Browsing by Author "Simon, Valeska"
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Item Both quiescent and proliferating cells circulate in the blood of the invasive apple snail Pomacea canaliculata(2020) Rodriguez, Cristian; Simon, Valeska; Conget, Paulette; Vega, Israel AGastropod hematopoiesis occurs at specialized tissues in some species, but the evidence also suggests that hemocyte generation is maybe widespread in the connective tissues or the blood system in others. In Ampullariidae (Caenogastropoda), both the kidney and the lung contain putative hematopoietic cells, which react to immune challenges. In the current study, we wanted to explore if hematopoiesis occurs in the blood of Pomacea canaliculata. Thus, we obtained circulating hemocytes from donor animals and tested their ability to proliferate in the blood of conspecific recipients. We tracked cell proliferation by labeling the donors' hemocytes with the fluorescent cell proliferation marker carboxyfluorescein diacetate succinimidyl ester (CFSE). Transferred CFSE-labeled hemocytes survived and proliferated into the recipients' circulation for at least 17 days. We also determined the cell cycle status of circulating hemocytes by using the propidium iodide (PI) and acridine orange (AO) staining methods. Flow cytometry analyses showed that most PI-stained hemocytes were in the G1 phase (~96%), while a lower proportion of cells were through the G2/S-M transition (~4%). When we instead used AO-staining, we further distinguished a subpopulation of cells (~5%) of low size, complexity-granularity, and RNA content. We regarded this subpopulation as quiescent cells. In separate experimental sets, we complemented these findings by assessing in circulating hemocytes two evolutionary conserved features of quiescent, undifferentiated cells. First, we used JC-1 staining to determine the mitochondrial membrane potential (Ψm) of circulating hemocytes, which is expected to be low in quiescent cells. Most hemocytes (~87%) showed high aggregation of JC-1, which indicates a high Ψm. Besides that, a small hemocyte subpopulation (~11%) showed low aggregation of the dye, thus indicating a low Ψm. It is known that the transition from a quiescent to a proliferating state associates with an increase of the Ψm. The specificity of these changes was here controlled by membrane depolarization with the Ψm disruptor CCCP. Second, we stained hemocytes with Hoechst33342 dye to determine the efflux activity of ABC transporters, which participate in the multixenobiotic resistance system characteristic of undifferentiated cells. Most hemocytes (>99%) showed a low dye-efflux activity, but a small proportion of cells (0.06-0.12%) showed a high dye-efflux activity, which was significantly inhibited by 100 and 500 μM verapamil, and thus is indicative of an undifferentiated subpopulation of circulating hemocytes. Taken together, our results suggest that, among circulating hemocytes, there are cells with the ability to proliferate or to stay in a quiescent state and behave as progenitor cells later, either in the circulation or the hematopoietic tissues/organs.Publication Evaluation of the Immune Response Induced by CoronaVac 28-Day Schedule Vaccination in a Healthy Population Group(2022) Escobar, Alejandro; Reyes, Felipe; Acevedo, Mónica; Alonso, Luis; Valiente, Fernando; Soto, Ricardo; Portillo, Hugo; Gatica, Jimena; Flores, Iván; Nova, Estefanía; Barrera, Carlos; Bono, María; Vargas, Leonardo; Simon, Valeska; Leiva, Elias; Vial, Cecilia; Hormazábal, Juan; Jimena, Lina; Valdés, Daniel; Sandino, Ana; Imarai, Mónica; Acuña, ClaudioCoronaVac vaccine from Sinovac Life Science is currently being used in several countries. In Chile, the effectiveness of preventing hospitalization is higher than 80% with a vaccination schedule. However, to date, there are no data about immune response induction or specific memory. For this reason, we recruited 15 volunteers without previous suspected/diagnosed COVID-19 and with negative PCR over time to evaluate the immune response to CoronaVac 28 and 90 days after the second immunization (dpi). The CoronaVac administration induces total and neutralizing anti-spike antibodies in all vaccinated volunteers at 28 and 90 dpi. Furthermore, using ELISpot analysis to assay cellular immune responses against SARS-CoV-2 spike protein, we found an increase in IFN-gamma- and Granzyme B-producing cells in vaccinated volunteers at 28 and 90 dpi. Together, our results indicate that CoronaVac induces a robust humoral immune response and cellular immune memory of at least 90 dpi.Item Highly Differentiated, Resting Gn-Specific Memory CD8+ T Cells Persist Years after Infection by Andes Hantavirus(2010) Manigold, Tobias; Mori, Andres; Graumann, Rebecca; Llop, Elena; Simon, Valeska; Ferres, Marcela; Valdivieso, Francisca; Castillo, Constanza; Hjelle, Brian; Vial, PabloIn man, infection with South American Andes virus (ANDV) causes hantavirus cardiopulmonary syndrome (HCPS). HCPS due to ANDV is endemic in Southern Chile and much of Argentina and increasing numbers of cases are reported all over South America. A case-fatality rate of about 36% together with the absence of successful antiviral therapies urge the development of a vaccine. Although T-cell responses were shown to be critically involved in immunity to hantaviruses in mouse models, no data are available on the magnitude, specificity and longevity of ANDV-specific memory T-cell responses in patients. Using sets of overlapping peptides in IFN-c ELISPOT assays, we herein show in 78 Chilean convalescent patients that Gnderived epitopes were immunodominant as compared to those from the N- and Gc-proteins. Furthermore, while the relative contribution of the N-specific response significantly declined over time, Gn-specific responses remained readily detectable ex vivo up to 13 years after the acute infection. Tetramer analysis further showed that up to 16.8% of all circulating CD3+ CD8+ T cells were specific for the single HLA-B*3501-restricted epitope Gn465–473 years after the acute infection. Remarkably, Gn465–473–specific cells readily secreted IFN-c, granzyme B and TNF-a but not IL-2 upon stimulation and showed a ‘revertant’ CD45RA+ CD272CD282CCR72CD1272 effector memory phenotype, thereby resembling a phenotype seen in other latent virus infections. Most intriguingly, titers of neutralizing antibodies increased over time in 10/17 individuals months to years after the acute infection and independently of whether they were residents of endemic areas or not. Thus, our data suggest intrinsic, latent antigenic stimulation of Gn-specific T-cells. However, it remains a major task for future studies to proof this hypothesis by determination of viral antigen in convalescent patients. Furthermore, it remains to be seen whether Gn-specific T cells are critical for viral control and protective immunity. If so, Gn-derived immunodominant epitopes could be of high value for future ANDV vaccinesPublication Serological study of CoronaVac vaccine and booster doses in Chile: immunogenicity and persistence of anti-SARS-CoV-2 spike antibodies(2022) Vargas, Leonardo; Valdivieso, Nicolás; Tempio, Fabián; Simon, Valeska; Sauma, Daniela; Valenzuela, Lucía; Beltrán, Caroll; Castillo- Delgado, Loriana; Contreras-Benavides, Ximena; Acevedo, Mónica L.; Acevedo, Johanna; Gonzalez, Rafael I.; Valiente-Echeverría, Fernando; Soto-Rifo, Ricardo; Rosemblatt, Mario; López, Mercedes; Osorio, Fabiola; Bono, María RosaBackground: Chile was severely affected by COVID19 outbreaks but was also one of the first countries to start a nationwide program to vaccinate against the disease. Furthermore, Chile became one of the fastest countries to inoculate a high percentage of the target population and implemented homologous and heterologous booster schemes in late 2021 to prevent potential immunological waning. The aim of this study is to compare the immunogenicity and time course of the humoral response elicited by the CoronaVac vaccine in combination with homologous versus heterologous boosters. Methods: We compared the immunogenicity of two doses of CoronaVac and BNT162b2 vaccines and one homologous or heterologous booster through an ELISA assay directed against the ancestral spike protein of SARS-CoV-2. Sera were collected from individuals during the vaccination schedule and throughout the implementation of homologous and heterologous booster programs in Chile. Results: Our findings demonstrate that a two-dose vaccination scheme with CoronaVac induces lower levels of anti-SARS-CoV-2 spike antibodies than BNT162b2 in a broad age range (median age 42 years; interquartile range (IQR) 27-61). Furthermore, antibody production declines with time in individuals vaccinated with CoronaVac and less noticeably, with BNT162b2. Analysis of booster schemes revealed that individuals vaccinated with two doses of CoronaVac generate immunological memory against the SARS-CoV-2 ancestral strain, which can be re-activated with homologous or heterologous (BNT162b2 and ChAdOx1) boosters. Nevertheless, the magnitude of the antibody response with the heterologous booster regime was considerably higher (induction fold BNT162b2: 11.2x; ChAdoX1; 12.4x; CoronaVac: 6.0x) than the responses induced by the homologous scheme. Both homologous and heterologous boosters induced persistent humoral responses (median 122 days, IQR (108-133)), although heterologous boosters remained superior in activating a humoral response after 100 days. Conclusions: Two doses of CoronaVac induces antibody titers against the SARS-CoV-2 ancestral strain which are lower in magnitude than those induced by the BNT162b2 vaccine. However, the response induced by CoronaVac can be greatly potentiated with a heterologous booster scheme with BNT162b2 or ChAdOx1 vaccines. Furthermore, the heterologous and homologous booster regimes induce a durable antibody response which does not show signs of decay 3 months after the booster dose.Item The Antidiabetic Effect of MSCs is not Impaired by Insulin Prophylaxis and is not Improved by a Second Dose of Cells(2011) Ezquer, Fernando; Ezquer, Marcelo; Simon, Valeska; Conget, PauletteType 1 diabetes mellitus (T1D) is due to autoimmune destruction of pancreatic beta-cells. Previously, we have shown that intravenously administered bone marrow-derived multipotent mesenchymal stromal cells (MSCs) allows pancreatic islet recovery, improves insulin secretion and reverts hyperglycemia in low doses streptozotocin (STZ)-induced diabetic mice. Here we evaluate whether insulin prophylaxis and the administration of a second dose of cells affect the antidiabetic therapeutic effect of MSC transplantation. Insulitis and subsequent elimination of pancreatic beta-cells was promoted in C57BL/6 mice by the injection of 40 mg/kg/day STZ for five days. Twenty-four days later, diabetic mice were distributed into experimental groups according to if they received or not insulin and/or one or two doses of healthy donor-derived MSCs. Three and half months later: glycemia, pancreatic islets number, insulinemia, glycated hemoglobin level and glucose tolerance were determined in animals that did not received exogenous insulin for the last 1.5 months. Also, we characterized MSCs isolated from mice healthy or diabetic. The therapeutic effect of MSC transplantation was observed in diabetic mice that received or not insulin prophylaxis. Improvements were similar irrespective if they received one or two doses of cells. Compared to MSCs from healthy mice, MSCs from diabetic mice had the same proliferation and adipogenic potentials, but were less abundant, with altered immunophenotype and no osteogenic potential. Our preclinical results should be taken into account when designing phase II clinical trials aimed to evaluate MSC transplantation in patients with T1D. Cells should be isolated form healthy donor, insulin prophylaxis could be maintained and a second dose, after an elapse of two months, appears unnecessary in the medium-term.