Uremia and Inadequate Oxygen Supply Induce Eryptosis and Intracellular Hypoxia in Red Blood Cells.

BACKGROUND/AIMS: Chronic kidney disease is frequently accompanied by anemia, hypoxemia, and hypoxia. It has become clear that the impaired erythropoietin production and altered iron homeostasis are not the sole causes of renal anemia. Eryptosis is a process of red blood cells (RBC) death, like apoptosis of nucleated cells, characterized by Ca2+ influx and phosphatidylserine (PS) exposure to the outer RBC membrane leaflet. Eryptosis can be induced by uremic toxins and occurs before senescence, thus shortening RBC lifespan and aggravating renal anemia. We aimed to assess eryptosis and intracellular oxygen levels of RBC from hemodialysis patients (HD-RBC) and their response to hypoxia, uremia, and uremic toxins uptake inhibition. METHODS: Using flow cytometry, RBC from healthy individuals (CON-RBC) and HD-RBC were subjected to PS (Annexin-V), intracellular Ca2+ (Fluo-3/AM) and intracellular oxygen (Hypoxia Green) measurements, at baseline and after incubation with uremic serum and/or hypoxia (5% O2), with or without ketoprofen. Baseline levels of uremic toxins were quantified in serum and cytosol by high performance liquid chromatography. RESULTS: Here, we show that HD-RBC have less intracellular oxygen and that it is further decreased post-HD. Also, incubation in 5% O2 and uremia triggered eryptosis in vitro by exposing PS. Hypoxia itself increased the PS exposure in HD-RBC and CON-RBC, and the addition of uremic serum aggravated it. Furthermore, inhibition of the organic anion transporter 2 with ketoprofen reverted eryptosis and restored the levels of intracellular oxygen. Cytosolic levels of the uremic toxins pCS and IAA were decreased after dialysis. CONCLUSION: These findings suggest the participation of uremic toxins and hypoxia in the process of eryptosis and intracellular oxygenation.

Uremia and Hypoxia Independently Induce Eryptosis and Erythrocyte Redox Imbalance.

BACKGROUND/AIMS: Red blood cell (RBC) death could contribute to anemia in chronic kidney disease (CKD) patients. Recent observational research has suggested a relationship between RBC death (eryptosis) and hypoxemia in hemodialysis patients. Thus, we studied the isolated and joint effects of a uremic toxin (indoxyl sulfate; IS) and hypoxia on RBC biology. METHODS: We incubated RBC from healthy donors with IS at concentrations of 0.01mM, 0.09mM and 0.17mM under both normoxic (21% O2) and hypoxic (5% O2) conditions for 24 hours. Eryptosis was evaluated by RBC phosphatidylserine (PS) exposure, cell volume, and cytosolic calcium which were quantified by Annexin-V+, forward scatter, and Fluo-3AM+ binding, respectively. RBC redox balance was reported by reactive oxygen species (ROS) production and intracellular reduced glutathione (GSH). Analyses were performed by flow cytometry. RESULTS: Hypoxia induced a 2-fold ROS production compared to normoxia. PS exposure and cytosolic calcium increased, while cell volume decreased by hypoxia and likewise by IS. IS increased ROS production in a dose-dependent manner under conditions of both normoxia and hypoxia. The same conditions promoted a GSH decrease with IS intensifying the hypoxia-induced effects. CONCLUSION: In summary, our results indicate that the concurrent presence of hypoxia and uremia augments RBC death and may therefore, contribute to the genesis of anemia in CKD.

The role of the lectin pathway of the complement system in SARS-CoV-2 lung injury.

Although some evidence showed the activation of complement systems in COVID-19 patients, proinflammatory status and lectin pathway remain unclear. Thus, the present study aimed to demonstrate the role of MBL and ficolin-3 in the complement system activation and compared to pandemic Influenza A virus H1N1 subtype infection (H1N1pdm09) and control patients. A total of 27 lungs formalin-fixed paraffin-embedded samples (10 from H1N1 group, 6 from the COVID-19 group, and 11 from the control group) were analyzed by immunohistochemistry using anti-IL-6, TNF-alfa, CD163, MBL e FCN3 antibodies. Genotyping of target polymorphisms in the MBL2 gene was performed by real-time PCR. Proinflammatory cytokines such as IL-6 and TNF-alpha presented higher tissue expression in the COVID-19 group compared to H1N1 and control groups. The same results were observed for ICAM-1 tissue expression. Increased expression of the FCN3 was observed in the COVID-19 group and H1N1 group compared to the control group. The MBL tissue expression was higher in the COVID-19 group compared to H1N1 and control groups. The genotypes AA for rs180040 (G/A), GG for rs1800451 (G/A) and CC for rs5030737 (T/C) showed a higher prevalence in the COVID-19 group. The intense activation of the lectin pathway, with particular emphasis on the MBL pathway, together with endothelial dysfunction and a massive proinflammatory cytokines production, possibly lead to a worse outcome in patients infected with SARS-Cov-2. Moreover, 3 SNPs of our study presented genotypes that might be correlated with high MBL tissue expression in the COVID-19 pulmonary samples.

The Role of Eryptosis in the Pathogenesis of Renal Anemia: Insights From Basic Research and Mathematical Modeling.

Red blood cells (RBC) are the most abundant cells in the blood. Despite powerful defense systems against chemical and mechanical stressors, their life span is limited to about 120 days in healthy humans and further shortened in patients with kidney failure. Changes in the cell membrane potential and cation permeability trigger a cascade of events that lead to exposure of phosphatidylserine on the outer leaflet of the RBC membrane. The translocation of phosphatidylserine is an important step in a process that eventually results in eryptosis, the programmed death of an RBC. The regulation of eryptosis is complex and involves several cellular pathways, such as the regulation of non-selective cation channels. Increased cytosolic calcium concentration results in scramblase and floppase activation, exposing phosphatidylserine on the cell surface, leading to early clearance of RBCs from the circulation by phagocytic cells. While eryptosis is physiologically meaningful to recycle iron and other RBC constituents in healthy subjects, it is augmented under pathological conditions, such as kidney failure. In chronic kidney disease (CKD) patients, the number of eryptotic RBC is significantly increased, resulting in a shortened RBC life span that further compounds renal anemia. In CKD patients, uremic toxins, oxidative stress, hypoxemia, and inflammation contribute to the increased eryptosis rate. Eryptosis may have an impact on renal anemia, and depending on the degree of shortened RBC life span, the administration of erythropoiesis-stimulating agents is often insufficient to attain desired hemoglobin target levels. The goal of this review is to indicate the importance of eryptosis as a process closely related to life span reduction, aggravating renal anemia.

Neutrophils activated by BJcuL, a C-type lectin isolated from Bothrops jararacussu venom, decrease the invasion potential of neuroblastoma SK-N-SH cells in vitro.

BACKGROUND: Neuroblastoma is a pediatric tumor with a mortality rate of 40% in the most aggressive cases. Tumor microenvironment components as immune cells contribute to the tumor progression; thereby, the modulation of immune cells to a pro-inflammatory and antitumoral profile could potentialize the immunotherapy, a suggested approach for high-risk patients. Preview studies showed the antitumoral potential of BJcuL, a C- type lectin isolated from Bothrops jararacussu venom. It was able to induce immunomodulatory responses, promoting the rolling and adhesion of leukocytes and the activation of neutrophils. METHODS: SK-N-SH cells were incubated with conditioned media (CM) obtained during the treatment of neutrophils with BJcuL and fMLP, a bacteria-derived peptide highly effective for activating neutrophil functions. Then we evaluated the effect of the same stimulation on the co-cultivation of neutrophils and SK-N-SH cells. Tumor cells were tested for viability, migration, and invasion potential. RESULTS: In the viability assay, only neutrophils treated with BJcuL (24 h) and cultivated with SK-N-SH were cytotoxic. Migration of tumor cells decreased when incubated directly (p < 0.001) or indirectly (p < 0.005) with untreated neutrophils. When invasion potential was evaluated, neutrophils incubated with BJcuL reduced the total number of colonies of SK-N-SH cells following co-cultivation for 24 h (p < 0.005). Treatment with CM resulted in decreased anchorage-free survival following 24 h of treatment (p < 0.001). CONCLUSION: Data demonstrated that SK-N-SH cells maintain their migratory potential in the face of neutrophil modulation by BJcuL, but their invasive capacity was significantly reduced.

Antitumoral effects of Amblyomma sculptum Berlese saliva in neuroblastoma cell lines involve cytoskeletal deconstruction and cell cycle arrest.

The antitumor properties of ticks salivary gland extracts or recombinant proteins have been reported recently, but little is known about the antitumor properties of the secreted components of saliva. The goal of this study was to investigate the in vitro effect of the saliva of the hard tick Amblyomma sculptum on neuroblastoma cell lines. SK-N-SK, SH-SY5Y, Be(2)-M17, IMR-32, and CHLA-20 cells were susceptible to saliva, with 80% reduction in their viability compared to untreated controls, as demonstrated by the methylene blue assay. Further investigation using CHLA-20 revealed apoptosis, with approximately 30% of annexin-V positive cells, and G0/G1-phase accumulation (>60%) after treatment with saliva. Mitochondrial membrane potential (Δψm) was slightly, but significantly (p < 0.05), reduced and the actin cytoskeleton was disarranged, as indicated by fluorescent microscopy. The viability of human fibroblast (HFF-1 cells) used as a non-tumoral control decreased by approximately 40%. However, no alterations in cell cycle progression, morphology, and Δψm were observed in these cells. The present work provides new perspectives for the characterization of the molecules present in saliva and their antitumor properties.

Cancer stem cell markers ALDH1 and CD44+/CD24- phenotype and their prognosis impact in invasive ductal carcinoma.

Breast cancer is a very heterogeneous disease. The intrinsic molecular subtypes can explain the intertumoral heterogeneity and the cancer stem cell (CSC) hypothesis can explain the intratumoral heterogeneity of this kind of tumor. CD44+/CD24- phenotype and ALDH1 expression are the major CSC markers described in invasive breast cancer. In the present study, 144 samples of invasive breast carcinoma, no special type were distributed in 15 tissue microarrays (TMA) and then evaluated for expression of the CD44+/CD24- phenotype and ALDH1 to understand the importance of these CSC markers and the clinical aspects of breast cancer. The samples were classified into four molecular subtypes according to clinicopathological criteria: Luminal A, Luminal B, HER2, and Basal-like. A statistical association was found between the molecular subtypes and the CSC markers, with HER2 the most frequent subtype for both markers. ALDH1 was also associated with other poor prognostic variables, such as a high histological grade and larger tumors, but it was not associated with the patients' prognosis in this sample and nor was the CD44+/CD24- phenotype in a multivariate analysis. There are still many controversies about the role of these markers in breast cancer molecular subtypes. The identification of these populations of cells, through immunohistochemical markers, can help to better understand the CSC theory in clinical practice and, in the near future, contribute to developing new target therapies.

Neutrophils activated by BJcuL, a C-type lectin isolated from Bothrops jararacussu venom, decrease the invasion potential of neuroblastoma SK-N-SH cells in vitro

ABSTRACT Background: Neuroblastoma is a pediatric tumor with a mortality rate of 40% in the most aggressive cases. Tumor microenvironment components as immune cells contribute to the tumor progression; thereby, the modulation of immune cells to a pro-inflammatory and antitumoral profile could potentialize the immunotherapy, a suggested approach for high-risk patients. Preview studies showed the antitumoral potential of BJcuL, a C- type lectin isolated from Bothrops jararacussu venom. It was able to induce immunomodulatory responses, promoting the rolling and adhesion of leukocytes and the activation of neutrophils. Methods: SK-N-SH cells were incubated with conditioned media (CM) obtained during the treatment of neutrophils with BJcuL and fMLP, a bacteria-derived peptide highly effective for activating neutrophil functions. Then we evaluated the effect of the same stimulation on the co-cultivation of neutrophils and SK-N-SH cells. Tumor cells were tested for viability, migration, and invasion potential. Results: In the viability assay, only neutrophils treated with BJcuL (24 h) and cultivated with SK-N-SH were cytotoxic. Migration of tumor cells decreased when incubated directly (p 0.001) or indirectly (p 0.005) with untreated neutrophils. When invasion potential was evaluated, neutrophils incubated with BJcuL reduced the total number of colonies of SK-N-SH cells following co-cultivation for 24 h (p 0.005). Treatment with CM resulted in decreased anchorage-free survival following 24 h of treatment (p 0.001). Conclusion: Data demonstrated that SK-N-SH cells maintain their migratory potential in the face of neutrophil modulation by BJcuL, but their invasive capacity was significantly reduced.

Neutrophils activated by BJcuL, a C-type lectin isolated from Bothrops jararacussu venom, decrease the invasion potential of neuroblastoma SK-N-SH cells in vitro

Neuroblastoma is a pediatric tumor with a mortality rate of 40% in the most aggressive cases. Tumor microenvironment components as immune cells contribute to the tumor progression; thereby, the modulation of immune cells to a pro-inflammatory and antitumoral profile could potentialize the immunotherapy, a suggested approach for high-risk patients. Preview studies showed the antitumoral potential of BJcuL, a C- type lectin isolated from Bothrops jararacussu venom. It was able to induce immunomodulatory responses, promoting the rolling and adhesion of leukocytes and the activation of neutrophils. Methods: SK-N-SH cells were incubated with conditioned media (CM) obtained during the treatment of neutrophils with BJcuL and fMLP, a bacteria-derived peptide highly effective for activating neutrophil functions. Then we evaluated the effect of the same stimulation on the co-cultivation of neutrophils and SK-N-SH cells. Tumor cells were tested for viability, migration, and invasion potential. Results: In the viability assay, only neutrophils treated with BJcuL (24 h) and cultivated with SK-N-SH were cytotoxic. Migration of tumor cells decreased when incubated directly (p < 0.001) or indirectly (p < 0.005) with untreated neutrophils. When invasion potential was evaluated, neutrophils incubated with BJcuL reduced the total number of colonies of SK-N-SH cells following co-cultivation for 24 h (p < 0.005). Treatment with CM resulted in decreased anchorage-free survival following 24 h of treatment (p < 0.001). Conclusion: Data demonstrated that SK-N-SH cells maintain their migratory potential in the face of neutrophil modulation by BJcuL, but their invasive capacity was significantly reduced.(AU)

The lectin BJcuL induces apoptosis through TRAIL expression, caspase cascade activation and mitochondrial membrane permeability in a human colon adenocarcinoma cell line.

It has been demonstrated that the cytotoxic effect of BJcuL, the lectin isolated from Bothrops jararacussu venom, on human gastric carcinoma is accompanied by the inhibition of extracellular matrix adhesion, cytoskeleton disassembly and apoptosis induction. The present study aimed to evaluate the apoptosis mechanisms triggered by the BJcuL interaction with specific glycans on the surface of HT29 human colon adenocarcinoma cells. The results demonstrated that BJcuL interacts with glycoligands targets on the cell, which were inhibited in the presence of d-galactose. It shows a dose-dependently cytotoxic effect that is inhibited in the presence of d-galactose. A dose-dependent cell aggregation decrease was also observed for the HT29 cells. Analysis of cell proliferation inhibition was assessed by anti-PCNA and demonstrated that lectin diminishes PCNA expression when compared with untreated cells. Differences in apoptotic marker expression estimated by immunohistochemistry revealed that the lectin promotes an increase in TRAIL expression, leading to an increase in the expression of FADD, caspase-8 and Bax. Besides the increased expression of apoptosis-related proteins, our results revealed that the lectin promotes a mitochondrial respiration decrease and a 75% increase in the amount of cytochrome c released. Together these results suggest that the cytotoxicity of BJcuL can sensitize pro-apoptotic proteins in the cytoplasm and mitochondria, leading to the apoptotic cascade.

Antitumoral effects of Amblyomma sculptum Berlese saliva in neuroblastoma cell lines involve cytoskeletal deconstruction and cell cycle arrest / Efeito antitumoral da saliva do carrapato Amblyomma sculptum Berlese em células de neuroblastoma envolve desconstrução do citoesqueleto e parada do ciclo celular

Abstract The antitumor properties of ticks salivary gland extracts or recombinant proteins have been reported recently, but little is known about the antitumor properties of the secreted components of saliva. The goal of this study was to investigate the in vitro effect of the saliva of the hard tick Amblyomma sculptum on neuroblastoma cell lines. SK-N-SK, SH-SY5Y, Be(2)-M17, IMR-32, and CHLA-20 cells were susceptible to saliva, with 80% reduction in their viability compared to untreated controls, as demonstrated by the methylene blue assay. Further investigation using CHLA-20 revealed apoptosis, with approximately 30% of annexin-V positive cells, and G0/G1-phase accumulation (>60%) after treatment with saliva. Mitochondrial membrane potential (Δψm) was slightly, but significantly (p < 0.05), reduced and the actin cytoskeleton was disarranged, as indicated by fluorescent microscopy. The viability of human fibroblast (HFF-1 cells) used as a non-tumoral control decreased by approximately 40%. However, no alterations in cell cycle progression, morphology, and Δψm were observed in these cells. The present work provides new perspectives for the characterization of the molecules present in saliva and their antitumor properties.

Resumo As propriedades antitumorais de extratos de glândulas salivares de carrapatos ou proteínas recombinantes foram relatadas recentemente, mas pouco se sabe sobre as propriedades antitumorais dos componentes secretados da saliva. O objetivo deste estudo foi investigar o efeito in vitro da saliva bruta do carrapato duro Amblyomma sculptum sobre as linhagens celulares de neuroblastoma. Células SK-N-SK, SH-SY5Y, Be(2)-M17, IMR-32 e CHLA-20 foram suscetíveis à saliva, com redução de 80% na sua viabilidade em comparação com controles não tratados, como demonstrado pelo ensaio de Azul de Metileno. Investigações posteriores utilizando CHLA-20 revelaram apoptose, com aproximadamente 30% de células positivas para anexina-V, e G0/G1 (> 60%) após tratamento com saliva. O potencial de membrana mitocondrial (Δψm) foi reduzido significativamente (p <0,05), e o citoesqueleto de actina foi desestruturado, como indicado pela microscopia de fluorescência. A viabilidade do fibroblasto humano (células HFF-1), usado como controle não tumoral, diminuiu em aproximadamente 40%. No entanto, não foram observadas alterações na progressão do ciclo celular, morfologia e Δψm nestas células. O presente trabalho fornece novas perspectivas para a caracterização das moléculas presentes na saliva e suas propriedades antitumorais.

BJcuL, a lectin purified from Bothrops jararacussu venom, induces apoptosis in human gastric carcinoma cells accompanied by inhibition of cell adhesion and actin cytoskeleton disassembly.

We show that BJcuL, a lectin purified from Bothrops jararacussu venom, exerts cytotoxic effects to gastric carcinoma cells MKN45 and AGS. This effect was due to the direct interaction with specific glycans on the cells surface and was observed by cell viability decrease, disorganization of actin filaments and apoptosis. In addition, BJcuL was able to reduce tumor cell adhesion to matrigel, what was inhibited by specific carbohydrate or partially inhibited when cells were pre-incubated with matrigel. Our results suggest that BJcuL was able to promote apoptosis in both tumor cells lines and therefore has a prospect for potential use in cancer therapy.