Cell Function Research Articles

Exploring the mechanism of interaction of glipizide with DNA: Combined in vitro and bioinformatics approach

DNA, vital for biological processes, encodes hereditary data for protein synthesis, shaping cell structure and function. Since revealing its structure, DNA has become a target for various therapeutically vital molecules, spanning antidiabetic to anticancer drugs. These agents engage with DNA-associated proteins, DNA–RNA hybrids, or bind directly to the DNA helix, triggering diverse downstream effects. These interactions disrupt vital enzymes and proteins essential for maintaining cell structure and function. Analysing drug–DNA interactions has significantly advanced our understanding of drug mechanisms. Glipizide, an antidiabetic drug, is known to cause DNA damage in adipocytes. However, its extract mechanism of DNA interaction is unknown. This study delves into the interaction between glipizide and DNA utilizing various biophysical tools and computational technique to gain insights into the interaction mechanism. Analysis of UV–visible and fluorescence data reveals the formation of complex between DNA and glipizide. The binding affinity of glipizide to DNA was of moderate strength. Examination of thermodynamic parameters at different temperatures suggests that the binding was entropically spontaneous and energetically favourable. Various experiments such as thermal melting assays, viscosity measurement, and dye displacement assays confirmed the minor grove nature of binding of glipizide with DNA. Molecular dynamics studies confirmed the glipizide forms stable complex with DNA when simulated by mimicking the physiological conditions. The binding was mainly favoured by hydrogen bonds and glipizide slightly reduced nucleotide fluctuations of DNA. The study deciphers the mechanism of interaction of glipizide with DNA at molecular levels.

Tumor-Derived Exosomes Promote the Angiogenic Function of Vascular Endothelial Cells by Activating the miR-423-5p/EFNA3 Signaling Pathway

Angiogenesis plays a key role in promoting the growth and metastasis of breast tumors. Tumor exosomes (EXs) contribute to angiogenesis in various tumor tissues by transferring their carried RNAs. MiR-423-5p was enriched in multiple tumors and implicated in tumor growth. In this study, we investigated the roles and underlying mechanisms of tumor-derived EXs and their carried miR-423-5p in regulating human umbilical vein endothelial cell (HUVEC) functions. EXs derived from MCF-7 cells (MCF-7 EXs) or with miR-423-5p knockdown (MCF-7 EXsSimiR-423-5p) were collected and incubated with ECs, and then the proliferation, migration, and tube formation abilities of ECs were detected. We found that miR-423-5p was enriched in breast cancer, MCF-7 cell lines and their derived EXs. After coculture with HUVECs, MCF-7 EXs merged into HUVECs and subsequently increased the miR-423-5p expression, proliferation, migration, and tube formation abilities of HUVECs, paralleling the increased EFNA3 and Notch1 expression, which was partially abolished by miR-423-5p knockdown. Altogether, our data suggest that MCF-7 EXs enriched with miR-423-5p promote the angiogenic function of vascular endothelial cells by activating the miR-423-5p/EFNA3/Akt signaling pathway.

Clinicopathological Characteristics, Prognosis, and Correlated Tumor Cell Function of Tropomodulin-3 in Pancreatic Adenocarcinoma.

Pancreatic adenocarcinoma (PAAD) is a frequent malignant tumor with a high mortality rate. Searching for novel biomarkers that can influence its prognosis may help patients. It has been shown that tropomodulin-3 (TMOD3) may influence tumor progression, but its role in pancreatic cancer is not clear. We aimed to explore the expression and prognostic value of TMOD3 in PAAD. We used bioinformatics analysis to analyze the relationship between TMOD3 expression and clinicopathological features and prognosis and verified it with clinical data from tissue microarray. We also conducted in vitro cell experiments to explore the effects of TMOD3 on the function of PAAD cells. TMOD3 expression was found to be significantly higher in PAAD tissues than in matched paracancerous tissues (P < 0.05). Meanwhile, high TMOD3 expression was associated with significantly poorer overall survival (P < 0.05). Analysis of relevant clinicopathological characteristics data obtained from TCGA showed that high TMOD3 expression correlated with age, TNM stage, N stage, and M stage (P < 0.05). Analysis of correlation data obtained from tissue microarrays showed that high TMOD3 expression was associated with lymph node invasion, nerve invasion, macrovascular invasion, and TNM stage (P < 0.05). In addition, siRNA knockdown of TMOD3 significantly reduced the migration and invasion of PAAD cells. Our study shows that TMOD3 may be associated with the progression of PAAD cells, and that it is an independent risk factor for poor pathological features and prognosis of PAAD. It may be helpful as a prognostic indicator of clinical outcomes in PAAD patients.

Hypoglycemic effect and the mechanism of action of a polysaccharide from sweet corncob in a high-fat diet and streptozotocin-induced diabetic mice

Type 2 diabetes mellitus (T2DM) is a metabolic disease caused by a glycolipid metabolism disorder and islet β-cell dysfunction. SCP-80-I is a biologically active water-soluble polysaccharide isolated from sweet corncob, an agricultural byproduct. The hypoglycemic effects of SCP-80-I on T2DM mice and its mechanisms were investigated in this study. SCP-80-I was found to significantly reduce blood glucose and lipid deposition levels in T2DM mice, as well as decrease serum leptin and increase adiponectin secretion. Interestingly, real time-polymerase chain reaction (RT-PCR) and Western blotting results revealed that SCP-80-I could regulate the expression of several glycolipid metabolisms and insulin secretion genes and proteins, including 5'-AMP-activated protein kinase (AMPK), carnitine palmitoyltransferase I (CPT1), and acetyl coenzyme A carboxylase (ACC) in the liver and AMPK, sirtuin 1 (Sirt1), peroxisome proliferator-activated receptorycoactivator-1 (PGC-1α), and uncoupling protein 2 (UCP2) in the pancreas. To have a hypoglycemic effect, SCP-80-I regulated glycolipid metabolism and islet cell function in the liver by regulating the AMPK/ACC/CPT1 signaling pathway and the AMPK/Sirt1/PGC-1α and AMPK/Sirt1/UCP2 signaling pathways. These findings improve our understanding of polysaccharides derived from sweet corncob and the use of SCP-80-I in the production of hypoglycemic foods.

Characteristics and biological properties of Imeglimin hydrochloride, A Novel antidiabetic agent: A systematic review.

WHO indicates that diabetes will become the 7th leading reason for death by 2030. The physiopathology of dysfunctioning is associated with obesity, weight gain and predominantly insulin resistance in insulin-sensitive cells and continuous deterioration of pancreatic beta cell function. Imeglimin is an investigational novel oral anti-diabetic drug Objectives: The motive of the review is to comprehensively explore the chemistry, biological and analytical analysis of the Imeglimin hydrochloride. To enhance the understanding a systematic review was conducted by forming a database of relevant existing studies from electronic resources like Web of Science, ScienceDirect and PubMed. The methodology is reflected in the PRISMA design. The drug was approved in the year 2021 for therapeutic purposes in Japan. It is the novel and first approved drug for this type of Anti-diabetic treatment. It is a small molecular drug whose molecular weight is 191.6 grams per mole utilized for oral administration. Imeglimin is thought to have both activities, as the amount of glucose is dependent on insulin secretory impact and insulin sensitivity is increased. Therapeutic, pharmacological, and analytical considerations for the novel drug Imeglimin hydrochloride are discussed in this review.

Dysregulated Lipid Metabolism Networks Modulate T-cell Function in People with Relapsing Remitting Multiple Sclerosis.

Altered cholesterol, oxysterol, sphingolipid, and fatty acid concentrations are reported in blood, cerebrospinal fluid, and brain tissue of people with relapsing remitting multiple sclerosis (RRMS) and are linked to disease progression and treatment responses. CD4+ T cells are pathogenic in RRMS, and defective T cell function could be mediated in part by liver X receptors (LXRs) - nuclear receptors that regulate lipid homeostasis and immunity. RNA-sequencing and pathway analysis identified that genes within the 'lipid metabolism' and 'signalling of nuclear receptors' pathways were dysregulated in CD4+ T cells isolated from RRMS patients compared with healthy donors. While LXRB and genes associated with cholesterol metabolism were upregulated, other T cell LXR-target genes, including genes involved in cellular lipid uptake (inducible degrader of the LDL receptor, IDOL), and the rate-limiting enzyme for glycosphingolipid biosynthesis (UDP-glucosylceramide synthase, UGCG) were downregulated in T cells from patients with RRMS compared to healthy donors. Correspondingly, plasma membrane glycosphingolipids were reduced, and cholesterol levels increased in RRMS CD4+ T cells, an effect partially recapitulated in healthy T cells by in vitro culture with T cell receptor stimulation in the presence of serum from RRMS patients. Notably, stimulation with LXR-agonist GW3965 normalised membrane cholesterol levels, and reduced proliferation and IL17 cytokine production in RRMS CD4+ T-cells. Thus, LXR-mediated lipid metabolism pathways were dysregulated in T cells from patients with RRMS and could contribute to RRMS pathogenesis. Therapies that modify lipid metabolism could help restore immune cell function.

Identification of positively selected genes in Mycobacterium tuberculosis from southern Xinjiang Uygur autonomous region of China

BackgroundTuberculosis (TB), mainly caused by Mycobacterium tuberculosis (Mtb), remains a serious public health problem. Increasing evidence supports that selective evolution is an important force affecting genomic determinants of Mtb phenotypes. It is necessary to further understand the Mtb selective evolution and identify the positively selected genes that probably drive the phenotype of Mtb.MethodsThis study mainly focused on the positive selection of 807 Mtb strains from Southern Xinjiang of China using whole genome sequencing (WGS). PAML software was used for identifying the genes and sites under positive selection in 807 Mtb strains.ResultsLineage 2 (62.70%) strains were the dominant strains in this area, followed by lineage 3 (19.45%) and lineage 4 (17.84%) strains. There were 239 codons in 47 genes under positive selection, and the genes were majorly associated with the functions of transcription, defense mechanisms, and cell wall/membrane/envelope biogenesis. There were 28 codons (43 mutations) in eight genes (gyrA, rpoB, rpoC, katG, pncA, embB, gid, and cut1) under positive selection in multi-drug resistance (MDR) strains but not in drug-susceptible (DS) strains, in which 27 mutations were drug-resistant loci, 9 mutations were non-drug-resistant loci but were in drug-resistant genes, 2 mutations were compensatory mutations, and 5 mutations were in unknown drug-resistant gene of cut1. There was a codon in Rv0336 under positive selection in L3 strains but not in L2 and L4 strains. The epitopes of T and B cells were both hyper-conserved, particularly in the T-cell epitopes.ConclusionThis study revealed the ongoing selective evolution of Mtb. We found some special genes and sites under positive selection which may contribute to the advantage of MDR and L3 strains. It is necessary to further study these mutations to understand their impact on phenotypes for providing more useful information to develop new TB interventions.

RUNX1 regulates promoter activity in the absence of cognate DNA binding motifs.

Runt-related transcription factor 1 (RUNX1) plays an important role in normal haematopoietic cell development and function, and its function is frequently disrupted in leukaemia. RUNX1 is widely recognised as a sequence-specific DNA binding factor that recognises the motif 5'-TG(T/C)GGT-3' in promoter and enhancer regions of its target genes. Moreover, RUNX1 fusion proteins, such as RUNX1-ETO formed by the t(8;21) translocation, retain the ability to recognise and bind to this sequence to elicit atypical gene regulatory effects on bona fide RUNX1 targets. However, our analysis of publicly available RUNX1 chromatin immunoprecipitation sequencing (ChIP-Seq) data has provided evidence challenging this dogma, revealing that this motif-specific model of RUNX1 recruitment and function is incomplete. Our analyses revealed that the majority of RUNX1 genomic localisation occurs outside of promoters, that 20% of RUNX1 binding sites lack consensus RUNX motifs, and that binding in the absence of a cognate binding site is more common in promoter regions compared to distal sites. Reporter assays demonstrate that RUNX1 can drive promoter activity in the absence of a recognised DNA binding motif, in contrast to RUNX1-ETO. RUNX1-ETO supresses activity when it is recruited to promoters containing a sequence specific motif, while interestingly, it binds but does not repress promoters devoid of a RUNX1 recognition site. These data suggest that RUNX1 regulation of target genes occurs through multiple mechanisms depending on genomic location, the type of regulatory element and mode of recruitment.

Iron Is Critical for Mucosal-Associated Invariant T Cell Metabolism and Effector Functions.

Mucosal-Associated Invariant T (MAIT) cells are a population of innate T cells that play a critical role in host protection against bacterial and viral pathogens. Upon activation, MAIT cells can rapidly respond via both TCR-dependent and -independent mechanisms, resulting in robust cytokine production. The metabolic and nutritional requirements for optimal MAIT cell effector responses are still emerging. Iron is an important micronutrient and is essential for cellular fitness, in particular cellular metabolism. Iron is also critical for many pathogenic microbes, including those that activate MAIT cells. However, iron has not been investigated with respect to MAIT cell metabolic or functional responses. In this study, we show that human MAIT cells require exogenous iron, transported via CD71 for optimal metabolic activity in MAIT cells, including their production of ATP. We demonstrate that restricting iron availability by either chelating environmental iron or blocking CD71 on MAIT cells results in impaired cytokine production and proliferation. These data collectively highlight the importance of a CD71-iron axis for human MAIT cell metabolism and functionality, an axis that may have implications in conditions where iron availability is limited.

Aging of alveolar type 2 cells induced by Lonp1 deficiency exacerbates pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive and chronic disease that significantly impacts patient quality of life, and its incidence is on the rise. The pathogenesis of IPF remains poorly understood. Alveolar type 2 (AT2) cells are crucial in the onset and progression of IPF, yet the specific mechanisms involved are not well defined. Lon protease 1 (LONP1), known for its critical roles in various diseases, has an unclear function in IPF. Our research investigated the impact of Lonp1 gene deletion on AT2 cell functionality and its subsequent effect on IPF development. We generated a bleomycin-induced pulmonary fibrosis mouse model with a targeted Lonp1 knockout in AT2 cells and assessed the consequences on AT2 cell function and fibrosis progression. Additionally, we constructed the MLE12 cells with stable Lonp1 knockdown and utilized transcriptome sequencing to identify pathways altered by the Lonp1 knockdown. Our results indicated that mice with AT2 cell-specific Lonp1 knockout exhibited more severe fibrosis compared to controls. These mice exhibited a reduction in AT2 and AT1 cell populations, along with an increase in p53- and p21-positive AT2 cells. Lonp1 knockdown in MLE12 cells led to the upregulation of aging-associated pathways, with fibroblast growth factor 2 (Fgf2) gene emerging as a central gene interconnecting these pathways. Therefore, loss of Lonp1 appears to promote AT2 cell aging and exacerbate bleomycin-induced pulmonary fibrosis. Fgf2 emerges as a pivotal downstream gene associated with cellular senescence. This study uncovers the role of the Lonp1 gene in pulmonary fibrosis, presenting a novel target for investigating the pathological mechanisms and potential therapeutic approaches for IPF.

THEMIS2 Impairs Antitumor Activity of NK Cells by Suppressing Activating NK Receptor Signaling.

NK cells are cytotoxic innate lymphocytes that play a critical role in antitumor immunity. NK cells recognize target cells by using a repertoire of activating NK receptors and exert the effector functions. Although the magnitude of activation signals through activating NK receptors controls NK cell function, it has not been fully understood how these activating signals are modulated in NK cells. In this study, we found that a scaffold protein, THEMIS2, inhibits activating NK receptor signaling. Overexpression of THEMIS2 attenuated the effector function of human NK cells, whereas knockdown of THEMIS2 enhanced it. Mechanistically, THEMIS2 binds to GRB2 and phosphorylated SHP-1 and SHP-2 at the proximity of activating NK receptors DNAM-1 and NKG2D. Knockdown of THEMIS2 in primary human NK cells promoted the effector functions. Furthermore, Themis2-deficient mice showed low metastatic burden in an NK cell-dependent manner. These findings demonstrate that THEMIS2 has an inhibitory role in the antitumor activity of NK cells, suggesting that THEMIS2 might be a potential therapeutic target for NK cell-mediated cancer immunotherapy.

The Clinical Significance and Involvement in Molecular Cancer Processes of Chemokine CXCL1 in Selected Tumors

Chemokines play a key role in cancer processes, with CXCL1 being a well-studied example. Due to the lack of a complete summary of CXCL1’s role in cancer in the literature, in this study, we examine the significance of CXCL1 in various cancers such as bladder, glioblastoma, hemangioendothelioma, leukemias, Kaposi’s sarcoma, lung, osteosarcoma, renal, and skin cancers (malignant melanoma, basal cell carcinoma, and squamous cell carcinoma), along with thyroid cancer. We focus on understanding how CXCL1 is involved in the cancer processes of these specific types of tumors. We look at how CXCL1 affects cancer cells, including their proliferation, migration, EMT, and metastasis. We also explore how CXCL1 influences other cells connected to tumors, like promoting angiogenesis, recruiting neutrophils, and affecting immune cell functions. Additionally, we discuss the clinical aspects by exploring how CXCL1 levels relate to cancer staging, lymph node metastasis, patient outcomes, chemoresistance, and radioresistance.

Tracking of Nascent Matrix Deposition during Muscle Stem Cell Activation across Lifespan Using Engineered Hydrogels.

Adult stem cells occupy a niche that contributes to their function, but how stem cells rebuild their microenvironment after injury remains an open-ended question. Herein, biomaterial-based systems and metabolic labeling are utilized to evaluate how skeletal muscle stem cells deposit extracellular matrix. Muscle stem cells and committed myoblasts are observed to generate less nascent matrix than muscle resident fibro-adipogenic progenitors. When cultured on substrates that matched the stiffness of physiological uninjured and injured muscles, muscle stem cells increased nascent matrix deposition with activation kinetics. Reducing the ability to deposit nascent matrix by an inhibitor of vesicle trafficking (Exo-1) attenuated muscle stem cell function and mimicked impairments observed from muscle stem cells isolated from old muscles. Old muscle stem cells are observed to deposit less nascent matrix than young muscle stem cells, which is rescued with therapeutic supplementation of insulin-like growth factors. These results highlight the role of nascent matrix production with muscle stem cell activation.

Mammalian Cell Membrane Hybrid Polymersomes for mRNA Delivery.

Cell membranes are structures essential to the cell function and adaptation. Recent studies have targeted cell membranes to identify their protective and interactive properties. Leveraging these attributes of cellular membranes and their application to vaccine delivery is gaining increasing prominence. This study aimed to fuse synthetic polymeric nanoparticles with cell membranes to develop cell membrane hybrid polymersomes (HyPSomes) for enhanced vaccine delivery. We designed a platform to hybridize cell membranes with methoxy-poly(ethylene glycol)-block-polylactic acid nanoparticles by using the properties of both components. The formed HyPSomes were optimized by using dynamic light scattering, transmission electron microscopy, and Förster resonance energy transfer, and their stability was confirmed. The synthesized HyPSomes replicated the antigenic surface of the source cells and possessed the stability and efficacy of synthetic nanoparticles. These HyPSomes demonstrated enhanced cellular uptake and translation efficiency and facilitated endosome escape. HyPSomes showed outstanding capabilities for the delivery of foreign mRNAs to antigen-presenting cells. HyPSomes may serve as vaccine delivery systems by bridging the gap between synthetic and natural systems. These systems could be used in other contexts, e.g., diagnostics and drug delivery.

Hit discovery of potential CDK8 inhibitors and analysis of amino acid mutations for cancer therapy through computer-aided drug discovery

Cyclin-dependent kinase 8 (CDK8) has emerged as a promising target for inhibiting cancer cell function, intensifying efforts towards the development of CDK8 inhibitors as potential cancer therapeutics. Mutations in CDK8, a protein kinase, are also implicated as a primary factor associated with tumor formation. In this study, we identified potential inhibitors through virtual screening for CDK8 and single amino acid mutations in CDK8, namely D173A (Aspartate 173 mutate to Alanine), D189N (Aspartate 189 mutate to Asparagine), T196A (Threonine 196 mutate to Alanine) and T196D (Threonine 196 mutate to Aspartate). Four databases (CHEMBEL, ZINC, MCULE, and MolPort) containing 65,209,131 molecules have been searched to identify new inhibitors for CDK8 and its single mutations. In the first step, structure-based pharmacophore modeling in the Pharmit server was used to select the compounds to know the inhibitors. Then molecules with better predicted drug-like molecule properties were selected. The final filter used to select more effective inhibitors among the previously selected molecules was molecular docking. Finally, 13 hits for CDK8, 11 hits for D173A, 11 hits for D189N, 15 hits for T196A, and 12 hits for T196D were considered potential inhibitors. A majority of the virtual screening hits exhibited satisfactorily predict pharmacokinetic characteristics and toxicity properties.

Semmaphorin 3 A causes immune suppression by inducing cytoskeletal paralysis in tumour-specific CD8+ T cells.

Semaphorin-3A (SEMA3A) functions as a chemorepulsive signal during development and can affect T cells by altering their filamentous actin (F-actin) cytoskeleton. The exact extent of these effects on tumour-specific T cells are not completely understood. Here we demonstrate that Neuropilin-1 (NRP1) and Plexin-A1 and Plexin-A4 are upregulated on stimulated CD8+ T cells, allowing tumour-derived SEMA3A to inhibit T cell migration and assembly of the immunological synapse. Deletion of NRP1 in both CD4+ and CD8+ T cells enhance CD8+ T-cell infiltration into tumours and restricted tumour growth in animal models. Conversely, over-expression of SEMA3A inhibit CD8+ T-cell infiltration. We further show that SEMA3A affects CD8+ T cell F-actin, leading to inhibition of immune synapse formation and motility. Examining a clear cell renal cell carcinoma patient cohort, we find that SEMA3A expression is associated with reduced survival, and that T-cells appear trapped in SEMA3A rich regions. Our study establishes SEMA3A as an inhibitor of effector CD8+ T cell tumour infiltration, suggesting that blocking NRP1 could improve T cell function in tumours.

CRISPR-Cas gene knockouts to optimize engineered T cells for cancer immunotherapy.

While CAR-T and tgTCR-T therapies have exhibited noteworthy and promising outcomes in hematologic and solid tumors respectively, a set of distinct challenges remains. Consequently, the quest for novel strategies has become imperative to safeguard and more effectively release the full functions of engineered T cells. These factors are intricately linked to the success of adoptive cell therapy. Recently, CRISPR-based technologies have emerged as a major breakthrough for maintaining T cell functions. These technologies have allowed the discovery of T cells' negative regulators such as specific cell-surface receptors, cell-signaling proteins, and transcription factors that are involved in the development or maintenance of T cell dysfunction. By employing a CRISPR-genic invalidation approach to target these negative regulators, it has become possible to prevent the emergence of hypofunctional T cells. This review revisits the establishment of the dysfunctional profile of T cells before delving into a comprehensive summary of recent CRISPR-gene invalidations, with each invalidation contributing to the enhancement of engineered T cells' antitumor capacities. The narrative unfolds as we explore how these advancements were discovered and identified, marking a significant advancement in the pursuit of superior adoptive cell therapy.

β cell acetate production and release are negligible

ABSTRACT Background Studies suggest that short chain fatty acids (SCFAs), which are primarily produced from fermentation of fiber, regulate insulin secretion through free fatty acid receptors 2 and 3 (FFA2 and FFA3). As these are G-protein coupled receptors (GPCRs), they have potential therapeutic value as targets for treating type 2 diabetes (T2D). The exact mechanism by which these receptors regulate insulin secretion and other aspects of pancreatic β cell function is unclear. It has been reported that glucose-dependent release of acetate from pancreatic β cells negatively regulates glucose stimulated insulin secretion. While these data raise the possibility of acetate’s potential autocrine action on these receptors, these findings have not been independently confirmed, and multiple concerns exist with this observation, particularly the lack of specificity and precision of the acetate detection methodology used. Methods Using Min6 cells and mouse islets, we assessed acetate and pyruvate production and secretion in response to different glucose concentrations, via liquid chromatography mass spectrometry. Results Using Min6 cells and mouse islets, we showed that both intracellular pyruvate and acetate increased with high glucose conditions; however, intracellular acetate level increased only slightly and exclusively in Min6 cells but not in the islets. Further, extracellular acetate levels were not affected by the concentration of glucose in the incubation medium of either Min6 cells or islets. Conclusions Our findings do not substantiate the glucose-dependent release of acetate from pancreatic β cells, and therefore, invalidate the possibility of an autocrine inhibitory effect on glucose stimulated insulin secretion.

Purified Granulocyte Concentrates from Buffy Coats with Extended Storage Time

&lt;b&gt;&lt;i&gt;Background:&lt;/i&gt;&lt;/b&gt; Granulocyte concentrates (GCs) are usually prepared by single-donor apheresis after G-CSF pretreatment and have to be transfused within 24 h after cell collection because of the rapid decrease in pH and cell survival due to high lactate production by red blood cell contamination. GCs pooled from buffy coats of whole blood donations could improve the availability of these products. Methods to reduce red blood cell and platelet contamination may improve storability. We developed a manufacturing process for pooled GCs and investigated cell viability and functionality over time. &lt;b&gt;&lt;i&gt;Methods:&lt;/i&gt;&lt;/b&gt; Six ABO blood group-identical buffy coats were pooled. Subsequently, the red blood cells spontaneously sedimented after the addition of hydroxyethyl starch. The resulting leukocyte-enriched supernatant was washed twice with saline to reduce platelets and was resuspended in ABO-identical donor plasma. The leukocyte concentrate was transferred to a platelet storage bag and stored up to 72 h at 20–24°C w/o agitation. Cell count and viability, pH, blood gases, phagocytosis, and oxidative burst activity were monitored. &lt;b&gt;&lt;i&gt;Results:&lt;/i&gt;&lt;/b&gt; The number of red blood cells and platelets was reduced to 0.4% and 6.1% of the baseline levels. About 50% of the original present leukocytes could be extracted (&lt;i&gt;n&lt;/i&gt; = 76). In the course of 72 h of storage, there were no significant changes in white blood cell counts (&lt;i&gt;p&lt;/i&gt; = 0.12). The viability exceeded 98% during the entire period. The rate of granulocytes performing phagocytosis and oxidative burst remained above 95% anytime. &lt;b&gt;&lt;i&gt;Conclusion:&lt;/i&gt;&lt;/b&gt; GCs prepared from pooled buffy coats provide a precious alternative to granulocytes obtained from apheresis. Reduction of red blood cells and platelets by more than 90% extends the maximum shelf life of GCs from 24 h to 72 h. For a therapeutic dose of at least 1 × 10&lt;sup&gt;10&lt;/sup&gt; granulocytes, 15–20 buffy coats are required.

Thymic Mimetic Cells: Ontogeny as Immunology.

Medullary thymic epithelial cells (mTECs) generate immunological self-tolerance by ectopically expressing peripheral-tissue antigens (PTAs) within the thymus to preview the peripheral self to maturing Tcells. Recent work, drawing inspiration from old histological observations, has shown that subtypes of mTECs, collectively termed mimetic cells, co-opt developmental programs from throughout the organism to express biologically coherent groups of PTAs. Here, we review key aspects of mimetic cells, especially as they relate to the larger contexts of molecular, cellular, developmental, and evolutionary biology. We highlight lineage-defining transcription factors as key regulators of mimetic cells and speculate as to what other factors, including Aire and the chromatin potential of mTECs, permit mimetic cell differentiation and function. Last, we consider what mimetic cells can teach us about not only the thymus but also other tissues.