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Purpose of Review: This review summarizes an overview of stem cell therapy for the treatment of chronic limb-threatening ischemia (CLTI), with a focus on recent advances and ongoing challenges. Recent Findings: Extensive research has aimed to identify ideal cell types and sources for effective and widely feasible cell-based regenerative therapy, highlighting mesenchymal stem cells (MSCs) as the most promising candidates for treating limb ischemia. Mechanistically, secreted factors released from MSCs, including exosomes, significantly contribute to their proangiogenic and anti-inflammatory effects. In addition, mitochondrial transfer plays a crucial role in promoting tissue repair. New approaches including cell engineering, induced pluripotent stem cell (iPSC) technology, and single-cell omics offer new avenues for enhancing the efficacy of MSC-based cell therapies. Summary: Despite accumulating research indicating its beneficial effects, significant challenges remain before the widespread adoption of stem cell-based therapies for CLTI. Continued research is essential to translate the therapeutic potential of stem cells into routine clinical practice. [ABSTRACT FROM AUTHOR]

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare and aggressive neoplastic process of precursor plasmacytoid dendritic cells. The diagnostic evaluation of this heterogenous entity is challenging, requiring a comprehensive approach of incorporating clinical, morphologic, immunohistochemical, and molecular/cytogenetic evaluations. Optimal management of BPDCN remains controversial, and clinical outcomes continues to be poor. Pediatric cases of BPDCN are rare and to our knowledge, this is the second case of BPDCN described in a Hispanic child, first one was described outside the US in Peru. Here, we report a case of a juvenile patient of Hispanic origin presenting with cutaneous and bone marrow involvement and initially misdiagnosed as a cutaneous infection that resulted in subsequent delaying of necessary chemotherapy for 2 months. Biopsy of the lesion showed diffuse infiltration of immature cells involving the dermis with classical sparring of epidermis. A huge panel of immunohistochemical stains were performed to reach the diagnosis of BPDCN. Staging bone marrow biopsy also revealed involvement by BPDCN. Treatment was not only delayed in this patient but also due to the rarity of BPDCN in pediatric population, the subsequent therapeutic decisions were challenging for the primary oncology team as it was based solely on published literature on adult population. Our case report will not only add one more case in the pediatric age group, but also will also emphasize that although BPDCN has a grave prognosis in the elderly, timely diagnosis with prompt treatment is the key to complete remission in pediatric BPDCN population. [ABSTRACT FROM AUTHOR]

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare and aggressive neoplastic process of precursor plasmacytoid dendritic cells. The diagnostic evaluation of this heterogenous entity is challenging, requiring a comprehensive approach of incorporating clinical, morphologic, immunohistochemical, and molecular/cytogenetic evaluations. Optimal management of BPDCN remains controversial, and clinical outcomes continues to be poor. Pediatric cases of BPDCN are rare and to our knowledge, this is the second case of BPDCN described in a Hispanic child, first one was described outside the US in Peru. Here, we report a case of a juvenile patient of Hispanic origin presenting with cutaneous and bone marrow involvement and initially misdiagnosed as a cutaneous infection that resulted in subsequent delaying of necessary chemotherapy for 2 months. Biopsy of the lesion showed diffuse infiltration of immature cells involving the dermis with classical sparring of epidermis. A huge panel of immunohistochemical stains were performed to reach the diagnosis of BPDCN. Staging bone marrow biopsy also revealed involvement by BPDCN. Treatment was not only delayed in this patient but also due to the rarity of BPDCN in pediatric population, the subsequent therapeutic decisions were challenging for the primary oncology team as it was based solely on published literature on adult population. Our case report will not only add one more case in the pediatric age group, but also will also emphasize that although BPDCN has a grave prognosis in the elderly, timely diagnosis with prompt treatment is the key to complete remission in pediatric BPDCN population.

Abstract Background To investigate the long-term effects of combining bone marrow mesenchymal stem cells (MSCs) with mononuclear cells (MCs) in the treatment of type 2 diabetes mellitus (T2DM). Methods T2DM patients were divided into the combination group (Dual MSC + MC, n = 33), the mononuclear cell group (MC-Only, n = 32) and the control group (Control, n = 31). All groups were treated with insulin and metformin. The Dual MSC + MC group additionally received MSC and MC infusion and the MC-Only group additionally received MC infusion. The patients were followed up for 8 years. The primary endpoint was the C-peptide area under the curve (C-p AUC) at 1 year. This study was registered with clinicaltrial.gov (NCT01719640). Results A total of 97 patients were included and 89 completed the follow-up. The area under the curve of C-peptide of the Dual MSC + MC group and the MC-Only group was significantly increased (50.6% and 32.8%, respectively) at 1 year. After eight years of follow-up, the incidence of macrovascular complications was 13.8% (p = 0.009) in the Dual MSC + MC group and 21.4% (p = 0.061) in the MC-Only group, while it was 44.8% in the Control group. The incidence of diabetic peripheral neuropathy (DPN) was 10.3% (p = 0.0015) in the Dual MSC + MC group, 17.9% (p = 0.015) in the MC-Only group, and 48.3% in the Control group. Conclusions The combination of MSC and MC therapy can reduce the incidence of chronic diabetes complications and improves metabolic control with mild side effects in T2DM patients.

Schizophrenia is a mental illness of complex etiology. Recently, there has been increased interest in the role of the immune system in the pathophysiology of mental disorders. The concept of neuroinflammation in neurodevelopmental disorders is gaining widespread interest, including the role of Toll-like receptors.Schizophrenia is associated with an increase in the concentration of cfDNA in human blood, and the composition of cfDNA fragments changes significantly compared to cellular DNA: GC-rich fragments of the ribosomal repeat accumulate and base oxidation occurs. Similar changes, but less pronounced, also occur for cfDNA from healthy donors.To confirm the hypothesis about the possible participation of cfDNA in the inflammation induction, we studied the effect of cfDNA samples on cultured mononuclear cells.Unlike cellular DNA, cfDNA(SZ) and cfDNA(K) stimulate transcription of the TLR9 gene in mononuclear cells. After 1 hour the amount of TLR9 RNA increases by 2.9 and 3.3 times compared to the control. After 24 hours, the TLR9 RNA level decreases slightly, but is still 2-3 times higher than the control level. After 1 hour, TLR9 protein increases by 1.5 and 1.7 times, respectively, and further increased after 24h of culture.An increase TLR9 protein expression correlates with an increase of the transcription factor NF-kB in lymphocytes and is accompanied by an increase in proinflammatory cytokine IL8 RNA, the transcription of IL8 is controlled by the NF-kB factor.Thus, cfDNA(SZ) and cfDNA(K) stimulate the TLR9-NF-kB-proinflammatory cytokine signaling pathway in lymphocytes. The effect of cfDNA also depends on the concentration of these fragments in the extracellular environment. Since the concentrations of cfDNA in the blood of patients with schizophrenia are significantly increased compared to healthy donors, we should expect a much higher level of activation of the TLR9-NF-kB signaling pathway in the body cells of sick people.Samples of cfDNA from patients with schizophrenia have a pronounced biological effect on cells of the immune system, stimulating the synthesis of pro-inflammatory cytokines by activating the TLR9-NF-kB-proinflammatory cytokines signaling pathway. High levels of cfDNA in blood plasma may be one of the reasons for the induction and maintenance of low-level inflammation in schizophrenia.

Background/Objectives: Extracorporeal photopheresis (ECP) is a well-established and efficacious cell therapy for a range of diseases. The objective of this retrospective study was to compare the new Amicus Blue inline system with the Therakos Cellex inline system and the Spectra Optia offline system in terms of collection efficiency, mononuclear cell (MNC) yield of mononuclear cell products (MCPs), processing time and correlation between MCP cell count and peripheral blood count of patients. Methods: This retrospective study compared 127 procedures utilizing the Spectra Optia offline system, 93 procedures employing the Amicus Blue inline system, and 81 procedures applying the Therakos Cellex inline system. The MNCs were subjected to flow cytometry analysis for CD45 and CD14 expression in order to ascertain the precise composition of the collected lymphocyte and monocyte fractions. Results: The Therakos inline system demonstrated the highest MNC collection efficiency (Therakos: 74.42 ± 1.82; Optia: 65.79 ± 1.48; Amicus: 56.32 ± 2.80; p < 0.01). Regarding the content of collected MNCs (×10⁶/kg body weight), the Spectra Optia offline system was superior to the other systems (Optia: 42.69 ± 1.42; Therakos: 31.21 ± 1.66; Amicus: 27.56 ± 1.54; p < 0.01). Conclusions: This study represents the first direct comparison of the new Amicus Blue inline system with the two most commonly used ECP systems in the same patient cohort of a single center. The data show that the Amicus Blue inline system collects sufficient MNCs to perform an ECP, but it has a significantly lower CE than the other systems and a significantly lower amount of collected MNCs than the Spectra Optia offline system. [ABSTRACT FROM AUTHOR]

Background: To investigate the long-term effects of combining bone marrow mesenchymal stem cells (MSCs) with mononuclear cells (MCs) in the treatment of type 2 diabetes mellitus (T2DM). Methods: T2DM patients were divided into the combination group (Dual MSC + MC, n = 33), the mononuclear cell group (MC-Only, n = 32) and the control group (Control, n = 31). All groups were treated with insulin and metformin. The Dual MSC + MC group additionally received MSC and MC infusion and the MC-Only group additionally received MC infusion. The patients were followed up for 8 years. The primary endpoint was the C-peptide area under the curve (C-p AUC) at 1 year. This study was registered with clinicaltrial.gov (NCT01719640). Results: A total of 97 patients were included and 89 completed the follow-up. The area under the curve of C-peptide of the Dual MSC + MC group and the MC-Only group was significantly increased (50.6% and 32.8%, respectively) at 1 year. After eight years of follow-up, the incidence of macrovascular complications was 13.8% (p = 0.009) in the Dual MSC + MC group and 21.4% (p = 0.061) in the MC-Only group, while it was 44.8% in the Control group. The incidence of diabetic peripheral neuropathy (DPN) was 10.3% (p = 0.0015) in the Dual MSC + MC group, 17.9% (p = 0.015) in the MC-Only group, and 48.3% in the Control group. Conclusions: The combination of MSC and MC therapy can reduce the incidence of chronic diabetes complications and improves metabolic control with mild side effects in T2DM patients. [ABSTRACT FROM AUTHOR]

Objective To select the cell isolation method, seeding number, and coating conditions for culture flasks to induce and amplify rat bone marrow mononuclear cells （BMMNCs） into endothelial progenitor cells （BM-EPCs）, aiming to establish an efficient, high-yield, and high-purity method for the separation, culture, induction, and amplification of BMEPCs derived from bone marrow. Methods Two-week-old male SD rats were used and euthanized by cervical dislocation, then we isolated the tibia and femur bilaterally to collect bone marrow cell suspensions. We prepared Percoll cell separation solutions at concentrations of 30%, 50%, 60%, and 70%, and separated rat BMMNCs seed cells using the Percoll density gradient centrifugation method, and calculated the ratio of viable cells. The obtained BMMNCs were divided into six groups （1×105, 5×105, 1×106, 2. 5×106, 5×106, and 1×107 cells） and were inoculated in the 25 cm² sterile culture flasks. After 7 days of culture, we observed the number of cell colonies formed in each group under a microscope and calculated the number of colonies formed per 106 cells. Logistic fitting curves were plotted using GraphPad Prism 9. 5 software to determine correla‑ tion based on the R² coefficient, and the P-values were used to include statistically significant seeding numbers. We then used R language to define calculation functions and iteratively found the optimal BMMNCs seeding number based on the known total number of seed cells and the correlation function limitations. Fibronectin （FN） solutions at concentrations of 20 nmol/L, 50 nmol/L, and 100 nmol/L were prepared, and culture flasks were coated for 2, 6, 12, and 24 h with blank solu‑ tions without FN as controls. We inoculated BMMNCs that did not adhere after 48 h into FN-coated culture flasks and cul‑ tured them for 3 days to calculate the number of colonies formed in each group, and determined the optimal FN concentration and coating time. BMMNCs that did not adhere after 48 h were inoculated in the 25 cm² culture flasks, and were induced with EGM-2 complete medium, and we observed the colony formation and induction expansion process under a microscope. BM-EPCs cultured for 14 days were identified for purity using dual-positive staining and flow cytometry. Results The Per‑ coll separation method clearly separated BMMNCs into five layers, with the highest ratio of viable cells between the 30% and 50% Percoll layers. The optimal seeding number for BMMNCs was 2. 5×106 cells. FN solutions at 50 nmol/L for 24 h or 100 nmol/L for 6 h effectively promoted colony formation. After 7 days of cell culture, well-formed cobblestone-like cells were observed, indicating that BMMNCs had been successfully induced into well-formed BM-EPCs. The percentage of Dil-AcLDL/FITC-UEA-1 dual-positive cells was 91. 89%±5. 77%, with CD31+KDR positive rate of 90. 73%±0. 61%, CD14 posi‑ tive rate of 0. 53%±0. 17%, and CD45 positive rate of 0. 77%±0. 34%, indicating that the obtained BM-EPCs were of high purity. Conclusions In the process of inducing and amplifying rat BMMNCs into BM-EPCs, Percoll density gradient cen‑ trifugation can be used to isolate BMMNCs. The optimal cell seeding number for BMMNCs is 2. 5×106 cells, and the culture flask coating conditions are 50 nmol/L FN solution for 24 h or 100 nmol/L FN solution for 6 h. The obtained BM-EPCs through separation, culture, induction, and amplification methods are of good morphology and of high purity. [ABSTRACT FROM AUTHOR]

Various exogenous factors, such as microbiological and chemical contamination condition food security. Salmonella Typhimurium (S. Typhimurium) is the cause of salmonellosis. This bacterium utilizes phagocytosis to create bacterial reservoirs. On the other hand, exposure to chemical contaminants, such as pesticides, increases susceptibility to numerous infections. Therefore, this research aims to evaluate the effect of co-exposure to diazoxon and S. Typhimurium on the in vitro infection dynamics. For this purpose, human mononuclear cells were pre-exposed in vitro to diazoxon and then challenged with S. Typhimurium at 1, 8, and 24 h. Bacterial internalization, actin polymerization, and reactive oxygen species (ROS) were analyzed. Obtained data show that mononuclear cells previously exposed to diazoxon exhibit greater internalization of S. Typhimurium. Likewise, greater ROS production and an increase in actin polymerization were observed. Therefore, in the proposed scenario, obtained data suggest that co-exposure to diazoxon and S. Typhimurium increases susceptibility to acquiring an illness. [ABSTRACT FROM AUTHOR]

Background: Recent research has identified alternative transcript variants of LMNA/C (LMNA, LMNC, LMNAΔ10, and LMNAΔ50) and insulin receptors (INSRs) as potential biomarkers for various types of cancer. The objective of this study was to assess the expression of LMNA/C and INSR transcript variants in peripheral blood mononuclear cells (PBMCs) of leukemia patients to investigate their potential as diagnostic biomarkers. Methods: Quantitative TaqMan reverse transcriptase polymerase chain reaction (RT-qPCR) was utilized to quantify the mRNA levels of LMNA/C (LMNA, LMNC, LMNAΔ10, and LMNAΔ50) as well as INSR (IR-A and IR-B) variants in PBMCs obtained from healthy individuals (n = 32) and patients diagnosed with primary leukemias (acute myeloid leukemia (AML): n = 17; acute lymphoblastic leukemia (ALL): n = 8; chronic myeloid leukemia (CML): n = 5; and chronic lymphocytic leukemia (CLL): n = 15). Results: Only LMNA and LMNC transcripts were notably present in PBMCs. Both exhibited significantly decreased expression levels in leukemia patients compared to the healthy control group. Particularly, the LMNC:LMNA ratio was notably higher in AML patients. Interestingly, IR-B expression was not detectable in any of the PBMC samples, precluding the calculation of the IR-A:IR-B ratio as a diagnostic marker. Despite reduced expression across all types of leukemia, IR-A levels remained detectable, indicating its potential involvement in disease progression. Conclusions: This study highlights the distinct expression patterns of LMNA/C and INSR transcript variants in PBMCs of leukemia patients. The LMNC:LMNA ratio shows promise as a potential diagnostic indicator for AML, while further research is necessary to understand the role of IR-A in leukemia pathogenesis and its potential as a therapeutic target. [ABSTRACT FROM AUTHOR]

Autologous bone marrow mononuclear cells (BMMNCs) infused after severe traumatic brain injury have shown promise for treating the injury. We evaluated their impact in children, particularly their hypothesized ability to preserve the blood–brain barrier and diminish neuroinflammation, leading to structural CNS preservation with improved outcomes. We performed a randomized, double-blind, placebo-sham-controlled Bayesian dose-escalation clinical trial at two children's hospitals in Houston, TX and Phoenix, AZ, USA (NCT01851083). Patients 5–17 years of age with severe traumatic brain injury (Glasgow Coma Scale score ≤ 8) were randomized to BMMNC or placebo (3:2). Bone marrow harvest, cell isolation and infusion were completed by 48 h post-injury. A Bayesian continuous reassessment method was used with cohorts of size 3 in the BMMNC group to choose the safest between two doses. Primary end points were quantitative brain volumes using MRI and microstructural integrity of the corpus callosum (diffusivity and oedema measurements) at 6 months and 12 months. Long-term functional outcomes and ventilator days, intracranial pressure monitoring days, intensive care unit days and therapeutic intensity measures were compared between groups. Forty-seven patients were randomized, with 37 completing 1-year follow-up (23 BMMNC, 14 placebo). BMMNC treatment was associated with an almost 3-day (23%) reduction in ventilator days, 1-day (16%) reduction in intracranial pressure monitoring days and 3-day (14%) reduction in intensive care unit (ICU) days. White matter volume at 1 year in the BMMNC group was significantly preserved compared to placebo [decrease of 19 891 versus 40 491, respectively; mean difference of −20 600, 95% confidence interval (CI): −35 868 to −5332; P = 0.01], and the number of corpus callosum streamlines was reduced more in placebo than BMMNC, supporting evidence of preserved corpus callosum connectivity in the treated groups (−431 streamlines placebo versus −37 streamlines BMMNC; mean difference of −394, 95% CI: −803 to 15; P = 0.055), but this did not reach statistical significance due to high variability. We conclude that autologous BMMNC infusion in children within 48 h after severe traumatic brain injury is safe and feasible. Our data show that BMMNC infusion led to: (i) shorter intensive care duration and decreased ICU intensity; (ii) white matter structural preservation; and (iii) enhanced corpus callosum connectivity and improved microstructural metrics. [ABSTRACT FROM AUTHOR]

Background/Aim: H. pylori infection can promote a systemic inflammatory syndrome, eventually leading to intestinal metaplasia and gastric cancer. The aim of our study was to investigate the possible association between dyslipidemia and histopathological features of H. pylori gastritis. Patients and Methods: An observational, retrospective study was conducted over the period 2017-2022 on symptomatic patients with a positive rapid urease test. A total of 121 patients who underwent upper gastrointestinal endoscopy with stomach biopsy were enrolled in this study. Based on the updated Sydney System, we investigated the association between neutrophils, mononuclear cells, intestinal metaplasia, or gastric atrophy and altered lipid profiles. Results: A high prevalence of H. pylori infection was noticed in the studied group upon the application of the rapid urease test, being associated with dyslipidemia regardless of patient sex. All the endoscopic diagnoses (acute, chronic, or atrophic chronic gastritis, metaplasia) correlated with the histopathological features. Mononuclear cells and metaplasia were more likely to be found in H. pylori-positive patients with dyslipidemia, which is consistent with acute and chronic inflammation caused by H. pylori in the gastric mucosa. Conclusion: Although our study was conducted on a small scale, it offers new insights and details regarding H. pylori infection and histopathological features. Mononuclear cells and metaplasia were associated with an altered lipid profile in H. pylori-positive patients. These findings warrant future investigation, such as the evolution of gastric biopsies and lipid profiles before and after eradication. [ABSTRACT FROM AUTHOR]

Multiple myeloma (MM) is a plasma cell neoplasm that is characterized by the clonal proliferation of malignant plasma cells in the bone marrow. It is considered the second most common hematological malignancy which accounts for approximately 1% - 2% of all cancers and among 10% of hematological malignancies. Autologous peripheral blood stem cell Transplantation (PBSCT) is the best treatment for MM. Since the optimum harvested stem cell yield is a crucial factor for sufficient engraftment, the enumeration of Mononuclear cell (MNC) count in peripheral blood and harvested CD 34+ stem cell count can be considered as the best predictive markers for the best timing of apheresis which positively correlates with engraftment outcome of PBSCT. MNC count can be obtained using either a hematological analyzer or peripheral blood smear while flow cytometry is the advanced technology that can be used to enumerate CD 34+ stem cell count other than peripheral blood smear. The unavailability of a flow cytometer, the expensiveness of this method, and the lack of trained personnel regarding this new technology, especially in lower-middle-income countries cause disturbance in the enumeration of stem cells. In such a situation, this review describes the importance of establishing an association between peripheral blood MNCs and harvested CD 34+ cells. Furthermore, this association facilitates conducting effective PBSCT for MM patients even in the absence of a flow cytometer and eventually, it focuses on decentralizing the treatment of PBSCT.

The SARS-CoV-2 P.1 variant, responsible for an outbreak in Manaus, Brazil, is distinguished by 12 amino acid differences in the S protein, potentially increasing its ACE-2 affinity and immune evasion capability. We investigated the innate immune response of this variant compared to the original B.1 strain, particularly concerning cytokine production. Blood samples from three severe COVID-19 patients were analyzed post-infection with both strains. Results showed no significant difference in cytokine production of mononuclear cells and neutrophils for either variant. While B.1 had higher cytopathogenicity, neither showed viral replication in mononuclear cells. Structural analyses of the S protein highlighted physicochemical variations, which might be linked to the differences in infectivity between the strains. Our studies point to the increased infectivity of P.1 could stem from altered immunogenicity and receptor-binding affinity. [ABSTRACT FROM AUTHOR]

The evolutionarily conserved mevalonate pathway plays an important role in the synthesis of cholesterol and isoprenoid compounds. Mevalonate kinase (MVK) and phosphomevalonate kinase (PMVK) enzymes regulate key rate‐limiting steps in this pathway by sequentially phosphorylating mevalonic acid to yield downstream metabolites that regulate protein prenylation and cell signaling. Biallelic pathogenic variants in MVK cause a spectrum of rare autoinflammatory disorders that encompass milder forms of hyper‐IgD syndrome (HIDS) at one end and the more severe mevalonic aciduria on the other. In contrast, pathogenic variants reported in PMVK are heterozygous and associated with porokeratosis, a skin disorder with no systemic manifestations. Recently, biallelic variants in PMVK were reported as a cause for an autoinflammatory disorder for the first time in two unrelated patients. In this study, we describe a child with recurrent arthritis and a HIDS‐like phenotype harboring a novel homozygous variant c.398 C>T (p.Ala133Val) in PMVK. Mononuclear cells isolated from the patient showed significantly elevated production of interleukin 1β, a key cytokine that shapes the inflammatory response in HIDS. Protein modeling studies suggested potential defects in PMVK enzyme activity. These results posit a further expanding of the genotypic spectrum of autoinflammatory disease to include biallelic PMVK variants. [ABSTRACT FROM AUTHOR]

Objective: To observe the histomorphological alterations induced by consumption of energy drinks on skeletal muscles of Albino rats. Study Design:An experimental study. Place and Duration of Study: The study was conducted by the Department of Anatomy in collaboration with the animal house of Liaquat national hospital and medical college (LNH&MC), Karachi from November 2 to December 1 2020. Materials and Methods: For the study, 30 adult male Wistar Albino rats weighing between 140 and 200 grammes were allocated evenly into 3 groups. Group A served as control, kept on a regular laboratory diet. Group B served as a low dose treated group receiving energy drink at a dose of 7.5ml/day while group C received a high dose of drink i-e 15ml/day via gastric tube. All the animals were sacrificed following completion of the experimental period and were subjected to microscopic examination for histo-morphological study. Data was analyzed by using SPSS version 25. Results: The Hematoxylin and Eosin (H&E) stained sections revealed significant structural and parenchymal damage in skeletal muscle tissue. The regular parallel arrangement of skeletal muscle fibers was lost with the disappearance of nuclei. Dilated and congested blood vessels were observed in the treated tissues. Infiltration of mononuclear cells was also observed suggesting the inflammatory changes in the tissues of animals treated with caffeinated beverages in the present study. Conclusion: The consumption of energy drinks produces a significant histo-morphological alteration in the skeletal muscles of Wistar Albino rats

Abstract Background Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are used in regenerative medicine and related research involving immunomodulatory, anti-inflammatory, anti-fibrotic and regenerative functions. Isolation of BM-MSCs from samples obtained during total hip arthroplasty (THA) is routinely possible. Advanced age and comorbidities of the majority of patients undergoing THA limit their applicability. Our study aimed to evaluate the potential of bone marrow obtained during periacetabular osteotomy (PAO) as a novel source of BM-MSCs from young donors by analyzing cell yield and cell characteristics. Methods Bone samples were obtained from the anterior Os ilium or superior Os pubis during PAO and from the femoral cavity during primary THA. Isolation of bone marrow-derived mononuclear cells (BM-MNCs) was performed by density gradient centrifugation. The samples from PAO and THA patients were compared in terms of BM-MSC yield, colony formation and the proportion of BM-MSCs within the BM-MNC population using flow cytometry analysis. The cells were characterized based on the expression of BM-MSC-specific surface markers. The functionality of the cells was compared by quantifying post-thaw viability, metabolic activity, proliferation capacity, senescence-associated beta galactosidase (SA-β-gal) expression, trilineage differentiation potential and major secretome proteins. Results Isolation of BM-MNCs was possible in a reliable and reproducible manner when using bone from PAO containing more than 0.24 g bone marrow. PAO patients were younger than patients of the THA group. Bone obtained during PAO contained less bone marrow and led to a lower BM-MSC number after the first cell culture passage compared to BM-MSCs obtained during THA. BM-MSCs from PAO samples are characterized by a higher proliferation capacity. This results in a higher yield in cell culture passage two, when normalized to the sample weight. BM-MSCs from PAO patients showed increased secretion of TGF-β1, TIMP2, and VEGF upon osteogenic differentiation. BM-MSCs from PAO and THA patients revealed similar results regarding the onset of SA-β-gal expression and trilineage differentiation capacity. Conclusions We suggest that bone obtained during PAO is a promising novel source for BM-MSCs from young donors. Limited absolute cell yield due to low sample weight must be considered in early cell culture passages and might be critical for the range of clinical applications possible for BM-MSCs from this source. The higher proliferation capacity and increased growth factor secretion of BM-MSCs from young donors may be beneficial for future regenerative cell therapies, in vitro models, and tissue engineering.

Background/Objectives: Extracorporeal photopheresis (ECP) is a well-established and efficacious cell therapy for a range of diseases. The objective of this retrospective study was to compare the new Amicus Blue inline system with the Therakos Cellex inline system and the Spectra Optia offline system in terms of collection efficiency, mononuclear cell (MNC) yield of mononuclear cell products (MCPs), processing time and correlation between MCP cell count and peripheral blood count of patients. Methods: This retrospective study compared 127 procedures utilizing the Spectra Optia offline system, 93 procedures employing the Amicus Blue inline system, and 81 procedures applying the Therakos Cellex inline system. The MNCs were subjected to flow cytometry analysis for CD45 and CD14 expression in order to ascertain the precise composition of the collected lymphocyte and monocyte fractions. Results: The Therakos inline system demonstrated the highest MNC collection efficiency (Therakos: 74.42 ± 1.82; Optia: 65.79 ± 1.48; Amicus: 56.32 ± 2.80; p < 0.01). Regarding the content of collected MNCs (×10⁶/kg body weight), the Spectra Optia offline system was superior to the other systems (Optia: 42.69 ± 1.42; Therakos: 31.21 ± 1.66; Amicus: 27.56 ± 1.54; p < 0.01). Conclusions: This study represents the first direct comparison of the new Amicus Blue inline system with the two most commonly used ECP systems in the same patient cohort of a single center. The data show that the Amicus Blue inline system collects sufficient MNCs to perform an ECP, but it has a significantly lower CE than the other systems and a significantly lower amount of collected MNCs than the Spectra Optia offline system.

Background: Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are used in regenerative medicine and related research involving immunomodulatory, anti-inflammatory, anti-fibrotic and regenerative functions. Isolation of BM-MSCs from samples obtained during total hip arthroplasty (THA) is routinely possible. Advanced age and comorbidities of the majority of patients undergoing THA limit their applicability. Our study aimed to evaluate the potential of bone marrow obtained during periacetabular osteotomy (PAO) as a novel source of BM-MSCs from young donors by analyzing cell yield and cell characteristics. Methods: Bone samples were obtained from the anterior Os ilium or superior Os pubis during PAO and from the femoral cavity during primary THA. Isolation of bone marrow-derived mononuclear cells (BM-MNCs) was performed by density gradient centrifugation. The samples from PAO and THA patients were compared in terms of BM-MSC yield, colony formation and the proportion of BM-MSCs within the BM-MNC population using flow cytometry analysis. The cells were characterized based on the expression of BM-MSC-specific surface markers. The functionality of the cells was compared by quantifying post-thaw viability, metabolic activity, proliferation capacity, senescence-associated beta galactosidase (SA-β-gal) expression, trilineage differentiation potential and major secretome proteins. Results: Isolation of BM-MNCs was possible in a reliable and reproducible manner when using bone from PAO containing more than 0.24 g bone marrow. PAO patients were younger than patients of the THA group. Bone obtained during PAO contained less bone marrow and led to a lower BM-MSC number after the first cell culture passage compared to BM-MSCs obtained during THA. BM-MSCs from PAO samples are characterized by a higher proliferation capacity. This results in a higher yield in cell culture passage two, when normalized to the sample weight. BM-MSCs from PAO patients showed increased secretion of TGF-β1, TIMP2, and VEGF upon osteogenic differentiation. BM-MSCs from PAO and THA patients revealed similar results regarding the onset of SA-β-gal expression and trilineage differentiation capacity. Conclusions: We suggest that bone obtained during PAO is a promising novel source for BM-MSCs from young donors. Limited absolute cell yield due to low sample weight must be considered in early cell culture passages and might be critical for the range of clinical applications possible for BM-MSCs from this source. The higher proliferation capacity and increased growth factor secretion of BM-MSCs from young donors may be beneficial for future regenerative cell therapies, in vitro models, and tissue engineering. [ABSTRACT FROM AUTHOR]

Simple Summary: There is a lack of biomarkers for predicting the response to chemotherapy in gastric cancer (GC). This study investigates the prognostic significance of PD-1 expression in CD8+ T cells in the blood of 68 patients with advanced GC receiving platinum-based chemotherapy. Patients were divided based on baseline frequencies of PD-1+CD8+ T cells and whether these frequencies increased or decreased on day 7 after treatment. While baseline frequencies of PD-1+CD8+ T cells had no impact on survival, the patients whose frequencies of PD-1+CD8+ T cells increased on day 7 from the treatment initiation showed significantly longer survival than those whose frequencies of PD-1+CD8+ T cells decreased. This difference was also reflected in the mean duration of response between the groups. These findings suggest that an early increase in frequencies of PD-1+CD8+ T cells could predict favorable prognoses and durable responses in patients with advanced GC receiving chemotherapy. The clinical significance of PD-1 expression in circulating CD8+ T cells in patients with gastric cancer (GC) receiving chemotherapy remains unelucidated. Therefore, we aimed to examine its prognostic significance in blood samples of 68 patients with advanced GC who received platinum-based chemotherapy. The correlation between peripheral blood mononuclear cells, measured using fluorescence-activated cell sorting, was evaluated. Patients were divided into two groups according to the changes in PD-1+CD8+ T-cell frequencies between day 0 and 7. They were categorized as increased or decreased PD-1+CD8+ T-cell groups. The increased PD-1+CD8+ T-cell group showed longer progression-free survival (PFS) and overall survival (OS) than the decreased PD-1+CD8+ T-cell group (PFS: 8.7 months vs. 6.1 months, p = 0.007; OS: 20.7 months vs. 10.8 months, p = 0.003). The mean duration of response was significantly different between the groups (5.7 months vs. 2.5 months, p = 0.041). Multivariate analysis revealed that an increase in PD-1+CD8+ T-cell frequency was an independent prognostic factor. We concluded that the early increase in PD-1+CD8+ T-cell frequency is a potential predictor of favorable prognoses and durable responses in patients with advanced GC receiving chemotherapy. [ABSTRACT FROM AUTHOR]

Aims: To investigate the correlations among cytokines and regulatory T cells (T-regs) in ankylosing spondylitis (AS) patients, and their changes after anti-tumour necrosis factor-α (TNF-α) treatment. Methods: We included 72 AS patients with detailed medical records, disease activity score (Bath Ankylosing Spondylitis Disease Activity Index), functional index (Bath Ankylosing Spondylitis Functional Index), and laboratory data (interleukin (IL)-2, IL-4, IL-10, TNF-α, interferon (IFN)-γ, transforming growth factor (TGF)-β, ESR, and CRP). Their peripheral blood mononuclear cells (PBMCs) were marked with anti-CD4, anti-CD25, and anti-FoxP3 antibodies, and triple positive T cells were gated by flow cytometry as T-regs. Their correlations were calculated and the changes after anti-TNF-α therapy were compared. Results: The frequency of T-regs in PBMCs was positively correlated to ESR and CRP in AS (r = 0.35 and 0.43; p = 0.032 and 0.027, respectively), and there was also a significant correlation between serum level of TNF-α and CRP (p = 0.041). The frequency of T-regs in PBMCs positively correlated to serum levels of TNF-α, IL-10, and TGF-β, while IL-2, IL-4, and IFN-γ showed opposite results. After anti-TNF-α treatment, there were significantly lower serum levels of TNF-α, IL-10, TGF-β, and frequency of T-regs in PBMCs among these AS patients (p = 0.026, 0.032, 0.029, and 0.037, respectively). Conclusion: In AS patients, proinflammatory cytokine may give positive feedback to induce more T-reg production and anti-inflammatory cytokine secretion to suppress this inflammatory status, and they can be reversed by anti-TNF-α therapy. However, the detailed interactions among T-regs and complex cytokine networks in autoinflammatory diseases still need more studies and further functional assay. Cite this article: Bone Joint Res 2023;12(2):133–137.

Abstract Background This study aimed to identify the density of mononuclear cells (MNCs) and CD34+ cells in the bone marrow of patients with three neurologic conditions. Methods The study included 88 patients with three neurologic conditions: 40 with cerebral palsy (CP) due to oxygen deprivation (OD), 23 with CP related to neonatal icterus (NI), and 25 with neurological sequelae after traumatic brain injury. Bone marrow aspiration was conducted from the patients’ bilateral anterior iliac crest under general anesthesia in an operating theater. MNCs were isolated by Ficoll gradient centrifugation and then infused intrathecally. Results There was a significant difference in the average MNC per ml and percentage of CD34+ cells by the type of disease, age group, and infusion time (p value

Cerebral palsy, the most common disability in childhood, is a devastating non-progressive ailment of the infants' brain with lifelong sequelae, e.g., spastic paresis, chronic pain, inability to walk, intellectual disability, behavioral disorders, for which there is no cure at present. CP may develop after pediatric brain damage caused, e.g., by hypoxic-ischemia, periventricular leukomalacia, intracranial hemorrhage, hypoxic-ischemic encephalopathy, trauma, stroke, and infection. About 17 million people worldwide live with cerebral palsy as a result of pediatric brain damage. This reflects both the magnitude of the personal, medical, and socioeconomic global burden of this brain disorder and the overt unmet therapeutic needs of the pediatric population. This review will focus on recent preclinical, clinical, and regulatory developments in cell therapy for infantile cerebral palsy by transplantation of cord blood derived mononuclear cells from bench to bedside. The body of evidence suggests that cord blood cell therapy of cerebral palsy in the autologous setting is feasible, effective, and safe, however, adequately powered phase 3 trials are overdue. [ABSTRACT FROM AUTHOR]

Endothelial Ca2+ signaling has important roles to play in maintaining pregnancy associated vasodilation in the utero-placenta. Inflammatory cytokines, often elevated in vascular complications of pregnancy, negatively regulate ATP-stimulated endothelial Ca2+ signaling and associated nitric oxide production. However, the role of direct engagement of immune cells on endothelial Ca2+ signaling and therefore endothelial function is unclear. To model immune-endothelial interactions, herein, we evaluate the effects of peripheral blood mononuclear cells (PBMCs) in short-term interaction with human umbilical vein endothelial cells (HUVECs) on agonist-stimulated Ca2+ signaling in HUVECs. We find that mononuclear cells (10:1 and 25:1 mononuclear: HUVEC) cause decreased ATP-stimulated Ca2+ signaling; worsened by activated mononuclear cells possibly due to increased cytokine secretion. Additionally, monocytes, natural killers, and T-cells cause decrease in ATP-stimulated Ca2+ signaling using THP-1 (monocyte), NKL (natural killer cells), and Jurkat (T-cell) cell lines, respectively. PBMCs with Golgi-restricted protein transport prior to interaction with endothelial cells display rescue in Ca2+ signaling, strongly suggesting that secreted proteins from PBMCs mediate changes in HUVEC Ca2+ signaling. We propose that endothelial cells from normal pregnancy interacting with PBMCs may model preeclamptic endothelial-immune interaction and resultant endothelial dysfunction. [ABSTRACT FROM AUTHOR]

Macrophages are important effectors of inflammation resolution that contribute to the elimination of pathogens and apoptotic cells and restoration of homeostasis. Pre-clinical studies have evidenced the anti-inflammatory and pro-resolving actions of GILZ (glucocorticoid-induced leucine zipper). Here, we evaluated the role of GILZ on the migration of mononuclear cells under nonphlogistic conditions and Escherichia coli-evoked peritonitis. TAT-GILZ (a cell-permeable GILZ-fusion protein) injection into the pleural cavity of mice induced monocyte/macrophage influx alongside increased CCL2, IL-10 and TGF-β levels. TAT-GILZ-recruited macrophages showed a regulatory phenotype, exhibiting increased expression of CD206 and YM1. During the resolving phase of E. coli-induced peritonitis, marked by an increased recruitment of mononuclear cells, lower numbers of these cells and CCL2 levels were found in the peritoneal cavity of GILZ-deficient mice (GILZ−/−) when compared to WT. In addition, GILZ−/− showed higher bacterial loads, lower apoptosis/efferocytosis counts and a lower number of macrophages with pro-resolving phenotypes. TAT-GILZ accelerated resolution of E. coli-evoked neutrophilic inflammation, which was associated with increased peritoneal numbers of monocytes/macrophages, enhanced apoptosis/efferocytosis counts and bacterial clearance through phagocytosis. Taken together, we provided evidence that GILZ modulates macrophage migration with a regulatory phenotype, inducing bacterial clearance and accelerating the resolution of peritonitis induced by E. coli. [ABSTRACT FROM AUTHOR]

The leading pathological mechanisms of Alzheimer's disease (AD) are amyloidosis and chronic inflammation. The study of new therapeutic drugs of the corresponding action, in particular miRNAs and curcominoids, as well as methods for their packaging, is topical. The aim of the work was to study the effect of miR- 101 + curcumin in a single liposome in a cellular AD model. AD model was made by incubating a suspension of mononuclear cells with aggregates of beta-amyloid peptide 1-40 (Ab40) for 1 h. The effect of the subsequent application of liposomal (L) preparations miR-101, curcumin (CUR), and miR-101 + CUR was analyzed over time of 1, 3, 6, and 12 h. A decrease in the level of endogenous Ab42 under the influence of L(miR-101 + CUR) was revealed during the entire incubation period (1-12 h), the first part of which was overlapped due to inhibition of mRNAAPP translation by miR-101 (1-3 h), and the second-by inhibition of mRNAAPP transcription by curcumin (3-12 h), the minimum concentration of Ab42 was recorded at 6 h. The cumulative effect of the combination drug L(miR-101 + CUR) was manifested in the suppression of the increase in the concentration of TNFa and IL-10 and a decrease in the concentration of IL-6 during the entire incubation period (1-12 h). Thus, miR-101 + CUR in one liposome enhanced each other's antiamyloidogenic and anti-inflammatory effects in a cellular AD model. [ABSTRACT FROM AUTHOR]

We suggest a novel approach for searching natural compounds with anti-aging and rejuvenation potential using cell cultures, with a high potential for the further in vivo applications. The present paper discusses ways of defining age for cell populations with large numbers of cells and suggests a method of assessing how young or old a cell population is based on a cell age profile approach. This approach uses experimental distributions of the cells over the cell cycle stages, acquired using flow cytometry. This paper discusses how such a profile should evolve under homeostatic maintenance of cell numbers in the proliferation niches. We describe promising results from experiments on a commercial substance claiming rejuvenating and anti-aging activity acting upon the cultures of human mononuclear cells and dermal fibroblasts. The chosen substance promotes a shift towards larger proportion of cells in synthesis and proliferation stages, and increases cell culture longevity. Further, we describe promising in vivo testing results of a selected food supplement. Based on the described concept of cell age profile and available test results, a strategy to search for natural compounds with regenerative, anti-aging and rejuvenation potential is suggested and proposed for wider and thorough testing. Proposed methodology of age assessment is rather generic and can be used for quantitative assessment of the anti-aging and rejuvenation potential of different interventions. Further research aimed at the tests of the suggested strategy using more substances and different interventions, and the thorough studies of molecular mechanisms related to the action of the substance used for testing the suggested search methodology, are needed. [ABSTRACT FROM AUTHOR]

Objective: To investigate the effect of MC-LR on the expressions of miR-146a and pro/anti-inflammatory cytokines in human peripheral blood mononuclear cells (PBMCs) and to further explore the role of miR-146a in the inflammatory responses caused by MC-LR. Methods: Serum samples from 1789 medical examiners were collected and detect the concentrations of MCs, and 30 serum samples with concentrations of MCs around P25, P50, and p75 were randomly selected for the detection of inflammatory factors. PBMCs from fresh peripheral blood extracted from these 90 medical examiners were subsequently tested for relative miR-146a expression. In vitro, the MC-LR were exposed to the PBMCs to detect the levels of inflammatory factors as well as the relative expression of miR-146a-5p. Then, a miRNA transfection assay was performed to verify the regulation of inflammatory factors by miR-146a-5p. Results: In population samples, the expression of inflammatory factors and miR-146a-5p increased with increasing MCs concentration. In vitro experiments showed that the expression of inflammatory factors and miR-146a-5p in PBMCs increased with MC-LR exposure time or exposure dose too. In addition, inhibiting the expression of miR-146a-5p in PBMCs reduced inflammatory factor levels. Conclusion: miR-146a-5p exerts a promoting effect on the MC-LR-induced inflammatory response by positively regulating inflammatory factor levels. [ABSTRACT FROM AUTHOR]

The SARS-CoV-2 P.1 variant, responsible for an outbreak in Manaus, Brazil, is distinguished by 12 amino acid differences in the S protein, potentially increasing its ACE-2 affinity and immune evasion capability. We investigated the innate immune response of this variant compared to the original B.1 strain, particularly concerning cytokine production. Blood samples from three severe COVID-19 patients were analyzed post-infection with both strains. Results showed no significant difference in cytokine production of mononuclear cells and neutrophils for either variant. While B.1 had higher cytopathogenicity, neither showed viral replication in mononuclear cells. Structural analyses of the S protein highlighted physicochemical variations, which might be linked to the differences in infectivity between the strains. Our studies point to the increased infectivity of P.1 could stem from altered immunogenicity and receptor-binding affinity.

It is unclear whether direct-acting antiviral drugs (DAAs) result in the complete eradication of HCV infection or whether some quantities of the virus may persist after achieving a sustained virologic response (SVR). Aim The aim of this work was to study the possibility of the persistence of HCV RNA in peripheral blood mononuclear cells (PBMCs) after achieving SVR following DAA treatment. This study included 100 patients infected with HCV genotype 4, who were candidates for receiving DAAs and who achieved SVR during follow-up, as determined at 12 and/or 24 weeks following the end of treatment. All patients were subjected to demographic, biochemical and hematological assessments. Detection of HCV RNA in the serum and PBMCs and determination of the HCV genotype were performed with real-time PCR. We detected HCV RNA in the PBMCs of 20 out of 100 (20%) patients infected with HCV genotype 4, who achieved SVR. However, the persistent viral load in the PBMCs was very low (range: 400–900 U/mL; mean ± SD: 645.45 ± 153 U/mL). Multiple logistic regression analysis showed that only the higher posttreatment levels of aspartate transaminase (AST) were significantly predictive of HCV RNA persistence in the PBMCs (OR: 1.29; 95% CI: 1.08–1.55). Additionally, according to the Cox proportional hazard model, liver cirrhosis was the only significant risk factor for the persistence of HCV infection in PBMCs (HR: 5.8; 95% CI: 1.3–26.1; P < 0.02). Our results indicated the persistence of HCV RNA in some HCV patients who achieved SVR after treatment with DAAs. [ABSTRACT FROM AUTHOR]

Adoptive immunotherapy using NK cells has become a promising therapeutic area. NK cells are a component of the innate immune system, act as key regulators, and have potent antitumor cytolytic activity. In order to be able to evaluate the therapeutic effect of adoptive NK cell immunotherapy at preclinical stages, there is a need for reliable protocols for in vitro production of NK cells. There are a large number of publications on methods for activating and generating human NK cells, including using feeder-cells and various cytokines. The article describes the experience of cultivation of NK cells from cancer patients or donors with feedercells and without feeder-cells (control group). The K562 cell line was used as a feeder after irradiation of two types: after gene modification of K562 (gmK562) with membrane-bound mbIL15, mbIL21 and without it. NK cells donors and cancer patients were mixed with K562 in a ratio of 1:1, 1:2 and 1:5 on 0, 7 and 14 days respectively. Daily morphological assessment showed that, NK cells donors and cancer patients began to proliferate and increase in size, while the viability of feeder cells began to decrease after 3 days of cultivation, and they were less than 20% on 21 days. NK cells of donors and cancer patients went into apoptosis, their viability level decreased to 70% in the control group (without feeder-cells) after 3 days of cultivation. A comparative evaluation of two different methods of obtaining human NK cells was carried out. It was shown when NK cells were isolated by magnetic selection, the proportion of CD3-CD56+CD16+ cells were more than 90%, and after the removal of adherent cells, it was at least 60%. When cultivating NK cells cancer patients (after magnetic separation) together with gmK562 on the 21st day, it was possible to increase the number of NK cells up to 85 times. When cultivating NK cells donors (after adhesion) together with non-genetically modified K562 cells on 21 days, it was possible to increase the number of NK cells up to 8 times. It was shown that in the supernatants collected during the cultivation of NK cells with feeder cells (both irradiated with K562 and genetically modified with K562), the concentrations of TNFα and IFNγ increased many times relative to the control group. The optimal conditions for culturing NK cells were experimentally selected to obtain a large number of NK cells.

Introduction. The incidence and mortality of malignant melanoma have increased steadily over the last decades; therefore, the development of novel diagnostic markers for malignant melanoma is of great importance. The purpose of the study was to assess whether the development of melanoma before any treatment is accompanied by the body changes and, in particular, DNA damage in the mononuclear cells of the peripheral blood of patients. Material and Methods. In 93 patients (26 men and 67 women) admitted to the N.N. Petrov National Medical Research Center of Oncology for surgical treatment of stage T1c-2a-b-3a-b4a-bN0-1 cutaneous malignant melanoma, and in 118 healthy people as a comparison group, the level of damage to DNA in peripheral blood mononuclear cells was studied using the “comet” method. All patients were divided into two groups: group 1 included 45 patients (13 men and 32 women) who were examined before a decision on treatment was made and group 2 consisted of 48 patients (13 men and 35 women) who previously underwent excision biopsy for melanoma. Results. The level of DNA damage in peripheral blood mononuclear cells, assessed by the comet assay, was found to be signifcantly higher in patients with melanoma than in the comparison group. Moreover, the increase in the level of DNA damage was similar both in patients with a primary tumor before starting any treatment and in those who previously underwent excision biopsy for melanoma. The relationship between the level of DNA damage in peripheral blood mononuclear cells and the morphological characteristics of the tumor cells was revealed. The Spearman correlation analysis showed that all parameters that determined DNA damage positively correlated with the thickness of melanoma according to the Breslow’s depth, and the percentage of DNA in the comet and the comet tail moment correlated with the stage of the disease. Conclusion. The development of cutaneous melanoma is accompanied by an increase in the level of DNA damage in peripheral blood mononuclear cells. The level of DNA damage in peripheral blood mononuclear cells refects the changes that occur in the patient’s body under the infuence of the tumor process, which may allow using this indicator as an additional criterion for the diagnosis and aggressiveness of melanoma.

Introduction: The isolation of captured peripheral blood mononuclear cells (PBMNCs) from leukoreduction filters (LRFs) can be of great importance in terms of bringing the lost cells back into use. Objective: The aim of this study was to evaluate various methods based on their potential to recover the peripheral blood cells from LRFs with a focus on mononuclear cells (MNCs). Method: For cell isolation from LRFs, three distinct methods (back-flushing, direct and vacuum pump) were compared through the calculation of the yield of isolated MNCs. The viability of extracted cells was determined by the flow cytometry technique. Moreover, the recovered MNCs were characterized regarding the presence of blood stem cell purification. The cell culture, microscopic observation, and immunophenotyping were employed to characterize the blood stem cells (hematopoietic, mesenchymal and progenitor endothelial stem cells). Results: The yield of isolation obtained in the back-flushing, direct and vacuum pump methods were 17.7 ± 1.28, 17.3 ± 0.96 and 21.2 ± 0.90 percent, respectively. Although the highest potential for total blood cell recovery belonged to the vacuum pump method, the lowest cell viability (85.73 ± 4.84%) was observed in this method. However, the isolation process of the back-flushing and direct methods had less effect on cell viability. The characterization of the isolated MNCs displayed that the dominant positive phenotype was for CD34/CD45, indicating hematopoietic stem cells. In addition, the endothelial stem/progenitor cells were significantly detected as CD31/CD133 positive cells. Conclusion: According to our results and considering the safety and efficiency potential of each of the applied methods, the back-flushing in comparison with the other methods can be considered a suitable procedure for MNC isolation from LRFs.

Abstract Introduction The current multi-center, randomized, double-blind study was conducted among children with cerebral palsy (CP) to assess the safety and efficacy of umbilical cord blood mononuclear cell (UCB-MNC). We performed the diffusion tensor imaging to assess the changes in the white matter structure. Methods Males and females aged 4 to 14 years old with spastic CP were included. Eligible participants were allocated in 4:1 ratio to be in the experimental or control groups; respectively. Individuals who were assigned in UCB-MNC group were tested for human leukocyte antigen (HLA) and fully-matched individuals were treated with UCB-MNCs. A single dose (5 × 106 /kg) UCB-MNCs were administered via intrathecal route in experimental group. The changes in gross motor function measure (GMFM)-66 from baseline to one year after treatment were the primary endpoints. The mean changes in modified Ashworth scale (MAS), pediatric evaluation of disability inventory (PEDI), and CP quality of life (CP-QoL) were also evaluated and compared between groups. The mean changes in fractional anisotropy (FA) and mean diffusivity (MD) of corticospinal tract (CST) and posterior thalamic radiation (PTR) were the secondary endpoints. Adverse events were safety endpoint. Results There were 72 included individuals (36 cases in each group). The mean GMFM-66 scores increased in experimental group; compared to baseline (+ 9.62; 95%CI: 6.75, 12.49) and control arm (β: 7.10; 95%CI: 2.08, 12.76; Cohen’s d: 0.62) and mean MAS reduced in individuals treated with UCB-MNCs compared to the baseline (-0.87; 95%CI: -1.2, -0.54) and control group (β: -0.58; 95%CI: -1.18, -0.11; Cohen’s d: 0.36). The mean PEDI scores and mean CP-QoL scores in two domains were higher in the experimental group compared to the control. The imaging data indicated that mean FA increased and MD decreased in participants of UCB-MNC group indicating improvements in white matter structure. Lower back pain, headaches, and irritability were the most common adverse events within 24 h of treatment that were related to lumbar puncture. No side effects were observed during follow-up. Conclusions This trial showed that intrathecal injection of UCB-MNCs were safe and effective in children with CP. Trial Registration The study was registered with ClinicalTrials.gov ( NCT03795974 ).

Abstract Background Measurement of serum CA125, an antigenic fragment of human mucin 16 (MUC16), is used to monitor the clinical progression of epithelial ovarian cancer (EOC). However, rather than simply a passive marker reflecting tumor burden, MUC16 may have a more active role by binding to immune cells and altering their tumor response. We developed a research tool to measure MUC16-binding to the surfaces of peripheral blood mononuclear cell (PBMC) subtypes and tested its research value using specimens collected serially from a woman being treated for high grade serous EOC. Methods Cryopreserved PBMCs were mixed with anti-CA125 antibody-labeled plasmonic gold nanoparticles (PNPs) to detect cell surface MUC16-binding along with fluorescent stains to identify B cells, NK cells, NK-T cells, T cells, and monocytes. From 3D darkfield images, a computer algorithm was applied to enumerate PNP-binding and fluorescence microscopy to identify cell lineage. Average MUC16-binding was determined by fitting a Poisson distribution to PNP-counts across similar cell types. MUC16-binding to cell types was correlated with treatment details, CA125 levels, and complete blood count (CBC) data. Results Over a 21-month period, monocytes had the highest level of MUC16-binding which was positively correlated with serum CA125 and inversely correlated with circulating monocyte and lymphocyte counts. Fluctuations of PNP-binding to NK cells were associated temporally with types of chemotherapy and surgical events. Levels of MUC16 bound to NK cells were positively correlated with levels of MUC16 bound to T and NK-T cells and inversely correlated with circulating platelets. Conclusions Assessment of MUC16-binding among cryopreserved PBMC cell types can be accomplished using darkfield and fluorescence microscopy. Correlations observed between level of binding by cell type with serum CA125, CBC data, and treatment details suggest that the new techniques may offer novel insights into EOC’s clinical course.

The leading pathological mechanisms of Alzheimer’s disease (AD) are amyloidosis and chronic inflammation. The study of new therapeutic drugs of the corresponding action, in particular miRNAs and curcominoids, as well as methods for their packaging, is topical. The aim of the work was to study the effect of miR-101 + curcumin in a single liposome in a cellular AD model. AD model was made by incubating a suspension of mononuclear cells with aggregates of beta-amyloid peptide 1–40 (Aβ40) for 1 h. The effect of the subsequent application of liposomal (L) preparations miR-101, curcumin (CUR), and miR-101 + CUR was analyzed over time of 1, 3, 6, and 12 h. A decrease in the level of endogenous Aβ42 under the influence of L(miR-101 + CUR) was revealed during the entire incubation period (1–12 h), the first part of which was overlapped due to inhibition of mRNAAPP translation by miR-101 (1–3 h), and the second-by inhibition of mRNAAPP transcription by curcumin (3–12 h), the minimum concentration of Aβ42 was recorded at 6 h. The cumulative effect of the combination drug L(miR-101 + CUR) was manifested in the suppression of the increase in the concentration of TNFα and IL-10 and a decrease in the concentration of IL-6 during the entire incubation period (1–12 h). Thus, miR-101 + CUR in one liposome enhanced each other’s antiamyloidogenic and anti- inflammatory effects in a cellular AD model.

Background: This study aimed to identify the density of mononuclear cells (MNCs) and CD34+ cells in the bone marrow of patients with three neurologic conditions. Methods: The study included 88 patients with three neurologic conditions: 40 with cerebral palsy (CP) due to oxygen deprivation (OD), 23 with CP related to neonatal icterus (NI), and 25 with neurological sequelae after traumatic brain injury. Bone marrow aspiration was conducted from the patients' bilateral anterior iliac crest under general anesthesia in an operating theater. MNCs were isolated by Ficoll gradient centrifugation and then infused intrathecally. Results: There was a significant difference in the average MNC per ml and percentage of CD34+ cells by the type of disease, age group, and infusion time (p value < 0.05). The multivariable regression model showed the percentage of CD34+ association with the outcome (gross motor function 88 items- GMFM-88) in patients with CP. Conclusions: The density of MNCs was 5.22 million cells per mL and 5.03% CD34+ cells in patients with three neurologic conditions. The highest density of MNCs in each ml of bone marrow was found in patients with CP due to OD, whereas the percentage of CD34+ cells was the highest among patients with CP related to NI. [ABSTRACT FROM AUTHOR]

1,25(OH)2D3 has anti‐inflammatory and growth inhibitory effects. Our study explored the effect of 1,25(OH)2D3 treatment on the expression of monocyte chemotactic protein‐1 (MCP‐1), hepatocyte growth factor (HGF), and insulin‐like growth factor‐1 (IGF‐1) by peripheral blood mononuclear cells (PBMCs), peritoneal fluid mononuclear cells (PFMCs), endometrial stromal cells (ESCs), and its effect on the proliferation of PBMCs and PFMCs of patients with endometriosis compared with controls. PBMCs, PFMCs, and ESCs were obtained from 10 endometriosis patients and 10 non‐endometriotic individuals. After treating cells with 0.1 μM of 1,25(OH)2D3 for 6, 24, and 48 h, the gene and protein expression of mentioned factors were evaluated by real‐time PCR and ELISA methods, respectively. 1,25(OH)2D3 treatment significantly reduced the protein expression of MCP‐1, HGF, and IGF‐1 in PBMCs and PFMCs of endometriotic patients at 48 h (p < 0.05–<0.01). Also, this treatment significantly reduced MCP‐1, HGF, and IGF‐1 gene and/or protein expression in EESCs and EuESCs at 24 and 48 h (p < 0.05–<0.01). 1,25(OH)2D3 treatment also reduced the proliferation of PBMCs and PFMCs of endometriotic patients compared with controls (p < 0.01). 1,25(OH)2D3 can be considered as a potentially effective agent in the prevention and treatment of endometriosis along with other therapies. [ABSTRACT FROM AUTHOR]

We investigated the features of the morphology and cytokine profiles of neuroblastoma SH-SY5Y cells, bone marrow-derived mesenchymal stromal/stem cells (BM-MSCs), and peripheral blood mononuclear cells (PBMCs) in double (BM-MSCs + SH-SY5Y cells) and triple (BM-MSCs + SH-SY5Y cells + PBMCs) co-cultures incubated on plastic and Matrigel. Cells in the co-cultures communicated by vesicular transport and by exchanging membrane and cytoplasmic components. The cytokine profile of double and triple co-cultures incubated on Matrigel and plastic had differences and showed the highest concentration of a number of chemokines/cytokines, such as CXCL8/IL-8, I-TAC/CXCL11, IP10/CXCL10, MDC/CCL22, MIP-1α/CCL3, IL-1β, ENA-78/CXCL5, Gro-α/CXCL1, MCP-1/CCL2, TERC/CCL25, CXCL8/IL-8, and IL-6. High concentrations of inflammatory chemokines/cytokines in the conditioned medium of triple co-culture form a chronic inflammation, which brings the presented co-cultivation system closer to a natural tumor. Triple co-cultures were more resistant to cisplatin (CDDP) than the double- and monoculture of SH-SY5Y. The mRNA levels of BCL2, BCL2L1, RAC1, CAV1, CASP3, and BAX genes were changed in cells after co-culturing and CDDP treatment in double and triple co-cultures. The expression of the BCL2, BAX, CAV1, and CASP3 proteins in SH-SY5Y cells after the triple co-culture and CAV1 and BAX protein expression in SH-SY5Y cells after the double co-culture were determined. This study demonstrated the nature of the cellular interactions between components of tumor niche and the intercellular influence on chemoresistance observed in our tumor model, which should enable the development of novel test systems for anti-tumor agents. [ABSTRACT FROM AUTHOR]

Background: Extracorporeal photopheresis (ECP) product characteristics are not well established. The aim of this study was to compare mononuclear cells (MNCs) collection using the new Amicus blue (AB) In-line ECP system to our standard Off-line ECP system using the Optia apheresis device and the MacoGenic G2 inactivation system (OM)., Study Design and Methods: We assessed the ECP products and procedure parameters, patient characteristics, and adverse events for both AB and OM systems in paired patients. Comparisons were made with t-test for paired samples., Results: Thirteen patients underwent 15 double, paired procedures using both ECP protocols and processing the same blood volume of 4000 mL. Total MNC collected in the product were 51.6 × 10 8 (95% CI 30.0-73.1) and 42.2 × 10 8 (95% CI 22.4-62.0) for the AB and OM, respectively (not significant). Both products were also similar regarding volume, MNC concentration, purity, and hematocrit. However, total platelet count (×10 11 ) was significantly lower in the AB products: 0.25 (95% CI 0.15-0.36) versus 1.2 (95% CI 0.9-1.5). The new AB system reduced significantly also the time invested and anticoagulant used per procedure compared with OM, albeit with similar collection efficiency and percentage of MNC captured per procedure. Hypocalcemia was the commonest adverse event with both systems, but it was not severe., Conclusions: The new AB system collected MNC products comparable to our current experience with OM, although in a significantly shorter time, with a reduced use of anticoagulant and lower contamination with platelets, which are all valuable advantages of the new system., (© 2025 AABB.)

Las periodontitis constituyen la primera causa de pérdida dentaria en la edad adulta y su tratamiento depende, casi por completo, del empleo de la implantación de sustitutos estructurales para lograr el potencial reparador necesario. El uso en los últimos años de la medicina celular regenerativa con células mononucleares, plaquetas y lisado plaquetario, han acelerado el proceso de cicatrización de los tejidos blandos y la regeneración ósea. Los resultados que se presentan derivan de una estrategia de superación profesional dirigida al periodoncista para el mejoramiento del desempeño en la aplicación de esta nueva terapéutica, como proyecto de investigación, en el Hospital Juan Bruno Zayas Alfonso de Santiago de Cuba, desde septiembre a diciembre del 2020. Su objetivo consiste en analizar la terapia periodontal regenerativa con hemocomponentes en Santiago de Cuba desde lo social y lo formativo./Periodontitis is the first cause of tooth loss in adulthood and its treatment depends, almost entirely, on the use of implantation of structural substitutes to achieve the necessary restorative potential. The use in recent years of regenerative cell medicine with mononuclear cells, platelets and platelet lysate, has accelerated the process of soft tissue healing and bone regeneration. The results presented are derived from a professional improvement strategy aimed at the periodontist to improve performance in the application of this new therapy, as a research project, at the Juan Bruno Zayas Alfonso Hospital in Santiago de Cuba, from September to December 2020. Its objective is to analyze regenerative periodontal therapy with blood components in Santiago de Cuba from a social and educational perspective.

The concomitant investigation of apoptosis (a regulated cell death) and autophagy (a conserved cell survival mechanism) in immune cells is rare. More detailed knowledge of these two types of self-consumption in circulating lymphocytes and monocytes would be important, since conditions such as fasting and acute exercise could promote health by a coordinated/linked modulation of autophagy and apoptosis in these mononuclear cells. In this study we performed flow cytometry to quantify numbers of apoptotic and autophagic mononuclear cells, lymphocytes and monocytes in fasting, standardized fed, and exercise conditions, using Annexin V, LC3B, and p62, respectively. We show that within total mononuclear cells lymphocytes are less apoptotic and autophagic than monocytes during fasting (p < 0.001, p < 0.05, respectively) and after acute exercise (p < 0.01, p < 0.05, respectively). Fasting increased circulating autophagic monocyte concentrations, but not lymphocytes compared to the fed control condition. Acute exercise elevated circulating autophagic lymphocyte concentrations, but not monocytes. Interestingly, Western blotting analysis of the fasting samples showed that higher LC3BII/I ratios were correlated with lower numbers of autophagic mononuclear cells (r = − 0.74, p = 0.02, n = 8), which could be attributed to the monocyte subgroup, but not lymphocytes. These results extend the current knowledge of the two types of self-consumption in circulating immune cells and underline their possible importance in pro-inflammatory monocytes during fasting and exercise as health promoting interventions. [ABSTRACT FROM AUTHOR]

Abstract Background Quality and Quantity culture media (QQ culture media) was reported to enhance vasculogenesis and angiogenesis function of mononuclear cells (MNCs) from healthy volunteers. In this study, MNCs from chronic limb-threatening ischemia (CLTI) patients were cultured in QQ culture media, and then investigated for angiogenesis-related phenotype and function. Methods Patients aged ≥ 18 years with CLTI caused by atherosclerosis of the lower extremities were prospectively recruited at Siriraj Hospital (Bangkok, Thailand) during July 2017–December 2018. Peripheral blood mononuclear cells (PBMNCs) were isolated from peripheral blood. PBMNCs were cultured in either QQ culture media or standard culture media. The number of CD34+CD133+ cells, CD206+ cells, CD4+CD25+CD127+ cells, colony formation assay, and human umbilical vein endothelial cell (HUVEC) tube formation assay in MNCs were compared between those cultured in QQ culture media and those cultured in standard culture media. Results Thirty-nine patients were included with a mean age of 69 ± 11 years. Diabetes mellitus was found in 25 (64%) patients. The percentage of CD34+CD133+ progenitor cells in MNCs cultured in QQ culture media and in MNCs cultured in standard culture media was 4.91 ± 5.30% and 0.40 ± 0.46%, respectively (p

Giant cell tumor (GCT) of bone is a distinctive neoplasm characterized by abundance of multinucleated giant cells scattered throughout the stroma of mononuclear cells. Its importance lies in recognizing and differentiating the characteristic histology, which at times may mimic several other bone tumors and endocrine disorders ranging from locally aggressive giant cell granulomas to hyperparathyroidism to malignant tumors. The jaw bones account for less than 1% of this lesion. We present a rare case of GCT of the mandible which occurred in a 19-year-old male. [ABSTRACT FROM AUTHOR]

Macrophages are important effectors of inflammation resolution that contribute to the elimination of pathogens and apoptotic cells and restoration of homeostasis. Pre-clinical studies have evidenced the anti-inflammatory and pro-resolving actions of GILZ (glucocorticoid-induced leucine zipper). Here, we evaluated the role of GILZ on the migration of mononuclear cells under nonphlogistic conditions and Escherichia coli-evoked peritonitis. TAT-GILZ (a cell-permeable GILZ-fusion protein) injection into the pleural cavity of mice induced monocyte/macrophage influx alongside increased CCL2, IL-10 and TGF-β levels. TAT-GILZ-recruited macrophages showed a regulatory phenotype, exhibiting increased expression of CD206 and YM1. During the resolving phase of E. coli-induced peritonitis, marked by an increased recruitment of mononuclear cells, lower numbers of these cells and CCL2 levels were found in the peritoneal cavity of GILZ-deficient mice (GILZ−/−) when compared to WT. In addition, GILZ−/− showed higher bacterial loads, lower apoptosis/efferocytosis counts and a lower number of macrophages with pro-resolving phenotypes. TAT-GILZ accelerated resolution of E. coli-evoked neutrophilic inflammation, which was associated with increased peritoneal numbers of monocytes/macrophages, enhanced apoptosis/efferocytosis counts and bacterial clearance through phagocytosis. Taken together, we provided evidence that GILZ modulates macrophage migration with a regulatory phenotype, inducing bacterial clearance and accelerating the resolution of peritonitis induced by E. coli.

Immunotherapy is an attractive therapeutic strategy for the treatment of osteosarcoma (OS). The unique features of γδ T cells have made them popular for cancer immunotherapy. Here, we expanded γδ T cells using human peripheral blood mononuclear cells (PBMCs) and investigated their therapeutic potential against OS cells. PBMCs from healthy donors were cultured for 10 days with CON medium (unstimulated control); EX media, CON with recombinant human interleukin-2 (rhIL-2) and zoledronate; and EX28 media, CON with rhIL-2, zoledronate, and CD3/CD28 activator. The expanded γδ T cells were isolated by magnetic cell separation or fluorescence-activated cell sorting, cultured with two OS cell lines (KHOS/NP and MG-63) at various cell ratios with or without doxorubicin or ifosfamide, and analyzed for cytotoxicity and cytokine secretion. The number of CD3+γδTCR+Vγ9+ triple-positive γδ T cells and concentrations of IFN-γ and TNF-α were highest in the rhIL-2 (100 IU) and zoledronate (1 μM) supplemented culture conditions. The CD3/CD28 agonist did not show any additional effects on γδ T cell expansion. The expanded γδ T cells exhibited potent in vitro cytotoxicity against OS in a ratio- and time-dependent manner. The γδ T cells may enhance the effect of chemotherapeutic agents against OS and may be a new treatment strategy, including chemo-immunotherapy, for OS. [ABSTRACT FROM AUTHOR]

Background: Ligation of intersphincteric fistula tract (LIFT) is a sphincter-saving procedure used for treatment of complex anal fistula. The current study aimed to assess the outcome of local injection of bone marrow mononuclear cells (BM-MNCs) in conjunction with LIFT as compared to LIFT alone in regards to healing rate, time to healing, and ultimate success rate.Methods: This was a prospective randomized trial on patients with trans-sphincteric anal fistula. Patients were randomly allocated to one of two equal groups: LIFT and LIFT with BM-MNC injection. The main outcome measures were healing at 10 weeks of follow-up, recurrence after healing, and complications.Results: Seventy patients (48 male and 22 female) of a mean age of 37.9 ± 10.4 years were included. The mean time to complete healing after LIFT + BM-MNCs was significantly shorter than after LIFT alone (20.5 ± 5.2 vs 28.04 ± 5.8 days; P < 0.0001). The ultimate success rates of both groups were similar (LIFT = 60% vs LIFT with BM-MNCs = 68.6%, P = 0.62). There was no significant difference in the mean operation time or complication rate between the two groups. Secondary extension and previous anal surgery were significant independent predictors of failure of healing.Conclusion: LIFT combined with BM-MNC injection was associated with a shorter time to complete healing than LIFT alone. However, BM-MNC injection did not have a significant impact on the overall healing and ultimate success rate. [ABSTRACT FROM AUTHOR]

Pomegranate peel extract (PoPEx) has been shown to have antioxidant and anti-inflammatory properties, but its effect on the adaptive immune system has not been sufficiently investigated. In this study, the treatment of human peripheral blood mononuclear cells (PBMC) with PoPEx (range 6.25–400 µg/mL) resulted in cytotoxicity at concentrations of 100 µg/mL and higher, due to the induction of apoptosis and oxidative stress, whereas autophagy was reduced. At non-cytotoxic concentrations, the opposite effect on these processes was observed simultaneously with the inhibition of PHA-induced PBMC proliferation and a significant decrease in the expression of CD4. PoPEx differently modulated the expression of activation markers (CD69, CD25, ICOS) and PD1 (inhibitory marker), depending on the dose and T-cell subsets. PoPEx (starting from 12.5 µg/mL) suppressed the production of Th1 (IFN-γ), Th17 (IL-17A, IL-17F, and IL-22), Th9 (IL-9), and proinflammatory cytokines (TNF-α and IL-6) in culture supernatants. Lower concentrations upregulated Th2 (IL-5 and IL-13) and Treg (IL-10) responses as well as CD4+CD25hiFoxp3+ cell frequency. Higher concentrations of PoPEx increased the frequency of IL-10- and TGF-β-producing T-cells (much higher in the CD4+ subset). In conclusion, our study suggested for the first time complex immunoregulatory effects of PoPEx on T cells, which could assist in the suppression of chronic inflammatory and autoimmune diseases. [ABSTRACT FROM AUTHOR]

Epilepsy is a chronic non-infectious disease of the brain, characterized primarily by recurrent unprovoked seizures, defined as an episode of disturbance of motor, sensory, autonomic, or mental functions resulting from excessive neuronal discharge. Despite the advances in the treatment achieved with the use of antiepileptic drugs and other non-pharmacological therapies, about 30% of patients suffer from uncontrolled seizures. This review summarizes the currently available methods of gene and cell therapy for epilepsy and discusses the development of these approaches. Currently, gene therapy for epilepsy is predominantly adeno-associated virus (AAV)-mediated delivery of genes encoding neuro-modulatory peptides, neurotrophic factors, enzymes, and potassium channels. Cell therapy for epilepsy is represented by the transplantation of several types of cells such as mesenchymal stem cells (MSCs), bone marrow mononuclear cells, neural stem cells, and MSC-derived exosomes. Another approach is encapsulated cell biodelivery, which is the transplantation of genetically modified cells placed in capsules and secreting various therapeutic agents. The use of gene and cell therapy approaches can significantly improve the condition of patient with epilepsy. Therefore, preclinical, and clinical studies have been actively conducted in recent years to prove the benefits and safety of these strategies. [ABSTRACT FROM AUTHOR]