Corrigendum: Neutrophil to lymphocyte ratio and principal component analysis offer prognostic advantage for dogs with mammary tumors.

[This corrects the article DOI: 10.3389/fvets.2023.1187271.].

Neutrophils versus Protozoan Parasites: Plasmodium, Trichomonas, Leishmania, Trypanosoma, and Entameoba.

Neutrophils are the most abundant polymorphonuclear granular leukocytes in human blood and are an essential part of the innate immune system. Neutrophils are efficient cells that eliminate pathogenic bacteria and fungi, but their role in dealing with protozoan parasitic infections remains controversial. At sites of protozoan parasite infections, a large number of infiltrating neutrophils is observed, suggesting that neutrophils are important cells for controlling the infection. Yet, in most cases, there is also a strong inflammatory response that can provoke tissue damage. Diseases like malaria, trichomoniasis, leishmaniasis, Chagas disease, and amoebiasis affect millions of people globally. In this review, we summarize these protozoan diseases and describe the novel view on how neutrophils are involved in protection from these parasites. Also, we present recent evidence that neutrophils play a double role in these infections participating both in control of the parasite and in the pathogenesis of the disease.

Neutrophil to lymphocyte ratio and principal component analysis offer prognostic advantage for dogs with mammary tumors.

Introduction: In veterinary medicine, cancer is the leading cause of death in companion animals, and mammary gland tumors represent the most common neoplasm in female dogs. Several epidemiological risk factors, such as age, breed, hormones, diet, and obesity have been reported to be relevant for canine mammary tumors. Nowadays, the gold standard for diagnosis of canine mammary tumors is the pathological examination of the suspected tissue. However, tumor grade can only be assessed after surgical removal or biopsy of the altered tissue. Therefore, in cases of tumors that could be surgically removed, it would be very helpful to be able to predict the biological behavior of the tumor, before performing any surgery. Since, inflammation constitutes part of the tumor microenvironment and it influences each step of tumorigenesis, cellular and biochemical blood markers of systemic inflammation, such as the neutrophil to lymphocyte ratio (NLR) and the albumin to globulin ratio (AGR) have been proposed as prognostic factors for human cancer development. The NLR and the AGR have not been explored enough as prognostic factors for cancer development in veterinary medicine. Methods: To determine the prognostic value of NLR in canine mammary tumors, clinical records including biochemistry and hematological studies of female dogs with mammary tumors and of control healthy dogs, were used to determine the pre-treatment NLR and AGR. Other clinical data included age, breed, tumor size, histological tumor grade, and survival time after surgery. Results and discussion: It was found that a higher pre-treatment NLR value (NLR > 5) associates with less survival rate. In contrast, the AGR did not show any predictive value on the malignancy of the tumor. However, by combining the NLR with AGR, age of the dog, and tumor size in a principal component analysis (PCA), the grade of the tumor and survival after surgery could be appropriately predicted. These data strongly suggest that pre-treatment NLR values have a prognostic value for the survival rate after surgery of dogs with mammary tumors.

Neutrophils Actively Contribute to Obesity-Associated Inflammation and Pathological Complications.

Obesity is characterized by an increase in body weight associated with an exaggerated enlargement of the adipose tissue. Obesity has serious negative effects because it is associated with multiple pathological complications such as type 2 diabetes mellitus, cardiovascular diseases, cancer, and COVID-19. Nowadays, 39% of the world population is obese or overweight, making obesity the 21st century epidemic. Obesity is also characterized by a mild, chronic, systemic inflammation. Accumulation of fat in adipose tissue causes stress and malfunction of adipocytes, which then initiate inflammation. Next, adipose tissue is infiltrated by cells of the innate immune system. Recently, it has become evident that neutrophils, the most abundant leukocytes in blood, are the first immune cells infiltrating the adipose tissue. Neutrophils then get activated and release inflammatory factors that recruit macrophages and other immune cells. These immune cells, in turn, perpetuate the inflammation state by producing cytokines and chemokines that can reach other parts of the body, creating a systemic inflammatory condition. In this review, we described the recent findings on the role of neutrophils during obesity and the initiation of inflammation. In addition, we discuss the involvement of neutrophils in the generation of obesity-related complications using diabetes as a prime example.

The Multiple Roles of Trogocytosis in Immunity, the Nervous System, and Development.

Trogocytosis is a general biological process that involves one cell physically taking small parts of the membrane and other components from another cell. In trogocytosis, one cell seems to take little "bites" from another cell resulting in multiple outcomes from these cell-cell interactions. Trogocytosis was first described in protozoan parasites, which by taking pieces of host cells, kill them and cause tissue damage. Now, it is known that this process is also performed by cells of the immune system with important consequences such as cell communication and activation, elimination of microbial pathogens, and even control of cancer cells. More recently, trogocytosis has also been reported to occur in cells of the central nervous system and in various cells during development. Some of the molecules involved in phagocytosis also participate in trogocytosis. However, the molecular mechanisms that regulate trogocytosis are still a mystery. Elucidating these mechanisms is becoming a research area of much interest. For example, why neutrophils can engage trogocytosis to kill Trichomonas vaginalis parasites, but neutrophils use phagocytosis to eliminate already death parasites? Thus, trogocytosis is a significant process in normal physiology that multiple cells from different organisms use in various scenarios of health and disease. In this review, we present the basic principles known on the process of trogocytosis and discuss the importance in this process to host-pathogen interactions and to normal functions in the immune and nervous systems.

Quantitative proteomic analysis of extracellular vesicle subgroups isolated by an optimized method combining polymer-based precipitation and size exclusion chromatography.

The molecular characterization of extracellular vesicles (EVs) has revealed a great heterogeneity in their composition at a cellular and tissue level. Current isolation methods fail to efficiently separate EV subtypes for proteomic and functional analysis. The aim of this study was to develop a reproducible and scalable isolation workflow to increase the yield and purity of EV preparations. Through a combination of polymer-based precipitation and size exclusion chromatography (Pre-SEC), we analyzed two subsets of EVs based on their CD9, CD63 and CD81 content and elution time. EVs were characterized using transmission electron microscopy, nanoparticle tracking analysis, and Western blot assays. To evaluate differences in protein composition between the early- and late-eluting EV fractions, we performed a quantitative proteomic analysis of MDA-MB-468-derived EVs. We identified 286 exclusive proteins in early-eluting fractions and 148 proteins with a differential concentration between early- and late-eluting fractions. A density gradient analysis further revealed EV heterogeneity within each analyzed subgroup. Through a systems biology approach, we found significant interactions among proteins contained in the EVs which suggest the existence of functional clusters related to specific biological processes. The workflow presented here allows the study of EV subtypes within a single cell type and contributes to standardizing the EV isolation for functional studies.

Immune Response to the Enteric Parasite Entamoeba histolytica.

Entamoeba histolytica is a protozoan parasite responsible for amoebiasis, a disease with a high prevalence in developing countries. Establishing an amoebic infection involves interplay between pathogenic factors for invasion and tissue damage, and immune responses for protecting the host. Here, we review the pathogenicity of E. histolytica and summarize the latest knowledge on immune response and immune evasion mechanisms during amoebiasis.

Phagocytosis: Our Current Understanding of a Universal Biological Process.

Phagocytosis is a cellular process for ingesting and eliminating particles larger than 0.5 µm in diameter, including microorganisms, foreign substances, and apoptotic cells. Phagocytosis is found in many types of cells and it is, in consequence an essential process for tissue homeostasis. However, only specialized cells termed professional phagocytes accomplish phagocytosis with high efficiency. Macrophages, neutrophils, monocytes, dendritic cells, and osteoclasts are among these dedicated cells. These professional phagocytes express several phagocytic receptors that activate signaling pathways resulting in phagocytosis. The process of phagocytosis involves several phases: i) detection of the particle to be ingested, ii) activation of the internalization process, iii) formation of a specialized vacuole called phagosome, and iv) maturation of the phagosome to transform it into a phagolysosome. In this review, we present a general view of our current understanding on cells, phagocytic receptors and phases involved in phagocytosis.

Blastula stage specification of avian neural crest.

Cell fate specification defines the earliest steps towards a distinct cell lineage. Neural crest, a multipotent stem cell population, is thought to be specified from the ectoderm, but its varied contributions defy canons of segregation potential and challenges its embryonic origin. Aiming to resolve this conflict, we have assayed the earliest specification of neural crest using blastula stage chick embryos. Specification assays on isolated chick epiblast explants identify an intermediate region specified towards the neural crest cell fate. Furthermore, low density culture suggests that the specification of intermediate cells towards the neural crest lineage is independent of contact mediated induction and Wnt-ligand induced signaling, but is, however, dependent on transcriptional activity of ß-catenin. Finally, we have validated the regional identity of the intermediate region towards the neural crest cell fate using fate map studies. Our results suggest a model of neural crest specification within a restricted epiblast region in blastula stage chick embryos.

Pathogenic Entamoeba histolytica, but not Entamoeba dispar, induce neutrophil extracellular trap (NET) formation.

Amoebiasis is an infection of global importance, caused by the eukaryotic parasite Entamoeba histolytica. Pathogenic E. histolytica is associated worldwide with over a million cases of amoebic dysentery, colitis, and amoebic liver abscess. In contrast, the nonpathogenic Entamoeba dispar does not cause these diseases, although it is commonly found in the same areas as pathogenic amoeba. Entamoeba histolytica infection is usually associated with infiltrating neutrophils. These neutrophils appear to play a defensive role against this parasite, by mechanisms not completely understood. Recently, our group reported that neutrophil extracellular traps (NET) are produced in response to E. histolytica trophozoites. But, there is no information on whether nonpathogenic E. dispar can also induce NET formation. In this report, we explored the possibility that E. dispar leads to NET formation. Neutrophils were stimulated by E. histolytica trophozoites or by E. dispar trophozoites, and NET formation was assessed by video microscopy. NET induced by E. histolytica were important for trapping and killing amoebas. In contrast, E. dispar did not induce NET formation in any condition. Also E. dispar did not induce neutrophil degranulation or reactive oxygen species production. In addition, E. histolytica-induced NET formation required alive amoebas and it was inhibited by galactose, N-acetylgalactosamine, and lactose. These data show that only alive pathogenic E. histolytica activates neutrophils to produce NET, and suggest that recognition of the parasite involves a carbohydrate with an axial HO- group at carbon 4 of a hexose.

Entamoeba histolytica Induce Signaling via Raf/MEK/ERK for Neutrophil Extracellular Trap (NET) Formation.

Amoebiasis, the disease caused by Entamoeba histolytica is the third leading cause of human deaths among parasite infections. E. histolytica was reported associated with around 100 million cases of amoebic dysentery, colitis and amoebic liver abscess that lead to almost 50,000 fatalities worldwide in 2010. E. histolytica infection is associated with the induction of inflammation characterized by a large number of infiltrating neutrophils. These neutrophils have been implicated in defense against this parasite, by mechanisms not completely described. The neutrophil antimicrobial mechanisms include phagocytosis, degranulation, and formation of neutrophil extracellular traps (NETs). Recently, our group reported that NETs are also produced in response to E. histolytica trophozoites. But, the mechanism for NETs induction remains unknown. In this report we explored the possibility that E. histolytica leads to NETs formation via a signaling pathway similar to the pathways activated by PMA or the Fc receptor FcγRIIIb. Neutrophils were stimulated by E. histolytica trophozoites and the effect of various pharmacological inhibitors on amoeba-induced NETs formation was assessed. Selective inhibitors of Raf, MEK, and NF-κB prevented E. histolytica-induced NET formation. In contrast, inhibitors of PKC, TAK1, and NADPH-oxidase did not block E. histolytica-induced NETs formation. E. histolytica induced phosphorylation of ERK in a Raf and MEK dependent manner. These data show that E. histolytica activates a signaling pathway to induce NETs formation, that involves Raf/MEK/ERK, but it is independent of PKC, TAK1, and reactive oxygen species (ROS). Thus, amoebas activate neutrophils via a different pathway from the pathways activated by PMA or the IgG receptor FcγRIIIb.

Control of Phagocytosis by Microbial Pathogens.

Phagocytosis is a fundamental process of cells to capture and ingest foreign particles. Small unicellular organisms such as free-living amoeba use this process to acquire food. In pluricellular organisms, phagocytosis is a universal phenomenon that all cells are able to perform (including epithelial, endothelial, fibroblasts, etc.), but some specialized cells (such as neutrophils and macrophages) perform this very efficiently and were therefore named professional phagocytes by Rabinovitch. Cells use phagocytosis to capture and clear all particles larger than 0.5 µm, including pathogenic microorganisms and cellular debris. Phagocytosis involves a series of steps from recognition of the target particle, ingestion of it in a phagosome (phagocytic vacuole), maturation of this phagosome into a phagolysosome, to the final destruction of the ingested particle in the robust antimicrobial environment of the phagolysosome. For the most part, phagocytosis is an efficient process that eliminates invading pathogens and helps maintaining homeostasis. However, several pathogens have also evolved different strategies to prevent phagocytosis from proceeding in a normal way. These pathogens have a clear advantage to perpetuate the infection and continue their replication. Here, we present an overview of the phagocytic process with emphasis on the antimicrobial elements professional phagocytes use. We also summarize the current knowledge on the microbial strategies different pathogens use to prevent phagocytosis either at the level of ingestion, phagosome formation, and maturation, and even complete escape from phagosomes.

Phagocytosis: A Fundamental Process in Immunity.

One hundred years have passed since the death of Élie Metchnikoff (1845-1916). He was the first to observe the uptake of particles by cells and realized the importance of this process for the host response to injury and infection. He also was a strong advocate of the role of phagocytosis in cellular immunity, and with this he gave us the basis for our modern understanding of inflammation and the innate and acquired immune responses. Phagocytosis is an elegant but complex process for the ingestion and elimination of pathogens, but it is also important for the elimination of apoptotic cells and hence fundamental for tissue homeostasis. Phagocytosis can be divided into four main steps: (i) recognition of the target particle, (ii) signaling to activate the internalization machinery, (iii) phagosome formation, and (iv) phagolysosome maturation. In recent years, the use of new tools of molecular biology and microscopy has provided new insights into the cellular mechanisms of phagocytosis. In this review, we present a general view of our current knowledge on phagocytosis. We emphasize novel molecular findings, particularly on phagosome formation and maturation, and discuss aspects that remain incompletely understood.

Evaluación de la citotoxicidad de tres cementos selladores endodóncicos utilizados en cirugía periapical: estudio in vitro / Cytotoxicity assessment of three endodontic sealing cements used in periapical surgery. In vitro study

Introducción: Existen diversos materiales de retroobturación, pero poco se sabe de su toxicidad sobre fibroblastos gingivales. Objetivo: Evaluar la citotoxicidad de tres materiales de retroobturación sobre fibroblastos gingivales humanos y fibroblastos de la línea L929. Material y métodos: Los medios condicionados de los materiales de retroobturación EndoSequence® BC RRMTM (ERRM), trióxido mineral agregado MTA Angelus® blanco (MTA) y material de restauración intermedia (IRM®) se obtuvieron en fresco, al tiempo de fraguado, y después de 1, 24 y 72 horas del tiempo de fraguado. La morfología celular fue evaluada por microscopia de luz y la viabilidad celular fue evaluada a través de la actividad metabólica mitocondrial con 3-(4,5-dimetiltiazol-2-il)-2,5-difenil bromuro de tetrazolio (MTT). El análisis estadístico se realizó por ANOVA. Resultados: El material ERRM no mostró efectos citotóxicos sobre los fibroblastos. Sin embargo, el MTA y el IRM® mostraron citotoxicidad moderada y alta, respectivamente. Esto revela que el MTA y el IRM® no son completamente inocuos. Conclusión: Los materiales biocerámicos como el ERRM pueden ser considerados los materiales de retroobturación más biocompatibles.

Introduction: Presently there are many retrofilling materials in themarket, nevertheless, little is known about their toxicity on gingival fibroblasts. Objective: To assess cytotoxicity of three materials tohuman gingival fibroblasts and L929 mouse fibroblasts cell line. Material and methods: EndoSequence® BC RRMTM (ERRM; rootrepair material), white MTA Angelus® (MTA) and intermediaterestoration material (IRM®) conditioned media were obtained whenmaterials were freshly mixed, at setting time and after 1, 24 and72 hours of setting time. Cell morphology was assessed with lightmicroscopy and cell viability was assessed through mitochondrialmetabolic activity with 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Statistical analysis was conducted withANOVA. Results: We found that ERRM material did not exhibitcytotoxic effects on used fibroblast, nevertheless, MTA and IRM® respectively exhibited moderate and severe cytotoxicity, thusindicating the materials were not fully harmless. Conclusion: Bioceramic cements like ERRM could be considered the mostcompatible retrofilling-materials.

Transforming Growth Factor-ß-Activated Kinase 1 Is Required for Human FcγRIIIb-Induced Neutrophil Extracellular Trap Formation.

Neutrophils (PMNs) are the most abundant leukocytes in the blood. PMN migrates from the circulation to sites of infection where they are responsible for antimicrobial functions. PMN uses phagocytosis, degranulation, and formation of neutrophil extracellular traps (NETs) to kill microbes. Several stimuli, including bacteria, fungi, and parasites, and some pharmacological compounds, such as Phorbol 12-myristate 13-acetate (PMA), are efficient inducers of NETs. Antigen-antibody complexes are also capable of inducing NET formation. Recently, it was reported that FcγRIIIb cross-linking induced NET formation similarly to PMA stimulation. Direct cross-linking of FcγRIIA or integrins did not promote NET formation. FcγRIIIb-induced NET formation presented different kinetics from PMA-induced NET formation, suggesting differences in signaling. Because FcγRIIIb also induces a strong activation of extracellular signal-regulated kinase (ERK) and nuclear factor Elk-1, and the transforming growth factor-ß-activated kinase 1 (TAK1) has recently been implicated in ERK signaling, in the present report, we explored the role of TAK1 in the signaling pathway activated by FcγRIIIb leading to NET formation. FcγRIIIb was stimulated by specific monoclonal antibodies, and NET formation was evaluated in the presence or absence of pharmacological inhibitors. The antibiotic LL Z1640-2, a selective inhibitor of TAK1 prevented FcγRIIIb-induced, but not PMA-induced NET formation. Both PMA and FcγRIIIb cross-linking induced phosphorylation of ERK. But, LL Z1640-2 only inhibited the FcγRIIIb-mediated activation of ERK. Also, only FcγRIIIb, similarly to transforming growth factor-ß-induced TAK1 phosphorylation. A MEK (ERK kinase)-specific inhibitor was able to prevent ERK phosphorylation induced by both PMA and FcγRIIIb. These data show for the first time that FcγRIIIb cross-linking activates TAK1, and that this kinase is required for triggering the MEK/ERK signaling pathway to NETosis.

Stereological Quantification of Cell-Cycle Kinetics and Mobilization of Epithelial Stem Cells during Wound Healing.

We describe a stereology method to obtain reliable estimates of the total number of proliferative and migratory epithelial cells after wounding. Using pulse and chase experiments with halogenated thymidine analogs such as iododeoxyuridine (IdU) and chlorodeoxyuridine (CldU), it is possible to track epithelial populations with heterogeneous proliferative characteristics through skin compartments. The stereological and tissue processing methods described here apply widely to address important questions of skin stem-cell biology.

Differential Use of Human Neutrophil Fcγ Receptors for Inducing Neutrophil Extracellular Trap Formation.

Neutrophils (PMN) are the most abundant leukocytes in the blood. PMN migrate from the circulation to sites of infection, where they are responsible for antimicrobial functions. PMN use phagocytosis, degranulation, and formation of neutrophil extracellular traps (NETs) to kill microbes. NETs are fibers composed of chromatin and neutrophil-granule proteins. Several pathogens, including bacteria, fungi, and parasites, and also some pharmacological stimuli such as phorbol 12-myristate 13-acetate (PMA) are efficient inducers of NETs. Antigen-antibody complexes are also capable of inducing NET formation. However the particular Fcγ receptor involved in triggering this function is a matter of controversy. In order to provide some insight into what Fcγ receptor is responsible for NET formation, each of the two human Fcγ receptors was stimulated individually by specific monoclonal antibodies and NET formation was evaluated. FcγRIIa cross-linking did not promote NET formation. Cross-linking other receptors such as integrins also did not promote NET formation. In contrast FcγRIIIb cross-linking induced NET formation similarly to PMA stimulation. NET formation was dependent on NADPH-oxidase, PKC, and ERK activation. These data show that cross-linking FcγRIIIb is responsible for NET formation by the human neutrophil.

Neutrophil Functions in Periodontal Homeostasis.

Oral tissues are constantly exposed to damage from the mechanical effort of eating and to microorganisms, mostly bacteria. In healthy gingiva tissue remodeling and a balance between bacteria and innate immune cells are maintained. However, excess of bacteria biofilm (plaque) creates an inflammation state that recruits more immune cells, mainly neutrophils to the gingiva. Neutrophils create a barrier for bacteria to reach inside tissues. When neutrophils are insufficient, bacteria thrive causing more inflammation that has been associated with systemic effects on other conditions such as atherosclerosis, diabetes, and cancer. But paradoxically when neutrophils persist, they can also promote a chronic inflammatory state that leads to periodontitis, a condition that leads to damage of the bone-supporting tissues. In periodontitis, bone loss is a serious complication. How a neutrophil balance is needed for maintaining healthy oral tissues is the focus of this review. We present recent evidence on how alterations in neutrophil number and function can lead to inflammatory bone loss, and how some oral bacteria signal neutrophils to block their antimicrobial functions and promote an inflammatory state. Also, based on this new information, novel therapeutic approaches are discussed.