Blood Clot Dynamics and Fibrinolysis Impairment in Cancer: The Role of Plasma Histones and DNA.

BACKGROUND: Blood viscoelasticity and plasma protein levels can play an important role in the diagnosis and prognosis of cancer. However, the role of histones and DNA in modulating blood clot properties remains to be investigated. This study investigates the differences in blood viscoelasticity and plasma protein levels among cancer patients, individuals with other diseases, and healthy individuals. METHODS: Blood samples were collected from 101 participants, including 45 cancer patients, 22 healthy individuals, and 34 individuals with other diseases. Rheological properties of clots formed in vitro by reconstituted elements of fibrinogen or plasma were analyzed with an Anton Paar Rheometer, USA. Plasma protein levels of D-dimer, TPA, EPCR, fibrinogen, and histone H3 were measured through ELISA. Blood clots were formed with or without DNA and histones (H3) by adding thrombin and calcium to plasma samples, and were evaluated for viscoelasticity, permeability, and degradation. RESULTS: Cancer patients show higher blood viscoelasticity and plasma D-dimer levels compared to healthy individuals and individuals with other diseases. Our in vitro analysis showed that the addition of histone to the plasma results in a significant decrease in viscoelasticity and mean fiber thickness of the clot formed thereafter. In parallel studies, using plasma from patients, DNA and histones were detected in fibrin clots and were associated with less degradation by t-PA. Moreover, our results show that the presence of DNA and histones not only increases clots' permeability, but also makes them more prone to degradation. CONCLUSIONS: Plasma histones and DNA affect the structure of the clot formed and induce defective fibrinolysis. Moreover, the increased viscoelastic properties of plasma from cancer patients can be used as potential biomarkers in cancer prognosis.

Why do patients with DR-TB do not complete their treatment? Findings of a qualitative study from Pakistan.

BACKGROUND: One of the major reasons for unsuccessful treatment outcomes among patients with drug-resistant tuberculosis (DR-TB) is the high rate of loss to follow-up (LTFU). However, in Pakistan, no qualitative study has been conducted to explore the perceptions of LTFU patients with regard to DR-TB treatment, the problems they face and the reasons for LTFU in detail. METHODS: This was a qualitative study that involved semistructured, indepth, face-to-face interviews of 39 LTFU patients with DR-TB. All interviews were carried out in Pakistan's national language 'Urdu' using an interview guide in two phases: the first phase was from December 2020 to February 2021 among patients with extensively drug-resistant tuberculosis and the second phase from July 2021 to September 2021 among patients with multidrug-resistant tuberculosis. RESULTS: The inductive thematic analysis of audio-recorded interviews generated the following four key themes, which were the major reasons reported by the participants of the current study to have led to LTFU: (1) patient-related factors, such as lack of awareness about the total duration of DR-TB treatment, fatigue from previous multiple failed episodes, lack of belief in treatment efficacy and perception of DR-TB as a non-curable disease; (2) medication-related factors, such as use of injectables, high pill burden, longer duration and adverse events; (3) socioeconomic factors, such as gender discrimination, poor socioeconomic conditions, non-supportive family members, social isolation and unemployment; and (4) service provider-related factors, such as distant treatment centres, non-availability of a qualified person, lack of adequate counselling and poor attitude of healthcare professionals. CONCLUSION: In the current study, patients' perceptions about DR-TB treatment, socioeconomic condition, medication and service provider-related factors emerged as barriers to the successful completion of DR-TB treatment. Increasing patients' awareness about the duration of DR-TB treatment, interacting sessions with successfully treated patients, availability of rapid drug susceptibility testing facilities at treatment centres, decentralising treatment and using the recently recommended all-oral regimen may further decrease the rate of LTFU.

Impact of a pharmacist-led educational intervention on knowledge, attitude, and practice toward the rational use of antibiotics among healthcare workers in a secondary care hospital in Punjab, Pakistan.

Introduction: Growing antimicrobial resistance (AMR) and decreasing efficacy of the available antimicrobials have become a significant public health concern. The antimicrobial stewardship program (ASP) ensures the appropriate use of antimicrobials and mitigates resistance prevalence through various interventions. One of the core components of the ASP is to educate healthcare workers (HWs). Therefore, this study aims to identify the impact of a pharmacist-led educational intervention targeting knowledge, attitude, and practices regarding rational antibiotic use among healthcare professionals in a secondary care hospital in Punjab. Methods: This is a single-center, questionnaire-based, pre-post interventional study conducted over a six-month time period. Data analysis was conducted using SPSS version 26. Results: Regarding the pre-interventional knowledge, attitude, and practice (KAP) score of the respondents, 90.3% had a good knowledge score, 81.5% had a positive attitude, and 72.3% of HWs (excluding doctors) had a good practice score. Additionally, 74.6% of the doctors had a good practice score. After educational intervention, there was a significant improvement in the knowledge, attitude, and practice of the respondent HWs (p-value <0.001). Furthermore, males have higher knowledge scores compared to females in the pre- and post-intervention stages (p-value <0.05), and doctors differ from nurses regarding knowledge scores in both pre- and post-intervention stages. Conclusion: Considering educational programs as the backbone of the ASP, it is imperative to sustain efforts in the ongoing educational programs of HWs to foster high awareness and adherence to the ASP among HWs.

Extracellular Vesicles in Transplantation.

Extracellular vesicles (EVs) have been extensively studied in the last two decades. It is now well documented that they can actively participate in the activation or regulation of immune system functions through different mechanisms, the most studied of which include protein-protein interactions and miRNA transfers. The functional diversity of EV-secreting cells makes EVs potential targets for immunotherapies through immune cell-derived EV functions. They are also a potential source of biomarkers of graft rejection through donor cells or graft environment-derived EV content modification. This review focuses on preclinical studies that describe the role of EVs from different cell types in immune suppression and graft tolerance and on the search for biomarkers of rejection.

Development of extracellular vesicle-based medicinal products: A position paper of the group "Extracellular Vesicle translatiOn to clinicaL perspectiVEs - EVOLVE France".

Extracellular vesicles (EV) are emergent therapeutic effectors that have reached clinical trial investigation. To translate EV-based therapeutic to clinic, the challenge is to demonstrate quality, safety, and efficacy, as required for any medicinal product. EV research translation into medicinal products is an exciting and challenging perspective. Recent papers, provide important guidance on regulatory aspects of pharmaceutical development, defining EVs for therapeutic applications and critical considerations for the development of potency tests. In addition, the ISEV Task Force on Regulatory Affairs and Clinical Use of EV-based Therapeutics as well as the Exosomes Committee from the ISCT are expected to contribute in an active way to the development of EV-based medicinal products by providing update on the scientific progress in EVs field, information to patients and expert resource network for regulatory bodies. The contribution of our work group "Extracellular Vesicle translatiOn to clinicaL perspectiVEs - EVOLVE France", created in 2020, can be positioned in complement to all these important initiatives. Based on complementary scientific, technical, and medical expertise, we provide EV-specific recommendations for manufacturing, quality control, analytics, non-clinical development, and clinical trials, according to current European legislation. We especially focus on early phase clinical trials concerning immediate needs in the field. The main contents of the investigational medicinal product dossier, marketing authorization applications, and critical guideline information are outlined for the transition from research to clinical development and ultimate market authorization.

Injured tissues favor cancer cell implantation via fibrin deposits on scar zones.

AIM: Evaluation of fibrin role on cancer cells implantation in injured tissues and studying the molecular mechanism of cancer cell interaction with the peritoneal damage. MATERIAL AND METHODS: Mouse colon cancer (CT26) and human mesothelial cells (HMCs) were used. CT26 cells were implanted on injured peritoneal zones. Icodextrin was used as a lubricant. For in vitro studies, fibrin clots from human plasma were used. The cell-fibrin interaction was observed by optical, electronic, and confocal microscopies. Aprotinin was used as a plasmin inhibitor. Hemostasis impact quantified by (1) the fibrin degradation product D-Dimer and PAR expression in HMCs; (2) the expression of plasminogen activator (PA) and its inhibitor (PAI-1) in cancer cells by qPCR and in supernatants through ELISA after in vitro HMC incubation with 2U of thrombin for 24 h. RESULTS: (i) Cancer cell lines were adhered and implanted into the wound area in vivo in both the incision and peeling zones of the peritoneum and on the fibrin network in vitro. (ii) Icodextrin significantly inhibited cancer nodule formation in the scar and the incision or peritoneal damaged zones after surgery. (iii) In in vitro studies, cancer cell interaction with the fibrin clot generated a lysed area, causing an increase in plasmin-dependent fibrinolysis measured by D-dimer levels in the supernatants that was inhibited by aprotinin. (iv) Aprotinin inhibited cell-fibrin interaction and invasion. (v) Thrombin upregulates PAI-1 and downregulates PA expression in HMC. CONCLUSION: Injured tissues favor cancer cell implantation through generated fibrin. Fibrin-cancer cells adhesion can be inhibited by icodextrin.

Mitosis in Cancer Cell Increases Immune Resistance via High Expression of HLA-G and PD-L1.

Cancer is a result of "aggressive" division and uncontrolled proliferation of the abnormal cells that survive attack by immune cells. We investigated the expression of HLA-G and PD-L1 with the different stages of cancer cell division along with their role in the interaction of immune cells in vitro. Ovarian cancer (OVCAR-3) and chronic myeloid leukemia cell line (K-562) are used for this study. The correlation of protein expression with percentage of cells in each phase (G1, S and G2 phase) was evaluated through FACS. Cells were synchronized in G1, G2 and mitotic phase to evaluate gene (RT-qPCR) and protein expression (FACS). Real-time immune cell attack (RTICA) analysis with PBMCs (peripheral blood mono-nuclear cells) and cancer cells were performed. We found that cells expressing higher levels of HLA-G and PD-L1 are mainly in G2 phase and those expressing lower levels are mainly in G1 phase. Evidently, the higher expression of the two proteins was observed when synchronized in mitotic phase as compared to low expression when synchronized in G1 phase. RTICA analysis showed the presence of HLA-G delayed the lysis of the cells. In conclusion, the cancer cell can escape from immune cells in division stage that suggests the impact of mitosis index for cancer immunotherapy.

Differentiation of cancer cells upregulates HLA­G and PD­L1.

A tumor contains special types of cells that have characteristics similar to stem cells that aid in tumor initiation, evasion and proliferation and are often resistant to chemotherapy. These cancer stem cells can be differentiated to eradicate their stemness and proliferative capacity by differentiating agents. This study investigated the effect of differentiation on the expression of two immune checkpoint inhibitors, human leukocyte antigen­G (HLA­G) and programmed death ligand­1 (PD­L1). Two cancer cell lines (OVCAR­3­NIH and KATO­III) were treated with adipocyte and neurocyte differentiation media for 14 days. Bone­marrow derived mesenchymal stem cells (BM­MSCs) were used as control healthy stem cells. We found that the cancer cell lines (OVCAR­3­NIH and KATO­III) when subjected to differentiation lost their proliferation ability. BM­MSC proliferation was not halted but was decreased in the adipocyte differentiation media. There was no decrease in the CD90 stem cell marker in the BM­MSCs; however, both cancer cell lines showed decreased CD90 stem cell marker. A significant increase in HLA­G was noted for both the cancer cell lines following adipocyte differentiation. No effect was found for BM­MSCs. Moreover, an increase in PD­L1 in cancer cell lines was found following neurocyte differentiation. Moreover, we found that differentiation resulted in decreased PD­L1 expression in BM­MSCs. Differentiation therapy of cancer stem cells may result in increased immunosuppression ability, hence causing hindrance in the removal of cancer cells. Moreover, the differentiation of healthy stem cells can result in increased immunogenic reactivity owing to a decrease in PD­L1 expression.

High Expression of HLA-G in Ovarian Carcinomatosis: The Role of Interleukin-1ß.

The present study focuses on the influence of the tumor microenvironment on the expression of HLA-G in ovarian cancer and its impact on immune cells. We used carcinomatosis fluids (n = 16) collected from patients diagnosed with epithelial ovarian cancer, detected by an increase in CA125 levels. Our results indicate that HLA-G is expressed by 1) ascitic cell clusters, 2) stromal cells (hospicells) extracted from cancer cell clusters, and 3) cancer cell lines and tumor cells. The origin of HLA-G was linked to inflammatory cytokines present in the cancer microenvironment. In parallel, the ascitic fluid of patients with ovarian cancer contains soluble HLA-G (sHLA-G). The mesothelial cell layer and submesothelial tissues, as well as the immune cell infiltrate, do not secrete HLA-G. In contrast, sHLA-G is absorbed by peritoneal tissues along with mesothelial layers as well as immune cell infiltrates. We demonstrated that interleukin-1ß along with TGF-ß can be a major HLA-G-inducing factor that upregulates HLA-G expression through the NF-κB pathway. The level of HLA-G in ascites correlated positively with the expression of T regulatory (T-regs) cells, while it negatively correlated with the expression of natural killer and memory cells in tumor-infiltrating immune cells. In conclusion, the production of HLA-G is associated with the presence of inflammatory cytokines and is strongly correlated with microenvironment tolerant cells such as T-regs and diminution of NK and memory T cells.