Red Blood Cell Lysis Research Articles

Rare Cell Population Analysis in Early-Stage Breast Cancer Patients.

Circulating rare cells participate in breast cancer evolution as systemic components of the disease and thus, are a source of theranostic information. Exploration of cancer-associated rare cells is in its infancy. We aimed to investigate and classify abnormalities in the circulating rare cell population among early-stage breast cancer patients using fluorescence marker identification and cytomorphology. In addition, we sought to determine the dependency of these markers on the presence of tumors. We evaluated the validity of a multi-rare-cell detection platform and demonstrated the utility of a specific rare cell subset as a novel approach to characterize the breast cancer system. Sampling was conducted both before and after tumor resection. Linearity of the Rarmax platform was established using a spike-in approach. The platform includes red blood cell lysis, leukocyte depletion and high-resolution fluorescence image recording. Rare cell analysis was conducted on 28 samples (before and after surgery) from 14 patients with breast cancer, 20 healthy volunteers and 9 noncancer control volunteers. In-depth identification of rare cells, including circulating tumor cells, endothelial-like cells, erythroblasts, and inflammation-associated cells, was performed using a phenotype and morphology-based classification system. The platform performed linearly over a range of 5 to 950 spiked cells, with an average recovery of 84.6%. Circulating epithelial and endothelial-like cell subsets have been demonstrated to be associated with or independent of cancer with tumor presence. Furthermore, certain cell profile patterns may be associated with treatment-related adverse effects. The sensitivity in detecting tumor-presence and cancer-associated abnormality before surgery was 43% and 85.7%, respectively, and the specificity was 100% and 96.6%, respectively. This study supports the idea of a cancer-associated rare cell abnormality to represent tumor entities as well as systemic cancer. The latter is independent of the apparent clinical cancer.

A novel environmentally friendly thermochemical process for Ti64 alloy surface modification for biomedical implants

This study develops an environmentally friendly novel thermochemical surface modification process of Ti64 alloy by utilizing citric acid and hydrogen peroxide to improve the mechanical properties, biocompatibility, and bioactivity for biomedical applications. The novel process was developed thermochemically, followed by simulated mineralization in Hanks Balanced Salt Solution (HBSS). The study investigates the effects of thermochemicals on surface characteristics by evaluating the influence of eco-friendly chemicals on modifying surfaces and finding the optimal pH value. The findings demonstrated that the modification effectively enhanced the Ti64 alloy mechanical properties with a significant increase in average microhardness by approximately 71 % and a reduction in wear rate by approximately 64.29 %, compared to untreated Ti64 alloy. The surface modification with pH (5,7, and 9) revealed absorptive properties as evidenced by a contact angle below 90°, indicating a hydrophilic surface, enhancing cells' attachment to biomaterials. After soaking Ti64 alloy in HBSS, a uniform coating layer of around 20.5 μm formed, leading to increased bioactivity, as evidenced by a Ca/P ratio of 1.67, comparable to hydroxyapatite in human bone. The hemolysis ratio of 0.027 % at pH 7 indicates little red blood cell (RBC) lysis, indicating increased biocompatibility. The corrosion rate was enhanced with pH (5,7, and 9) approximately (1.975 × 10−2, 1.078 × 10−2, and 1.615 × 10−2) mm/year, respectively. These findings indicate that the novel process with neutral pH (7) is optimal for surface modification as it is the most effective in enhancing the biocompatibility and bioactivity of the Ti64 alloy, making it suitable for biomedical implants.

Abstract A001: Analytical validation of a novel multiomic metastatic breast cancer liquid biopsy test that combines ctDNA analysis with CTC HER2-ultralow and ER co-expression, as well as single-cell chromosomal instability

Abstract Background: Metastatic breast cancer (MBC) subtype characterization is critical for effective treatment. The DefineMBC™ multiomic blood biopsy clinical diagnostic combines ctDNA and circulating tumor cell (CTC) analyses to characterize MBC patients when a tissue biopsy is not feasible. Innovation in the DefineMBC 2.0 5-channel immunofluorescent (IF) CTC characterization assay now measures ultralow (UL) levels of HER2 protein expression, ER protein expression, and chromosomal instability (CI) as well as ERBB2 amplification via single- cell sequencing. Cell-level HER2/ER co-detection enables monitoring of receptor conversion during a patient’s MBC treatment journey. As trastuzumab-deruxtecan (T-DxD) has become standard of care for HER2-low disease, distinguishing HER2-UL from HER2[-] by DefineMBC’s liquid biopsy approach may identify additional patients who can benefit from such therapy. This addresses an unmet need given the known shortcomings of tissue biopsy IHC in the metastatic setting as recently noted by communications from NCCN, ASCO, and the College of American Pathologists. Methods: Assay development including analytical validation studies utilized biologically relevant cell lines of epithelial origin and known expression levels of HER2 and ER (MDA-MB-453: HER2[+] & ER[-]; MCF-7: HER2-low & ER[+]; and MCF-7/ERBB2 knockout: HER2[-]) that were spiked into healthy donor blood. Additionally, patients with various forms of MBC were characterized with the new assay. As with all samples, following red blood cell lysis, nucleated cells were deposited on glass slides at high density (∼3E6 WBC/slide). Slides underwent IF staining for cytokeratins (CK), CD31, CD45, HER2, ER, and Hoechst to localize nuclear DNA, followed by scanning and machine learning-based rare cell detection. CK[+], CD31[-]/CD45[-] cells were classified as CTC candidates and assessed for HER2/ER expression. Pathologist-selected CTCs were isolated for single-cell whole genome sequencing and analyzed using a proprietary CTC DNA copy number pipeline to detect CI and amplification at the ERBB2 locus. Results: Analytical validation studies demonstrated the limit of detection at 1 CTC per 12E6 WBC (∼1.6 mL of blood) with a linear range of 1- 300 CTCs per slide. Sensitivity, specificity, accuracy, and precision metrics of the HER2 and ER IF assays were 96%, 96%, 96%, 2% CV; 91%, 95%, 93%, 3% CV; respectively. CTC enumeration precision was validated at greater than 80% for enumeration bins of 0, 1-10, 11-50, and>50 CTCs per sample. During patient testing 7 patients previously called HER2[-] are now designated as consistent with HER2-low by the pathologist, suggesting improved test performance in future clinical validation studies is likely. Conclusions: Advancement of our comprehensive cancer profiling platform now includes ER/HER2-UL/CI co-detection on enumerated CTCs in MBC patients. Clinical studies currently in progress will reveal whether HER2-UL/HER2[+] detection on CTCs will identify patients better suited for T-DxD or trastuzumab, respectively. Citation Format: Raj Srikrishnan, Jordan Ritchie, Alessandra Cunsolo, Andrew Kunihiro, Brandon Guillory, Sara Tin, Zhuo Meng, Ernest T Lam, Bharat Annaldas, Evan Schwab, Nilesh Dharajiya, Timothy Pluard, Martin Blankfard, David Bourdon. Analytical validation of a novel multiomic metastatic breast cancer liquid biopsy test that combines ctDNA analysis with CTC HER2-ultralow and ER co-expression, as well as single-cell chromosomal instability [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A001.

An evaluation of the analytical and biological robustness of a method for quantifying iron in individual red blood cells via single-cell tandem ICP-mass spectrometry

This work evaluated the analytical and biological robustness of iron (Fe) determination in individual red blood cells (RBCs) via single-cell ICP-MS (SC-ICP-MS). RBCs were separated from other whole blood constituents using Ficoll-Paque™ density gradient centrifugation. While fixation with paraformaldehyde (PFA) led to RBC lysis, the use of glutaraldehyde (GA) left the RBCs intact and permitted storage of RBC suspensions in ultra-pure water for up to 16 months at 4 °C. GA-fixation also rendered the RBCs sufficiently robust to maintain integrity during their introduction into the ICP by means of nebulization of dilute suspensions. Obtaining quantitative data on a cell-per-cell basis required determination of the transport efficiency using the particle size method and external calibration against aqueous Fe standard solutions. The average Fe content per RBC obtained using SC-ICP-MS (103 fg/cell) agreed well with the value obtained using solution-based ICP-MS obtained after cell pellet digestion and with values obtained from literature. Variation of the cell number density in the suspensions analyzed between 1.5 × 105 and 6.0 × 105 cells per mL did not affect the result. Identical results from one-week interval blood drawings from healthy individuals demonstrate biological consistency. Compared to bulk analysis, the SC-ICP-MS approach offers the added value of providing information on the cell-to-cell heterogeneity in Fe content.

Probiotic and Postbiotic Potentials of Enterococcus faecalis EF-2001: A Safety Assessment.

Probiotics, which are live microorganisms that, when given in sufficient quantities, promote the host's health, have drawn a lot of interest for their ability to enhance gut health. Enterococcus faecalis, a member of the human gut microbiota, has shown promise as a probiotic candidate due to its functional attributes. However, safety concerns associated with certain strains warrant comprehensive evaluation before therapeutic application. In this study, E. faecalis EF-2001, originally isolated from fecal samples of a healthy human infant, was subjected to a multi-faceted assessment for its safety and probiotic potential. In silico analysis, CAZyme, biosynthetic, and stress-responsive proteins were identified. The genome lacked biogenic amine genes but contained some essential amino acid and vitamin synthetic genes, and carbohydrate-related enzymes essential for probiotic properties. The negligible difference of 0.03% between the 1st and 25th generations indicates that the genetic information of the E. faecalis EF-2001 genome remained stable. The live E. faecalis EF-2001 (E. faecalis EF-2001L) demonstrated low or no virulence potential, minimal D-Lactate production, and susceptibility to most antibiotics except some aminoglycosides. No bile salt deconjugation or biogenic amine production was observed in an in vitro assay. Hemolytic activity assessment showed a β-hemolytic pattern, indicating no red blood cell lysis. Furthermore, the EF-2001L did not produce gelatinase and tolerated simulated gastric and intestinal fluids in an in vitro study. Similarly, heat-killed E. faecalis EF-2001 (E. faecalis EF-2001HK) exhibits tolerance in both acid and base conditions in vitro. Further, no cytotoxicity of postbiotic EF-2001HK was observed in human colorectal adenocarcinoma HT-29 cells. These potential properties suggest that probiotic and postbiotic E. faecalis EF-2001 could be considered safe and retain metabolic activity suitable for human consumption.

Comparative Properties of Helical and Linear Amphipathicity of Peptides Composed of Arginine, Tryptophan, and Valine.

The persistence of antibiotic resistance has incited a strong interest in the discovery of agents with novel antimicrobial mechanisms. The direct killing of multidrug-resistant bacteria by cationic antimicrobial peptides (AMPs) underscores their importance in the fight against infections associated with antibiotic resistance. Despite a vast body of AMP literature demonstrating a plurality in structural classes, AMP engineering has been largely skewed toward peptides with idealized amphipathic helices (H-amphipathic). In contrast to helical amphipathicity, we designed a series of peptides that display the amphipathic motifs in the primary structure. We previously developed a rational framework for designing AMP libraries of H-amphipathic peptides consisting of Arg, Trp, and Val (H-RWV, with a confirmed helicity up to 88% in the presence of membrane lipids) tested against the most common MDR organisms. In this study, we re-engineered one of the series of the H-RWV peptides (8, 10, 12, 14, and 16 residues in length) to display the amphipathicity in the primary structure by side-by-side (linear) alignment of the cationic and hydrophobic residues into the 2 separate linear amphipathic (L-amphipathic) motifs. We compared the 2 series of peptides for antibacterial activity, red blood cell (RBC) lysis, killing and membrane-perturbation properties. The L-RWV peptides achieved the highest antibacterial activity at a minimum length of 12 residues (L-RWV12, minimum optimal length or MOL) with the lowest mean MIC of 3-4 µM, whereas the MOL for the H-RWV series was reached at 16 residues (H-RWV16). Overall, H-RWV16 displayed the lowest mean MIC at 2 µM but higher levels of RBC lysis (25-30%), while the L-RWV series displayed minor RBC lytic effects at the test concentrations. Interestingly, when the S. aureus strain SA719 was chosen because of its susceptibility to most of the peptides, none of the L-RWV peptides demonstrated a high level of membrane perturbation determined by propidium iodide incorporation measured by flow cytometry, with <50% PI incorporation for the L-RWV peptides. By contrast, most H-RWV peptides displayed almost up to 100% PI incorporation. The results suggest that membrane perturbation is not the primary killing mechanism of the L-amphipathic RWV peptides, in contrast to the H-RWV peptides. Taken together, the data indicate that both types of amphipathicity may provide different ideal pharmacological properties that deserve further investigation.

The Protective Role of Transcription Factor Nrf2 in Murine Macrophage Activation Syndrome.

Macrophage activation syndrome (MAS) is characterized by multi-lineage cytopenias, hypercytokinemia, and tissue hemophagocytosis. Transcription factor Nrf2 is a master regulator of redox homeostasis. In this work we aim to investigate the role of Nrf2 in murine hyperinflammation and the mechanisms by which Nrf2 activation by red blood cell products regulates pro-inflammatory cytokine production. We induce murine MAS in wildtype and Nrf2 knockout (Nrf2 -/-) mice by repeat administration of TLR9-agonist CpG. Clinical and biochemical markers of disease were measured including complete blood counts, liver and spleen pathology, serum free heme, ferritin, and cytokine profiles. In vitro bone marrow derived macrophages and dendritic cells were used to investigate regulation of CpG-induced cytokine expression by oxidized red blood cells and hemin. Patients with hyperinflammatory disease have higher levels of Nrf2 gene expression. Mice with CpG-induced hyperinflammation have elevated systemic lipid peroxidation which is exacerbated in Nrf2 -/- mice. Compared to wildtype controls, Nrf2 -/- mice develop significantly worse organomegaly, organ pathology, and reticulocytosis. Nrf2 -/- mice have exacerbated hypercytokinemia in cytokines central MAS physiology: IL-12, IFNg, and IL-10. In vitro we found that oxidized red blood cell lysates and hemin are able to suppress IL-12 transcription and protein production from bone marrow derived dendritic cells in a Nrf2-dependent manner. Together our findings show that transcription factor Nrf2 is highly expressed in patients with hyperinflammatory disease and demonstrate a protective role for Nrf2 in a murine model of MAS in part due to Nrf2-mediated suppression of proinflammatory cytokine production.

Neutrophil Diversity (Immature, Aged, and Low-Density Neutrophils) and Functional Plasticity: Possible Impacts of Iron Overload in β-Thalassemia.

Neutrophil dysfunction is a form of immune suppression in patients with β-thalassemia (Beta-thal), although data on this are limited. In this study, blood from patients and healthy volunteers was analyzed. Flow cytometry analysis demonstrated an increase in immature neutrophils (CD16- CD62L+) and aged (senescent) neutrophils (CD16+ CD62L-) in Beta-thal patients compared to healthy volunteers. The Beta-thal neutrophils demonstrated less prominent chemotaxis and phagocytosis than healthy neutrophils at the baseline. With phorbol myristate acetate (PMA) or lipopolysaccharide (LPS) stimulations, some of the indicators, including the flow cytometry markers (CD11b, CD62L, CD66b, CD63, apoptosis, and reactive oxygen species) and neutrophil extracellular traps (NETs; detected by anti-citrullinated histone 3 immunofluorescence), were lower than the control. Additionally, low-density neutrophils (LDNs), which are found in the peripheral blood mononuclear cell (PBMC) fraction, were observed in Beta-thal patients but not in the control group. The expression of CD11b, CD66b, CD63, arginase I, and ROS in LDNs was higher than the regular normal-density neutrophils (NDNs). The proliferation rate of CD3+ T cells isolated from the PBMC fraction of healthy volunteers was higher than that of the cells from patients with Beta-thal. The incubation of red blood cell (RBC) lysate plus ferric ions with healthy NDNs transformed the NDNs into the aged neutrophils (decreased CD62L) and LDNs. In conclusion, iron overload induces neutrophil diversity along with some dysfunctions.

Abstract A031: Methylomic Biomarkers Associated with Prostate Cancer in Black Men

Abstract Introduction: The prostate cancer (PCa) disparity remains the highest cancer disparity in the US, with Black men facing an estimated 78% higher incidence and 230% higher mortality than White men. However, current understanding of risk factors for PCa such as socioeconomic status (SES), diet, and genetics cannot fully explain these disparities. Our objective is to examine DNA methylation (DNAm) markers in a cohort of Black men and assess their associations with PCa status. Methods: We randomly selected 100 Black men for DNAm profiling (50 cases diagnosed with clinically significant (Gleason Grade 3+) prostate cancer and 50 age-matched controls) from two larger cohorts. The cohorts recruited 400 Black men who were newly diagnosed with sporadic PCa as well as 400 controls matched on age and race. All men were 40-79 at recruitment and were enrolled from urology clinics at five Chicago-area medical institutions. All PCa cases underwent a transrectal prostate biopsy for abnormal prostate-specific antigen (PSA) or digital rectal exam and had a histologically confirmed prostate cancer and were not previously diagnosed with PCa. All men in the control group had been recruited from community prostate cancer screening events or were referred to participating urology clinics and had a negative prostate biopsy for a PSA &amp;lt;10ng/ml and were not diagnosed with PCa for 2 years of follow-up. Men with prior PCa diagnosis or conditions known to affect PSA levels were excluded. After enrollment and informed consent, all men provided demographic and lifestyle data via three questionnaires, followed by venipuncture for DNA collection. These specimens were obtained using buffy coat extraction after red blood cell lysis, centrifugation, and pipetting off the overlying plasma and buffy coat for freezing followed by Illumina EPIC arrays for DNAm assays. We examined DNAm markers in gene promoter regions associated with PCa using logistic regression and adjusted for multiple testing using Bonferroni correction. All models controlled for age, BMI, smoking status, first degree family history of PCa, blood cell types, and technical control variables. Results: We found 2,180 CpG sites in gene promoter regions associated with any PCa in this population. The top ten strongest associations identified in our models include loci on genes that have already been associated with prostate cancer progression (MAFG, FAM65B, OSM, PRKAG2), risk (SLC11A1, LAT), and metastasis (S1PR4) as well as immune system functioning (RAP1GAP2). Conclusions: Our initial findings suggest that DNAm markers may be useful predictors of clinically significant PCa status among Black men. Additional research is currently ongoing to expand the sample size of the DNAm profiling and to explore links between these loci and social determinants of health, as well as associations with more advanced (i.e., higher Gleason Grade) PCa. Citation Format: Brian T. Joyce, Yishu Qu, Jun Wang, Kai Zhang, Zequn Sun, Lifang Hou, Adam B. Murphy. Methylomic Biomarkers Associated with Prostate Cancer in Black Men [abstract]. In: Proceedings of the 17th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2024 Sep 21-24; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2024;33(9 Suppl):Abstract nr A031.

Haemolysis overestimates plasma oxidative stress biomarkers in free-ranging roe deer

Quantifying oxidative stress has garnered extensive interest in evolutionary ecology and physiology since proposed as a mediator of life histories. However, while the theoretical framework of oxidative stress ecology is well-supported by laboratory-based studies, results obtained in wild populations on oxidative damage and antioxidant biomarkers have shown inconsistent trends. We propose that red blood cell lysis could be a source of bias affecting measurements of oxidative stress biomarkers, distorting the conclusions drawn from them. Using an experimental approach consisting of enriching plasma from roe deer with lysed red blood cells, we show that the values of commonly used oxidative stress biomarkers linearly increase with the degree of haemolysis – assayed by haemoglobin concentration. This result concerns oxidized proteins (carbonyls) and lipids (TBARS), as well as enzymatic (superoxide dismutase) and non-enzymatic (trolox assay, OXY assay) antioxidant markers. Based on 707 roe deer blood samples collected in the field, we next show that the occurrence of haemolysis in plasma samples is negatively related to age. Finally, we illustrate that considering the variance explained by age-related haemolysis improves explanatory models for inter-individual variability in plasma oxidative stress biomarkers, without substantially altering the estimates of the parameters studied here. Our results raise the question of the veracity of the conclusions if the degree of haemolysis in plasma is not considered in animal models such as roe deer, for which the occurrence and severity of haemolysis vary according to individual characteristics. We recommend measuring and controlling for the degree of haemolysis be considered in future studies that investigate the causes and consequences of oxidative stress in ecophysiological studies.

Pleomorphic Liposarcoma Cytologically Detected in Hemosiderotic Pleural Effusion: Pitfalls Mitigated by Cytologic Clues and Cellblock Immunocytochemistry.

Sarcomatous serous effusions are uncommon and diagnostically challenging. Dedifferentiated and pleomorphic liposarcomas are rare tumors in pleural effusions revealing highly pleomorphic tumor cells mimicking carcinoma, mesothelioma, melanoma, and other sarcomas. Hematothoracic effusions further complicate the cytologic diagnosis. Correct cytologic recognition is important. We report pleomorphic liposarcoma cytologically detected in effusion fluid in a 56-year-old man who presented with a massive unilateral pleural effusion. ThinPrep showed hemorrhagic effusion fluid characterized by lysed red blood cells, foamy macrophages, and siderophages intermixed with highly pleomorphic predominantly naked mononuclear and giant nuclei. The aggregated siderophages and vacuolated macrophages could be mistaken for tumor cells, whereas the bare nuclei may be missed as nonspecific degenerate changes. Cellblock sections showed highly pleomorphic mononuclear and multinucleated giant tumor cells with diagnostic lipoblasts, intermixed with foamy macrophages and siderophages. Cellblock immunocytochemistry showed staining for vimentin and S-100 protein in the tumor cells. Other lineage-specific immunomarkers were negative. CD68 and calretinin revealed frequent background macrophages and scarce mesothelial cells. The tumor cells were negative for MDM2 and CDK4. The entertained cytopathologic diagnosis was pleomorphic liposarcoma. Core needle biopsy was procured from the mass. The histopathologic features and immunoprofile of the tissue specimen matched the cytopathologic and immunocytochemical findings confirming the cytologic diagnosis of pleomorphic liposarcoma. Pleomorphic liposarcoma is an unexpected cytologically challenging finding in effusions, particularly when compounded by pitfalls introduced by hemosiderotic fluid. Attention to certain cytologic clues mitigate pitfalls. Cellblock is a valuable diagnostic tool when integrated with relevant negative and positive immunocytochemical markers.

Optimization of pre-enrichment strategies for mouse hematopoietic stem cell isolation and metabolomic analysis

Blood cell production arises from the activity of hematopoietic stem cells (HSCs), defined by their self-renewal capacity and ability to give rise to all mature blood cell types. The mouse remains one of the most studied species in hematological research, and markers to define and isolate mouse HSCs are well-established. Given the very low frequency of HSCs in the bone marrow, stem cell pre-enrichment by red blood cell lysis and magnetic cell separation is often performed as part of the isolation process to reduce sorting times. Several pre-enrichment strategies are available, differing in their speed, degree of enrichment, final cell yield, and cost. In the current study, we performed a side-by-side comparison and provide a decision tree to help researchers select a pre-enrichment strategy for mouse HSC isolation depending on their downstream application. We then compared different pre-enrichment techniques in combination with metabolomics analysis of HSCs, where speed, yield and temperature during pre-enrichment are crucial factors, and found that the choice of pre-enrichment strategy significantly impacts the number of metabolites detected and levels of individual metabolites in HSCs.

Tangential flow filtration-facilitated purification of human red blood cell membrane fragments and its preferential use in removing unencapsulated material from resealed red blood cell ghosts compared to centrifugation.

The biodistribution of many therapeutics is controlled by the immune system. In addition, some molecules are cytotoxic when not encapsulated inside of larger cellular structures, such as hemoglobin (Hb) encapsulation inside of red blood cells (RBCs). To counter immune system recognition and cytotoxicity, drug delivery systems based on red blood cell membrane fragments (RBCMFs) have been proposed as a strategy for creating immunoprivileged therapeutics. However, the use of RBCMFs for drug delivery applications requires purification of RBCMFs at large scale from lysed RBCs free of their intracellular components. In this study, we were able to successfully use tangential flow filtration (TFF) to remove >99% of cell-free Hb from lysed RBCs at high concentrations (30%-40% v/v), producing RBCMFs that were 2.68 ± 0.17 μm in diameter. We were also able to characterize the RBCMFs more thoroughly than prior work, including measurement of particle zeta potential, along with individual TFF diacycle data on the cell-free Hb concentration in solution and time per diacycle, as well as concentration and size of the RBCMFs. In addition to purifying RBCMFs from lysed RBCs, we utilized a hypertonic solution to reseal purified RBCMFs encapsulating a model protein (Hb) to yield resealed Hb-encapsulated RBC ghosts (Hb-RBCGs). TFF was then compared against centrifugation as an alternative method for removing unencapsulated Hb from Hb-RBCGs, and the effects that each washing method on the resulting Hb-RBCG biophysical properties was assessed.

Exploring the destructive synergy between IL-33 and Suilysin hemolysis on blood-brain barrier stability.

Streptococcus suis type 2 (SS2) is a zoonotic pathogen capable of eliciting meningitis, presenting significant challenges to both the swine industry and public health. Suilysin (Sly), one of SS2 most potent virulence determinants, releases a surfeit of inflammatory agents following red blood cell lysis. Notably, while current research on Sly role in SS2-induced meningitis predominantly centers on its interaction with the blood-brain barrier (BBB), the repercussions of Sly hemolytic products on BBB function have largely been sidestepped. In this vein, our study delves into the ramifications of Sly-induced hemolysis on BBB integrity. We discern that Sly hemolytic derivatives exacerbate the permeability of Sly-induced in vitro BBB models. Within these Sly hemolytic products, Interleukin-33 (IL-33) disrupts the expression and distribution of Claudin-5 in brain microvascular endothelial cells, facilitating the release of Interleukin-6 (IL-6) and Interleukin-8 (IL-8), thereby amplifying BBB permeability. Preliminary mechanistic insights suggest that IL-33-driven expression of IL-6 and IL-8 is orchestrated by the p38-mitogen-activated protein kinase signaling, whereas matrix metalloproteinase 9 mediates IL-33-induced suppression of Claudin-5. To validate these in vitro findings, an SS2-infected mouse model was established, and upon intravenous administration of growth stimulation expressed gene 2 (ST2) antibodies, in vivo results further underscored the pivotal role of the IL-33/ST2 axis during SS2 cerebral invasion. In summation, this study pioneerly illuminates the involvement of Sly hemolytic products in SS2-mediated BBB compromise and spotlights the instrumental role and primary mechanism of IL-33 therein. These insights enrich our comprehension of SS2 meningitis pathogenesis, laying pivotal groundwork for therapeutic advancements against SS2-induced meningitis.IMPORTANCEThe treatment of meningitis caused by Streptococcus suis type 2 (SS2) has always been a clinical challenge. Elucidating the molecular mechanisms by which SS2 breaches the blood-brain barrier (BBB) is crucial for the development of meningitis therapeutics. Suilysin (Sly) is one of the most important virulence factors of SS2, which can quickly lyse red blood cells and release large amounts of damage-associated molecular patterns, such as hemoglobin, IL-33, cyclophilin A, and so on. However, the impact of these hemolytic products on the function of BBB is unknown and ignored. This study is the first to investigate the effect of Sly hemolytic products on BBB function. The data are crucial for the study of the pathogenesis of SS2 meningitis and can provide an important reference for the development of meningitis therapeutics.

NanoGraphene Clot: A New Fibrinogen-Mimic Hemostatic Material.

Trauma is the leading cause of death for adults under the age of 44. Internal bleeding remains a significant challenge in medical emergencies, necessitating the development of effective hemostatic materials that could be administered by paramedics before a patient is in the hospital and treated by surgeons. In this study, we introduce a graphene oxide (GO)-based PEGylated synthetic hemostatic nanomaterial with an average size of 211 ± 83 nm designed to target internal bleeding by mimicking the role of fibrinogen. Functionalization of GO-g-PEG with peptides derived from the α-chain of fibrinogen, such as GRGDS, or the γ-chain of fibrinogen, such as HHLGGAKQAGDV:H12, was achieved with peptide loadings of 72 ± 6 and 68 ± 15 μM, respectively. In vitro studies with platelet-rich plasma (PRP) under confinement demonstrated aggregation enhancement of 39 and 24% for GO-g-PEG-GRGDS and GO-g-PEG-H12, respectively, compared to buffer, while adenosine diphosphate (ADP) alone induced a 5% aggregation. Compared to the same materials in the absence of ADP, GO-g-PEG-GRGDS achieved a 47% aggregation enhancement, while GO-g-PEG-H12 a 25% enhancement. This is particularly important for injectable hemostats and highlights the fact that our nanographene-based materials can only act as hemostats in the presence of agonists, reducing the possibility of unwanted clotting during circulation. Further studies on collagen-coated wells under dynamic flow revealed statistically significant augmentation of PRP fluorescence signal using GRGDS- or H12-coated GO-g-PEG compared to controls. Hemolysis studies showed <1% lysis of red blood cells (RBCs) at the highest PEGylated nanographene concentration. Finally, whole human blood coagulation studies reveal faster and more pronounced clotting using our nanohemostats vs PBS control from 3 min and below (blood is clotted with 10% CaCl2 within 4-5 min), with the biggest differences to be shown at 2 and 1 min. At 1 min, the clot weight was found to be ∼45% of that between 4 and 5 min, while no clot was formed in PBS-treated blood. Reduction of CaCl2 to 5 and 3%, or utilization of prostaglandin E1, an anticoagulant, still leads to clots but of smaller weight. The findings highlight the potential of our fibrinogen-mimic PEGylated nanographene as a promising non-hemolytic injectable scaffold for targeting internal bleeding, offering insights into its platelet aggregation capabilities under confinement and under dynamic flow as well as its pronounced coagulation abilities.

Assessing the probiotic potential and safety of newly isolated Bacillus coagulans VHBAX‐04: in vitro and in silico evaluations

SummaryThe human gastrointestinal tract hosts a diverse and intricate microbial community, a pivotal contributor to human well‐being. An effective strategy for exploring the diversity and potential probiotic attributes of Bacillus species is to isolate them from faecal samples. In this investigation, we scrutinised the in vitro probiotic characteristics of Bacillus coagulans VHBAX‐04, an isolate derived from human faeces. These attributes encompassed acid and gastric juice tolerance, resistance to bile salts and intestinal juice, adhesion capabilities, safety profile, antibiotic resistance pattern, the absence of toxin genes, and antimicrobial efficacy. Furthermore, we conducted in silico analyses to evaluate its probiotic potential. The notable absence of genes associated with mucin degradation, red blood cell lysis, gelatin hydrolysis, or toxin production in B. coagulans VHBAX‐04 certifies its safety as a probiotic. Additionally, the distinct antibiotic resistance pattern of strain VHBAX‐04 suggests its potential for co‐administration with antibiotics. In conclusion, this study underscores the suitability of B. coagulans VHBAX‐04 as a viable probiotic option. Isolation and Evaluation of Bacillus coagulans WBX04: A Probiotic Strain from Human Feces Studied through 16S rRNA Sequencing and In Vitro Tests.

Study on Circulating Tumor Cell Detection System Based on Microfluidic Chip

To achieve high throughput and high detection rate of circulating tumor cells (CTCs) in human peripheral blood, and to provide efficient and accurate early screening for cancer patients. A microfluidic chip with the integration of sorting, enrichment and detection was designed, and CTCs at the single cell level were detected by fluorescence detection system to obtain the number of CTCs in samples. The peripheral blood samples after lysed red blood cells were used for 6 experiments. When the injection rate reached 0.2 mL/h, CTCs could reach the best detection rate of 78.6%, and the correlation coefficient within the group was above 0.8. CTCs detection system can achieve high detection rate and has good reliability, which can provide a reliable reference for clinical research in related fields.

Deeper insights into heart failure with preserved ejection fraction through rna-sequencing of circulating endothelial cells - advanced protocol establishment enables complete transcriptome analysis

Abstract Funding Acknowledgements None. Heart failure with preserved ejection fraction (HFpEF) continues to pose a significant challenge with a bleak prognosis and a rising incidence, particularly among the aging population in Western countries. An in-depth comprehension of cellular-level pathogenesis, with a specific emphasis on the pivotal role of cellular senescence in endothelial dysfunction, is imperative for the evolution of innovative therapeutic interventions. Many previous approaches to investigate the genesis of HFpEF are based only on HFpEF-mouse models, which cannot directly be transferred to humans. The isolation of circulating endothelial cells from peripheral blood samples represents a more translatable, feasible, less invasive way to study endothelial dysfunction, a central component of HFpEF-pathogenesis, in human material for the identification of new therapeutical targets. This study focuses on establishing a new protocol for isolating circulating endothelial cells (CEC) in HFpEF patients to address endothelial dysfunction and pinpoint novel therapeutic targets through a comprehensive whole transcriptome analysis. Methodologically, CEC isolation from whole blood samples was achieved using a newly established Fluorescence-Activated Cell Sorting (FACS) panel. HFpEF patients were recruited based on their HFA-PEFF score, with non-diseased controls comprising both young and old subjects meticulously matched for sex and age. Subsequently, the isolated CECs were subjected to transcriptome analysis. Our new protocol is based on red blood cell lysis followed by magnetic cell sorting to deplete CD45+ cells. This is followed by staining with fluorescent-conjugated monoclonal antibodies. This FACS panel includes known endothelial-specific markers including CD11b- and CD45-, CD146+, CD34+ and CD31+ as well as DAPI- as a nucleus marker. The prepared CECs were sorted using FACS and a specially developed gating. We isolated the RNA in preparation for sequencing. Our protocol was validated by immunofluorescence staining of isolated CECs (markers: CD31, vWF, CD146, Phalloidin) and PCR analysis (markers: CD31, CD146 and vWF). In summary, we have successfully established a new, robust and highly targeted workflow for consistently isolating circulating endothelial cells from blood and performing RNA sequencing on the collected samples. This is a key advance towards a deeper understanding of the pathophysiology of HFpEF. The application of our methodology may enable the characterization of patients with HFpEF at the transcriptome level. The whole transcriptome analyses provide potential starting points for further in vitro and in vivo studies. As we extend our investigations to larger patient cohorts in the future, there is potential for the identification of novel therapeutic targets.

β-Thalassemia gene editing therapy: Advancements and difficulties.

β-Thalassemia is the world's number 1 single-gene genetic disorder and is characterized by suppressed or impaired production of β-pearl protein chains. This results in intramedullary destruction and premature lysis of red blood cells in peripheral blood. Among them, patients with transfusion-dependent β-thalassemia face the problem of long-term transfusion and iron chelation therapy, which leads to clinical complications and great economic stress. As gene editing technology improves, we are seeing the dawn of a cure for the disease, with its reduction of ineffective erythropoiesis and effective prolongation of survival in critically ill patients. Here, we provide an overview of β-thalassemia distribution and pathophysiology. In addition, we focus on gene therapy and gene editing advances. Nucleic acid endonuclease tools currently available for gene editing fall into 3 categories: zinc finger nucleases, transcription activator-like effector nucleases, and regularly interspaced short palindromic repeats (CRISPR-Cas9) nucleases. This paper reviews the exploratory applications and exploration of emerging therapeutic tools based on 3 classes of nucleic acid endonucleases in the treatment of β-thalassemia diseases.

Biodistribution of Native and Nanoformulated Innate Defense Regulator Peptide 1002.

Innate defense regulator-1002 (IDR-1002) is a synthetic peptide with promising immunomodulatory and antibiofilm properties. An appreciable body of work exists around its mechanism of action at the cellular and molecular level, along with its efficacy across several infection and inflammation models. However, little is known about its absorption, distribution, and excretion in live organisms. Here, we performed a comprehensive biodistribution assessment with a gallium-67 radiolabeled derivative of IDR-1002 using nuclear tracing techniques. Various dose levels of the radiotracer (2-40 mg/kg) were administered into the blood, peritoneal cavity, and subcutaneous tissue, or instilled into the lungs. The peptide was well tolerated at all subcutaneous and intraperitoneal doses, although higher levels were associated with delayed absorption kinetics and precipitation of the peptide within the tissues. Low intratracheal doses were rapidly absorbed systemically, and small increases in the dose level were lethal. Intravenous doses were rapidly cleared from the blood at lower levels, and upon escalation, were toxic with a high proportion of the dose accumulating within the lung tissue. To improve biocompatibility and prolong its circulation within the blood, IDR-1002 was further formulated onto high molecular weight hyperbranched polyglycerol (HPG) polymers. Constructs prepared at 5:1 and 10:1 peptide-to-polymer ratios were colloidally stable, maintained the biological profile of the peptide payload and helped reduce red blood cell lysis. The 5:1 construct circulated well in the blood, but higher peptide loading was associated with rapid clearance by the reticuloendothelial system. Many peptides face pharmacokinetic and biocompatibility challenges, but formulations such as those with HPG have the potential to overcome these limitations.