Transcriptome profiling of macrophages persistently infected with human respiratory syncytial virus and effect of recombinant Taenia solium calreticulin on immune-related genes.

Introduction: Human respiratory syncytial virus (hRSV) is a main cause of bronchiolitis in infants and its persistence has been described in immunocompromised subjects. However, limited evidence has been reported on the gene expression triggered by the hRSV and the effect of recombinant Taenia solium-derived calreticulin (rTsCRT). Methods: Using a comprehensive microarray approach, we analyzed the transcriptome profile of a macrophage cell line that has supported hRSV persistence for over 150 passages. We compared the gene expression of persistently infected and non-infected macrophages. We also evaluated the effect of rTsCRT on hRSV-infected macrophage gene transcription, as well as on cytokine production and number of copies of the persistent hRSV genome. Results: Our analysis showed that hRSV long-term virus infection significantly alters mRNA expression of antiviral, inflammatory, as well as arginine and lipid metabolism-associated genes, revealing a transcriptional signature that suggests a mixed M1/M2 phenotype. The resulting host-virus equilibrium allows for the regulation of viral replication, while evading the antiviral and proinflammatory responses. Interestingly, rTsCRT stimulus upregulated Tnfα, Il6 and Nos2 mRNA. We found increased levels of both proinflammatory cytokines and nitrite levels in the conditioned media of persistent macrophages treated with rTsCRT. This increase was associated with a significant reduction in viral genome copies. Discussion: hRSV persistently infected macrophages retain responsiveness to external stimuli and demonstrate that the profound changes induced by viral persistence are potentially reversible. Our observations contribute to the understanding of the mechanisms related to hRSV persistence in macrophages and have implications for the development of targeted therapies to eliminate persistent infections or reduce the negative effects related with chronic inflammatory diseases associated with hRSV infection.

Plant extracts modulate cellular stress to inhibit replication of mouse Coronavirus MHV-A59.

The Covid-19 infection outbreak led to a global epidemic, and although several vaccines have been developed, the appearance of mutations has allowed the virus to evade the immune response. Added to this is the existing risk of the appearance of new emerging viruses. Therefore, it is necessary to explore novel antiviral therapies. Here, we investigate the potential in vitro of plant extracts to modulate cellular stress and inhibit murine hepatitis virus (MHV)-A59 replication. L929 cells were treated with P2Et (Caesalpinia spinosa) and Anamu SC (Petiveria alliacea) plant extracts during infection and virus production, ROS (reactive oxygen species), UPR (unfolded protein response), and autophagy were assessed. P2Et inhibited virus replication and attenuated both ROS production and UPR activation induced during infection. In contrast, the sustained presence of Anamu SC during viral adsorption and replication was required to inhibit viral infection, tending to induce pro-oxidant effects, and increasing UPR gene expression. Notably, the loss of the PERK protein resulted in a slight decrease in virus yield, suggesting a potential involvement of this UPR pathway during replication. Intriguingly, both extracts either maintained or increased the calreticulin surface exposure induced during infection. In conclusion, our findings highlight the development of antiviral natural plant extracts that differentially modulate cellular stress.

CARL y MPL en pacientes cubanos con trombocitemia esencial y mielofibrosis primaria / CARL and MPL in Cuban patients with essential thrombocythemia and primary mielofibrosi

Introducción: La trombocitemia esencial y la mielofibrosis primaria comparten la presencia de las mutaciones JAK2, CALR y MPL. En total, están presentes en poco más del 90 % de los pacientes con estas enfermedades. Objetivos: Determinar el comportamiento de las mutaciones más frecuentes en los genes MPL y CALR en pacientes cubanos. Métodos: Se realizó un estudio ambispectivo, descriptivo y longitudinal en el Instituto de Hematología e Inmunología de Cuba, entre los años 2010 y 2020. Se incluyeron todos los pacientes con sospecha de trombocitemia esencial y de mielofibrosis primaria con muestras de ADN válidas. Se les identificaron las mutaciones CALR y MPL por PCR en tiempo real. Resultados: De los 53 pacientes estudiados, el 67,9 % fueron diagnosticados con trombocitemia esencial, el 22,6 % con mielofibrosis primaria. En el 90,6 % se pudo detectar alguna de las mutaciones conductoras; el 67,9 % fueron positivos a la mutación JAK2V617F, el 13,2 % a las mutaciones en el gen que codifica para la calreticulina y en el 9,4 % se identificaron mutaciones en el gen MPL. Conclusiones: El comportamiento de las mutaciones conductoras JAK2V617F, CALR y MPL en la muestra de pacientes cubanos con trombocitemia esencial y mielofibrosis primaria estuvo en correspondencia con lo descrito en la mayoría de las investigaciones.

Introduction: Essential thrombocythemia and primary myelofibrosis share the presence of JAK2, CALR and MPL mutations. In total, they comprise slightly more than 90 % of patients with these diseases. Objectives: To determine the behavior of the most frequent mutations in MPL and CALR genes in Cuban patients. Methods: An ambispective, descriptive and longitudinal study was performed at the Institute of Hematology and Immunology of Cuba, between 2010 and 2020. All patients with suspected essential thrombocythemia and primary myelofibrosis with valid DNA samples were included. CALR and MPL mutations were identified by real-time PCR. Results: Of the 53 patients studied, 67.9% were diagnosed with essential thrombocythemia, and 22.6% with primary myelofibrosis. In 90.6% it was possible to detect any of the driver mutations: 67.9% were positive for the JAK2V617F mutation, 13.2% for mutations in the gene coding for calreticulin and in 9.4% mutations in the MPL gene were identified. Conclusions: The behavior of the driver mutations JAK2V617F, CALR and MPL in the sample of Cuban patients with essential thrombocythemia and primary myelofibrosis was in correspondence with what is described in the majority of the investigations.

Calreticulin Expression in Human Carcinomas: A Systematic Review and Meta-Analysis.

OBJECTIVE: The present study performed a systematic review and meta-analysis of observational studies on whether calreticulin levels could represent a prognostic factor in carcinoma patients. Calreticulin (CRT) is a multifunctional protein in the endoplasmic reticulum that can play distinct roles in different cancers. METHODS: The search was performed in PubMed, Scopus, the Cochrane Library, Web of Science, Lilacs, Science Direct, Embase, Bireme, and SciELO databases. After a full-text evaluation, only 14 articles remained. The RoBANS tool assessed the risk of bias. The meta-analysis was performed with R software, and the odds ratio (OR) was the effect measure. The random effects model was chosen, and the quality of evidence was evaluated according to GRADE. RESULT: The most frequent carcinomas were in the breasts and the colon. CRT expression varied according to carcinoma origin and type, but these diseases had a prevalence of high CRT levels, indicating tumor progression. The high CRT levels were associated with lymph node metastasis (OR = 3.06 [1.71; 5.48]/p = 0.0002/I2 = 0%). All included articles had a blinding bias. CONCLUSION: High CRT levels may represent a prognostic factor for metastatic lymph nodes in carcinoma patients.

Identification of Proteomic Biomarkers in Proliferative Verrucous Leukoplakia through Liquid Chromatography With Tandem Mass Spectrometry.

Proliferative verrucous leukoplakia (PVL) is an oral potentially malignant disorder associated with high risk of malignant transformation. Currently, there is no treatment available, and restrictive follow-up of patients is crucial for a better prognosis. Oral leukoplakia (OL) shares some clinical and microscopic features with PVL but exhibits different clinical manifestations and a lower rate of malignant transformation. This study aimed to investigate the proteomic profile of PVL in tissue and saliva samples to identify potential diagnostic biomarkers with therapeutic implications. Tissue and saliva samples obtained from patients with PVL were compared with those from patients with oral OL and controls. Label-free liquid chromatography with tandem mass spectrometry was employed, followed by qualitative and quantitative analyses, to identify differentially expressed proteins. Potential biomarkers were identified and further validated using immunohistochemistry. Staining intensity scan analyses were performed on tissue samples from patients with PVL, patients with OL, and controls from Brazil, Spain, and Finland. The study revealed differences in the immune system, cell cycle, DNA regulation, apoptosis pathways, and the whole proteome of PVL samples. In addition, liquid chromatography with tandem mass spectrometry analyses showed that calreticulin (CALR), receptor of activated protein C kinase 1 (RACK1), and 14-3-3 Tau-protein (YWHAQ) were highly expressed in PVL samples. Immunohistochemistry validation confirmed increased CARL expression in PVL compared with OL. Conversely, RACK1 and YWHA were highly expressed in oral potentially malignant disorder compared to the control group. Furthermore, significant differences in CALR and RACK1 expression were observed in the OL group when comparing samples with and without oral epithelial dysplasia, unlike the PVL. This research provides insights into the molecular mechanisms underlying these conditions and highlights potential targets for future diagnostic and therapeutic approaches.

The many faces of parasite calreticulin.

Calreticulin from parasites and its vertebrate hosts share ~50% identity and many of its functions are equally conserved. However, the existing amino acid differences can affect its biological performance. Calreticulin plays an important role in Ca2+ homeostasis and as a chaperone involved in the correct folding of proteins within the endoplasmic reticulum. Outside the endoplasmic reticulum, calreticulin is involved in several immunological functions such as complement inhibition, enhancement of efferocytosis, and immune upregulation or inhibition. Several parasite calreticulins have been shown to limit immune responses and promote infectivity, while others are strong immunogens and have been used for the development of potential vaccines that limit parasite growth. Furthermore, calreticulin is essential in the dialogue between parasites and hosts, inducing Th1, Th2 or regulatory responses in a species-specific manner. In addition, calreticulin participates as initiator of endoplasmic reticulum stress in tumor cells and promotion of immunogenic cell death and removal by macrophages. Direct anti-tumoral activity has also been reported. The highly immunogenic and pleiotropic nature of parasite calreticulins, either as positive or negative regulators of the immune response, render these proteins as valuable tools to modulate immunopathologies and autoimmune disorders, as well as a potential treatment of neoplasms. Moreover, the disparities in the amino acid composition of parasite calreticulins might provide subtle variations in the mechanisms of action that could provide advantages as therapeutic tools. Here, we review the immunological roles of parasite calreticulins and discuss possible beneficial applications.

Silver nanoparticles induce a non-immunogenic tumor cell death.

Immunogenic cell death (ICD) is a form of cell death characterized by the release of danger signals required to trigger an adaptive immune response against tumor-associated antigens. Silver nanoparticles (AgNP) display anti-proliferative and cytotoxic effects in tumor cells, but it has not been previously studied whether AgNP act as an ICD inductor. The present study evaluated the in vitro release of calreticulin as a damage-associated molecular pattern (DAMP) associated with the cytotoxicity of AgNP and their in vivo anti-cancer effects. In vitro, mouse CT26 colon carcinoma and MCA205 fibrosarcoma cells were exposed to AgNP and then cell proliferation, adhesion, and release of calreticulin were determined. The results indicated there were time- and concentration-related anti-proliferative effects of AgNP in both the CT26 and MCA205 lines. Concurrently, changes in cell adhesion were detected mainly in the CT26 cells. Regarding DAMP detection, a significant increase in calreticulin was observed only in CT26 cells treated with doxorubicin and AgNP; however, no differences were found in the MCA205 cells. In vivo, the survival and growth of subcutaneous tumors were monitored after vaccination of mice with cell debris from tumor cells treated with AgNP or after intra-tumoral administration of AgNP to established tumors. Consequently, anti-tumoral prophylactic immunization with AgNP-dead cells failed to protect mice from tumor re-challenge; intra-tumor injection of AgNP did not induce a significant effect. In conclusion, there was a noticeable anti-tumoral effect of AgNP in vitro in both CT26 and MCA205 cell lines, accompanied by the release of calreticulin in CT26 cells. In vivo, immunization with cell debris derived from AgNP-treated tumor cells failed to induce a protective immune response in the cancer model mice. Clearly, further research is needed to determine if one could combine AgNP with other ICD inducers to improve the anti-tumor effect of these nanoparticles in vivo.

Oocyte in vitro maturation with eugenol improves the medium antioxidant capacity and total cell number per blastocyst.

This study investigates the impact of eugenol (EU) supplementation on bovine oocyte in vitro maturation (IVM) and antioxidant capacity, as well as in vitro embryo production and quality after conventional in vitro fertilization (IVF). A total of 1077 cumulus oocyte complexes were cultured in TCM-199+ without EU supplementation (control treatment) or supplemented with EU at the concentrations of 10 µM (EU-10), 20 µM (EU-20), or 40 µM (EU-40). After IVM, the oocytes were subjected to IVF and embryo culture. The addition of EU at 40 µM to the IVM medium improved (P < 0.05) the antioxidant capacity and cleavage rate when compared to the control treatment. Moreover, a positive correlation (r = 0.61, P < 0.03) was observed between cleavage rate and EU concentration. The addition of EU at concentrations of 10 and 20 µM decreased (P < 0.05) the calreticulin (CALR) levels in expanded blastocysts when compared to the control treatment and EU-40 treatment. However, the EU-10 and EU-20 treatments had a greater (P < 0.05) mean total cell number (TCN) per expanded blastocyst when compared to the control treatment and EU-40 treatment. In conclusion, the addition of EU to the enriched culture medium during IVM of bovine oocytes improved the antioxidant capacity of the spent medium, as well as the cleavage rate and embryonic quality (i.e., TCN/expanded blastocyst), and reduced the endoplasmic reticulum stress (i.e., CALR levels) in the embryos. Thus, we recommend enriching the IVM medium with 10 µM EU for in vitro bovine embryo production.

Immunogenic analysis of epitope-based vaccine candidate induced by photodynamic therapy in MDA-MB-231 triple-negative breast cancer cells.

BACKGROUND: Photodynamic therapy (PDT) is used to treat tumors through selective cytotoxic effects. PDT induces damage-associated molecular patterns (DAMPs) expression, which can cause an immunogenic death cell (IDC). In this study we identified potential immunogenic epitopes generated by PDT on triple-negative breast cancer cell line (MDA-MB-231). METHODS: MDA-MB-231 cells were exposed to PDT using ALA (160 µg/mL)/630 nm at 8 J/cm2. Membrane proteins were extracted and separated by 2D PAGE. Proteins overexpressed were identified by LC-MS/MS and analyzed in silico through a peptide-HLA docking in order to identify the epitopes with more immunogenicity and antigenicity properties, as well as lower allergenicity and toxicity activity. The selected peptides were evaluated in response to macrophage activation and cytokine release by flow cytometry. RESULTS: Differential proteins were overexpressed in the cells treated with PDT. A group of 16 peptides were identified from them, established in a rigorous selection by measuring antigenicity, immunogenicity, allergenicity, and toxicity in silico. The final selection was based on molecular dynamics, where 2 peptides showed the highest stability regarding to the RMSD value. These peptides were obtained from the proteins calreticulin and HSP90. The cytokine analysis evidenced macrophage activation by the releasing of TNF. CONCLUSION: Two peptides were identified from calreticulin and HSP90; proteins induced by PDT in MDA-MB-231 cells. Both epitopes showed immunogenic potential as a peptide-based vaccine for triple-negative breast cancer.

Increased CD47 and MHC Class I Inhibitory Signals Expression in Senescent CD1 Primary Mouse Lung Fibroblasts.

Cellular senescence is more than a proliferative arrest in response to various stimuli. Senescent cells (SC) participate in several physiological processes, and their adequate removal is essential to maintain tissue and organism homeostasis. However, SC accumulation in aging and age-related diseases alters the tissue microenvironment leading to deterioration. The immune system clears the SC, but the specific scenarios and mechanisms related to recognizing and eliminating them are unknown. Hence, we aimed to evaluate the existence of three regulatory signals of phagocytic function, CD47, major histocompatibility complex class I (MHC-I), and calreticulin, present in the membrane of SC. Therefore, primary fibroblasts were isolated from CD1 female mice lungs, and stress-induced premature senescence (SIPS) was induced with hydrogen peroxide. Replicative senescence (RS) was used as a second senescent model. Our results revealed a considerable increment of CD47 and MHC-I in RS and SIPS fibroblasts. At the same time, no significant changes were found in calreticulin, suggesting that those signals might be associated with evading immune system recognition and thus averting senescent cells clearance.

The Interactions of Parasite Calreticulin With Initial Complement Components: Consequences in Immunity and Virulence.

Because of its capacity to increase a physiologic inflammatory response, to stimulate phagocytosis, to promote cell lysis and to enhance pathogen immunogenicity, the complement system is a crucial component of both the innate and adaptive immune responses. However, many infectious agents resist the activation of this system by expressing or secreting proteins with a role as complement regulatory, mainly inhibitory, proteins. Trypanosoma cruzi, the causal agent of Chagas disease, a reemerging microbial ailment, possesses several virulence factors with capacity to inhibit complement at different stages of activation. T. cruzi calreticulin (TcCalr) is a highly-conserved, endoplasmic reticulum-resident chaperone that the parasite translocates to the extracellular environment, where it exerts a variety of functions. Among these functions, TcCalr binds C1, MBL and ficolins, thus inhibiting the classical and lectin pathways of complement at their earliest stages of activation. Moreover, the TcCalr/C1 interaction also mediates infectivity by mimicking a strategy used by apoptotic cells for their removal. More recently, it has been determined that these Calr strategies are also used by a variety of other parasites. In addition, as reviewed elsewhere, TcCalr inhibits angiogenesis, promotes wound healing and reduces tumor growth. Complement C1 is also involved in some of these properties. Knowledge on the role of virulence factors, such as TcCalr, and their interactions with complement components in host-parasite interactions, may lead toward the description of new anti-parasite therapies and prophylaxis.

In vitro Treatment of a Murine Mammary Adenocarcinoma Cell Line with Recombinant Trypanosoma cruzi Calreticulin Promotes Immunogenicity and Phagocytosis.

American Trypanosomiasis, a parasitic disease produced by Trypanosoma cruzi (T. cruzi), endemic in Latin America, infects about 6 million people. During the chronic stage of the infection, approximately 30% of infected people will develop Chagas Disease, the clinical manifestation. Few decades ago it was reported that, during the chronic stage, the parasite interferes with the development of solid tumors. However, the identification of parasite molecules responsible for such effects remained elusive. Years later, we described T.cruzi Calreticulin (TcCalr), an endoplasmic reticulum resident chaperone that infective trypomastigotes translocate to the parasite exterior, where it displays anticomplement activities. Most likely, at least some of these activities are related with the antitumor properties of TcCalr, as shown in in vitro, ex vivo, in ovum, and in vivo models. In this context we, we have seen that in vivo subcutaneous peritumoral inoculation of rTcCalr enhances local infiltration of T cells and slows tumor development. Based on these precedents, we propose that in vitro treatment of a mammary adenocarcinoma (TA3 cell line) with rTcCalr, will enhance tumor immunogenicity. In agreement with this proposal, we have shown that: i). rTcCalr binds to TA3 cells in a concentration-dependent fashion, ii). C1q binds to TA3 cells in an rTcCalr-dependent fashion, confirmed by the reversion attained using anti-TcS (a central TcCalr domain that binds C1) F(ab')2 antibody fragments, iii). incubation of TA3 cells with rTcCalr, promotes cell phagocytosis by murine macrophages and, iv). rTcCalr decreases the membrane expression of MHC class II, m-Dectin-1, Galectin-9 and PD-L1, while increasing the expression of Rae-1γ. In synthesis, herein we show that in vitro treatment of a murine mammary adenocarcinoma with rTcCalr enhances phagocytosis and modulates the expression of a variety of membrane molecules that correlates with increased tumor immunogenicity.

In Vitro Employment of Recombinant Taenia solium Calreticulin as a Novel Strategy Against Breast and Ovarian Cancer Stem-like Cells.

BACKGROUND AND AIMS: Calreticulin is a chaperone and master regulator of intracellular calcium homeostasis. Several additional functions have been discovered. Human and parasite calreticulin have been shown to suppress mammary tumor growth in vivo. Here, we explored the capacity of recombinant Taenia solium calreticulin (rTsCRT) to modulate cancer cell growth in vitro. METHODS: We used different concentrations of rTsCRT to treat cancer cell lines and analyzed viability and colony formation capacity. We also tested the combination of the IC20 or IC50 doses of rTsCRT and of the chemotherapeutic drug 5-fluorouracil on MCF7 and SKOV3 cell lines. As a control, the non-tumorigenic cell line MCF10-A was employed. The effect of the drug combinations was also assessed in cancer stem-like cells. Additionally, scavenger receptor ligands were employed to identify the role of this receptor in the rTsCRT anti-tumoral effect. RESULTS: rTsCRT has a dose-dependent in vitro anti-tumoral effect, being SKOV3 the most sensitive cell line followed by MCF7. When rTsCRT/5-fluorouracil were used, MCF7 and SKOV3 showed a 60% reduction in cell viability; colony formation capacity was also diminished. Treatment of cancer stem-like cells from MCF7 showed a higher reduction in cell viability, while those from SKOV3 were more sensitive to colony disaggregation. Finally, pharmacological inhibition of the scavenger receptor, abrogated the reduction in viability induced by rTsCRT in both the parental and stem-like cells. CONCLUSION: Our data suggest that rTsCRT alone or in combination with 5-fluorouracil inhibits the growth of breast and ovarian cancer cell lines through its interaction with scavenger receptors.

Análisis de expresión de calreticulina en vena safena parva / Calreticulin expression analysis in small saphenous vein

La proteína chaperona Calreticulina (CRT), ha sido identificada en retículo endoplásmico (RE) y últimamente en la matriz extracelular (MEC) de predentina y arterias, atribuyéndole diferentes funciones extracelulares entre las que destacan la adhesión celular, regulación de la MEC y prevención en la formación de trombos. El objetivo del estudio fue identificar la presencia de CRT en MEC de vena safena parva. Se extrajo una muestra de vena safena parva de un espécimen masculino y luego fue procesada por medios histológicos e inmunohistoquímicos para identificar su presencia. Mediante técnicas de inmunohistoquímica se pudo evidenciar la presencia de CRT en la MEC de la adventicia de vena safena parva. La presencia de CRT en MEC de safena parva orienta a que CRT tienen funciones de tipo extracelular en esta localización, pero es necesario realizar estudios más precisos para dilucidar sus principales funciones en la zona.

Calreticulin (CRT) protein, has been identified in the endoplasmic reticulum (ER) and lately in the extracellular matrix (ECM) of predentine and arteries. It is responsible for different extracellular functions, such as cell adhesion, ECM regulation, and the prevention of thrombosis. The aim was to identify the presence of CRT in ECM of small saphenous vein. A sample of small saphenous vein from a male specimen was extracted and then processed by histological and immunohistochemical assays to identify its presence. The presence of CRT in the ECM of the small saphenous vein was observed by immunohistochemical techniques. The presence of CRT in the small saphenous vein ECM, indicates that CRT have extracellular functions in this area, however, more precise studies are necessary to determine its main functions.

Structural bases that underline Trypanosoma cruzi calreticulin proinfective, antiangiogenic and antitumor properties.

Microbes have developed mechanisms to resist the host immune defenses and some elicit antitumor immune responses. About 6 million people are infected with Trypanosoma cruzi, the protozoan agent of Chagas' disease, the sixth neglected tropical disease worldwide. Eighty years ago, G. Roskin and N. Klyuyeva proposed that T. cruzi infection mediates an anti-cancer activity. This observation has been reproduced by several other laboratories, but no molecular basis has been proposed. We have shown that the highly pleiotropic chaperone calreticulin (TcCalr, formerly known as TcCRT), translocates from the parasite ER to the exterior, where it mediates infection. Similar to its human counterpart HuCALR (formerly known as HuCRT), TcCalr inhibits C1 in its capacity to initiate the classical pathway of complement activation. We have also proposed that TcCalr inhibits angiogenesis and it is a likely mediator of antitumor effects. We have generated several in silico structural TcCalr models to delimit a peptide (VC-TcCalr) at the TcCalr N-domain. Chemically synthesized VC-TcCalr did bind to C1q and was anti-angiogenic in Gallus gallus chorioallantoic membrane assays. These properties were associated with structural features, as determined in silico. VC-TcCalr, a strong dipole, interacts with charged proteins such as collagen-like tails and scavenger receptors. Comparatively, HuCALR has less polarity and spatial stability, probably due to at least substitutions of Gln for Gly, Arg for Lys, Arg for Asp and Ser for Arg that hinder protein-protein interactions. These differences can explain, at least in part, how TcCalr inhibits the complement activation pathway and has higher efficiency as an antiangiogenic and antitumor agent than HuCALR.

Expresión de calreticulina en placenta canina / Calreticulin expression in canine placenta

La placenta es un anexo embrionario de los mamíferos que tiene por función principal el intercambio de nutrientes y gases y proteger al concepto de un potencial daño inmune provocado por diferencias alogénicas en los Complejos Principales de Histocompatibilidad paternos. Se han descrito diversas proteínas asociadas a su función, siendo Calreticulina una de ellas. Si bien existen estudios de la presencia de Calreticulina en placenta humana, no existen reportes de esta proteína en la placenta canina. Se obtuvieron muestras de placenta canina de las que se extrajo el contenido proteico total y se determinó la presencia de Calreticulina por western blot e inmunohistoquímica. Los resultados mostraron presencia de Calreticulina en placenta canina con un peso molecular aparente de 60 kDa, concordante con lo descrito para la molécula por otros autores. El análisis inmunohistoquímico mostró que Calreticulina canina está presente principalmente en el trofoblasto de las vellosidades, no existiendo diferencias en cuanto a su localización al compararla con placenta humana, pese a sus diferencias morfológicas e histológicas. Esta información permitirá establecer un protocolo estandarizado de extracción de Calreticulina desde placenta, así como orientar acerca de los posibles roles de esta molécula en la placenta.

The placenta is an embryonic organ present in mammals, whose main functions are the exchange of nutrients and gases and to protect the fetus from potential immune damage mediated by paternal and maternal allogeneic differences in the Major Histocompatibility Complex. Several proteins associated with its function have been described, being Calreticulin one of them. Although there are studies on the presence of Calreticulin in human placenta, there are no reports of this protein in canine placenta. Samples from canine placenta were obtained, proteins extracted and Calreticulin was subsequently detected by western blot and immunohistochemistry. The results showed the presence of Calreticulin in canine placenta with an apparent molecular weight of 60 kDa, in agreement with the results from other authors. The immunohistochemical analysis showed that canine Calreticulin is present mainly in the trophoblast of the villi, and there is no difference in its localization when compared with a blood-filled placenta such as human one, despite its morphological and histological differences. We also propose a standardized protocol for the extraction of Calreticulin from placenta, given its abundant expression in this organ. Future studies are aimed at elucidating possible roles of this protein in placenta.

Calreticulin in phagocytosis and cancer: opposite roles in immune response outcomes.

Calreticulin (CRT) is a pleiotropic and highly conserved molecule that is mainly localized in the endoplasmic reticulum. Recently, CRT has gained special interest for its functions outside the endoplasmic reticulum where it has immunomodulatory properties. CRT translocation to the cell membrane serves as an "eat me" signal and promotes efferocytosis of apoptotic cells and cancer cell removal with completely opposite outcomes. Efferocytosis results in a silenced immune response and homeostasis, while removal of dying cancer cells brought about by anthracycline treatment, ionizing-irradiation or photodynamic therapy results in immunogenic cell death with activation of the innate and adaptive immune responses. In addition, CRT impacts phagocyte activation and cytokine production. The effects of CRT on cytokine production depend on its conformation, species specificity, degree of oligomerization and/or glycosylation, as well as its cellular localization and the molecular partners involved. The controversial roles of CRT in cancer progression and the possible role of the CALR gene mutations in myeloproliferative neoplasms are also addressed. The release of CRT and its influence on the different cells involved during efferocytosis and immunogenic cell death points to additional roles of CRT besides merely acting as an "eat me" signal during apoptosis. Understanding the contribution of CRT in physiological and pathological processes could give us some insight into the potential of CRT as a therapeutic target.

Calreticulina: aspectos moleculares y técnicas para su detección / Calreticulin: molecular aspects and techniques for its detection

Las neoplasias mieloproliferativas crónicas (NMPC) son enfermedades clonales caracterizadas por un aumento en el número de células maduras circulantes; estas incluyen: policitemia vera (PV), trombocitemia esencial (TE), mielofibrosis primaria (MFP), entre otras. Una de las principales características moleculares de estas tres entidades es la ausencia del gen de fusión BCR/ABL. La primera mutación relacionada directamente con estas neoplasias fue detectada en el gen JAK2; a partir de su descubrimiento, otras mutaciones en los genes del receptor de trombopoyetina (MPL) y calreticulina (CALR) han sido fuertemente relacionadas con la presentación de la enfermedad. La calreticulina es una proteína del retículo endoplásmico con diversas funciones a nivel celular como la homeostasis del calcio y la actividad de chaperona. Hasta la fecha se ha identificado un gran número de mutaciones en el gen CALR. La mayoría de ellas son inserciones y deleciones que generan cambios a nivel proteico con implicaciones importantes en el curso clínico y pronóstico de las neoplasias. Debido a su alta frecuencia y fuerte asociación con las NMPC, las mutaciones de CALR se incluyen como criterio mayor para el diagnóstico de estas entidades. Por este motivo, se han desarrollado varias técnicas encaminadas a la detección rápida, eficiente, sensible y especifica de esta mutación como: la secuenciación, el análisis de fragmentos y el análisis de fusión de alta resolución. El conocimiento e implementación de estas técnicas en los laboratorios clínicos constituye un avance importante para el diagnóstico y la evolución de los pacientes(AU)

Chronic myeloproliferative neoplasms (NMPC) are clonal diseases characterized by an increase in the number of mature circulating cells; these diseases include: polycythemia vera (PV), essential thrombocythemia (ET), primary myelofibrosis (MFP) among others. One of the main molecular characteristics of these three entities is the absence of the BCR/ABL fusion gene. The first mutation related to this group of neoplasms was detected in the JAK2 gene; since its discovery, other mutations in thrombopoietin receptor (MPL) and calreticulin (CALR) genes have been strongly related with the presentation of the disease. Calreticulin is an endoplásmic reticulum protein with different functions in the cell such as calcium homeostasis and the chaperone activity. To date, a large number of mutations have been identified in CALR gene most of them are insertions and deletions that generate changes in the protein that generate important implications in the clinical course and prognosis of neoplasms. Due to its high frequency and strong association with NMPC, CALR mutations are included as a major criteria for the diagnosis of these entities. For this reason, several techniques have been developed aimed at the rapid, efficient, sensitive and specific detection of this mutation as: sequencing, fragment analysis and high resolution fusion analysis. The knowledge and implementation of these techniques in clinical laboratories is an important advance for the diagnosis and in the evolution of patients(AU)

Arginylated Calreticulin Increases Apoptotic Response Induced by Bortezomib in Glioma Cells.

After retrotranslocation from the endoplasmic reticulum to the cytoplasm, calreticulin is modified by the enzyme arginyltransferase-1 (ATE1). Cellular levels of arginylated calreticulin (R-CRT) are regulated in part by the proteasomal system. Under various stress conditions, R-CRT becomes associated with stress granules (SGs) or reaches the plasma membrane (PM), where it participates in pro-apoptotic signaling. The mechanisms underlying the resistance of tumor cells to apoptosis induced by specific drugs remain unclear. We evaluated the regulatory role of R-CRT in apoptosis of human glioma cell lines treated with the proteasome inhibitor bortezomib (BT). Two cell lines (HOG, MO59K) displaying distinctive susceptibility to apoptosis induction were studied further. BT efficiency was found to be correlated with a subcellular distribution of R-CRT. In MO59K (apoptosis-resistant), R-CRT was confined to SGs formed following BT treatment. In contrast, HOG (apoptosis-susceptible) treated with BT showed lower SG formation and higher levels of cytosolic and PM R-CRT. Increased R-CRT level was associated with enhanced mobilization of intracellular Ca2+ and with sustained apoptosis activation via upregulation of cell death receptor DR5. R-CRT overexpression in the cytoplasm of MO59K rendered the cells susceptible to BT-induced, DR5-mediated cell death. Our findings suggest that R-CRT plays an essential role in the effect of BT treatment on tumor cells and that ATE1 is a strong candidate target for future studies of cancer diagnosis and therapy.

Differential expression and molecular interactions of chromosome region maintenance 1 and calreticulin exportins in breast cancer cells.

Chromosome region maintenance 1 (CRM-1) and calreticulin (CALR) are two proteins that act as exportins for some nuclear receptors, in addition to other critical functions for cellular homeostasis. In several cancer types, CRM-1 and CALR are upregulated suggesting an imbalance in their functions. However, the regulation of CRM-1 and CALR, and their biological implications, are not completely known. Here, we evaluated the interplay between the levels of CRM-1 and CALR, and estrogen receptor alpha (ERα) status, in breast cancer cells. CRM-1 and CALR were upregulated in mammary tumors relative to normal mammary tissue. Furthermore, the mRNA and protein levels of CRM-1 and CALR were higher in breast cancer cells lacking ERα, in comparison with those that express ERα. Additionally, both proteins were distributed in the nucleus and cytoplasm in the two cell types. Importantly, we identified novel interactions for these exportins. First, we showed an interaction between CRM-1 and CALR, and then we identified that SUN1 and SUN2, two proteins localized in the nuclear envelop, were able to interact specifically with CRM-1, but not CALR. Interestingly, SUN1 and SUN2 expression seemed to be decreased in breast cancer, thereby affecting the interactions of these proteins with CRM-1, and possibly its actions as an exportin. Thus, our data suggest that expression levels for CRM-1 and CALR, the interaction between these exportins, and specific interactions of SUN1 and SUN2 with CRM-1 but not CALR, may be central elements in nucleo-cytoplasmic transport. Furthermore, deregulation of these elements may have serious implications in the progression of breast and other types of cancer.