Identification of miRNAs Present in Cell- and Plasma-Derived Extracellular Vesicles-Possible Biomarkers of Colorectal Cancer.

Globally, an increasing prevalence of colorectal cancer (CRC) prompts a need for the development of new methods for early tumor detection. MicroRNAs (also referred to as miRNAs) are short non-coding RNA molecules that play a pivotal role in the regulation of gene expression. MiRNAs are effectively transferred to extracellular vesicle (EVs) membrane sacs commonly released by cells. Our study aimed to examine the expression of miRNAs in four CRC cell lines and EVs derived from them (tumor EVs) in comparison to the normal colon epithelium cell line and its EVs. EVs were isolated by ultracentrifugation from the culture supernatant of SW480, SW620, SW1116, HCT116 and normal CCD841CoN cell lines and characterized according to the MISEV2023 guidelines. MiRNAs were analyzed by small RNA sequencing and validated by quantitative PCR. The performed analysis revealed 22 common miRNAs highly expressed in CRC cell lines and effectively transferred to tumor EVs, including miR-9-5p, miR-182-5p, miR-196b-5p, miR-200b-5p, miR-200c-3p, miR-425-5p and miR-429, which are associated with development, proliferation, invasion and migration of colorectal cancer cells, as well as in vesicle maturation and transport-associated pathways. In parallel, normal cells expressed miRNAs, such as miR-369 and miR-143, which play a role in proinflammatory response and tumor suppression. The analysis of selected miRNAs in plasma-derived EVs and tumor samples from CRC patients showed the similarity of miRNA expression profile between the patients' samples and CRC cell lines. Moreover, miR-182-5p, miR-196-5p, miR-425-5p and miR-429 were detected in several EV samples isolated from patients' plasma. Our results suggest that miR-182-5p, miR-196b-5p and miR-429 are differentially expressed between EVs from CRC patients and healthy donors, which might have clinical implications.

What we know about alterations in immune cells during sepsis in veterinary animals?

Sepsis is still one of the most common causes of death of animals and humans. It is marked by an aberrant immune response to infection, resulting in extensive inflammation, organ dysfunction, and, in severe instances, organ failure. Recognizable symptoms and markers of sepsis encompass substantial elevations in body temperature, respiratory rate, hemoglobin levels, and alterations in immune cell counts, including neutrophils, monocytes, and basophils, along with increases in certain acute-phase proteins. In contrast to human medicine, veterinarians must take into account some species differences. This article provides a comprehensive overview of changes in the immune system during sepsis, placing particular emphasis on species variations and exploring potential future drugs and interventions. Hence, understanding the intricate balance of the immune responses during sepsis is crucial to develop effective treatments and interventions to improve the chances of recovery in animals suffering from this serious condition.

Development of a prototypic, field-usable diagnostic tool for the detection of gram-positive cocci-induced mastitis in cattle.

BACKGROUND: Bovine mastitis is one of the most widespread diseases affecting cattle, leading to significant losses for the dairy industry. Currently, the so-called gold standard in mastitis diagnosis involves determining the somatic cell count (SCC). Apart from a number of advantages, this method has one serious flaw: It does not identify the etiological factor causing a particular infection, making it impossible to introduce targeted antimicrobial therapy. This can contribute to multidrug-resistance in bacterial species. The diagnostic market lacks a test that has the advantages of SCC and also recognizes the species of pathogen causing the inflammation. Therefore, the aim of our study was to develop a lateral flow immunoassay (LFIA) based on elongation factor Tu for identifying most prevalent Gram-positive cocci responsible for causing mastitis including Streptococcus uberis, Streptococcus agalactiae and Staphylococcus aureus. RESULTS: As a result, we showed that the assay for S. uberis detection demonstrated a specificity of 89.02%, a sensitivity of 43.59%, and an accuracy of 80.3%. In turn, the second variant - assay for Gram-positive cocci reached a specificity of 95.59%, a sensitivity of 43.28%, and an accuracy of 78.33%. CONCLUSIONS: Our study shows that EF-Tu is a promising target for LFIA and we have delivered evidence that further evaluation could improve test parameters and fill the gap in the mastitis diagnostics market.

Molecular and phenotypic identification of bacterial species isolated from cows with mastitis from three regions of Poland.

BACKGROUND: Bovine mastitis is a widespread disease affecting dairy cattle worldwide and it generates substantial losses for dairy farmers. Mastitis may be caused by bacteria, fungi or algae. The most common species isolated from infected milk are, among others, Streptococcus spp., Escherichia coli, Staphylococcus aureus and non-aureus staphylococci and mammaliicocci. The aim of this paper is to determine the frequency of occurrence of bacterial species in milk samples from cows with mastitis from three regions of Poland: the north-east, the south-west and the south. To this end 203 milk samples taken from cows with a clinical form (CM) of mastitis (n = 100) and healthy animals (n = 103) were examined, which included culture on an appropriate medium followed by molecular detection of E. coli, S. aureus, Streptococcus agalactiae and Streptococcus uberis, as one of the most common species isolated from mastitis milk. RESULTS: The results obtained indicated that S. uberis was the most commonly cultivated CM species (38%, n = 38), followed by S. aureus (22%, n = 22), E. coli (21%, n = 21) and S. agalactiae (18%, n = 18). Similar frequencies in molecular methods were obtained for S. uberis (35.1%) and S. aureus (28.0%). The variation of sensitivity of both methods may be responsible for the differences in the E. coli (41.0%, p = 0.002) and S. agalactiae (5.0%, p = 0.004) detection rates. Significant differences in composition of species between three regions of Poland were noted for E. coli incidence (p < 0.001), in both the culture and molecular methods, but data obtained by the PCR method indicated that this species was the least common in north-eastern Poland, while the culture method showed that in north-eastern Poland E. coli was the most common species. Significant differences for the molecular method were also observed for S. uberis (p < 0.001) and S. aureus (p < 0.001). Both species were most common in southern and south-western Poland. CONCLUSIONS: The results obtained confirm the need to introduce rapid molecular tests for veterinary diagnostics, as well as providing important epidemiological data, to the best of our knowledge data on Polish cows in selected areas of Poland is lacking.

The Immunology of Psoriasis-Current Concepts in Pathogenesis.

Psoriasis is one of the most common inflammatory skin diseases with a chronic, relapsing-remitting course. The last decades of intense research uncovered a pathological network of interactions between immune cells and other types of cells in the pathogenesis of psoriasis. Emerging evidence indicates that dendritic cells, TH17 cells, and keratinocytes constitute a pathogenic triad in psoriasis. Dendritic cells produce TNF-α and IL-23 to promote T cell differentiation toward TH17 cells that produce key psoriatic cytokines IL-17, IFN-γ, and IL-22. Their activity results in skin inflammation and activation and hyperproliferation of keratinocytes. In addition, other cells and signaling pathways are implicated in the pathogenesis of psoriasis, including TH9 cells, TH22 cells, CD8+ cytotoxic cells, neutrophils, γδ T cells, and cytokines and chemokines secreted by them. New insights from high-throughput analysis of lesional skin identified novel signaling pathways and cell populations involved in the pathogenesis. These studies not only expanded our knowledge about the mechanisms of immune response and the pathogenesis of psoriasis but also resulted in a revolution in the clinical management of patients with psoriasis. Thus, understanding the mechanisms of immune response in psoriatic inflammation is crucial for further studies, the development of novel therapeutic strategies, and the clinical management of psoriasis patients. The aim of the review was to comprehensively present the dysregulation of immune response in psoriasis with an emphasis on recent findings. Here, we described the role of immune cells, including T cells, B cells, dendritic cells, neutrophils, monocytes, mast cells, and innate lymphoid cells (ILCs), as well as non-immune cells, including keratinocytes, fibroblasts, endothelial cells, and platelets in the initiation, development, and progression of psoriasis.

Wounds of Companion Animals as a Habitat of Antibiotic-Resistant Bacteria That Are Potentially Harmful to Humans-Phenotypic, Proteomic and Molecular Detection.

Skin wounds and their infections by antibiotic-resistant bacteria (ARB) are very common in small animals, posing the risk of acquiring ARB by pet owners or antibiotic resistance gene (ARG) transfer to the owners' microbiota. The aim of this study was to identify the most common pathogens infecting wounds of companion animals, assess their antibiotic resistance, and determine the ARGs using culture-based, molecular, and proteomic methods. A total of 136 bacterial strains were isolated from wound swabs. Their species was identified using chromogenic media, followed by MALDI-TOF spectrometry. Antibiotic resistance was tested using disc diffusion, and twelve ARGs were detected using PCRs. The dominant species included Staphylococcus pseudintermedius (9.56%), E. coli, and E. faecalis (both n = 11, 8.09%). Enterobacterales were mostly resistant to amoxicillin/clavulanic acid (68.3% strains), all Pseudomonas were resistant to ceftazidime, piperacillin/tazobactam, imipenem, and tylosin, Acinetobacter were mostly resistant to tylosin (55.5%), all Enterococcus were resistant to imipenem, and 39.2% of Staphylococci were resistant to clindamycin. Among ARGs, strA (streptomycin resistance), sul3 (sulfonamide resistance), and blaTEM, an extended-spectrum beta-lactamase determinant, were the most frequent. The risk of ARB and ARG transfer between animals and humans causes the need to search for new antimicrobial therapies in future veterinary medicine.

Emergence and potential transmission route of avian influenza A (H5N1) virus in domestic cats in Poland, June 2023.

In June 2023, a fatal disease outbreak in cats occurred in Poland. Most cases tested in Poland (29 of 47) were positive for highly pathogenic avian influenza (HPAI) A (H5N1) virus. Genetic analyses revealed clade 2.3.4.4b with point mutations indicative of initial mammalian hosts adaptations. Cat viral sequences were highly similar (n = 21), suggesting a potential common infection source. To investigate possible infection routes, our group tested food samples from affected households. HPAI H5N1 virus was detected in one poultry meat sample.

Isolation of Low Endotoxin Content Extracellular Vesicles Derived from Cancer Cell Lines.

Extracellular vesicles (EVs) are a heterogeneous population of membrane vesicles released by cells in vitro and in vivo. Their omnipresence and significant role as carriers of biological information make them intriguing study objects, requiring reliable and repetitive protocols for their isolation. However, realizing their full potential is difficult as there are still many technical obstacles related to their research (like proper acquisition). This study presents a protocol for the isolation of small EVs (according to the MISEV 2018 nomenclature) from the culture supernatant of tumor cell lines based on differential centrifugation. The protocol includes guidelines on how to avoid contamination with endotoxins during the isolation of EVs and how to properly evaluate them. Endotoxin contamination of EVs can significantly hinder subsequent experiments or even mask their true biological effects. On the other hand, the overlooked presence of endotoxins may lead to incorrect conclusions. This is of particular importance when referring to cells of the immune system, including monocytes, because monocytes constitute a population that is especially sensitive to endotoxin residues. Therefore, it is highly recommended to screen EVs for endotoxin contamination, especially when working with endotoxin-sensitive cells such as monocytes, macrophages, myeloid-derived suppressor cells, or dendritic cells.

Myeloid-Derived Suppressor Cells as Key Players and Promising Therapy Targets in Prostate Cancer.

Prostate cancer (PC) is the second most often diagnosed malignancy in men and one of the major causes of cancer death worldwide. Despite genetic predispositions, environmental factors, including a high-fat diet, obesity, a sedentary lifestyle, infections of the prostate, and exposure to chemicals or ionizing radiation, play a crucial role in PC development. Moreover, due to a lack of, or insufficient T-cell infiltration and its immunosuppressive microenvironment, PC is frequently classified as a "cold" tumor. This is related to the absence of tumor-associated antigens, the lack of T-cell activation and their homing into the tumor bed, and the presence of immunological cells with regulatory functions, including myeloid-derived suppressor cells (MDSCs), regulatory T cells (Treg), and tumor-associated macrophages (TAMs). All of them, by a variety of means, hamper anti-tumor immune response in the tumor microenvironment (TME), stimulating tumor growth and the formation of metastases. Therefore, they emerge as potential anti-cancer therapy targets. This article is focused on the function and role of MDSCs in the initiation and progression of PC. Clinical trials directly targeting this cell population or affecting its biological functions, thus limiting its pro-tumorigenic activity, are also presented.

Mo-MDSCs are pivotal players in colorectal cancer and may be associated with tumor recurrence after surgery.

Colorectal cancer (CRC) is the third most common malignancy. Its development and progression is associated with natural immunosuppression related, among others, to myeloid derived suppressor cells (MDSCs). Overall, 54 patients in different stage of CRC, before any treatment were recruited into the study. The analysis included flow cytometry evaluation of blood MDSCs subsets, correlation their level with the tumor stage and T cell subsets. In the case of 11 patients, MDSCs level was evaluated before and 3 days after surgery, and these patients were monitored for cancer recurrence over 5 years. The results showed that frequency of circulating MDSCs subsets is increased significantly in CRC patients, with highest level detected in most advanced tumor stages. Moreover, only monocytic MDSCs (Mo-MDSCs) positively correlate with regulatory Treg, and negatively with tumor Her2/neu specific CD8+ T cells. Circulating MDSCs, in contrast to tumor resident (mostly Mo-MDSCs), are negative for PD-L1 expression. Additionally, after surgery the blood level of Mo-MDSCs increases significantly, and this is associated with tumor recurrence during a 5-year follow-up. In conclusion, Mo-MDSCs are pivotal players in CRC-related immunosuppression and may be associated with the risk of tumor recurrence after surgery.

Mild and Asymptomatic COVID-19 Convalescents Present Long-Term Endotype of Immunosuppression Associated With Neutrophil Subsets Possessing Regulatory Functions.

The SARS-CoV-2 infection [coronavirus disease 2019 (COVID-19)] is associated with severe lymphopenia and impaired immune response, including expansion of myeloid cells with regulatory functions, e.g., so-called low-density neutrophils, containing granulocytic myeloid-derived suppressor cells (LDNs/PMN-MDSCs). These cells have been described in both infections and cancer and are known for their immunosuppressive activity. In the case of COVID-19, long-term complications have been frequently observed (long-COVID). In this context, we aimed to investigate the immune response of COVID-19 convalescents after a mild or asymptomatic course of disease. We enrolled 13 convalescents who underwent a mild or asymptomatic infection with SARS-CoV-2, confirmed by a positive result of the PCR test, and 13 healthy donors without SARS-CoV-2 infection in the past. Whole blood was used for T-cell subpopulation and LDNs/PMN-MDSCs analysis. LDNs/PMN-MDSCs and normal density neutrophils (NDNs) were sorted out by FACS and used for T-cell proliferation assay with autologous T cells activated with anti-CD3 mAb. Serum samples were used for the detection of anti-SARS-CoV-2 neutralizing IgG and GM-CSF concentration. Our results showed that in convalescents, even 3 months after infection, an elevated level of LDNs/PMN-MDSCs is still maintained in the blood, which correlates negatively with the level of CD8+ and double-negative T cells. Moreover, LDNs/PMN-MDSCs and NDNs showed a tendency for affecting the production of anti-SARS-CoV-2 S1 neutralizing antibodies. Surprisingly, our data showed that in addition to LDNs/PMN-MDSCs, NDNs from convalescents also inhibit proliferation of autologous T cells. Additionally, in the convalescent sera, we detected significantly higher concentrations of GM-CSF, indicating the role of emergency granulopoiesis. We conclude that in mild or asymptomatic COVID-19 convalescents, the neutrophil dysfunction, including propagation of PD-L1-positive LDNs/PMN-MDSCs and NDNs, is responsible for long-term endotype of immunosuppression.

Granulocytes and Cells of Granulocyte Origin-The Relevant Players in Colorectal Cancer.

Colorectal cancer (CRC) is one of the most common malignancy and cause of cancer death worldwide, and it still remains a therapeutic challenge for western medicine. There is strong evidence that, in addition to genetic predispositions, environmental factors have also a substantial impact in CRC development. The risk of CRC is attributed, among others to dietary habits, alcohol consumption, whereas physical activity, food containing dietary fiber, dairy products, and calcium supplements have a protective effect. Despite progress in the available therapies, surgery remains a basic treatment option for CRC. Implementation of additional methods of treatment such as chemo- and/or targeted immunotherapy, improved survival rates, however, the results are still far from satisfactory. One of the reasons may be the lack of deeper understanding of the interactions between the tumor and different types of cells, including tumor infiltrating granulocytes. While the role of neutrophils is quite well explored in many cancers, role of eosinophils and basophils is often underestimated. As part of this review, we focused on the function of different granulocyte subsets in CRC, emphasizing the beneficial role of eosinophils and basophils, as well as dichotomic mode of neutrophils action. In addition, we addressed the current knowledge on cells of granulocyte origin, specifically granulocytic myeloid derived suppressor cells (Gr-MDSCs) and their role in development and progression of CRC.

Myeloid-Derived Suppressor Cells in Colorectal Cancer.

Colorectal cancer (CRC) remains one of the most common malignancies diagnosed worldwide. The pathogenesis of CRC is complex and involves, among others, accumulation of genetic predispositions and epigenetic factors, dietary habits, alterations in gut microbiota, and lack of physical activity. A growing body of evidence suggests that immune cells play different roles in CRC, comprising both pro- and anti-tumorigenic functions. Immunosuppression observed during cancer development and progression is a result of the orchestration of many cell types, including myeloid-derived suppressor cells (MDSCs). MDSCs, along with other cells, stimulate tumor growth, angiogenesis, and formation of metastases. This article focuses on MDSCs in relation to their role in the initiation and progression of CRC. Possible forms of immunotherapies targeting MDSCs in CRC are also discussed.

Perioperative changes in lymphocyte subpopulations in patients undergoing surgery for colorectal cancer.

Surgical procedure has immense impact on the immune balance. However, little is known about perioperative changes in T regulatory and Th17 lymphocyte subpopulations in patients undergoing colorectal resection. Patients with resectable colon cancer were enrolled in the study. Blood samples were obtained on two occasions, i.e. before the procedure and two days after the surgery. We also recruited a control group of young, healthy individuals. Lymphocyte subpopulations were analyzed with the use of flow cytometry. Investigated subpopulations consisted of total lymphocyte count, CD4+, CD8+, T regulatory Foxp3+ (Tregs), Th17 lymphocytes and white blood cell (WBC) count. There were significant differences in immune cell levels before and after the surgery. Reduction was recorded in the CD4+, CD8+, Tregs and total lymphocyte counts (p=0.002, p=0.01, p=0.008 and p=0.001, respectively). Increase was observed in total WBC and Th17 cells, however, Th17 lymphocytes did not reach statistical significance (p=0.01 and p=0.5, respectively). In conclusion, surgical intervention caused changes in all lymphocyte subpopulations investigated in patients undergoing surgery for colorectal cancer. However, it seemed to be an effect of perioperative trauma. Further studies are needed to investigate the impact of surgical intervention on lymphocyte subpopulations.

The level of myeloid-derived suppressor cells positively correlates with regulatory T cells in the blood of children with transient hypogammaglobulinaemia of infancy.

INTRODUCTION: Transient hypogammaglobulinaemia of infancy (THI) is a primary immunodeficiency characterised by low levels of immunoglobulin G (often with concomitant decrease of IgA and sometimes also of IgM) with still unknown exact reason. A delayed normalisation of the immunoglobulin level in THI may be associated with a transiently elevated number of regulatory T cells (Treg). Although in cancer and chronic inflammation it was shown that the level of Treg cells can be increased by myeloid-derived suppressor cells (MDSCs), until now no studies have been performed in the context of the role of MDSCs in THI and their correlation with Treg cells. Consequently, we aimed to determine the occurrence of MDSCs in the peripheral blood of children with THI and correlate their level with the level of Treg cells. MATERIAL AND METHODS: Flow cytometry analyses of Mo-MDSCs and Gr-MDSCs, characterised as HLA-DR-CD11b+CD15-CD14+ and HLA-DR-CD11b+CD15+CD14-, respectively, and Treg (CD4+CD25+Foxp3+) cells were performed. RESULTS: The proportion of Mo-MDSCs and Gr-MDSCs was significantly higher in the group of THI patients with elevated level of Treg cells (from the 95% confidence interval level of healthy controls). The cells with Mo-MDSC and Gr-MDSC characteristics positively correlated with the level of Treg cells. Moreover, children with a higher proportion of circulating Treg cells, and thereby higher level of MDSCs, showed delayed normalisation of IgG level and recovery. CONCLUSIONS: These findings show for the first time that MDSCs may be involved in the pathomechanism of THI, probably acting through the induction of Treg cells.