A Genetically Encoded Dark-to-Bright Biosensor for Visualisation of Granzyme-Mediated Cytotoxicity.

Granzyme B (GZMB) is a key enzyme released by cytotoxic T lymphocytes (CTL) and natural killer (NK) cells to induce apoptosis in target cells. We designed a novel fluorogenic biosensor which is able to assess GZMB activity in a specific and sensitive manner. This cleavage-responsive sensor for T cell activity level (CRSTAL) is based on a fluorescent protein that is only activated upon cleavage by GZMB or caspase-8. CRSTAL was tested in stable cell lines and demonstrated a strong and long-lasting fluorescence signal upon induction with GZMB. It can detect GZMB activity not only by overexpression of GZMB in target cells but also following transfer of GZMB and perforin from effector cells during cytotoxicity. This feature has significant implications for cancer immunotherapy, particularly in monitoring the efficacy of chimeric antigen receptor (CAR)-T cells. CAR-T cells are a promising therapy option for various cancer types, but monitoring their activity in vivo is challenging. The development of biosensors like CRSTAL provides a valuable tool for monitoring of CAR-T cell activity. In summary, CRSTAL is a highly sensitive biosensor that can detect GZMB activity in target cells, providing a means for evaluating the cytotoxic activity of immune cells and monitoring T cell activity in real time.

Facts and prospects of peptide in targeted therapy and immune regulation against triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer. Due to the lack of specific therapeutic targets, treatment options are limited, and the recurrence and metastasis rate is high, the overall survival of patients is poor. However, with the discovery of some new targets and the corresponding immune regulation after targeting these targets, TNBC has a new hope in treatment. The peptide has a simple structure, strong binding affinity, and high stability, and has great potential in targeted therapy and immune regulation against TNBC. This review will discuss how single peptides and peptide combinations target triple-negative breast cancer to exert immunomodulatory effects. Among them, single peptides target specific receptors on TNBC cells, act as decoys to target key ligands in the regulatory pathway, and target TME-related cells. The combinations of peptides work in the form of cancer vaccines, engineered exosomes, microRNAs and other immune-related molecular pathways, immune checkpoint inhibitors, chimeric antigen receptor T cells, and drug-peptide conjugates. This article is mainly dedicated to exploring new treatment methods for TNBC to improve the curative effect and prolong the survival time of patients.

An inference model gives insights into innate immune adaptation and repertoire diversity.

The innate immune system is the body's first line of defense against infection. Natural killer (NK) cells, a vital part of the innate immune system, help to control infection and eliminate cancer. Studies have identified a vast array of receptors that NK cells use to discriminate between healthy and unhealthy cells. However, at present, it is difficult to explain how NK cells will respond to novel stimuli in different environments. In addition, the expression of different receptors on individual NK cells is highly stochastic, but the reason for these variegated expression patterns is unclear. Here, we studied the recognition of unhealthy target cells as an inference problem, where NK cells must distinguish between healthy targets with normal variability in ligand expression and ones that are clear "outliers." Our mathematical model fits well with experimental data, including NK cells' adaptation to changing environments and responses to different target cells. Furthermore, we find that stochastic, "sparse" receptor expression profiles are best able to detect a variety of possible threats, in agreement with experimental studies of the NK cell repertoire. While our study was specifically motivated by NK cells, our model is general and could also apply more broadly to explain principles of target recognition for other immune cell types.

Pentacyclic Triterpenoid Phytochemicals with Anticancer Activity: Updated Studies on Mechanisms and Targeted Delivery.

Pentacyclic triterpenoids (TTs) represent a unique family of phytochemicals with interesting properties and pharmacological effects, with some representatives, such as betulinic acid (BA) and betulin (B), being mainly investigated as potential anticancer molecules. Considering the recent scientific and preclinical investigations, a review of their anticancer mechanisms, structure-related activity, and efficiency improved by their insertion in nanolipid vehicles for targeted delivery is presented. A systematic literature study about their effects on tumor cells in vitro and in vivo, as free molecules or encapsulated in liposomes or nanolipids, is discussed. A special approach is given to liposome-TTs and nanolipid-TTs complexes to be linked to microbubbles, known as contrast agents in ultrasonography. The production of such supramolecular conjugates to deliver the drugs to target cells via sonoporation represents a new scientific and applicative direction to improve TT efficiency, considering that they have limited availability as lipophilic molecules. Relevant and recent examples of in vitro and in vivo studies, as well as the challenges for the next steps towards the application of these complex delivery systems to tumor cells, are discussed, as are the challenges for the next steps towards the application of targeted delivery to tumor cells, opening new directions for innovative nanotechnological solutions.

Screening of subclinical functional hemoglobin and red blood cell abnormalities among blood donors of Fayoum University Hospital in Egypt: Are RET-He, and IRF useful screening tools?

BACKGROUND: The effectiveness of red cell transfusion in a given blood unit that relied on both quantity and quality of donated cells undoubtedly affects prognostic outcomes. OBJECTIVE: We aimed to determine the frequency of subclinical functional hemoglobin and red cell abnormalities in donated blood of Fayoum University Hospital in Egypt. Additionally, to assess the usefulness of reticulocyte mean hemoglobin content (RET-He) and immature reticulocyte fraction (IRF) as screening measures for such abnormalities. MATERIAL AND METHODS: This cross-sectional study enrolled 200 volunteer blood donors who met the national standard criterion of blood donation. Complete blood count with reticulocyte parameters, serum ferritin, sickling test, G6PD assay, Mentzer index, and naked-eye single tube red cell osmotic fragility test were carried out. RESULTS: Functional red cell abnormalities represented 44 % of this cohort. Out of them, 4.5 % had iron deficiency, 11 % had a positive sickling test, 19 % had G6PD deficiency, and 9.5 % had suspicious thalassemia. The sensitivity and specificity test for RET-He in selective identification of functional hemoglobin abnormalities in donated blood were 83.3 % and 61.2 %, respectively at a cutoff value of 26.9. Though there was no statistically significant effect of RET-He on the selective detection of G6PD deficiency, IRF had a statistically significant high level with a p-value of 0.04. CONCLUSION: Subclinical functional red cell abnormalities seem to be prevalent among blood donors. Reticulocyte/ erythrocyte indices could be useful screening tools for red cell abnormalities. Further studies are required for assessing the impact of transfusing such abnormalities to neonates and other critical recipients.

The NK cell checkpoint NKG2A maintains expansion capacity of human NK cells.

Human natural killer (NK) cells are cytotoxic effector cells that are increasingly harnessed in cancer immunotherapy. NKG2A/CD94 is an inhibitory receptor on NK cells that has established regulatory functions in the direct interaction with target cells when engaged with its ligand, the non-classical HLA class I molecule HLA-E. Here, we confirmed NKG2A as a checkpoint molecule in primary human NK cells and identified a novel role for NKG2A in maintaining NK cell expansion capacity by dampening both proliferative activity and excessive activation-induced cell death. Maintenance of NK cell expansion capacity might contribute to the preferential accumulation of human NKG2A+ NK cells after hematopoietic cell transplantation and enrichment of functionally impaired NK cells in human cancers. Functional silencing of NKG2A for cancer immunotherapy is highly attractive but will need to consider that this might also lead to a reduced survival by driving activation-induced cell death in targeted NK cells.

In silico and in vitro study of the adhesion dynamics of erythrophagocytosis in sickle cell disease.

Erythrophagocytosis occurring in the spleen is a critical process for removing senescent and diseased red blood cells (RBCs) from the microcirculation. Although some progress has been made in understanding how the biological signaling pathways mediate the phagocytic processes, the role of the biophysical interaction between RBCs and macrophages, particularly under pathological conditions such as sickle cell disease, has not been adequately studied. Here, we combine computational simulations with microfluidic experiments to quantify RBC-macrophage adhesion dynamics under flow conditions comparable to those in the red pulp of the spleen. We also investigate the RBC-macrophage interaction under normoxic and hypoxic conditions. First, we calibrate key model parameters in the adhesion model using microfluidic experiments for normal and sickle RBCs under normoxia and hypoxia. We then study the adhesion dynamics between the RBC and the macrophage. Our simulation illustrates three typical adhesion states, each characterized by a distinct dynamic motion of the RBCs, namely firm adhesion, flipping adhesion, and no adhesion (either due to no contact with macrophages or detachment from the macrophages). We also track the number of bonds formed when RBCs and macrophages are in contact, as well as the contact area between the two interacting cells, providing mechanistic explanations for the three adhesion states observed in the simulations and microfluidic experiments. Furthermore, we quantify, for the first time to our knowledge, the adhesive forces between RBCs (normal and sickle) and macrophages under different oxygenated conditions. Our results show that the adhesive forces between normal cells and macrophages under normoxia are in the range of 33-58 pN and 53-92 pN for sickle cells under normoxia and 155-170 pN for sickle cells under hypoxia. Taken together, our microfluidic and simulation results improve our understanding of the biophysical interaction between RBCs and macrophages in sickle cell disease and provide a solid foundation for investigating the filtration function of the splenic macrophages under physiological and pathological conditions.

Catch bonds in sickle cell disease: Shear-enhanced adhesion of red blood cells to laminin.

Could the phenomenon of catch bonding-force-strengthened cellular adhesion-play a role in sickle cell disease, where abnormal red blood cell (RBC) adhesion obstructs blood flow? Here, we investigate the dynamics of sickle RBCs adhering to a surface functionalized with the protein laminin (a component of the extracellular matrix around blood vessels) under physiologically relevant microscale flow. First, using total internal reflectance microscopy we characterize the spatial fluctuations of the RBC membrane above the laminin surface before detachment. The complex dynamics we observe suggest the possibility of catch bonding, where the mean detachment time of the cell from the surface initially increases to a maximum and then decreases as a function of shear force. We next conduct a series of shear-induced detachment experiments on blood samples from 25 sickle cell disease patients, quantifying the number and duration of adhered cells under both sudden force jumps and linear force ramps. The experiments reveal that a subset of patients does indeed exhibit catch bonding. By fitting the data to a theoretical model of the bond dynamics, we can extract the mean bond lifetime versus force for each patient. The results show a striking heterogeneity among patients, both in terms of the qualitative behavior (whether or not there is catch bonding) and in the magnitudes of the lifetimes. Patients with large bond lifetimes at physiological forces are more likely to have certain adverse clinical features, like a diagnosis of pulmonary arterial hypertension and intracardiac shunts. By introducing an in vitro platform for fully characterizing RBC-laminin adhesion dynamics, our approach could contribute to the development of patient-specific antiadhesive therapies for sickle cell disease. The experimental setup is also easily generalizable to studying adhesion dynamics in other cell types, for example, leukocytes or cancer cells, and can incorporate disease-relevant environmental conditions like oxygen deprivation.

Red Blood Cell Vitamin C Concentration and Its Effect on Deformability in Pediatric Sickle Cell Disease.

Red cell rigidity is common in sickle cell disease (SCD). The contribution of oxidative stress on deformability remains unknown. This study investigated red blood cell (RBC) vitamin C concentrations in pediatric SCD (n=43) compared with healthy controls ( n =23) and developed a protocol to raise RBC vitamin C concentrations to measure the effect on deformability. Sickle cell RBC vitamin C concentrations seem low (20.5 µM, SD: 16.2 vs. 51.7 µM, SD: 15.8; P <0.0001). Vitamin C can be successfully loaded into sickle cell RBCs but seems to have minimal effect on deformability. Future studies are needed to understand the clinical implications of vitamin C deficiency in pediatric SCD.

Mechanosensitive Pannexin 1 Activity Is Modulated by Stomatin in Human Red Blood Cells.

Pannexin 1 (PANX1) was proposed to drive ATP release from red blood cells (RBCs) in response to stress conditions. Stomatin, a membrane protein regulating mechanosensitive channels, has been proposed to modulate PANX1 activity in non-erythroid cells. To determine whether stomatin modulates PANX1 activity in an erythroid context, we have (i) assessed the in situ stomatin-PANX1 interaction in RBCs, (ii) measured PANX1-stimulated activity in RBCs expressing stomatin or from OverHydrated Hereditary Stomatocytosis (OHSt) patients lacking stomatin, and in erythroid K562 cells invalidated for stomatin. Proximity Ligation Assay coupled with flow imaging shows 27.09% and 6.13% positive events in control and OHSt RBCs, respectively. The uptake of dyes 5(6)-Carboxyfluorescein (CF) and TO-PRO-3 was used to evaluate PANX1 activity. RBC permeability for CF is 34% and 11.8% in control and OHSt RBCs, respectively. PANX1 permeability for TO-PRO-3 is 35.72% and 18.42% in K562 stom+ and stom- clones, respectively. These results suggest an interaction between PANX1 and stomatin in human RBCs and show a significant defect in PANX1 activity in the absence of stomatin. Based on these results, we propose that stomatin plays a major role in opening the PANX1 pore by being involved in a caspase-independent lifting of autoinhibition.

Case Report: Novel STIM1 Gain-of-Function Mutation in a Patient With TAM/STRMK and Immunological Involvement.

Gain-of-function (GOF) mutations in STIM1 are responsible for tubular aggregate myopathy and Stormorken syndrome (TAM/STRMK), a clinically overlapping multisystemic disease characterised by muscle weakness, miosis, thrombocytopaenia, hyposplenism, ichthyosis, dyslexia, and short stature. Several mutations have been reported as responsible for the disease. Herein, we describe a patient with TAM/STRMK due to a novel L303P STIM1 mutation, who not only presented clinical manifestations characteristic of TAM/STRMK but also manifested immunological involvement with respiratory infections since childhood, with chronic cough and chronic bronchiectasis. Despite the seemingly normal main immunological parameters, immune cells revealed GOF in calcium signalling compared with healthy donors. The calcium flux dysregulation in the immune cells could be responsible for our patient's immune involvement. The patient's mother carried the mutation but did not exhibit TAM/STRMK, manifesting an incomplete penetrance of the mutation. More cases and evidence are necessary to clarify the dual role of STIM1 in immune system dysregulation and myopathy.

STIM1 and ORAI1 mutations leading to tubular aggregate myopathies are sensitive to the Store-operated Ca2+-entry modulators CIC-37 and CIC-39.

Gain-of-function mutations on STIM1 and ORAI1 genes are responsible for an increased store-operated calcium entry, and underlie the characteristic symptoms of three overlapping ultra-rare genetic disorders (i.e tubular aggregate myopathy, Stormorken syndrome, York platelet syndrome) that can be grouped as tubular aggregate myopathies. These mutations lead to a wide spectrum of defects, which usually include muscle weakness and cramps. Negative modulators of store-operated Ca2+-entry targeting wild-type STIM1 and ORAI1 have entered clinical trials for a different array of disorders, including pancreatitis, COVID-19, cancer, and autoimmune disorders and, while efficacy data is awaited, safety data indicates tolerability of this STIM1/ORAI1 mutations are amenable to pharmacological intervention. If this were so, given that there are no approved treatments or clinical trials ongoing for these rare disorders, it could be envisaged that these agents could also rehabilitate tubular aggregate myopathy patients. In the present contribution we characterized the Ca2+-entry patterns induced by eleven STIM1 and three ORAI1 mutations in heterologous systems or in patient-derived cells, i.e. fibroblasts and myotubes, and evaluated the effect of CIC-37 and CIC-39, two novel store-operated calcium entry modulators. Our data show that all STIM1 and ORAI1 gain-of-function mutations tested, with the possible exception of the R304Q STIM1 mutation, are amenable to inhibition, albeit with slightly different sensitivities, paving the way to the development of SOCE modulators in tubular aggregate myopathies.

Black Americans' willingness to participate in pediatric sickle cell clinical trials: A retrospective, systematic review.

Black individuals are underrepresented in randomized clinical trials (RCTs). Willingness to participate is a frequently cited explanation. However, the few studies that have investigated willingness to participate demonstrated no difference between Black individuals and other groups. We sought to measure willingness to participate by focusing on sickle cell disease (SCD), in which approximately 90% of affected individuals are Black. We conducted an analysis of 17 RCTs. A level of clarity was defined and correlated with each article's transparency in reporting patient enrollment data. Calculated measures of acceptance ranged from 32% to 93.5%. Calculated completion rates ranged from 58.8% to 100%. Weighted measures of acceptance and completion were 59.1% and 83.8%, respectively. Our study is limited by focusing solely on studies pertinent to SCD and only a minority of publications reviewed provided sufficient patient enrollment data. Yet, our results suggest that decreased willingness to participate does not account for underrepresentation of Black individuals.

Impact of small fractions of abnormal erythrocytes on blood rheology.

Red blood cell (RBC) populations are inherently heterogeneous, given mature RBC lack the transcriptional machinery to re-synthesize proteins affected during in vivo aging. Clearance of older, less functional cells thus aids in maintaining consistent hemorheological properties. Scenarios occur, however, where portions of mechanically impaired RBC are re-introduced into blood (e.g., damaged from circulatory support, blood transfusion) and may alter whole blood fluid behavior. Given such perturbations are associated with poor clinical outcomes, determining the tolerable level of abnormal RBC in blood is valuable. Thus, the current study aimed to define the critical threshold of blood fluid properties to re-infused physically-impaired RBC. Cell mechanics of RBC were impaired through membrane cross-linking (glutaraldehyde) or intracellular oxidation (phenazine methosulfate). Mechanically impaired RBC were progressively re-introduced into the native cell population. Negative alterations of cellular deformability and high shear blood viscosity were observed following additions of only 1-5% rigidified RBC. Low-shear blood viscosity was conversely decreased following addition of glutaraldehyde-treated cells; high-resolution microscopy of these mixed cell populations revealed decreased capacity to form reversible aggregates and decreased aggregate size. Mixed RBC populations, when exposed to supraphysiological shear, presented with compounded mechanical impairment. Collectively, key determinants of blood flow behavior are sensitive to mechanical perturbations in RBC, even when only 1-5% of the cell population is affected. Given this fraction is well-below the volume of rigidified RBC introduced during circulatory support or transfusion practice, it is plausible that some adverse events following surgery and/or transfusion may be related to impaired blood fluidity.

The relevance of macrocytosis induction during neoadjuvant dose-dense chemotherapy in breast cancer patients.

The aim of this study was to explore the red blood cell changes that occur during neoadjuvant dose-dense chemotherapy (NAC) of breast cancer. Also, we investigated the role of macrocytosis as a predictive biomarker for pathological complete response and disease-free survival (DFS) in these patients. A retrospective analysis of 82 breast cancer patients' data treated with anthracycline-cyclophosphamide-paclitaxel (AC-T) NAC in three oncology institutions in south Croatia from 2013 to 2020 was carried out. During chemotherapy mean corpuscular volume increased with time, with a median increase of 7.25 fl. Macrocytosis was induced in 38% of patients overall. Development of macrocytosis did not correlate with DFS [hazard ratio = 0.525; 95% confidence interval (CI), 0.074-3.768; P = 0.525]. Higher percentage of patients in macrocytosis group achieved PCR, 39% vs. 29% in no macrocytosis group, but this difference was not statistically significant. The relevance of macrocytosis induction during dose-dense neoadjuvant chemotherapy in breast cancer should be further explored.