high expression yields. Escherichia coli is considered the workhorse for this purpose, given its fast growth rate and feasible manipulation. However, bacterial inclusion body formation remains a challenge for further protein purification. We analyzed and optimized the expression conditions for three different proteins: an anti-MICA scFv, MICA, and p19 subunit of IL-23. We used a response surface methodology based on a three-level Box-Behnken design, which included three factors: post-induction temperature, post-induction time and IPTG concentration. Comparing this information with soluble protein data in a principal component analysis revealed that insoluble and soluble proteins have different optimal conditions for post-induction temperature, post-induction time, IPTG concentration and in amino acid sequence features. Finally, we optimized the refolding conditions of the least expressed protein, anti-MICA scFv, using a fast dilution protocol with different additives, obtaining soluble and active scFv for binding assays. These results allowed us to obtain higher yields of proteins expressed in inclusion bodies. Further studies using the system proposed in this study may lead to the identification of optimal environmental factors for a given protein sequence, favoring the acceleration of bioprocess development and structural studies.

Development of a new promoter to avoid the silencing of genes in the production of recombinant antibodies in chinese hamster ovary cells.

BACKGROUND: The production of recombinant proteins in mammalian cell lines is one of the most important areas in biopharmaceutical industry. Viral transcriptional promoters are widely used to express recombinant proteins in mammalian cell lines. However, these promoters are susceptible to silencing, thus limiting protein productivity. Some CpG islands can avoid the silencing of housekeeping genes; for that reason, they have been used to increase the production of recombinant genes in cells of animal origin. In this study, we evaluated the CpG island of the promoter region of the ß-actin gene of Cricetulus griseous (Chinese hamster), associated to the Cytomegalovirus (CMV) promoter, to increase recombinant antibodies production in Chinese Hamster Ovary (CHO) cells. RESULTS: We focused on the non-coding region of CpG island, which we called RegCG. RegCG behaved as a promoter, whose transcriptional activity was mainly commanded by the CAAT and CArG boxes of the proximal promoter. However, the transcription started mainly at the intronic region before the proximal transcription start site. While the CMV promoter was initially more powerful than RegCG, the latter promoter was more resistant to silencing than the CMV promoter in stable cell lines, and its activity was improved when combined with the CMV promoter. Thereby, the chimeric promoter was able to maintain the expression of recombinant antibodies in stable clones for 40 days at an average level 4 times higher than the CMV promoter. Finally, the chimeric promoter showed compatibility with a genetic amplification system by induction with methotrexate in cells deficient in the dihydrofolate reductase gene. CONCLUSIONS: We have generated an efficient synthetic hybrid transcription promoter through the combination of RegCG with CMV, which, in stable cell lines, shows greater activity than when both promoters are used separately. Our chimeric promoter is compatible with a genetic amplification system in CHO DG44 cells and makes possible the generation of stable cell lines with high production of recombinant antibodies. We propose that this promoter can be a good alternative for the generation of clones expressing high amount of recombinant proteins, essential for industrial applications.

Regulation of Tolerogenic Features on Dexamethasone-Modulated MPLA-Activated Dendritic Cells by MYC.

The potential of tolerogenic dendritic cells (tolDCs) to shape immune responses and restore tolerance has turn them into a promising therapeutic tool for cellular therapies directed toward immune regulation in autoimmunity. Although the cellular mechanisms by which these cells can exert their regulatory function are well-known, the mechanisms driving their differentiation and function are still poorly known, and the variety of stimuli and protocols applied to differentiate DCs toward a tolerogenic phenotype makes it even more complex to underpin the molecular features involved in their function. Through transcriptional profiling analysis of monocyte-derived tolDCs modulated with dexamethasone (Dex) and activated with monophosphoryl lipid A (MPLA), known as DM-DCs, we were able to identify MYC as one of the transcriptional regulators of several genes differentially expressed on DM-DCs compared to MPLA-matured DCs (M-DCs) and untreated/immature DCs (DCs) as revealed by Ingenuity Pathway Analysis (IPA) upstream regulators evaluation. Additionally, MYC was also amidst the most upregulated genes in DM-DCs, finding that was confirmed at a transcriptional as well as at a protein level. Blockade of transactivation of MYC target genes led to the downregulation of tolerance-related markers IDO1 and JAG1. MYC blockade also led to downregulation of PLZF and STAT3, transcription factors associated with immune regulation and inhibition of DC maturation, further supporting a role of MYC as an upstream regulator contributing to the regulatory phenotype of DM-DCs. On the other hand, we had previously shown that fatty acid oxidation, oxidative metabolism and zinc homeostasis are amongst the main biological functions represented in DM-DCs, and here we show that DM-DCs exhibit higher intracellular expression of ROS and Zinc compared to mature M-DCs and DCs. Taken together, these findings suggest that the regulatory profile of DM-DCs is partly shaped by the effect of the transcriptional regulation of tolerance-inducing genes by MYC and the modulation of oxidative metabolic processes and signaling mediators such as Zinc and ROS.

Transcription factor engineering in CHO cells for recombinant protein production.

The continuous increase of approved biopharmaceutical products drives the development of more efficient recombinant protein expression systems. Chinese hamster ovary (CHO) cells are the mainstay for this purpose but have some drawbacks, such as low levels of expression. Several strategies have been applied to increase the productivity of CHO cells with different outcomes. Transcription factor (TF) engineering has emerged as an interesting and successful approach, as these proteins can act as master regulators; the expression and function of a TF can be controlled by small molecules, and it is possible to design tailored TFs and promoters with desired features. To date, the majority of studies have focused on the use of TFs with growth, metabolic, cell cycle or endoplasmic reticulum functions, although there is a trend to develop new, synthetic TFs. Moreover, new synthetic biological approaches are showing promising advances for the development of specific TFs, even with tailored ligand sensitivity. In this article, we summarize the strategies to increase recombinant protein expression by modulating and designing TFs and with advancements in synthetic biology. We also illustrate how this class of proteins can be used to develop more robust expression systems.

Mild hypothermia upregulates myc and xbp1s expression and improves anti-TNFα production in CHO cells.

Chinese hamster ovary (CHO) cells are the most frequently used host for commercial production of therapeutic proteins. However, their low protein productivity in culture is the main hurdle to overcome. Mild hypothermia has been established as an effective strategy to enhance protein specific productivity, although the causes of such improvement still remain unclear. The self-regulation of global transcriptional regulatory factors, such as Myc and XBP1s, seems to be involved in increased the recombinant protein production at low temperature. This study evaluated the impact of low temperature in CHO cell cultures on myc and xbp1s expression and their effects on culture performance and cell metabolism. Two anti-TNFα producing CHO cell lines were selected considering two distinct phenotypes: i.e. maximum cell growth, (CN1) and maximum specific anti-TNFα production (CN2), and cultured at 37, 33 and 31°C in a batch system. Low temperature led to an increase in the cell viability, the expression of the recombinant anti-TNFα and the production of anti-TNFα both in CN1 and CN2. The higher production of anti-TNFα in CN2 was mainly associated with the large expression of anti-TNFα. Under mild hypothermia myc and xbp1s expression levels were directly correlated to the maximal viable cell density and the specific anti-TNFα productivity, respectively. Moreover, cells showed a simultaneous metabolic shift from production to consumption of lactate and from consumption to production of glutamine, which were exacerbated by reducing culture temperature and coincided with the increased anti-TNFα production. Our current results provide new insights of the regulation of myc and xbp1s in CHO cells at low temperature, and suggest that the presence and magnitude of the metabolic shift might be a relevant metabolic marker of productive cell line.

Triatoma infestans Calreticulin: Gene Cloning and Expression of a Main Domain That Interacts with the Host Complement System.

Triatoma infestans is an important hematophagous vector of Chagas disease, a neglected chronic illness affecting approximately 6 million people in Latin America. Hematophagous insects possess several molecules in their saliva that counteract host defensive responses. Calreticulin (CRT), a multifunctional protein secreted in saliva, contributes to the feeding process in some insects. Human CRT (HuCRT) and Trypanosoma cruzi CRT (TcCRT) inhibit the classical pathway of complement activation, mainly by interacting through their central S domain with complement component C1. In previous studies, we have detected CRT in salivary gland extracts from T. infestans We have called this molecule TiCRT. Given that the S domain is responsible for C1 binding, we have tested its role in the classical pathway of complement activation in vertebrate blood. We have cloned and characterized the complete nucleotide sequence of CRT from T. infestans, and expressed its S domain. As expected, this S domain binds to human C1 and, as a consequence, it inhibits the classical pathway of complement, at its earliest stage of activation, namely the generation of C4b. Possibly, the presence of TiCRT in the salivary gland represents an evolutionary adaptation in hematophagous insects to control a potential activation of complement proteins, present in the massive blood meal that they ingest, with deleterious consequences at least on the anterior digestive tract of these insects.

Treatment with Dexamethasone and Monophosphoryl Lipid A Removes Disease-Associated Transcriptional Signatures in Monocyte-Derived Dendritic Cells from Rheumatoid Arthritis Patients and Confers Tolerogenic Features.

Tolerogenic dendritic cells (TolDCs) are promising tools for therapy of autoimmune diseases, such as rheumatoid arthritis (RA). Here, we characterize monocyte-derived TolDCs from RA patients modulated with dexamethasone and activated with monophosphoryl lipid A (MPLA), referred to as MPLA-tDCs, in terms of gene expression, phenotype, cytokine profile, migratory properties, and T cell-stimulatory capacity in order to explore their suitability for cellular therapy. MPLA-tDCs derived from RA patients displayed an anti-inflammatory profile with reduced expression of co-stimulatory molecules and high IL-10/IL-12 ratio, but were capable of migrating toward the lymphoid chemokines CXCL12 and CCL19. These MPLA-tDCs induced hyporesponsiveness of autologous CD4+ T cells specific for synovial antigens in vitro. Global transcriptome analysis confirmed a unique transcriptional profile of MPLA-tDCs and revealed that RA-associated genes, which were upregulated in untreated DCs from RA patients, returned to expression levels of healthy donor-derived DCs after treatment with dexamethasone and MPLA. Thus, monocyte-derived DCs from RA patients have the capacity to develop tolerogenic features at transcriptional as well as at translational level, when modulated with dexamethasone and MPLA, overcoming disease-related effects. Furthermore, the ability of MPLA-tDCs to impair T cell responses to synovial antigens validates their potential as cellular treatment for RA.

Clinical significance of tumor expression of major histocompatibility complex class I-related chains A and B (MICA/B) in gastric cancer patients.

Gastric cancer (GC) is the third most common cause of cancer death worldwide. Natural killer cells play an important role in the immune defense against transformed cells. They express the activating receptor NKG2D, whose ligands belong to the MIC and ULBP/RAET family. Although it is well established that these ligands are generally expressed in tumors, the association between their expression in the tumor and gastric mucosa and clinical parameters and prognosis of GC remains to be addressed. In the present study, MICA and MICB expression was analyzed, by flow cytometry, in 23 and 20 pairs of gastric tumor and adjacent non-neoplasic gastric mucosa, respectively. Additionally, ligands expression in 13 tumors and 7 gastric mucosa samples from GC patients were evaluated by immunohistochemistry. The mRNA levels of MICA in 9 pairs of tumor and mucosa were determined by quantitative PCR. Data were associated with the clinicopathological characteristics and the patient outcome. MICA expression was observed in 57% of tumors (13/23) and 44% of mucosal samples (10/23), while MICB was detected in 50% of tumors (10/20) and 45% of mucosal tissues (9/20). At the protein level, ligand expression was significantly higher in the tumor than in the gastric mucosa. MICA mRNA levels were also increased in the tumor as compared to the mucosa. However, clinicopathological analysis indicated that, in patients with tumors >5 cm, the expression of MICA and MICB in the tumor did not differ from that of the mucosa, and tumors >5 cm showed significantly higher MICA and MICB expression than tumors ≤5 cm. Patients presenting tumors >5 cm that expressed MICA and MICB had substantially shorter survival than those with large tumors that did not express these ligands. Our results suggest that locally sustained expression of MICA and MICB in the tumor may contribute to the malignant progression of GC and that expression of these ligands predicts an unfavorable prognosis in GC patients presenting large tumors.

A short protocol using dexamethasone and monophosphoryl lipid A generates tolerogenic dendritic cells that display a potent migratory capacity to lymphoid chemokines.

BACKGROUND: Generation of tolerogenic dendritic cells (TolDCs) for therapy is challenging due to its implications for the design of protocols suitable for clinical applications, which means not only using safe products, but also working at defining specific biomarkers for TolDCs identification, developing shorter DCs differentiation methods and obtaining TolDCs with a stable phenotype. We describe here, a short-term protocol for TolDCs generation, which are characterized in terms of phenotypic markers, cytokines secretion profile, CD4+ T cell-stimulatory ability and migratory capacity. METHODS: TolDCs from healthy donors were generated by modulation with dexamethasone plus monophosphoryl lipid A (MPLA-tDCs). We performed an analysis of MPLA-tDCs in terms of yield, viability, morphology, phenotypic markers, cytokines secretion profile, stability, allogeneic and antigen-specific CD4+ T-cell stimulatory ability and migration capacity. RESULTS: After a 5-day culture, MPLA-tDCs displayed reduced expression of costimulatory and maturation molecules together to an anti-inflammatory cytokines secretion profile, being able to maintain these tolerogenic features even after the engagement of CD40 by its cognate ligand. In addition, MPLA-tDCs exhibited reduced capabilities to stimulate allogeneic and antigen-specific CD4+ T cell proliferation, and induced an anti-inflammatory cytokine secretion pattern. Among potential tolerogenic markers studied, only TLR-2 was highly expressed in MPLA-tDCs when compared to mature and immature DCs. Remarkable, like mature DCs, MPLA-tDCs displayed a high CCR7 and CXCR4 expression, both chemokine receptors involved in migration to secondary lymphoid organs, and even more, in an in vitro assay they exhibited a high migration response towards CCL19 and CXCL12. CONCLUSION: We describe a short-term protocol for TolDC generation, which confers them a stable phenotype and migratory capacity to lymphoid chemokines, essential features for TolDCs to be used as therapeutics for autoimmunity and prevention of graft rejection.

An efficient method for variable region assembly in the construction of scFv phage display libraries using independent strand amplification.

Phage display library technology is a common method to produce human antibodies. In this technique, the immunoglobulin variable regions are displayed in a bacteriophage in a way that each filamentous virus displays the product of a single antibody gene on its surface. From the collection of different phages, it is possible to isolate the virus that recognizes specific targets. The most common form in which to display antibody variable regions in the phage is the single chain variable fragment format (scFv), which requires assembly of the heavy and light immunoglobulin variable regions in a single gene. In this work, we describe a simple and efficient method for the assembly of immunoglobulin heavy and light chain variable regions in a scFv format. This procedure involves a two-step reaction: (1) DNA amplification to produce the single strand form of the heavy or light chain gene required for the fusion; and (2) mixture of both single strand products followed by an assembly reaction to construct a complete scFv gene. Using this method, we produced 6-fold more scFv encoding DNA than the commonly used splicing by overlap extension PCR (SOE-PCR) approach. The scFv gene produced by this method also proved to be efficient in generating a diverse scFv phage display library. From this scFv library, we obtained phages that bound several non-related antigens, including recombinant proteins and rotavirus particles.

Anti-TNF therapy in patients with rheumatoid arthritis decreases Th1 and Th17 cell populations and expands IFN-γ-producing NK cell and regulatory T cell subsets.

The aim of this work was to study the effect of anti-TNF treatment on CD4+ Th1, Th17 and regulatory T cells (Tregs), together with CD8+ T cells and NK cells from rheumatoid arthritis (RA) patients. For this purpose, 18 RA patients received adalimumab during 16weeks and their peripheral blood lymphocytes were assessed by flow cytometry at the beginning and at the end of the study. We found that the proportion of Th17 cells was directly correlated with Th1 cells, but inversely correlated with IFN-γ-producing NK cells. A decrease was observed in Th1, Th17 cells and IFN-γ-producing CD8+ T cells by anti-TNF therapy. Conversely, the proportion of Tregs increased, as did the percentage of IFN-γ-producing NK cells. We postulate that a rise in IFN-γ production due to recovery of NK cells' function, together with expanded Tregs, contribute to decrease the Th17 response in anti-TNF-treated RA patients.

Interleukin 10 decreases MICA expression on melanoma cell surface.

Natural-killer group 2, member D (NKG2D) binds to a variety of ligands, including the major histocompatibility complex (MHC) class I chain-related proteins (MIC) and UL16-binding proteins (ULBP). It is regarded as a co-activating receptor on NK cells, having an important role in the cell-mediated immune response to tumours. We studied the influence of interleukin (IL)-10 on the regulation of MIC and ULBP expression on melanoma cells, and its effect on the cytotoxic function of NK cells in vitro. Here, we show that, in the presence of IL-10, FMS mel and BL mel cell lines decreased MICA and ULBP2 surface expression, whereas MHC class I did not change substantially on the cell surface. MICA mRNA levels decreased in IL-10-treated FMS and IL-10-transduced BL cell lines. Interestingly, we observed that MICB surface expression and its mRNA levels increased upon IL-10 treatment in a melanoma cell line. These changes in NKG2D ligands surface expression patterns owing to IL-10 treatment resulted in an effect on lysis susceptibility mediated by lymphocyte-activated killer cells, as tumour cell lines that displayed a higher decrease of MICA on their surface had lower levels of lysis. In addition, expression of CD107a was downregulated on the surface of NK cells following stimulation with IL-10-treated FMS cells. Our results suggest a novel function for IL-10 in the modulation of NKG2D ligand expression and in the control of cytotoxicity mediated by NKG2D/NKG2D ligand axis.

Trypanosoma cruzi calreticulin: a possible role in Chagas' disease autoimmunity.

Trypanosoma cruzi (T. cruzi) is the causative agent of Chagas' disease, an endemic and chronic illness that affects 18 million people in Latin America. The mechanisms underlying its pathogenesis are controversial. There is a growing body of evidence supporting the view that T. cruzi infection elicits severe autoimmune responses in the host, which significantly contribute to the pathogenesis of Chagas' disease, and several recent studies have reported the presence of autoantibodies and effector T lymphocytes against parasite and self antigens in infected patients and experimentally infected animals. T. cruzi calreticulin (TcCRT) is a 45kDa protein, immunogenic in humans, rabbits and mice. It has a high degree of homology with human (HuCRT) and mouse calreticulin (MoCRT), which would explain why an immune response to TcCRT could contribute to autoimmune reactions in Chagas' disease. Anti-TcCRT antibodies generated in A/J mice immunized with recombinant TcCRT (rTcCRT) reacted with rHuCRT and bound to neonatal and adult isogenic cardiomyocytes cultured in vitro. Interestingly, histological alterations, such as edema formation and cell infiltrates, which include CD3(+) cells, were detected in heart sections from immunized animals. Therefore, in rTcCRT-immunized mice, an autoimmune reaction against host CRT, paralleled by histological cardiac alterations, suggests a role of the parasite molecule in the induction of immunologically mediated heart tissue damage. The data presented here propose that TcCRT participates in the induction of cardiac autoimmunity in Chagas' disease.

Does Trypanosoma cruzi calreticulin modulate the complement system and angiogenesis?

Calreticulin, a calcium-binding protein that is highly conserved in its multiple functions, is present in a wide spectrum of subcellular compartments in virtually every cell of higher organisms. In this article, we propose a dual role for parasite calreticulin, with emphasis on the Trypanosoma cruzi model. By modulating the vertebrate complement system, calreticulin might provide the parasite with an effective immune-escape mechanism. Alternatively, by inhibiting angiogenesis, the parasite molecule might protect the host from ongoing neoplasic aggressions. Many questions are still unanswered, particularly those regarding the consequences that these interactions could have in vivo for both the parasite and the host.

Role of calreticulin from parasites in its interaction with vertebrate hosts.

Although parasites range from protozoan to complex, evolutionary advanced arthropods, in general, a hallmark of parasite life cycles is their ability to adapt to changes in temperature, pH and host defense strategies. Calreticulin, a calcium-binding protein, highly conserved and multifunctional, is present in every cell of higher organisms, except erythrocytes. The surprising array of calreticulin-associated functions include lectin-like chaperoning, calcium storage and signaling, modulation of gene expression, cell adhesion, enhancement of phagocytosis of C1q or collectin opsonized apoptotic cells, inhibition of angiogenesis and tumoral growth, inhibition of perforin pore formation in T and NK cells, and inhibition of C1q-dependent complement activation. Likewise, calreticulin is present in a wide spectrum of sub cellular compartments. Parasite calreticulin shows a surprisingly high degree of conservation within the framework of its functional domains. Its role within the parasite/host relationship needs to be assessed further, in particular with regard to its impact on parasite infectivity, by helping to evade from its hosts' immune response. With special emphasis on calreticulin from Trypanosoma cruzi, the intracellular protozoan agent of American trypanosomiasis (Chagas' disease), we wish to exemplify and highlight the various implications of parasite calreticulin, within the pathophysiology of parasite-mediated human and animal disease.

The classical activation pathway of the human complement system is specifically inhibited by calreticulin from Trypanosoma cruzi.

The high resistance of Trypanosoma cruzi trypomastigotes, the causal agent of Chagas' disease, to complement involves several parasite strategies. In these in vitro studies, we show that T. cruzi calreticulin (TcCRT) and two subfragments thereof (TcCRT S and TcCRT R domains) bind specifically to recognition subcomponents of the classical and lectin activation pathways (i.e., to collagenous tails of C1q and to mannan-binding lectin) of the human complement system. As a consequence of this binding, specific functional inhibition of the classical pathway and impaired mannan-binding lectin to mannose were observed. By flow cytometry, TcCRT was detected on the surface of viable trypomastigotes and, by confocal microscopy, colocalization of human C1q with surface TcCRT of infective trypomastigotes was visualized. Taken together, these findings imply that TcCRT may be a critical factor contributing to the ability of trypomastigotes to interfere at the earliest stages of complement activation.

[New immunological weapons for medicine in the 21st Century: biological therapy based on the use of the latest generation monoclonal antibodies]. / Nuevas armas inmunológicas para la medicina del siglo XXI: terapia biológica basada en el uso de anticuerpos monoclonales de última generación.

The fusion of a murine B cell and a myeloma cell generates a hybridoma that produces monoclonal antibody (mAb). These murine mAb induce the HAMA (human anti-mouse antibodies) response. Murine mAb have been modified by genetic engineering, producing molecules with a higher proportion of human protein. At present, chimeric, humanized and fully human mAb are available. mAb block interactions between target molecules and their ligands or trigger the lyses of mAb-coated tumor cells. Numerous mAb have been developed using the recombinant DNA technology and several are available in the market. Trastuzumab, against HER2/neu, is useful in breast cancer; rituximab, against CD20 in B lymphocytes is useful in lymphoma; alemtuzumah, against CD52 is used in lymphoma and leukemia; daclizumab and basiliximab block the IL-2 receptor interaction and reduce acute rejection in kidney transplantation; abciximab, an antagonist of GPIIb/IIIa platelet receptor, is used in patients undergoing acute coronary syndromes. In autoimmunity diseases, blocking tumor necrosis factor by infliximab and adalimumab has demonstrated excellent results. Thus, infliximab is useful in the treatment of rheumatoid arthritis (RA), Crohn's disease and ulcerative colitis while adalimumab is the first fully human mAb available for RA. Infliximab and adalimumab reduce signs and symptoms in RA and they also interfere with progression of joint damage. Finally, the direct benefits of antagonist treatment can occur at the expense of a major adverse effect in some other biological function.